Structured Review

Roche enhancer mutated hek293t cell lines
Mutating the TT > A enhancer region by TALEN engineering impairs TNFAIP3 expression A. Quantitative RT-PCR analysis of TNFAIP3 mRNA expression normalized to control HMBS mRNA in control and clonal mutant <t>HEK293T</t> cell lines. B. Western blotting analysis of A20 protein normalized to β-actin in parental (control) and clonal mutant HEK293T cell lines. Statistical significance was calculated using paired student t-test. For all experiments: n = 3, *p
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1) Product Images from "TALEN-mediated enhancer knockout influences TNFAIP3 gene expression and mimics a molecular phenotype associated with systemic lupus erythematosus"

Article Title: TALEN-mediated enhancer knockout influences TNFAIP3 gene expression and mimics a molecular phenotype associated with systemic lupus erythematosus

Journal: Genes and immunity

doi: 10.1038/gene.2016.4

Mutating the TT > A enhancer region by TALEN engineering impairs TNFAIP3 expression A. Quantitative RT-PCR analysis of TNFAIP3 mRNA expression normalized to control HMBS mRNA in control and clonal mutant HEK293T cell lines. B. Western blotting analysis of A20 protein normalized to β-actin in parental (control) and clonal mutant HEK293T cell lines. Statistical significance was calculated using paired student t-test. For all experiments: n = 3, *p
Figure Legend Snippet: Mutating the TT > A enhancer region by TALEN engineering impairs TNFAIP3 expression A. Quantitative RT-PCR analysis of TNFAIP3 mRNA expression normalized to control HMBS mRNA in control and clonal mutant HEK293T cell lines. B. Western blotting analysis of A20 protein normalized to β-actin in parental (control) and clonal mutant HEK293T cell lines. Statistical significance was calculated using paired student t-test. For all experiments: n = 3, *p

Techniques Used: Expressing, Quantitative RT-PCR, Mutagenesis, Western Blot

Design of TALENs for targeting the TT > A enhancer A. Location of 5’ and 3’ TALEN nuclease binding sites. B. Western blotting of transfected HA-tagged 5’ TALEN arm and FLAG-tagged 3’ TALEN arm in HEK293T cells. C. Location of TALEN-mediated mutations of the TT > A enhancer in two HEK293T cell lines. Mutant 1 has a 26 base pair deletion of the TT > A enhancer including the NF-κB and SATB1 binding sites. Mutant 2 has a 9 base pair deletion and 4 base pair insertion (indel) of the TT > A enhancer that disrupts the NF-κB binding site. Red letters in A and C indicate the SLE risk TT > A polymorphisms.
Figure Legend Snippet: Design of TALENs for targeting the TT > A enhancer A. Location of 5’ and 3’ TALEN nuclease binding sites. B. Western blotting of transfected HA-tagged 5’ TALEN arm and FLAG-tagged 3’ TALEN arm in HEK293T cells. C. Location of TALEN-mediated mutations of the TT > A enhancer in two HEK293T cell lines. Mutant 1 has a 26 base pair deletion of the TT > A enhancer including the NF-κB and SATB1 binding sites. Mutant 2 has a 9 base pair deletion and 4 base pair insertion (indel) of the TT > A enhancer that disrupts the NF-κB binding site. Red letters in A and C indicate the SLE risk TT > A polymorphisms.

Techniques Used: TALENs, Binding Assay, Western Blot, Transfection, Mutagenesis

TALEN mutagenesis of the TT > A enhancer reduces interaction with the TNFAIP3 promoter A. Schematic representation of the allele-specific 3C and clonal assay analyses of the engineered heterozygous clonal mutant HEK293T cell lines. Wild type (WT) allele experiences higher frequencies of long-range DNA looping, thus higher proportions of 3C events are observed relative to Mutant 1 or Mutant 2 alleles. B. Genomic DNA isolated from 72 clonal expansions from Mutant 1 or Mutant 2 HEK293T cells without 3C was analyzed for wild type and mutant allele frequencies. The expected clonal frequency of approximately 50% wild type and 50% mutant allele was observed. C. Analysis of 72 mutant 1 or 2 clonal expansions from the 3C capture were analyzed for relative crosslinking frequencies between wild-type or mutated TT > A enhancer and TNFAIP3 promoter. P values were calculated using the Fisher’s exact test and are as indicated.
Figure Legend Snippet: TALEN mutagenesis of the TT > A enhancer reduces interaction with the TNFAIP3 promoter A. Schematic representation of the allele-specific 3C and clonal assay analyses of the engineered heterozygous clonal mutant HEK293T cell lines. Wild type (WT) allele experiences higher frequencies of long-range DNA looping, thus higher proportions of 3C events are observed relative to Mutant 1 or Mutant 2 alleles. B. Genomic DNA isolated from 72 clonal expansions from Mutant 1 or Mutant 2 HEK293T cells without 3C was analyzed for wild type and mutant allele frequencies. The expected clonal frequency of approximately 50% wild type and 50% mutant allele was observed. C. Analysis of 72 mutant 1 or 2 clonal expansions from the 3C capture were analyzed for relative crosslinking frequencies between wild-type or mutated TT > A enhancer and TNFAIP3 promoter. P values were calculated using the Fisher’s exact test and are as indicated.

Techniques Used: Mutagenesis, Clone Assay, Isolation

TALEN mutagenesis of the TT > A enhancer region delayed TNFα-inducible inactivation of NF-κB signaling A. Western blotting of phospho-IκBα at designated time points following TNFα stimulation of parental (wild type) and clonal mutant HEK293T cells. B. Densitometry analyses of Western blotting data represented in (A) normalized to β-actin levels; n = 3, P value calculated by two-way ANOVA and are as indicated.
Figure Legend Snippet: TALEN mutagenesis of the TT > A enhancer region delayed TNFα-inducible inactivation of NF-κB signaling A. Western blotting of phospho-IκBα at designated time points following TNFα stimulation of parental (wild type) and clonal mutant HEK293T cells. B. Densitometry analyses of Western blotting data represented in (A) normalized to β-actin levels; n = 3, P value calculated by two-way ANOVA and are as indicated.

Techniques Used: Mutagenesis, Western Blot

2) Product Images from "Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth"

Article Title: Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth

Journal: The EMBO Journal

doi: 10.15252/embj.201899322

Mechanically stretched human LECs have more VEGFR3‐β1 integrin and less ILK‐β1 integrin interactions LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 min. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (−) mechanical stretch ( n = 6 independent stretch chambers), * P = 0.039. Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 min and used for immunoprecipitation (IP) of HA‐tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates. Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (−) mechanical stretch; normalised to the respective amount of HA‐tagged β1 integrin ( n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers), * P = 0.0007. Data information: Data are presented as means ± SEM, unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.
Figure Legend Snippet: Mechanically stretched human LECs have more VEGFR3‐β1 integrin and less ILK‐β1 integrin interactions LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 min. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (−) mechanical stretch ( n = 6 independent stretch chambers), * P = 0.039. Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 min and used for immunoprecipitation (IP) of HA‐tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates. Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (−) mechanical stretch; normalised to the respective amount of HA‐tagged β1 integrin ( n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers), * P = 0.0007. Data information: Data are presented as means ± SEM, unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.

Techniques Used: Proximity Ligation Assay, Western Blot, Immunoprecipitation, Two Tailed Test

The lymphatic vascular effect of Ilk deletion strictly depends on β1 integrin Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/+ ;Itgb1 ∆/+ mouse embryo (referred to as “control”) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jugular lymph sac/primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 μm, respectively. Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/∆ ;Itgb1 ∆/+ embryo (referred to as “ILK β1 integrin K.O.”), with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jls/pTD. Scale bars: 500 and 100 μm, respectively. LSM images of cross‐sections through the jls/pTD of E13.5 control and ILK β1 integrin K.O. embryos stained for the proliferation marker phospho‐Histone H3. Arrows point to phospho‐Histone H3‐positive LECs. Scale bars: 20 μm. LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p‐Tyr) on stained cross‐sections through the jls/pTD of control and ILK β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1‐stained area. Scale bars: 10 μm. Number of LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. LEC proliferation as determined by the number of phospho‐Histone H3‐positive LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p‐Tyr) per LEC of E13.5 control or ILK β1 integrin K.O. embryos. Data information: Data are presented as means ± SEM, shown as percentage of control embryos with n = 5 embryos per genotype, unpaired two‐tailed Student's t ‐test.
Figure Legend Snippet: The lymphatic vascular effect of Ilk deletion strictly depends on β1 integrin Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/+ ;Itgb1 ∆/+ mouse embryo (referred to as “control”) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jugular lymph sac/primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 μm, respectively. Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/∆ ;Itgb1 ∆/+ embryo (referred to as “ILK β1 integrin K.O.”), with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jls/pTD. Scale bars: 500 and 100 μm, respectively. LSM images of cross‐sections through the jls/pTD of E13.5 control and ILK β1 integrin K.O. embryos stained for the proliferation marker phospho‐Histone H3. Arrows point to phospho‐Histone H3‐positive LECs. Scale bars: 20 μm. LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p‐Tyr) on stained cross‐sections through the jls/pTD of control and ILK β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1‐stained area. Scale bars: 10 μm. Number of LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. LEC proliferation as determined by the number of phospho‐Histone H3‐positive LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p‐Tyr) per LEC of E13.5 control or ILK β1 integrin K.O. embryos. Data information: Data are presented as means ± SEM, shown as percentage of control embryos with n = 5 embryos per genotype, unpaired two‐tailed Student's t ‐test.

Techniques Used: Staining, Marker, Proximity Ligation Assay, Two Tailed Test

ILK regulates α‐parvin expression in adult human LECs ILK and α‐parvin protein bands in Western blots of lysates from human LECs transfected with control siRNA or ILK siRNAs (ILK1‐3). The GAPDH protein band served as a loading control. ILK protein expression normalised to GAPDH protein expression of siRNA‐transfected human LECs ( n = 9 (control and ILK‐3), n = 10 (ILK‐1) and n = 8 (ILK‐2) independent transfections), * P = 0.0001 (control versus ILK‐1 and control versus ILK‐3), * P = 0.017 (control versus ILK‐2). α‐parvin protein expression normalised to GAPDH protein expression of transfected human LEC ( n = 9 (control, ILK‐2 and ILK‐3) and n = 10 (ILK‐1) independent transfections per siRNA), * P = 0.0002 (control versus ILK‐1), P = 0.075 (control versus ILK‐2), * P = 0.0001 (control versus ILK‐3). ILK and α‐parvin protein bands in Western blots of lysates from human LECs transfected with control siRNA or α‐parvin siRNAs (PARVA1‐3). The GAPDH protein band served as a loading control. α‐parvin protein expression normalised to GAPDH protein expression of transfected human LECs ( n = 16 (control and PARVA‐1), n = 11 (PARVA‐2) and n = 14 (PARVA‐3) independent transfections per siRNA), * P = 0.0001. ILK protein expression normalised to GAPDH protein expression of transfected human LECs ( n = 16 (control), n = 15 (PARVA‐1), n = 12 (PARVA‐2) and n = 14 (PARVA‐3) independent transfections per siRNA). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test. Kruskal–Wallis test with Dunn's multiple comparisons test was additionally performed as a non‐parametric test for (B) with * P = 0.011 (control versus ILK‐1) and * P = 0.0003 (control versus ILK‐3), (C) with * P = 0.006 (control versus ILK‐1) and * P = 0.0003 (control versus ILK‐3), (E) with * P = 0.013 (control versus ILK‐1) and * P = 0.0001 (control versus ILK‐2 and control versus ILK‐3) and (F). Source data are available online for this figure.
Figure Legend Snippet: ILK regulates α‐parvin expression in adult human LECs ILK and α‐parvin protein bands in Western blots of lysates from human LECs transfected with control siRNA or ILK siRNAs (ILK1‐3). The GAPDH protein band served as a loading control. ILK protein expression normalised to GAPDH protein expression of siRNA‐transfected human LECs ( n = 9 (control and ILK‐3), n = 10 (ILK‐1) and n = 8 (ILK‐2) independent transfections), * P = 0.0001 (control versus ILK‐1 and control versus ILK‐3), * P = 0.017 (control versus ILK‐2). α‐parvin protein expression normalised to GAPDH protein expression of transfected human LEC ( n = 9 (control, ILK‐2 and ILK‐3) and n = 10 (ILK‐1) independent transfections per siRNA), * P = 0.0002 (control versus ILK‐1), P = 0.075 (control versus ILK‐2), * P = 0.0001 (control versus ILK‐3). ILK and α‐parvin protein bands in Western blots of lysates from human LECs transfected with control siRNA or α‐parvin siRNAs (PARVA1‐3). The GAPDH protein band served as a loading control. α‐parvin protein expression normalised to GAPDH protein expression of transfected human LECs ( n = 16 (control and PARVA‐1), n = 11 (PARVA‐2) and n = 14 (PARVA‐3) independent transfections per siRNA), * P = 0.0001. ILK protein expression normalised to GAPDH protein expression of transfected human LECs ( n = 16 (control), n = 15 (PARVA‐1), n = 12 (PARVA‐2) and n = 14 (PARVA‐3) independent transfections per siRNA). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test. Kruskal–Wallis test with Dunn's multiple comparisons test was additionally performed as a non‐parametric test for (B) with * P = 0.011 (control versus ILK‐1) and * P = 0.0003 (control versus ILK‐3), (C) with * P = 0.006 (control versus ILK‐1) and * P = 0.0003 (control versus ILK‐3), (E) with * P = 0.013 (control versus ILK‐1) and * P = 0.0001 (control versus ILK‐2 and control versus ILK‐3) and (F). Source data are available online for this figure.

Techniques Used: Expressing, Western Blot, Transfection

ILK controls proliferation, VEGFR3 signalling and VEGFR3‐β1 integrin interactions in human LECs Images of adult human LECs after 1 h of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 μm. LEC proliferation as determined by the number of BrdU‐positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 3 independent transfections per siRNA), * P = 0.032 (control versus ILK‐1), * P = 0.005 (control versus ILK‐2), * P = 0.0003 (control versus ILK‐3). VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 4 (control siRNA, ILK‐1 siRNA and ILK‐3 siRNA) or n = 8 (ILK‐2 siRNA) independent transfections per siRNA), * P = 0.0001 (control versus each siRNA). LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK‐1), * P = 0.024 (control versus ILK‐2), * P = 0.001 (control versus ILK‐3). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test.
Figure Legend Snippet: ILK controls proliferation, VEGFR3 signalling and VEGFR3‐β1 integrin interactions in human LECs Images of adult human LECs after 1 h of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 μm. LEC proliferation as determined by the number of BrdU‐positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 3 independent transfections per siRNA), * P = 0.032 (control versus ILK‐1), * P = 0.005 (control versus ILK‐2), * P = 0.0003 (control versus ILK‐3). VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 4 (control siRNA, ILK‐1 siRNA and ILK‐3 siRNA) or n = 8 (ILK‐2 siRNA) independent transfections per siRNA), * P = 0.0001 (control versus each siRNA). LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK‐1), * P = 0.024 (control versus ILK‐2), * P = 0.001 (control versus ILK‐3). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test.

Techniques Used: BrdU Incorporation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Proximity Ligation Assay

Simplified model of mechanosensitive VEGFR3 signalling and ILK‐controlled lymphatic vascular growth In quiescent LECs, VEGFR3 and β1 integrin are physically separated. ILK directly or indirectly interacts with β1 integrin and connects it to the F‐actin cytoskeleton via intracellular proteins, such as α‐parvin, a component of the IPP complex. Upon mechanical stretch, the complex of β1 integrin and ILK (along with the entire IPP complex) transiently disrupts. This releases β1 integrin, resulting in its interaction with VEGFR3, and thus in increased VEGFR3 tyrosine phosphorylation (“P” in yellow circle). As a consequence, LEC proliferation and lymphatic vascular growth are induced. The absence of ILK results in permanent interaction between VEGFR3 and β1 integrin, leading to upregulated VEGFR3 tyrosine phosphorylation (“P” in yellow circle), LEC proliferation and non‐physiologic lymphatic vascular growth.
Figure Legend Snippet: Simplified model of mechanosensitive VEGFR3 signalling and ILK‐controlled lymphatic vascular growth In quiescent LECs, VEGFR3 and β1 integrin are physically separated. ILK directly or indirectly interacts with β1 integrin and connects it to the F‐actin cytoskeleton via intracellular proteins, such as α‐parvin, a component of the IPP complex. Upon mechanical stretch, the complex of β1 integrin and ILK (along with the entire IPP complex) transiently disrupts. This releases β1 integrin, resulting in its interaction with VEGFR3, and thus in increased VEGFR3 tyrosine phosphorylation (“P” in yellow circle). As a consequence, LEC proliferation and lymphatic vascular growth are induced. The absence of ILK results in permanent interaction between VEGFR3 and β1 integrin, leading to upregulated VEGFR3 tyrosine phosphorylation (“P” in yellow circle), LEC proliferation and non‐physiologic lymphatic vascular growth.

Techniques Used:

3) Product Images from "WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4. WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4"

Article Title: WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4. WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4

Journal: Cancer Medicine

doi: 10.1002/cam4.1837

Association of p27 with peripheral rearrangements of the actin cytoskeleton. A, siRNA oligonucleotides targeting p27 (sip27) or scrambled control siRNAs (Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐p27 antibody. B, MTT assays of S2‐013 and PANC‐1 cells transiently transfected with scrambled control siRNA, ACTN4 siRNA, or p27 siRNA were performed to evaluate cell viability. Data are representative of three independent experiments and are the means ±SD. ABS on Y ‐axis means absorbance at 490 nm and at 630 nm as reference measured with a microplate reader. C, Confocal immunofluorescence microscopic images. Scr‐transfected S2‐013 cells and sip27‐transfected S2‐013 cells were incubated on fibronectin and subsequently stained with anti‐p27 antibody (green) and phalloidin (red). Arrows, peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. D, Quantification of the data shown in B. The values represent the number of cells with fibronectin‐stimulated cell protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P
Figure Legend Snippet: Association of p27 with peripheral rearrangements of the actin cytoskeleton. A, siRNA oligonucleotides targeting p27 (sip27) or scrambled control siRNAs (Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐p27 antibody. B, MTT assays of S2‐013 and PANC‐1 cells transiently transfected with scrambled control siRNA, ACTN4 siRNA, or p27 siRNA were performed to evaluate cell viability. Data are representative of three independent experiments and are the means ±SD. ABS on Y ‐axis means absorbance at 490 nm and at 630 nm as reference measured with a microplate reader. C, Confocal immunofluorescence microscopic images. Scr‐transfected S2‐013 cells and sip27‐transfected S2‐013 cells were incubated on fibronectin and subsequently stained with anti‐p27 antibody (green) and phalloidin (red). Arrows, peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. D, Quantification of the data shown in B. The values represent the number of cells with fibronectin‐stimulated cell protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P

Techniques Used: Transfection, Western Blot, MTT Assay, Immunofluorescence, Incubation, Staining, Derivative Assay

Effects of WAVE2 and ACTN4 on p27 activity. A, Confocal immunofluorescence microscopic images. S2‐013 and HPNE cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐phosphorylated p27 (red) antibodies. Actin filaments were labeled with phalloidin (violet). Arrows, phosphorylated p27 in the nucleus. Blue, DAPI staining. Scale bar, 10 μm. B, S2‐013 and HPNE cells were incubated on fibronectin and fractionated into cytosolic (c) and nuclear (n) fractions. Western blotting of the fractions was performed using anti‐phosphorylated p27 and anti‐p27 antibodies. C, Scr‐transfected S2‐013 cells and siWAVE2‐transfected S2‐013 cells were incubated on fibronectin. Western blotting was performed using anti‐WAVE2, anti‐phosphorylated p27, and anti‐p27 antibodies. D, A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with scrambled control siRNA or WAVE2 siRNA with or without ACTN4 siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies
Figure Legend Snippet: Effects of WAVE2 and ACTN4 on p27 activity. A, Confocal immunofluorescence microscopic images. S2‐013 and HPNE cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐phosphorylated p27 (red) antibodies. Actin filaments were labeled with phalloidin (violet). Arrows, phosphorylated p27 in the nucleus. Blue, DAPI staining. Scale bar, 10 μm. B, S2‐013 and HPNE cells were incubated on fibronectin and fractionated into cytosolic (c) and nuclear (n) fractions. Western blotting of the fractions was performed using anti‐phosphorylated p27 and anti‐p27 antibodies. C, Scr‐transfected S2‐013 cells and siWAVE2‐transfected S2‐013 cells were incubated on fibronectin. Western blotting was performed using anti‐WAVE2, anti‐phosphorylated p27, and anti‐p27 antibodies. D, A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with scrambled control siRNA or WAVE2 siRNA with or without ACTN4 siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies

Techniques Used: Activity Assay, Immunofluorescence, Cell Culture, Labeling, Staining, Incubation, Western Blot, Transfection, Construct

Roles of WAVE2 in the translocation of ACTN4 to actin filaments in cell protrusions. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2, ACTN4, and actin. Rabbit IgG isotype control antibody was used as the control. B, Confocal immunofluorescence microscopic images. Oligonucleotides (siRNAs targeting WAVE2 (siWAVE2) or scrambled siRNAs (Scr) as the negative control) were transiently transfected into S2‐013 cells. Transfected cells were incubated on fibronectin and were subsequently stained with anti‐WAVE2 antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, ACTN4 bound to peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. C, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with WAVE2 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, exogenous WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 µm. D, Oligonucleotides (siRNAs targeting ACTN4 (siACTN4) or Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐ACTN4 antibody. E, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Blue, DAPI staining. Scale bars, 10 µm
Figure Legend Snippet: Roles of WAVE2 in the translocation of ACTN4 to actin filaments in cell protrusions. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2, ACTN4, and actin. Rabbit IgG isotype control antibody was used as the control. B, Confocal immunofluorescence microscopic images. Oligonucleotides (siRNAs targeting WAVE2 (siWAVE2) or scrambled siRNAs (Scr) as the negative control) were transiently transfected into S2‐013 cells. Transfected cells were incubated on fibronectin and were subsequently stained with anti‐WAVE2 antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, ACTN4 bound to peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. C, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with WAVE2 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, exogenous WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 µm. D, Oligonucleotides (siRNAs targeting ACTN4 (siACTN4) or Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐ACTN4 antibody. E, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Blue, DAPI staining. Scale bars, 10 µm

Techniques Used: Translocation Assay, Immunoprecipitation, Cell Culture, Western Blot, Immunofluorescence, Negative Control, Transfection, Incubation, Staining, Construct

Association of WAVE2 with ACTN4. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Rabbit IgG isotype control antibody was used as the control. Proteins within immunoprecipitates were examined on Western blots probed with anti‐WAVE2 antibody. B, Proteins in immunoprecipitates were examined with silver staining. Rabbit IgG isotype control antibody was used as the control. A 100‐kDa band is indicated by the arrow. C, The percent coverage for ACTN4 is represented by the identified peptides in the total protein sequence (accession number NP_004915). D, Immunoprecipitation of WAVE2 or ACTN4 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2 and ACTN4. Rabbit IgG isotype control antibody for WAVE2 and mouse IgG isotype control antibody for ACTN4 was used as controls. E, Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐ACTN4 (red) antibodies. Arrows, WAVE2 co‐localized with ACTN4 in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm
Figure Legend Snippet: Association of WAVE2 with ACTN4. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Rabbit IgG isotype control antibody was used as the control. Proteins within immunoprecipitates were examined on Western blots probed with anti‐WAVE2 antibody. B, Proteins in immunoprecipitates were examined with silver staining. Rabbit IgG isotype control antibody was used as the control. A 100‐kDa band is indicated by the arrow. C, The percent coverage for ACTN4 is represented by the identified peptides in the total protein sequence (accession number NP_004915). D, Immunoprecipitation of WAVE2 or ACTN4 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2 and ACTN4. Rabbit IgG isotype control antibody for WAVE2 and mouse IgG isotype control antibody for ACTN4 was used as controls. E, Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐ACTN4 (red) antibodies. Arrows, WAVE2 co‐localized with ACTN4 in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm

Techniques Used: Immunoprecipitation, Cell Culture, Western Blot, Silver Staining, Sequencing, Immunofluorescence, Labeling, Staining

Subcellular localization of WAVE2 in PDAC cells grown on fibronectin. Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 antibody (green). Actin filaments were labeled with phalloidin (red). Arrows, WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 μm
Figure Legend Snippet: Subcellular localization of WAVE2 in PDAC cells grown on fibronectin. Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 antibody (green). Actin filaments were labeled with phalloidin (red). Arrows, WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 μm

Techniques Used: Immunofluorescence, Cell Culture, Labeling, Staining

Roles of WAVE2 and ACTN4 in forming cell protrusions. A, Confocal immunofluorescence microscopic i mages showing phalloidin‐labeled peripheral actin structures (red) and DAPI‐labeled nuclei (blue) in scrambled control siRNA‐transfected S2‐013 and PANC‐1 cells, and WAVE2 siRNA‐transfected S2‐013 and PANC‐1 cells grown on fibronectin. Arrows, peripheral actin structures in cell protrusions. Scale bars, 10 µm. B, Quantification of data shown in A; the values represent the number of cells with protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P
Figure Legend Snippet: Roles of WAVE2 and ACTN4 in forming cell protrusions. A, Confocal immunofluorescence microscopic i mages showing phalloidin‐labeled peripheral actin structures (red) and DAPI‐labeled nuclei (blue) in scrambled control siRNA‐transfected S2‐013 and PANC‐1 cells, and WAVE2 siRNA‐transfected S2‐013 and PANC‐1 cells grown on fibronectin. Arrows, peripheral actin structures in cell protrusions. Scale bars, 10 µm. B, Quantification of data shown in A; the values represent the number of cells with protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P

Techniques Used: Immunofluorescence, Labeling, Transfection, Derivative Assay

4) Product Images from "Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis"

Article Title: Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis

Journal: Aging (Albany NY)

doi: 10.18632/aging.101740

Chronic rapamycin treatment disrupts spermatogenesis in male mice and inhibits assembly of mTOR complexes. Tissues from adult males were analyzed after weeks of daily i.p. injection with rapamycin or vehicle control beginning at age 7-8 weeks. ( a ) Western blot analysis of phosphorylated S6, AKT, PKCα, and the SGK substrate NDRG1 in testicular extracts from adult males (control, n=3; rapamycin, n=3). ( b ) Chronic rapamycin treatment impairs mTOR complex integrity and activity. Immunoblotting of mTOR immunoprecipitates from testis tissue from adult males (control, n=3; rapamycin, n=3). ( c ) Gross morphology of testis tissue from control or rapamycin (rapa) treated males. ( d ) Testis weight (control, n=5; rapamycin, n=4). Error bars represent SD (***P
Figure Legend Snippet: Chronic rapamycin treatment disrupts spermatogenesis in male mice and inhibits assembly of mTOR complexes. Tissues from adult males were analyzed after weeks of daily i.p. injection with rapamycin or vehicle control beginning at age 7-8 weeks. ( a ) Western blot analysis of phosphorylated S6, AKT, PKCα, and the SGK substrate NDRG1 in testicular extracts from adult males (control, n=3; rapamycin, n=3). ( b ) Chronic rapamycin treatment impairs mTOR complex integrity and activity. Immunoblotting of mTOR immunoprecipitates from testis tissue from adult males (control, n=3; rapamycin, n=3). ( c ) Gross morphology of testis tissue from control or rapamycin (rapa) treated males. ( d ) Testis weight (control, n=5; rapamycin, n=4). Error bars represent SD (***P

Techniques Used: Mouse Assay, Injection, Western Blot, Activity Assay

5) Product Images from "HIV-1 Vpr stimulates NF-?B and AP-1 signaling by activating TAK1"

Article Title: HIV-1 Vpr stimulates NF-?B and AP-1 signaling by activating TAK1

Journal: Retrovirology

doi: 10.1186/1742-4690-11-45

Vpr enhances the phosphorylation of TAK1 following HIV-1 infection. (A) Schematic representation of NLENY1-ES-IRES and NLENY1-ΔVpr viruses. A mutated start codon of vpr gene was introduced in NLENY1-ES to generate NLENY1-ΔVpr. (B-D) Virion-associated Vpr enhances the phosphorylation of TAK1 on Thr-187 in HIV-1 permissive cells. A total of 2 × 10 6 Jurkat cells (B) , THP-1 cells (C) , and THP-1 differentiated macrophage-like cells (D) were infected with VSV-G pseudotyped WT or ΔVpr viruses equivalent to 500 ng p24 in the presence of 5 μg/ml polybrene by spinoculation at 300 xg for 30 min. After another 1.5 hours, cells were pretreated with 15 nM Calyculin A for 5 min. Then cells were harvested by centrifugation at 1000 × g for 3 min at 4°C, washed twice with ice-cold 1xphosphate-buffered saline, lysed in 70 μl lysis buffer, and chilled on ice for 30 min with frequent agitation. Whole cell lysates were subjected to Western blotting and probed with anti-phospho-TAK1 (Thr-187), rabbit anti-TAK1, anti-p24, anti-Vpr, and anti-Tubulin antibodies. (E) Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors. PBMCs were activated with phytohemagglutinin (PHA; 5 mg/ml) and IL-2 (20 U/ml) for 24 h, followed by infection with VSV-G pseudotyped WT or ΔVpr (equivalent to 500 ng p24) in the presence of 5 μg/ml polybrene by spinoculation at 300 xg for 30 min. After two hours, cells were pretreated with 15 nM Calyculin A for 5 min. Whole cell lysates were examined in Western blotting with the indicated antibodies.
Figure Legend Snippet: Vpr enhances the phosphorylation of TAK1 following HIV-1 infection. (A) Schematic representation of NLENY1-ES-IRES and NLENY1-ΔVpr viruses. A mutated start codon of vpr gene was introduced in NLENY1-ES to generate NLENY1-ΔVpr. (B-D) Virion-associated Vpr enhances the phosphorylation of TAK1 on Thr-187 in HIV-1 permissive cells. A total of 2 × 10 6 Jurkat cells (B) , THP-1 cells (C) , and THP-1 differentiated macrophage-like cells (D) were infected with VSV-G pseudotyped WT or ΔVpr viruses equivalent to 500 ng p24 in the presence of 5 μg/ml polybrene by spinoculation at 300 xg for 30 min. After another 1.5 hours, cells were pretreated with 15 nM Calyculin A for 5 min. Then cells were harvested by centrifugation at 1000 × g for 3 min at 4°C, washed twice with ice-cold 1xphosphate-buffered saline, lysed in 70 μl lysis buffer, and chilled on ice for 30 min with frequent agitation. Whole cell lysates were subjected to Western blotting and probed with anti-phospho-TAK1 (Thr-187), rabbit anti-TAK1, anti-p24, anti-Vpr, and anti-Tubulin antibodies. (E) Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors. PBMCs were activated with phytohemagglutinin (PHA; 5 mg/ml) and IL-2 (20 U/ml) for 24 h, followed by infection with VSV-G pseudotyped WT or ΔVpr (equivalent to 500 ng p24) in the presence of 5 μg/ml polybrene by spinoculation at 300 xg for 30 min. After two hours, cells were pretreated with 15 nM Calyculin A for 5 min. Whole cell lysates were examined in Western blotting with the indicated antibodies.

Techniques Used: Infection, Centrifugation, Lysis, Western Blot, Isolation

6) Product Images from "Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation"

Article Title: Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

Journal: Molecular Systems Biology

doi: 10.15252/msb.20145880

Validation of BCL10 linear ubiquitylation A, B Validation of BCR stimulation-dependent linear ubiquitylation of BCL10. BCL10 was immunoprecipitated from BCR-stimulated and unstimulated control A20 cells and immunoblotted with LUB9 antibody that binds to linear ubiquitin (A, the upper panel). From the same cell lysates, linear ubiquitylated proteins were isolated using Met1-SUB and probed with BCL10 (A, the lower panel). The latter approach was also used to confirm BCR-induced linear ubiquitylation of BCL10 in A20.2J cells (B). The blots at the bottom of the figure show the expression levels of BCL10 and actin in the input material used for BCL10 immunoprecipitation and Met1-SUB pull-downs. C BCL10 ubiquitylation is sensitive to linear and K63 linkage-specific deubiquitylases. Linearly ubiquitylated proteins were isolated from BCR stimulated cells with Met1-SUB, treated with the indicated deubiquitylases, and subsequently immunostained with antibodies recognizing BCL10, or ubiquitin. The blots at the bottom of the figures show the expression levels of BCL10 and actin in the input material used for Met1-SUB pull-downs.
Figure Legend Snippet: Validation of BCL10 linear ubiquitylation A, B Validation of BCR stimulation-dependent linear ubiquitylation of BCL10. BCL10 was immunoprecipitated from BCR-stimulated and unstimulated control A20 cells and immunoblotted with LUB9 antibody that binds to linear ubiquitin (A, the upper panel). From the same cell lysates, linear ubiquitylated proteins were isolated using Met1-SUB and probed with BCL10 (A, the lower panel). The latter approach was also used to confirm BCR-induced linear ubiquitylation of BCL10 in A20.2J cells (B). The blots at the bottom of the figure show the expression levels of BCL10 and actin in the input material used for BCL10 immunoprecipitation and Met1-SUB pull-downs. C BCL10 ubiquitylation is sensitive to linear and K63 linkage-specific deubiquitylases. Linearly ubiquitylated proteins were isolated from BCR stimulated cells with Met1-SUB, treated with the indicated deubiquitylases, and subsequently immunostained with antibodies recognizing BCL10, or ubiquitin. The blots at the bottom of the figures show the expression levels of BCL10 and actin in the input material used for Met1-SUB pull-downs.

Techniques Used: Immunoprecipitation, Isolation, Expressing

BCR stimulation induces linear ubiquitylation of BCL10 A Strategy for the identification of BCR-regulated ubiquitylated proteins. SILAC-labeled A20 cells were stimulated with α-IgG F(ab′)2 for the indicated times, and ubiquitylated proteins were affinity-enriched using tandem ubiquitin-binding entities (TUBE)-based pull-downs B Interaction network of BCR-induced ubiquitylated proteins. The network shows interaction among the proteins that were significantly enriched in TUBE pull-downs from BCR-stimulated cells compared to proteins pulled down from mock-treated control cells. Blue circles indicate the proteins which were also identified as BCR-upregulated in our di-Gly dataset. The dotted box indicates members of the CBM complex. C BCR stimulation increases the abundance of Met1-linked ubiquitin (Met1-UB). The plot shows the SILAC ratios of peptide corresponding to Met1-UB chains in the TUBE pull-downs from (A). The error bars represent mean ± SEM of the SILAC ratios. D BCR stimulation increases the abundance of linear ubiquitin peptide. The MS spectrum shows the relative abundance of the peptide (GGMQIFVK) corresponding to Met1-UB in TUBE pull-downs from unstimulated cells, or after 5 or 15 min of BCR stimulation. E, F Identification of BCR-regulated linear ubiquitylated proteins. Schematic presentation of the strategy used for SILAC-based Met1-SUB pull-downs (E). The scatter plot shows proteins identified in Met1-SUB pull-downs (F). The gray background indicates proteins that were identified in the pull-downs independent of BCR stimulation, and the red dots indicate proteins that were enriched after 5 and 15 min of BCR stimulation.
Figure Legend Snippet: BCR stimulation induces linear ubiquitylation of BCL10 A Strategy for the identification of BCR-regulated ubiquitylated proteins. SILAC-labeled A20 cells were stimulated with α-IgG F(ab′)2 for the indicated times, and ubiquitylated proteins were affinity-enriched using tandem ubiquitin-binding entities (TUBE)-based pull-downs B Interaction network of BCR-induced ubiquitylated proteins. The network shows interaction among the proteins that were significantly enriched in TUBE pull-downs from BCR-stimulated cells compared to proteins pulled down from mock-treated control cells. Blue circles indicate the proteins which were also identified as BCR-upregulated in our di-Gly dataset. The dotted box indicates members of the CBM complex. C BCR stimulation increases the abundance of Met1-linked ubiquitin (Met1-UB). The plot shows the SILAC ratios of peptide corresponding to Met1-UB chains in the TUBE pull-downs from (A). The error bars represent mean ± SEM of the SILAC ratios. D BCR stimulation increases the abundance of linear ubiquitin peptide. The MS spectrum shows the relative abundance of the peptide (GGMQIFVK) corresponding to Met1-UB in TUBE pull-downs from unstimulated cells, or after 5 or 15 min of BCR stimulation. E, F Identification of BCR-regulated linear ubiquitylated proteins. Schematic presentation of the strategy used for SILAC-based Met1-SUB pull-downs (E). The scatter plot shows proteins identified in Met1-SUB pull-downs (F). The gray background indicates proteins that were identified in the pull-downs independent of BCR stimulation, and the red dots indicate proteins that were enriched after 5 and 15 min of BCR stimulation.

Techniques Used: Labeling, Binding Assay, Mass Spectrometry

Functional analysis of BCL10 linear ubiquitylation A HOIP interacts with BCL10. A20.2J HOIP −/− cells reconstituted with the full-length FLAG-HOIP were stimulated with α-IgG for the indicated times, HOIP was immunoprecipitated with α-FLAG antibody, and the immunoprecipitates were immunostained with BCL10 antibody. The amounts of immunoprecipitated HOIP, and equal expression of FLAG-HOIP and BCL10 in whole-cell lysates used for the IPs, are shown in the lower blots. The asterisk indicates unmodified BCL10. B HOIP is required for BCL10 linear ubiquitylation. A20.2J, A20.2J HOIP −/− , and A20.2J HOIP −/− cells expressing the HOIP full-length or the indicated HOIP mutants were stimulated with α-IgG followed by Met1-SUB pull-down and immunoblotting as described in Fig 7A . C TRAF6 is required for BCL10 linear ubiquitylation. A20.2J wild-type and TRAF6 −/− cells were stimulated with α-IgG for 10 min, and linear ubiquitylated proteins were pulled down with Met1-SUB and immunoblotted with BCL10 and ubiquitin antibodies. Equal expression of BCL10 and actin was verified in the input material. D HOIP and TRAF6 function is important for BCR-induced IκB phosphorylation. A20.2J wild-type, HOIP −/− , TRAF6 −/− , and HOIP −/− cells expressing HOIP ΔRBR were stimulated with α-IgG for the indicated time points, and lysates were immunostained with pIκB and IκB antibodies. Actin and vinculin staining serves as loading control. E, F BCL10 linear ubiquitin fusion protein activates NF-κB. HEK293T cells were co-transfected with increasing amount (0.5, 1 or 2 μg) of BCL10, or BCL10-LinUBL73P-4X construct together with pNF-κB Luc and pRL-TK Renilla. NF-κB transcriptional activity was measured 24 h later using Dual-Glo® Luciferase Assay System (Promega). In (F), NF-κB transcriptional activity was measured in HEK293T cells co-transfected with 1 μg of the indicated plasmids. Error bars indicate mean ± SEM of 3 (E) or 2 (F) independent experiments. Statistical significance was determined by two-tailed Student's t -test.
Figure Legend Snippet: Functional analysis of BCL10 linear ubiquitylation A HOIP interacts with BCL10. A20.2J HOIP −/− cells reconstituted with the full-length FLAG-HOIP were stimulated with α-IgG for the indicated times, HOIP was immunoprecipitated with α-FLAG antibody, and the immunoprecipitates were immunostained with BCL10 antibody. The amounts of immunoprecipitated HOIP, and equal expression of FLAG-HOIP and BCL10 in whole-cell lysates used for the IPs, are shown in the lower blots. The asterisk indicates unmodified BCL10. B HOIP is required for BCL10 linear ubiquitylation. A20.2J, A20.2J HOIP −/− , and A20.2J HOIP −/− cells expressing the HOIP full-length or the indicated HOIP mutants were stimulated with α-IgG followed by Met1-SUB pull-down and immunoblotting as described in Fig 7A . C TRAF6 is required for BCL10 linear ubiquitylation. A20.2J wild-type and TRAF6 −/− cells were stimulated with α-IgG for 10 min, and linear ubiquitylated proteins were pulled down with Met1-SUB and immunoblotted with BCL10 and ubiquitin antibodies. Equal expression of BCL10 and actin was verified in the input material. D HOIP and TRAF6 function is important for BCR-induced IκB phosphorylation. A20.2J wild-type, HOIP −/− , TRAF6 −/− , and HOIP −/− cells expressing HOIP ΔRBR were stimulated with α-IgG for the indicated time points, and lysates were immunostained with pIκB and IκB antibodies. Actin and vinculin staining serves as loading control. E, F BCL10 linear ubiquitin fusion protein activates NF-κB. HEK293T cells were co-transfected with increasing amount (0.5, 1 or 2 μg) of BCL10, or BCL10-LinUBL73P-4X construct together with pNF-κB Luc and pRL-TK Renilla. NF-κB transcriptional activity was measured 24 h later using Dual-Glo® Luciferase Assay System (Promega). In (F), NF-κB transcriptional activity was measured in HEK293T cells co-transfected with 1 μg of the indicated plasmids. Error bars indicate mean ± SEM of 3 (E) or 2 (F) independent experiments. Statistical significance was determined by two-tailed Student's t -test.

Techniques Used: Functional Assay, Immunoprecipitation, Expressing, Staining, Transfection, Construct, Activity Assay, Luciferase, Two Tailed Test

Strategy for proteomic analysis of BCR signaling Analysis of BCR-induced phosphorylation and ubiquitylation. A20 cells were isotopically labeled using the SILAC approach. Control “light” labeled cells were mock-treated, and “medium” and “heavy” labeled cells were stimulated with α-IgG F(ab′)2 for 5 and 15 min, respectively. Di-Gly-modified (ubiquitylated), tyrosine-phosphorylated peptides were enriched sequentially using di-Gly-lysine- and phosphotyrosine-specific antibodies. Phosphorylated peptides were separately enriched using TiO 2 -based chromatography. All samples were analyzed using high-resolution mass spectrometry. Strategy for analyzing the dynamics of BCR signalosomes. “Medium” and “heavy” SILAC-labeled A20 cells were stimulated with biotinylated α-IgG F(ab′)2 for 5 and 15 min, respectively. Control cells (labeled with “light” SILAC) were mock-treated. Proteins associated with biotinylated α-IgG F(ab′)2-bound BCR signalosomes were affinity-enriched using streptavidin, separated by SDS–PAGE, and analyzed by LC-MS/MS. Validation of BCR signaling activation. Stimulation of BCR signaling in A20 cells was confirmed using the indicated phosphorylation site-specific antibodies that recognize the activated forms of BTK, ERK1/2, and AKT kinases.
Figure Legend Snippet: Strategy for proteomic analysis of BCR signaling Analysis of BCR-induced phosphorylation and ubiquitylation. A20 cells were isotopically labeled using the SILAC approach. Control “light” labeled cells were mock-treated, and “medium” and “heavy” labeled cells were stimulated with α-IgG F(ab′)2 for 5 and 15 min, respectively. Di-Gly-modified (ubiquitylated), tyrosine-phosphorylated peptides were enriched sequentially using di-Gly-lysine- and phosphotyrosine-specific antibodies. Phosphorylated peptides were separately enriched using TiO 2 -based chromatography. All samples were analyzed using high-resolution mass spectrometry. Strategy for analyzing the dynamics of BCR signalosomes. “Medium” and “heavy” SILAC-labeled A20 cells were stimulated with biotinylated α-IgG F(ab′)2 for 5 and 15 min, respectively. Control cells (labeled with “light” SILAC) were mock-treated. Proteins associated with biotinylated α-IgG F(ab′)2-bound BCR signalosomes were affinity-enriched using streptavidin, separated by SDS–PAGE, and analyzed by LC-MS/MS. Validation of BCR signaling activation. Stimulation of BCR signaling in A20 cells was confirmed using the indicated phosphorylation site-specific antibodies that recognize the activated forms of BTK, ERK1/2, and AKT kinases.

Techniques Used: Labeling, Modification, Chromatography, Mass Spectrometry, SDS Page, Liquid Chromatography with Mass Spectroscopy, Activation Assay

Proteomic analysis of BCR signalosomes A The Venn diagram shows the overlap between the proteins that were enriched in BCR signalosomes at 5 and 15 min. B A network view of proteins present in BCR signalosome. Proteins are color-coded based on their association with BCR signalosomes at 5 or 15 min after BCR cross-linking. C Validation of the dynamic association of proteins with BCR signalosomes. A20 cells were stimulated with biotinylated α-IgG F(ab′)2 for the indicated time points, the signalosomes were isolated by streptavidin pull-downs, and enrichment of the indicated proteins was analyzed by immunoblotting. D, E ANKRD13A associates with BCR signalosomes. A20 cells were transiently transfected with GFP-tagged ANKRD13A WT and the ANRKD13A ΔUIM mutant (D), or ANKRD13A WT and ANKRD13A UIM3/4 mutant (E). BCR signalosomes were isolated as described in (C), and the enrichment of ANKRD13A was probed using α-GFP antibody. The expression of GFP-tagged ANKRD13A WT, ANRKD13A ΔUIM, and ANKRD13A UIM3/4 mutants in the input material was verified.
Figure Legend Snippet: Proteomic analysis of BCR signalosomes A The Venn diagram shows the overlap between the proteins that were enriched in BCR signalosomes at 5 and 15 min. B A network view of proteins present in BCR signalosome. Proteins are color-coded based on their association with BCR signalosomes at 5 or 15 min after BCR cross-linking. C Validation of the dynamic association of proteins with BCR signalosomes. A20 cells were stimulated with biotinylated α-IgG F(ab′)2 for the indicated time points, the signalosomes were isolated by streptavidin pull-downs, and enrichment of the indicated proteins was analyzed by immunoblotting. D, E ANKRD13A associates with BCR signalosomes. A20 cells were transiently transfected with GFP-tagged ANKRD13A WT and the ANRKD13A ΔUIM mutant (D), or ANKRD13A WT and ANKRD13A UIM3/4 mutant (E). BCR signalosomes were isolated as described in (C), and the enrichment of ANKRD13A was probed using α-GFP antibody. The expression of GFP-tagged ANKRD13A WT, ANRKD13A ΔUIM, and ANKRD13A UIM3/4 mutants in the input material was verified.

Techniques Used: Isolation, Transfection, Mutagenesis, Expressing

7) Product Images from "CCDC88A, a prognostic factor for human pancreatic cancers, promotes the motility and invasiveness of pancreatic cancer cells"

Article Title: CCDC88A, a prognostic factor for human pancreatic cancers, promotes the motility and invasiveness of pancreatic cancer cells

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-016-0466-0

Co-localization of CCDC88A with actin-filaments in cell protrusions. a . Immunoprecipitation (IP) of CCDC88A from S2-013 cells cultured on fibronectin. Proteins within the immunoprecipitates were examined by western blotting. The blots were probed with antibodies against CCDC88A and actin. Mouse IgG isotype control antibody was used as an isotype control. b . Confocal immunofluorescence microscopic images show nuclear DAPI staining ( blue ), abundant cytoplasmic CCDC88A, and the accumulation of CCDC88A ( green ) in membrane protrusions of fibronectin-stimulated S2-013 cells. Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Bar, 10 μm. c . Confocal immunofluorescence microscopic images of S2-013 and PANC-1 cells that were pretreated with 100 μM Cytochalasin D for 12 h and were then incubated on fibronectin. Cells were stained with anti-CCDC88A antibody ( green ). Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Blue, DAPI staining. Bars, 10 μm. d . Confocal immunofluorescence microscopic images. CCDC88A -siRNA transfected S2-013 and PANC-1 cells, which were transiently transfected with a myc-tagged CCDC88A-rescue construct, were pretreated with 100 μM Cytochalasin D for 12 h, and were subsequently incubated on fibronectin. Cells were stained with anti-myc antibody ( green ). Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Blue, DAPI staining. Bars, 10 μm
Figure Legend Snippet: Co-localization of CCDC88A with actin-filaments in cell protrusions. a . Immunoprecipitation (IP) of CCDC88A from S2-013 cells cultured on fibronectin. Proteins within the immunoprecipitates were examined by western blotting. The blots were probed with antibodies against CCDC88A and actin. Mouse IgG isotype control antibody was used as an isotype control. b . Confocal immunofluorescence microscopic images show nuclear DAPI staining ( blue ), abundant cytoplasmic CCDC88A, and the accumulation of CCDC88A ( green ) in membrane protrusions of fibronectin-stimulated S2-013 cells. Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Bar, 10 μm. c . Confocal immunofluorescence microscopic images of S2-013 and PANC-1 cells that were pretreated with 100 μM Cytochalasin D for 12 h and were then incubated on fibronectin. Cells were stained with anti-CCDC88A antibody ( green ). Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Blue, DAPI staining. Bars, 10 μm. d . Confocal immunofluorescence microscopic images. CCDC88A -siRNA transfected S2-013 and PANC-1 cells, which were transiently transfected with a myc-tagged CCDC88A-rescue construct, were pretreated with 100 μM Cytochalasin D for 12 h, and were subsequently incubated on fibronectin. Cells were stained with anti-myc antibody ( green ). Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Blue, DAPI staining. Bars, 10 μm

Techniques Used: Immunoprecipitation, Cell Culture, Western Blot, Immunofluorescence, Staining, Labeling, Incubation, Transfection, Construct

8) Product Images from "Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease"

Article Title: Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017953

Triton X-100 solubilization of striatal lysates from A53T α-Syn mutant and age-matched control animals. Striatal lysates from A53T α-Syn mutant mice and control, non-Tg mice [4 animals per group] were extracted with Triton X-100 as described in Methods. Proteins in Triton X-100-soluble and Triton X-100-insoluble fractions were measured by Western blots. Blots show representative gels while the bar graphs are composites summarized from all animals, expressed as percent of control, non-Tg mice. (A) β-actin was added as a loading control. (B) p-GSK-3β was normalized to GSK-3β from within each fraction on the blot. (C) pSer202, pSer262 and PSer396/404 were all normalized to total Tau in each fraction, and β-actin was added as a loading control. *,  P
Figure Legend Snippet: Triton X-100 solubilization of striatal lysates from A53T α-Syn mutant and age-matched control animals. Striatal lysates from A53T α-Syn mutant mice and control, non-Tg mice [4 animals per group] were extracted with Triton X-100 as described in Methods. Proteins in Triton X-100-soluble and Triton X-100-insoluble fractions were measured by Western blots. Blots show representative gels while the bar graphs are composites summarized from all animals, expressed as percent of control, non-Tg mice. (A) β-actin was added as a loading control. (B) p-GSK-3β was normalized to GSK-3β from within each fraction on the blot. (C) pSer202, pSer262 and PSer396/404 were all normalized to total Tau in each fraction, and β-actin was added as a loading control. *, P

Techniques Used: Mutagenesis, Mouse Assay, Western Blot

9) Product Images from "Characterization of the selective in vitro and in vivo binding properties of crenezumab to oligomeric Aβ"

Article Title: Characterization of the selective in vitro and in vivo binding properties of crenezumab to oligomeric Aβ

Journal: Alzheimer's Research & Therapy

doi: 10.1186/s13195-019-0553-5

Crenezumab binding to the hippocampal mossy fibers is Aβ dependent. Representative epifluorescent images of in vivo-dosed crenezumab (80 mg/kg) binding to the mossy fibers ( a ) of PS2APP mice (arrows). Immunostaining for BACE1 shows strong binding in the mossy fibers ( b ) that overlap with crenezumab staining ( c , merge). Scale bar = 50 μm. In vivo-dosed crenezumab (80 mg/kg) staining to the mossy fibers in the PS2APP/BACE1 WT/WT mice ( d ) was nearly completely absent in PS2APP/BACE1 KO/KO ( e ) compared with Ntg/BACE1 WT/WT ( f ) mice. Scale bar, 200 μm. g Significant differences in mossy fiber binding were found between the groups (ANOVA: F 2,8 = 29.16, p
Figure Legend Snippet: Crenezumab binding to the hippocampal mossy fibers is Aβ dependent. Representative epifluorescent images of in vivo-dosed crenezumab (80 mg/kg) binding to the mossy fibers ( a ) of PS2APP mice (arrows). Immunostaining for BACE1 shows strong binding in the mossy fibers ( b ) that overlap with crenezumab staining ( c , merge). Scale bar = 50 μm. In vivo-dosed crenezumab (80 mg/kg) staining to the mossy fibers in the PS2APP/BACE1 WT/WT mice ( d ) was nearly completely absent in PS2APP/BACE1 KO/KO ( e ) compared with Ntg/BACE1 WT/WT ( f ) mice. Scale bar, 200 μm. g Significant differences in mossy fiber binding were found between the groups (ANOVA: F 2,8 = 29.16, p

Techniques Used: Binding Assay, In Vivo, Mouse Assay, Immunostaining, Staining

In vivo-dosed crenezumab binds to (o)ligomeric Aβ, not to (mo)nomeric Aβ, in the hippocampal mossy fiber tract. Plasma and cerebellum PK levels 6 h after the final day of dosing (100 mg/kg daily for 5 d) with an anti-moAβ ( n = 3) or 5 days after a single injection of control IgG (anti-gD 40 mg/kg, n = 4) or crenezumab (80 mg/kg, n = 4) in PS2APP mice. ANOVA found a significant difference in plasma PK levels ( a ) ( F 2,8 = 86.90, p
Figure Legend Snippet: In vivo-dosed crenezumab binds to (o)ligomeric Aβ, not to (mo)nomeric Aβ, in the hippocampal mossy fiber tract. Plasma and cerebellum PK levels 6 h after the final day of dosing (100 mg/kg daily for 5 d) with an anti-moAβ ( n = 3) or 5 days after a single injection of control IgG (anti-gD 40 mg/kg, n = 4) or crenezumab (80 mg/kg, n = 4) in PS2APP mice. ANOVA found a significant difference in plasma PK levels ( a ) ( F 2,8 = 86.90, p

Techniques Used: In Vivo, Injection, Mouse Assay

Crenezumab recognizes Aβ oligomers from in vitro and in vivo sources. Pre-formed (m)onomericAβ 42 , (o)ligomericAβ 42 , or (a)ggregatedAβ 42 were run on native PAGE at 1000, 500, and 250 ng per lane to visualize Aβ 42 banding patterns ( a ). Note that aAβ was too large to enter the gel. Antibodies were incubated with pre-formed Aβ 42 oligomers overnight at 4 °C. To visualize nondenatured oligomers, immunoprecipitated (IP) eluates using were run on native PAGE. Crenezumab recognizes both low molecular weight oligomers between 20 and 50 kDa and high molecular weight (HMW) oligomers between 250 and 700 kDa ( b ). Anti-Aβ IPs from the soluble fraction of PS2APP mouse brain homogenates were run on native PAGE. Crenezumab recognizes HMW oligomers ( c ). 6E10 and 4G8 were used as detection antibodies on all blots
Figure Legend Snippet: Crenezumab recognizes Aβ oligomers from in vitro and in vivo sources. Pre-formed (m)onomericAβ 42 , (o)ligomericAβ 42 , or (a)ggregatedAβ 42 were run on native PAGE at 1000, 500, and 250 ng per lane to visualize Aβ 42 banding patterns ( a ). Note that aAβ was too large to enter the gel. Antibodies were incubated with pre-formed Aβ 42 oligomers overnight at 4 °C. To visualize nondenatured oligomers, immunoprecipitated (IP) eluates using were run on native PAGE. Crenezumab recognizes both low molecular weight oligomers between 20 and 50 kDa and high molecular weight (HMW) oligomers between 250 and 700 kDa ( b ). Anti-Aβ IPs from the soluble fraction of PS2APP mouse brain homogenates were run on native PAGE. Crenezumab recognizes HMW oligomers ( c ). 6E10 and 4G8 were used as detection antibodies on all blots

Techniques Used: In Vitro, In Vivo, Clear Native PAGE, Incubation, Immunoprecipitation, Molecular Weight

In vivo-dosed crenezumab binds in a halo around amyloid plaques and to dystrophic neurites in PS2APP mice. In vivo-dosed crenezumab (200 mg/kg, i.v.) was visualized 7 days postdose with anti-hIgG-Alexa594 antibody (red), and plaques were stained with methoxy-X04 (blue). Representative epifluorescent images of plaque-associated halo of staining by crenezumab alone ( c ) and with plaques ( d ) in the cortex. Note the absence of staining in the control-injected (control IgG, gD) mice around plaques ( a , b ). In the amygdala ( e – g ), confocal z-stacked images show crenezumab binding was prominent around the core of the plaque but in regions not covered by microglia ( e ) (labeled with Iba1, green). This staining pattern was reminiscent of dystrophic neurites and was confirmed by co-staining of crenezumab (80 mg/kg, i.v., red) with markers of dystrophic neurites including BACE1 (green, f ) and LAMP1 (green, g ). Arrowheads indicate example regions of overlap. In vivo-dosed crenezumab ( j , k , red, 120 mg/kg, IP) was localized to regions between methoxy-X04-labeled plaques ( h , k, blue) and GFP-labeled dendrites ( i , k , green) in the dentate gyrus of PS2APP-GFP (line M) mice (2 days postdose). Scale bar, 200 μm ( a – d ) and 50 μm ( e – g )
Figure Legend Snippet: In vivo-dosed crenezumab binds in a halo around amyloid plaques and to dystrophic neurites in PS2APP mice. In vivo-dosed crenezumab (200 mg/kg, i.v.) was visualized 7 days postdose with anti-hIgG-Alexa594 antibody (red), and plaques were stained with methoxy-X04 (blue). Representative epifluorescent images of plaque-associated halo of staining by crenezumab alone ( c ) and with plaques ( d ) in the cortex. Note the absence of staining in the control-injected (control IgG, gD) mice around plaques ( a , b ). In the amygdala ( e – g ), confocal z-stacked images show crenezumab binding was prominent around the core of the plaque but in regions not covered by microglia ( e ) (labeled with Iba1, green). This staining pattern was reminiscent of dystrophic neurites and was confirmed by co-staining of crenezumab (80 mg/kg, i.v., red) with markers of dystrophic neurites including BACE1 (green, f ) and LAMP1 (green, g ). Arrowheads indicate example regions of overlap. In vivo-dosed crenezumab ( j , k , red, 120 mg/kg, IP) was localized to regions between methoxy-X04-labeled plaques ( h , k, blue) and GFP-labeled dendrites ( i , k , green) in the dentate gyrus of PS2APP-GFP (line M) mice (2 days postdose). Scale bar, 200 μm ( a – d ) and 50 μm ( e – g )

Techniques Used: In Vivo, Mouse Assay, Staining, Injection, Binding Assay, Labeling

In vivo-dosed crenezumab binds to the mossy fibers in PS2APP mice. In vivo-dosed crenezumab, but not control IgG (anti-gD IgG4), dose-dependently binds to the mossy fiber axons in the hippocampus of PS2APP mice. Representative epifluorescent images of mossy fiber binding by crenezumab in PS2APP mice ( a ). Quantification of mossy fiber binding integrated density (IntDen) found a significant treatment effect ( b ) (ANOVA: F 4,19 = 50.10, p
Figure Legend Snippet: In vivo-dosed crenezumab binds to the mossy fibers in PS2APP mice. In vivo-dosed crenezumab, but not control IgG (anti-gD IgG4), dose-dependently binds to the mossy fiber axons in the hippocampus of PS2APP mice. Representative epifluorescent images of mossy fiber binding by crenezumab in PS2APP mice ( a ). Quantification of mossy fiber binding integrated density (IntDen) found a significant treatment effect ( b ) (ANOVA: F 4,19 = 50.10, p

Techniques Used: In Vivo, Mouse Assay, Binding Assay

In vivo-dosed crenezumab does not bind to vascular amyloid in PS2APP mice. Representative confocal × 40 images (z-stack maximum projection) of parenchymal amyloid plaques (arrow) and vascular amyloid (arrowhead) stained with methoxy-X04 ( a , c , blue). Note the selective staining of in vivo-dosed crenezumab (200 mg/kg, i.v.) ( b , c , red) to the peri-plaque region and the absence from the vascular amyloid. Scale bar, 100 μm
Figure Legend Snippet: In vivo-dosed crenezumab does not bind to vascular amyloid in PS2APP mice. Representative confocal × 40 images (z-stack maximum projection) of parenchymal amyloid plaques (arrow) and vascular amyloid (arrowhead) stained with methoxy-X04 ( a , c , blue). Note the selective staining of in vivo-dosed crenezumab (200 mg/kg, i.v.) ( b , c , red) to the peri-plaque region and the absence from the vascular amyloid. Scale bar, 100 μm

Techniques Used: In Vivo, Mouse Assay, Staining

10) Product Images from "Folic acid-decorated polyamidoamine dendrimer mediates selective uptake and high expression of genes in head and neck cancer cells"

Article Title: Folic acid-decorated polyamidoamine dendrimer mediates selective uptake and high expression of genes in head and neck cancer cells

Journal: Nanomedicine

doi: 10.2217/nnm-2016-0244

Characterization of dendrimers and dendrimer/plasmid polyplexes. (A) The size and ζ-potential of G4 dendrimer and its derivatives. (B) The cytocompatibility of G4 dendrimer and G4-FA in HN12 cells as determined by WST-1 assay. (C) The complexation stability of G4-FA/GFP plasmid at various weight ratios. (D) The ζ-potential of GFP plasmid and its polyplexes formed with dendrimers. Sample size (n) was in the range of 4–9 for the above experiments.
Figure Legend Snippet: Characterization of dendrimers and dendrimer/plasmid polyplexes. (A) The size and ζ-potential of G4 dendrimer and its derivatives. (B) The cytocompatibility of G4 dendrimer and G4-FA in HN12 cells as determined by WST-1 assay. (C) The complexation stability of G4-FA/GFP plasmid at various weight ratios. (D) The ζ-potential of GFP plasmid and its polyplexes formed with dendrimers. Sample size (n) was in the range of 4–9 for the above experiments.

Techniques Used: Plasmid Preparation, WST-1 Assay

11) Product Images from "Purine and pyrimidine metabolism: Convergent evidence on chronic antidepressant treatment response in mice and humans"

Article Title: Purine and pyrimidine metabolism: Convergent evidence on chronic antidepressant treatment response in mice and humans

Journal: Scientific Reports

doi: 10.1038/srep35317

Chronic paroxetine treatment induces differential alteration of ATIC, CPS2 and HPRT protein expression in mice. ( a ) Western blot and densitometry analyses of CPS2 and HPRT protein levels in the hippocampus, prefrontal cortex and erythrocytes. The image represents cropped blots showing the relevant protein bands. All gels and blots were run under the same experimental conditions. Cropping lines are indicated in full-length blots of Supplementary Fig. S9 . Hippocampal and erythrocytic ATIC, CPS2 and HPRT proteins showed significant expression level differences between the PLF and PSF groups, n = 5/group. ( b ) ATIC protein level showed moderate correlation with FST floating time. Correlation of ( c ) CPS2 and ( d ) HPRT protein levels with FST floating time was significant in the hippocampus and erythrocytes, n = 15. The cropped blots were used for the Fig. 4a. The gels have been run under the same experimental conditions. Protein expression levels were normalized with β-actin. Data are expressed as the mean ± SEM. * p
Figure Legend Snippet: Chronic paroxetine treatment induces differential alteration of ATIC, CPS2 and HPRT protein expression in mice. ( a ) Western blot and densitometry analyses of CPS2 and HPRT protein levels in the hippocampus, prefrontal cortex and erythrocytes. The image represents cropped blots showing the relevant protein bands. All gels and blots were run under the same experimental conditions. Cropping lines are indicated in full-length blots of Supplementary Fig. S9 . Hippocampal and erythrocytic ATIC, CPS2 and HPRT proteins showed significant expression level differences between the PLF and PSF groups, n = 5/group. ( b ) ATIC protein level showed moderate correlation with FST floating time. Correlation of ( c ) CPS2 and ( d ) HPRT protein levels with FST floating time was significant in the hippocampus and erythrocytes, n = 15. The cropped blots were used for the Fig. 4a. The gels have been run under the same experimental conditions. Protein expression levels were normalized with β-actin. Data are expressed as the mean ± SEM. * p

Techniques Used: Expressing, Mouse Assay, Western Blot

12) Product Images from "WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4. WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4"

Article Title: WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4. WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4

Journal: Cancer Medicine

doi: 10.1002/cam4.1837

Association of p27 with peripheral rearrangements of the actin cytoskeleton. A, siRNA oligonucleotides targeting p27 (sip27) or scrambled control siRNAs (Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐p27 antibody. B, MTT assays of S2‐013 and PANC‐1 cells transiently transfected with scrambled control siRNA, ACTN4 siRNA, or p27 siRNA were performed to evaluate cell viability. Data are representative of three independent experiments and are the means ±SD. ABS on Y ‐axis means absorbance at 490 nm and at 630 nm as reference measured with a microplate reader. C, Confocal immunofluorescence microscopic images. Scr‐transfected S2‐013 cells and sip27‐transfected S2‐013 cells were incubated on fibronectin and subsequently stained with anti‐p27 antibody (green) and phalloidin (red). Arrows, peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. D, Quantification of the data shown in B. The values represent the number of cells with fibronectin‐stimulated cell protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P
Figure Legend Snippet: Association of p27 with peripheral rearrangements of the actin cytoskeleton. A, siRNA oligonucleotides targeting p27 (sip27) or scrambled control siRNAs (Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐p27 antibody. B, MTT assays of S2‐013 and PANC‐1 cells transiently transfected with scrambled control siRNA, ACTN4 siRNA, or p27 siRNA were performed to evaluate cell viability. Data are representative of three independent experiments and are the means ±SD. ABS on Y ‐axis means absorbance at 490 nm and at 630 nm as reference measured with a microplate reader. C, Confocal immunofluorescence microscopic images. Scr‐transfected S2‐013 cells and sip27‐transfected S2‐013 cells were incubated on fibronectin and subsequently stained with anti‐p27 antibody (green) and phalloidin (red). Arrows, peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. D, Quantification of the data shown in B. The values represent the number of cells with fibronectin‐stimulated cell protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P

Techniques Used: Transfection, Western Blot, MTT Assay, Immunofluorescence, Incubation, Staining, Derivative Assay

Effects of WAVE2 and ACTN4 on p27 activity. A, Confocal immunofluorescence microscopic images. S2‐013 and HPNE cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐phosphorylated p27 (red) antibodies. Actin filaments were labeled with phalloidin (violet). Arrows, phosphorylated p27 in the nucleus. Blue, DAPI staining. Scale bar, 10 μm. B, S2‐013 and HPNE cells were incubated on fibronectin and fractionated into cytosolic (c) and nuclear (n) fractions. Western blotting of the fractions was performed using anti‐phosphorylated p27 and anti‐p27 antibodies. C, Scr‐transfected S2‐013 cells and siWAVE2‐transfected S2‐013 cells were incubated on fibronectin. Western blotting was performed using anti‐WAVE2, anti‐phosphorylated p27, and anti‐p27 antibodies. D, A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with scrambled control siRNA or WAVE2 siRNA with or without ACTN4 siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies
Figure Legend Snippet: Effects of WAVE2 and ACTN4 on p27 activity. A, Confocal immunofluorescence microscopic images. S2‐013 and HPNE cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐phosphorylated p27 (red) antibodies. Actin filaments were labeled with phalloidin (violet). Arrows, phosphorylated p27 in the nucleus. Blue, DAPI staining. Scale bar, 10 μm. B, S2‐013 and HPNE cells were incubated on fibronectin and fractionated into cytosolic (c) and nuclear (n) fractions. Western blotting of the fractions was performed using anti‐phosphorylated p27 and anti‐p27 antibodies. C, Scr‐transfected S2‐013 cells and siWAVE2‐transfected S2‐013 cells were incubated on fibronectin. Western blotting was performed using anti‐WAVE2, anti‐phosphorylated p27, and anti‐p27 antibodies. D, A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with scrambled control siRNA or WAVE2 siRNA with or without ACTN4 siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies

Techniques Used: Activity Assay, Immunofluorescence, Cell Culture, Labeling, Staining, Incubation, Western Blot, Transfection, Construct

Roles of WAVE2 in the translocation of ACTN4 to actin filaments in cell protrusions. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2, ACTN4, and actin. Rabbit IgG isotype control antibody was used as the control. B, Confocal immunofluorescence microscopic images. Oligonucleotides (siRNAs targeting WAVE2 (siWAVE2) or scrambled siRNAs (Scr) as the negative control) were transiently transfected into S2‐013 cells. Transfected cells were incubated on fibronectin and were subsequently stained with anti‐WAVE2 antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, ACTN4 bound to peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. C, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with WAVE2 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, exogenous WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 µm. D, Oligonucleotides (siRNAs targeting ACTN4 (siACTN4) or Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐ACTN4 antibody. E, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Blue, DAPI staining. Scale bars, 10 µm
Figure Legend Snippet: Roles of WAVE2 in the translocation of ACTN4 to actin filaments in cell protrusions. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2, ACTN4, and actin. Rabbit IgG isotype control antibody was used as the control. B, Confocal immunofluorescence microscopic images. Oligonucleotides (siRNAs targeting WAVE2 (siWAVE2) or scrambled siRNAs (Scr) as the negative control) were transiently transfected into S2‐013 cells. Transfected cells were incubated on fibronectin and were subsequently stained with anti‐WAVE2 antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, ACTN4 bound to peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. C, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with WAVE2 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, exogenous WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 µm. D, Oligonucleotides (siRNAs targeting ACTN4 (siACTN4) or Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐ACTN4 antibody. E, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Blue, DAPI staining. Scale bars, 10 µm

Techniques Used: Translocation Assay, Immunoprecipitation, Cell Culture, Western Blot, Immunofluorescence, Negative Control, Transfection, Incubation, Staining, Construct

Association of WAVE2 with ACTN4. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Rabbit IgG isotype control antibody was used as the control. Proteins within immunoprecipitates were examined on Western blots probed with anti‐WAVE2 antibody. B, Proteins in immunoprecipitates were examined with silver staining. Rabbit IgG isotype control antibody was used as the control. A 100‐kDa band is indicated by the arrow. C, The percent coverage for ACTN4 is represented by the identified peptides in the total protein sequence (accession number NP_004915). D, Immunoprecipitation of WAVE2 or ACTN4 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2 and ACTN4. Rabbit IgG isotype control antibody for WAVE2 and mouse IgG isotype control antibody for ACTN4 was used as controls. E, Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐ACTN4 (red) antibodies. Arrows, WAVE2 co‐localized with ACTN4 in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm
Figure Legend Snippet: Association of WAVE2 with ACTN4. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Rabbit IgG isotype control antibody was used as the control. Proteins within immunoprecipitates were examined on Western blots probed with anti‐WAVE2 antibody. B, Proteins in immunoprecipitates were examined with silver staining. Rabbit IgG isotype control antibody was used as the control. A 100‐kDa band is indicated by the arrow. C, The percent coverage for ACTN4 is represented by the identified peptides in the total protein sequence (accession number NP_004915). D, Immunoprecipitation of WAVE2 or ACTN4 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2 and ACTN4. Rabbit IgG isotype control antibody for WAVE2 and mouse IgG isotype control antibody for ACTN4 was used as controls. E, Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐ACTN4 (red) antibodies. Arrows, WAVE2 co‐localized with ACTN4 in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm

Techniques Used: Immunoprecipitation, Cell Culture, Western Blot, Silver Staining, Sequencing, Immunofluorescence, Labeling, Staining

Subcellular localization of WAVE2 in PDAC cells grown on fibronectin. Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 antibody (green). Actin filaments were labeled with phalloidin (red). Arrows, WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 μm
Figure Legend Snippet: Subcellular localization of WAVE2 in PDAC cells grown on fibronectin. Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 antibody (green). Actin filaments were labeled with phalloidin (red). Arrows, WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 μm

Techniques Used: Immunofluorescence, Cell Culture, Labeling, Staining

Roles of WAVE2 and ACTN4 in forming cell protrusions. A, Confocal immunofluorescence microscopic images showing phalloidin‐labeled peripheral actin structures (red) and DAPI‐labeled nuclei (blue) in scrambled control siRNA‐transfected S2‐013 and PANC‐1 cells, and WAVE2 siRNA‐transfected S2‐013 and PANC‐1 cells grown on fibronectin. Arrows, peripheral actin structures in cell protrusions. Scale bars, 10 µm. B, Quantification of data shown in A; the values represent the number of cells with protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P
Figure Legend Snippet: Roles of WAVE2 and ACTN4 in forming cell protrusions. A, Confocal immunofluorescence microscopic images showing phalloidin‐labeled peripheral actin structures (red) and DAPI‐labeled nuclei (blue) in scrambled control siRNA‐transfected S2‐013 and PANC‐1 cells, and WAVE2 siRNA‐transfected S2‐013 and PANC‐1 cells grown on fibronectin. Arrows, peripheral actin structures in cell protrusions. Scale bars, 10 µm. B, Quantification of data shown in A; the values represent the number of cells with protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P

Techniques Used: Immunofluorescence, Labeling, Transfection, Derivative Assay

13) Product Images from "TALEN-mediated enhancer knockout influences TNFAIP3 gene expression and mimics a molecular phenotype associated with systemic lupus erythematosus"

Article Title: TALEN-mediated enhancer knockout influences TNFAIP3 gene expression and mimics a molecular phenotype associated with systemic lupus erythematosus

Journal: Genes and immunity

doi: 10.1038/gene.2016.4

TALEN mutagenesis of the TT > A enhancer reduces interaction with the TNFAIP3 promoter A. Schematic representation of the allele-specific 3C and clonal assay analyses of the engineered heterozygous clonal mutant HEK293T cell lines. Wild type (WT) allele experiences higher frequencies of long-range DNA looping, thus higher proportions of 3C events are observed relative to Mutant 1 or Mutant 2 alleles. B. Genomic DNA isolated from 72 clonal expansions from Mutant 1 or Mutant 2 HEK293T cells without 3C was analyzed for wild type and mutant allele frequencies. The expected clonal frequency of approximately 50% wild type and 50% mutant allele was observed. C. Analysis of 72 mutant 1 or 2 clonal expansions from the 3C capture were analyzed for relative crosslinking frequencies between wild-type or mutated TT > A enhancer and TNFAIP3 promoter. P values were calculated using the Fisher’s exact test and are as indicated.
Figure Legend Snippet: TALEN mutagenesis of the TT > A enhancer reduces interaction with the TNFAIP3 promoter A. Schematic representation of the allele-specific 3C and clonal assay analyses of the engineered heterozygous clonal mutant HEK293T cell lines. Wild type (WT) allele experiences higher frequencies of long-range DNA looping, thus higher proportions of 3C events are observed relative to Mutant 1 or Mutant 2 alleles. B. Genomic DNA isolated from 72 clonal expansions from Mutant 1 or Mutant 2 HEK293T cells without 3C was analyzed for wild type and mutant allele frequencies. The expected clonal frequency of approximately 50% wild type and 50% mutant allele was observed. C. Analysis of 72 mutant 1 or 2 clonal expansions from the 3C capture were analyzed for relative crosslinking frequencies between wild-type or mutated TT > A enhancer and TNFAIP3 promoter. P values were calculated using the Fisher’s exact test and are as indicated.

Techniques Used: Mutagenesis, Clone Assay, Isolation

14) Product Images from "DR5 and caspase-8 are dispensable in ER stress-induced apoptosis"

Article Title: DR5 and caspase-8 are dispensable in ER stress-induced apoptosis

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2017.53

Caspase-8 activation during ER stress occurs downstream of BAX/BAK activation. ( a ) HCT116 cells were treated with thapsigargin (Tg) and harvested at the indicated time points and analysed by western blotting. ( b ) WT and Bax −/− /Bak −/− HCT116 cells were treated with thapsisgargin and analysed by western blotting. ( c and were identified by western blotting. Treatment with FAS ligand was used as a positive control for caspase-8 activation (upper panel). The experiment was repeated and blots were probed for BiP (marker of ER stress) and caspase-3 (bottom panel)
Figure Legend Snippet: Caspase-8 activation during ER stress occurs downstream of BAX/BAK activation. ( a ) HCT116 cells were treated with thapsigargin (Tg) and harvested at the indicated time points and analysed by western blotting. ( b ) WT and Bax −/− /Bak −/− HCT116 cells were treated with thapsisgargin and analysed by western blotting. ( c and were identified by western blotting. Treatment with FAS ligand was used as a positive control for caspase-8 activation (upper panel). The experiment was repeated and blots were probed for BiP (marker of ER stress) and caspase-3 (bottom panel)

Techniques Used: Activation Assay, Western Blot, Positive Control, Marker

15) Product Images from "Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth"

Article Title: Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth

Journal: The EMBO Journal

doi: 10.15252/embj.201899322

Mechanically stretched human LECs have more VEGFR3‐β1 integrin and less ILK‐β1 integrin interactions A–D LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 min. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 μm. E Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (−) mechanical stretch ( n = 6 independent stretch chambers), * P = 0.039. F Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 min and used for immunoprecipitation (IP) of HA‐tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates. G Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (−) mechanical stretch; normalised to the respective amount of HA‐tagged β1 integrin ( n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers), * P = 0.0007. Data information: Data are presented as means ± SEM, unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.
Figure Legend Snippet: Mechanically stretched human LECs have more VEGFR3‐β1 integrin and less ILK‐β1 integrin interactions A–D LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 min. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 μm. E Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (−) mechanical stretch ( n = 6 independent stretch chambers), * P = 0.039. F Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 min and used for immunoprecipitation (IP) of HA‐tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates. G Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (−) mechanical stretch; normalised to the respective amount of HA‐tagged β1 integrin ( n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers), * P = 0.0007. Data information: Data are presented as means ± SEM, unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.

Techniques Used: Proximity Ligation Assay, Western Blot, Immunoprecipitation, Two Tailed Test

ILK controls VEGFR3‐β1 integrin interactions in mouse embryos A–D LSM images of cross‐sections through the jugular lymph sac/primordial thoracic duct (jls/pTD) of an E13.5 control embryo stained for surface VEGFR3 and β1 integrin. Scale bar: 10 μm. E–H LSM images of proximity ligation assay (PLA) dots (arrows) composed of VEGFR3 and β1 integrin on cross‐sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 μm. I Quantification of VEGFR3/β1 integrin PLA dots normalised to Lyve1‐positive area of control or ILK K.O. embryos ( n = 6 embryos per genotype), * P = 0.005. Data information: Data are presented as means ± SEM, shown as percentage of control embryos, unpaired two‐tailed Student's t ‐test.
Figure Legend Snippet: ILK controls VEGFR3‐β1 integrin interactions in mouse embryos A–D LSM images of cross‐sections through the jugular lymph sac/primordial thoracic duct (jls/pTD) of an E13.5 control embryo stained for surface VEGFR3 and β1 integrin. Scale bar: 10 μm. E–H LSM images of proximity ligation assay (PLA) dots (arrows) composed of VEGFR3 and β1 integrin on cross‐sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 μm. I Quantification of VEGFR3/β1 integrin PLA dots normalised to Lyve1‐positive area of control or ILK K.O. embryos ( n = 6 embryos per genotype), * P = 0.005. Data information: Data are presented as means ± SEM, shown as percentage of control embryos, unpaired two‐tailed Student's t ‐test.

Techniques Used: Staining, Proximity Ligation Assay, Two Tailed Test

The lymphatic vascular effect of Ilk deletion strictly depends on β1 integrin A, B Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/+ ;Itgb1 ∆/+ mouse embryo (referred to as “control”) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jugular lymph sac/primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 μm, respectively. C, D Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/∆ ;Itgb1 ∆/+ embryo (referred to as “ILK β1 integrin K.O.”), with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jls/pTD. Scale bars: 500 and 100 μm, respectively. E–H LSM images of cross‐sections through the jls/pTD of E13.5 control and ILK β1 integrin K.O. embryos stained for the proliferation marker phospho‐Histone H3. Arrows point to phospho‐Histone H3‐positive LECs. Scale bars: 20 μm. I–L LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p‐Tyr) on stained cross‐sections through the jls/pTD of control and ILK β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1‐stained area. Scale bars: 10 μm. M Number of LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. N LEC proliferation as determined by the number of phospho‐Histone H3‐positive LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. O Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p‐Tyr) per LEC of E13.5 control or ILK β1 integrin K.O. embryos. Data information: Data are presented as means ± SEM, shown as percentage of control embryos with n = 5 embryos per genotype, unpaired two‐tailed Student's t ‐test.
Figure Legend Snippet: The lymphatic vascular effect of Ilk deletion strictly depends on β1 integrin A, B Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/+ ;Itgb1 ∆/+ mouse embryo (referred to as “control”) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jugular lymph sac/primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 μm, respectively. C, D Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/∆ ;Itgb1 ∆/+ embryo (referred to as “ILK β1 integrin K.O.”), with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jls/pTD. Scale bars: 500 and 100 μm, respectively. E–H LSM images of cross‐sections through the jls/pTD of E13.5 control and ILK β1 integrin K.O. embryos stained for the proliferation marker phospho‐Histone H3. Arrows point to phospho‐Histone H3‐positive LECs. Scale bars: 20 μm. I–L LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p‐Tyr) on stained cross‐sections through the jls/pTD of control and ILK β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1‐stained area. Scale bars: 10 μm. M Number of LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. N LEC proliferation as determined by the number of phospho‐Histone H3‐positive LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. O Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p‐Tyr) per LEC of E13.5 control or ILK β1 integrin K.O. embryos. Data information: Data are presented as means ± SEM, shown as percentage of control embryos with n = 5 embryos per genotype, unpaired two‐tailed Student's t ‐test.

Techniques Used: Staining, Marker, Proximity Ligation Assay, Two Tailed Test

ILK controls proliferation, VEGFR3 signalling and VEGFR3‐β1 integrin interactions in human LECs A, B Images of adult human LECs after 1 h of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 μm. C LEC proliferation as determined by the number of BrdU‐positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 3 independent transfections per siRNA), * P = 0.032 (control versus ILK‐1), * P = 0.005 (control versus ILK‐2), * P = 0.0003 (control versus ILK‐3). D VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 4 (control siRNA, ILK‐1 siRNA and ILK‐3 siRNA) or n = 8 (ILK‐2 siRNA) independent transfections per siRNA), * P = 0.0001 (control versus each siRNA). E, F LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 μm. G Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK‐1), * P = 0.024 (control versus ILK‐2), * P = 0.001 (control versus ILK‐3). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test.
Figure Legend Snippet: ILK controls proliferation, VEGFR3 signalling and VEGFR3‐β1 integrin interactions in human LECs A, B Images of adult human LECs after 1 h of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 μm. C LEC proliferation as determined by the number of BrdU‐positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 3 independent transfections per siRNA), * P = 0.032 (control versus ILK‐1), * P = 0.005 (control versus ILK‐2), * P = 0.0003 (control versus ILK‐3). D VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 4 (control siRNA, ILK‐1 siRNA and ILK‐3 siRNA) or n = 8 (ILK‐2 siRNA) independent transfections per siRNA), * P = 0.0001 (control versus each siRNA). E, F LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 μm. G Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK‐1), * P = 0.024 (control versus ILK‐2), * P = 0.001 (control versus ILK‐3). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test.

Techniques Used: BrdU Incorporation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Proximity Ligation Assay

Simplified model of mechanosensitive VEGFR3 signalling and ILK‐controlled lymphatic vascular growth In quiescent LECs, VEGFR3 and β1 integrin are physically separated. ILK directly or indirectly interacts with β1 integrin and connects it to the F‐actin cytoskeleton via intracellular proteins, such as α‐parvin, a component of the IPP complex. Upon mechanical stretch, the complex of β1 integrin and ILK (along with the entire IPP complex) transiently disrupts. This releases β1 integrin, resulting in its interaction with VEGFR3, and thus in increased VEGFR3 tyrosine phosphorylation (“P” in yellow circle). As a consequence, LEC proliferation and lymphatic vascular growth are induced. The absence of ILK results in permanent interaction between VEGFR3 and β1 integrin, leading to upregulated VEGFR3 tyrosine phosphorylation (“P” in yellow circle), LEC proliferation and non‐physiologic lymphatic vascular growth.
Figure Legend Snippet: Simplified model of mechanosensitive VEGFR3 signalling and ILK‐controlled lymphatic vascular growth In quiescent LECs, VEGFR3 and β1 integrin are physically separated. ILK directly or indirectly interacts with β1 integrin and connects it to the F‐actin cytoskeleton via intracellular proteins, such as α‐parvin, a component of the IPP complex. Upon mechanical stretch, the complex of β1 integrin and ILK (along with the entire IPP complex) transiently disrupts. This releases β1 integrin, resulting in its interaction with VEGFR3, and thus in increased VEGFR3 tyrosine phosphorylation (“P” in yellow circle). As a consequence, LEC proliferation and lymphatic vascular growth are induced. The absence of ILK results in permanent interaction between VEGFR3 and β1 integrin, leading to upregulated VEGFR3 tyrosine phosphorylation (“P” in yellow circle), LEC proliferation and non‐physiologic lymphatic vascular growth.

Techniques Used:

16) Product Images from "The molecular targets of diclofenac differs from ibuprofen to induce apoptosis and epithelial mesenchymal transition due to alternation on oxidative stress management p53 independently in PC3 prostate cancer cells"

Article Title: The molecular targets of diclofenac differs from ibuprofen to induce apoptosis and epithelial mesenchymal transition due to alternation on oxidative stress management p53 independently in PC3 prostate cancer cells

Journal: Prostate International

doi: 10.1016/j.prnil.2019.09.003

The effect of p53 on ibuprofen and diclofenac-treated PC3 cells on EMT and wound closure. (A) To the sample, 60 μg of whole cell lysate were loaded in 12% SDS–PAGE gels and probed with E-cadherin, N-cadherin, vimentin, and Snail antibodies. GAPDH was used as equal loading control. (B–C) Images of wound closure assays (100× magnification). Cells were seeded into 6-well cell culture plates, cultured in RPMI1640 supplemented with 10% FBS, and allowed to grow to near confluence. Confluent monolayers were wounded via 200 μl micropipette tip, then the cellular debris was gently washed away with 1× PBS. The wounded cell monolayer for both wt (B) and p53+ (C) PC3 cells were treated with each drug for 24 h. All pictures were taken after 24 h drug treatment. Wound closure assay is repeated by two-independent assays and representative images are shown. SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; EMT, epithelial mesenchymal transition; PBS, phosphate-buffered saline.
Figure Legend Snippet: The effect of p53 on ibuprofen and diclofenac-treated PC3 cells on EMT and wound closure. (A) To the sample, 60 μg of whole cell lysate were loaded in 12% SDS–PAGE gels and probed with E-cadherin, N-cadherin, vimentin, and Snail antibodies. GAPDH was used as equal loading control. (B–C) Images of wound closure assays (100× magnification). Cells were seeded into 6-well cell culture plates, cultured in RPMI1640 supplemented with 10% FBS, and allowed to grow to near confluence. Confluent monolayers were wounded via 200 μl micropipette tip, then the cellular debris was gently washed away with 1× PBS. The wounded cell monolayer for both wt (B) and p53+ (C) PC3 cells were treated with each drug for 24 h. All pictures were taken after 24 h drug treatment. Wound closure assay is repeated by two-independent assays and representative images are shown. SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; EMT, epithelial mesenchymal transition; PBS, phosphate-buffered saline.

Techniques Used: SDS Page, Cell Culture, Wound Closure Assay, Polyacrylamide Gel Electrophoresis

Ibuprofen and diclofenac caused cell cycle arrest and modulated AKT – FoxO signaling axis in both cell lines. (A) Wt and p53 + PC3 prostate cancer cells were treated for 24 h with ibuprofen (1 mM) or diclofenac (250 μM) (A). Cells were labeled with propidium iodide and analyzed by using a FACS flow cytometer (BD Accuri) for 10x10. The image shown is representative of two experiments. (B) 60 μg of whole cell lysate were loaded in 12% SDS–PAGE gels and probed with AKT, FoxO1, and FoxO3. GAPDH was used as loading control. Wt, wild type; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; FACS.
Figure Legend Snippet: Ibuprofen and diclofenac caused cell cycle arrest and modulated AKT – FoxO signaling axis in both cell lines. (A) Wt and p53 + PC3 prostate cancer cells were treated for 24 h with ibuprofen (1 mM) or diclofenac (250 μM) (A). Cells were labeled with propidium iodide and analyzed by using a FACS flow cytometer (BD Accuri) for 10x10. The image shown is representative of two experiments. (B) 60 μg of whole cell lysate were loaded in 12% SDS–PAGE gels and probed with AKT, FoxO1, and FoxO3. GAPDH was used as loading control. Wt, wild type; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; FACS.

Techniques Used: Labeling, FACS, Flow Cytometry, Cytometry, SDS Page, Polyacrylamide Gel Electrophoresis

Ibuprofen and diclofenac caused apoptosis mechanism differs by the presence of p53 expression. (A) To the sample, 60 μg of whole cell lysate were loaded in 12% SDS–PAGE gels and probed with Fas, full caspase-8, full caspase-2, TNF-R2, and RIP antibodies. GAPDH was used as loading control. (B) Same protocol (A) was used with procaspase-9 and procaspase-3. β-Actin was used as loading control. (C) Same protocol (A) was used with antiapoptotic Bcl-2 family members: Bcl-2, Mcl-1, and Bcl-x. GAPDH was used as loading control. (D) Same protocol (A) was used with proapoptotic protein antibodies: Bax, BimEL, Puma, Bak, Bid. (E) PC3 wt prostate cancer cells were subjected to lysis following drug treatment and immunoprecipitated with anti–Mcl-1 antibodies. Immunoprecipitated samples were observed at SDS–PAGE and Western blot analysis was performed for Bim. SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; wt, wild type.
Figure Legend Snippet: Ibuprofen and diclofenac caused apoptosis mechanism differs by the presence of p53 expression. (A) To the sample, 60 μg of whole cell lysate were loaded in 12% SDS–PAGE gels and probed with Fas, full caspase-8, full caspase-2, TNF-R2, and RIP antibodies. GAPDH was used as loading control. (B) Same protocol (A) was used with procaspase-9 and procaspase-3. β-Actin was used as loading control. (C) Same protocol (A) was used with antiapoptotic Bcl-2 family members: Bcl-2, Mcl-1, and Bcl-x. GAPDH was used as loading control. (D) Same protocol (A) was used with proapoptotic protein antibodies: Bax, BimEL, Puma, Bak, Bid. (E) PC3 wt prostate cancer cells were subjected to lysis following drug treatment and immunoprecipitated with anti–Mcl-1 antibodies. Immunoprecipitated samples were observed at SDS–PAGE and Western blot analysis was performed for Bim. SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; wt, wild type.

Techniques Used: Expressing, SDS Page, Lysis, Immunoprecipitation, Western Blot, Polyacrylamide Gel Electrophoresis

17) Product Images from "Chemosensitization by phenothiazines in human lung cancer cells: impaired resolution of γH2AX and increased oxidative stress elicit apoptosis associated with lysosomal expansion and intense vacuolation"

Article Title: Chemosensitization by phenothiazines in human lung cancer cells: impaired resolution of γH2AX and increased oxidative stress elicit apoptosis associated with lysosomal expansion and intense vacuolation

Journal: Cell Death & Disease

doi: 10.1038/cddis.2011.62

TFP augments the activation of both the extrinsic and the intrinsic apoptotic pathways after DNA damage. U1810 cells were exposed to DNA-damaging drugs (2.5 μ g/ml bleomycin, 20 μ M cisplatin) alone or in combination with TFP (10 μ M). For immunoblotting and FLICA assay, samples were collected 48 h post DNA-damaging treatment. ( a ) TFP augmented caspase-3 activation after bleomycin treatment (left); enhanced caspase-3 activation was detected predominantly in cells containing 4n DNA content (right). ( b ) TFP potentiated caspase-3 activation after cisplatin treatment. ( c ) TFP co-treatment resulted in increased cleavage of PARP. ( d ) TFP co-treatment resulted in increased cleavage of caspase-8. ( e ) TFP co-treatment resulted in increased cleavage of caspase-9. ( f ) TFP-co-treated cells contain increased levels of catalytically active caspase-9. For ( a , b and f ), mean and S.D. were compiled from three independent experiments performed in duplicates ( * P
Figure Legend Snippet: TFP augments the activation of both the extrinsic and the intrinsic apoptotic pathways after DNA damage. U1810 cells were exposed to DNA-damaging drugs (2.5 μ g/ml bleomycin, 20 μ M cisplatin) alone or in combination with TFP (10 μ M). For immunoblotting and FLICA assay, samples were collected 48 h post DNA-damaging treatment. ( a ) TFP augmented caspase-3 activation after bleomycin treatment (left); enhanced caspase-3 activation was detected predominantly in cells containing 4n DNA content (right). ( b ) TFP potentiated caspase-3 activation after cisplatin treatment. ( c ) TFP co-treatment resulted in increased cleavage of PARP. ( d ) TFP co-treatment resulted in increased cleavage of caspase-8. ( e ) TFP co-treatment resulted in increased cleavage of caspase-9. ( f ) TFP-co-treated cells contain increased levels of catalytically active caspase-9. For ( a , b and f ), mean and S.D. were compiled from three independent experiments performed in duplicates ( * P

Techniques Used: Activation Assay

Phenothiazines delay γ H2AX resolution in human lung cancer cells. Cells were exposed to DNA-damaging agents alone or in combination with TFP or NU7026 (both 10 μ M). ( a ) TFP significantly delayed the resolution of γ H2AX in U1810 and H23 cells exposed to 7.5 μ g/ml bleomycin (top left) and 5 μ g/ml bleomycin (top right), respectively. Phosphorylation of H2AX (Ser139) was quantified by flow cytometry using Alexa Fluor 488-conjugated antibodies. The histograms below highlight differences in cellular Alexa Fluor 488-associated fluorescence at 1 h post-bleomycin treatment: shaded, bleo; unshaded, bleo+TFP. For bleomycin-treated cells, the kinetics of γ H2AX resolution is indicated by fold changes in the geometric mean of Alexa Fluor 488-associated fluorescence. An arbitrary value of 1 is assigned to samples collected immediately after bleomycin treatment in the absence of TFP ( t =0 h). ( b ) TFP retarded the resolution of γ H2AX in U1810 cells exposed to 2.5 μ g/ml bleomycin. Phosphorylated H2AX was detected by immunoblotting. ( c ) TFPZ impaired the resolution of γ H2AX in U1810 cells 1 h after exposure to 15 μ g/ml bleomycin. ( d ) TFP impeded the resolution of γ H2AX in U1810 cells exposed to 50 μ M cisplatin. As cisplatin induced γ H2AX only in a subset of cells, the kinetics of γ H2AX resolution in cisplatin-treated cells is defined by changes in the percentage of cells exhibiting high Alexa Fluor 488-associated fluorescence ( γ H2AX High (%)). For ( a and d ), mean and S.D. were compiled from three independent experiments performed in duplicates ( * P
Figure Legend Snippet: Phenothiazines delay γ H2AX resolution in human lung cancer cells. Cells were exposed to DNA-damaging agents alone or in combination with TFP or NU7026 (both 10 μ M). ( a ) TFP significantly delayed the resolution of γ H2AX in U1810 and H23 cells exposed to 7.5 μ g/ml bleomycin (top left) and 5 μ g/ml bleomycin (top right), respectively. Phosphorylation of H2AX (Ser139) was quantified by flow cytometry using Alexa Fluor 488-conjugated antibodies. The histograms below highlight differences in cellular Alexa Fluor 488-associated fluorescence at 1 h post-bleomycin treatment: shaded, bleo; unshaded, bleo+TFP. For bleomycin-treated cells, the kinetics of γ H2AX resolution is indicated by fold changes in the geometric mean of Alexa Fluor 488-associated fluorescence. An arbitrary value of 1 is assigned to samples collected immediately after bleomycin treatment in the absence of TFP ( t =0 h). ( b ) TFP retarded the resolution of γ H2AX in U1810 cells exposed to 2.5 μ g/ml bleomycin. Phosphorylated H2AX was detected by immunoblotting. ( c ) TFPZ impaired the resolution of γ H2AX in U1810 cells 1 h after exposure to 15 μ g/ml bleomycin. ( d ) TFP impeded the resolution of γ H2AX in U1810 cells exposed to 50 μ M cisplatin. As cisplatin induced γ H2AX only in a subset of cells, the kinetics of γ H2AX resolution in cisplatin-treated cells is defined by changes in the percentage of cells exhibiting high Alexa Fluor 488-associated fluorescence ( γ H2AX High (%)). For ( a and d ), mean and S.D. were compiled from three independent experiments performed in duplicates ( * P

Techniques Used: Flow Cytometry, Cytometry, Fluorescence

18) Product Images from "Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus"

Article Title: Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus

Journal: Endocrinology

doi: 10.1210/en.2012-1748

siRNA-mediated knockdown of ERα inhibits E 2 -mediated repression of GILZ expression. A, ECC1 cells, transfected with ERα siRNA or NTC, were assessed for the extent of knockdown compared with NTC by QRT-PCR and Western blotting. GR expression
Figure Legend Snippet: siRNA-mediated knockdown of ERα inhibits E 2 -mediated repression of GILZ expression. A, ECC1 cells, transfected with ERα siRNA or NTC, were assessed for the extent of knockdown compared with NTC by QRT-PCR and Western blotting. GR expression

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Western Blot

ER depletion by ICI 182,780 inhibits E 2 -mediated repression of GILZ expression. A, Whole-cell lysates from ECC1 cells treated over a time course with 1 or 10 μ m ICI 182,780 (ICI) were subjected to Western blot analysis for ERα. The housekeeping
Figure Legend Snippet: ER depletion by ICI 182,780 inhibits E 2 -mediated repression of GILZ expression. A, Whole-cell lysates from ECC1 cells treated over a time course with 1 or 10 μ m ICI 182,780 (ICI) were subjected to Western blot analysis for ERα. The housekeeping

Techniques Used: Expressing, Western Blot

Glucocorticoid-responsive GILZ expression in ECC1. A, Expression of GILZ was measured by QRT-PCR in ECC1 cells treated for 6 or 24 h with 100 n m Dex, 10 n m E 2 , or 100 n m Dex and 10 n m E 2 (D + E 2 ). Values were normalized to the housekeeping gene Cyclophilin
Figure Legend Snippet: Glucocorticoid-responsive GILZ expression in ECC1. A, Expression of GILZ was measured by QRT-PCR in ECC1 cells treated for 6 or 24 h with 100 n m Dex, 10 n m E 2 , or 100 n m Dex and 10 n m E 2 (D + E 2 ). Values were normalized to the housekeeping gene Cyclophilin

Techniques Used: Expressing, Quantitative RT-PCR

GR and ERα expression in response to Dex and E 2 treatment in ECC1 cells. A, Expression of GR and ERα was measured by QRT-PCR. ECC1 cells treated with 100 n m Dex, 10 n m E 2 , or 100 n m Dex and 10 n m E 2 were normalized to the housekeeping
Figure Legend Snippet: GR and ERα expression in response to Dex and E 2 treatment in ECC1 cells. A, Expression of GR and ERα was measured by QRT-PCR. ECC1 cells treated with 100 n m Dex, 10 n m E 2 , or 100 n m Dex and 10 n m E 2 were normalized to the housekeeping

Techniques Used: Expressing, Quantitative RT-PCR

Kinetics of GILZ regulation in response to Dex and E 2 treatment. A, Expression of GILZ was measured by QRT-PCR in ECC1 cells treated for 0, 0.5, 1, 2, 4, 6, and 24 h with 100 n m Dex or 100 n m Dex and 10 n m E 2 (D + E 2 ). Graph shows mean ± sem .
Figure Legend Snippet: Kinetics of GILZ regulation in response to Dex and E 2 treatment. A, Expression of GILZ was measured by QRT-PCR in ECC1 cells treated for 0, 0.5, 1, 2, 4, 6, and 24 h with 100 n m Dex or 100 n m Dex and 10 n m E 2 (D + E 2 ). Graph shows mean ± sem .

Techniques Used: Expressing, Quantitative RT-PCR

Transcriptional regulation is a likely mechanism of E 2 -mediated GILZ antagonism. A, ECC1 cells were treated with 10 μ m cycloheximide for 1 h before hormone treatment. Cells were then administered 100 n m Dex, 10 n m E 2 , or 100 n m Dex and 10 n m E
Figure Legend Snippet: Transcriptional regulation is a likely mechanism of E 2 -mediated GILZ antagonism. A, ECC1 cells were treated with 10 μ m cycloheximide for 1 h before hormone treatment. Cells were then administered 100 n m Dex, 10 n m E 2 , or 100 n m Dex and 10 n m E

Techniques Used:

19) Product Images from "Determinants of Dengue Virus NS4A Protein Oligomerization"

Article Title: Determinants of Dengue Virus NS4A Protein Oligomerization

Journal: Journal of Virology

doi: 10.1128/JVI.00546-15

Differential responses of NS4A proteins following CHX treatment. 293T cells were transfected with NS4A-Flag with E50A (A) or G67A (B), with the WT construct as a control. At 24 h posttransfection, the cells were treated with 75 μg/ml CHX. Cell
Figure Legend Snippet: Differential responses of NS4A proteins following CHX treatment. 293T cells were transfected with NS4A-Flag with E50A (A) or G67A (B), with the WT construct as a control. At 24 h posttransfection, the cells were treated with 75 μg/ml CHX. Cell

Techniques Used: Transfection, Construct

20) Product Images from "AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia"

Article Title: AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia

Journal: Nature Communications

doi: 10.1038/ncomms8388

Ghrelin preserves cardiac function and reduces infarct size after myocardial infarction (MI). ( a ) Real-time PCR quantification of ghrelin mRNA in total ventricular RNA extracted 2 or 90 days after AAV9 injection and MI induction. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over endogenous ( n =5). ( b , c ) Intracardiac ( b ) and plasmatic ( c ) levels of acyl and des-acyl ghrelin evaluated by EIA 90 days after intracardiac vector injection ( n =4). ( d – g ) Echocardiographic analysis in AAV9-injected and control mice at 2, 7, 30, 60 and 90 days after MI. Left ventricular ejection fraction (LVEF; d ), left ventricular fractional shortening (LVFS; e ), anterior wall thickening (AWTK; f ) and diastolic LV internal diameter (LVID d; g ) of infarcted hearts treated either with AAV9-ghrelin or AAV9-control were measured ( n =12 per group). (h ) Representative M-mode echocardiographic images 90 days after AAV9-control or AAV9-ghrelin injection and MI induction. ( i ) Representative images of whole transverse sections after Azan-trichromic staining of hearts transduced with AAV9-control or AAV9-ghrelin. Fibrotic areas are stained in blue. ( j ) Quantification of infarct size expressed as percentage of LV ( n =12 per group). ( k ) Cardiac expression levels of the indicated genes in AAV9-control and AAV9-ghrelin-treated hearts, 90 days after MI. Values are normalized for GAPDH and expressed as fold over untreated ( n =6). All values are mean±s.e.m. Pairwise comparison was performed with the Student's t -test ( b , c , j ); one-way analysis of variance (ANOVA) and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( a , k ); two-way ANOVA was used in d – g . * P
Figure Legend Snippet: Ghrelin preserves cardiac function and reduces infarct size after myocardial infarction (MI). ( a ) Real-time PCR quantification of ghrelin mRNA in total ventricular RNA extracted 2 or 90 days after AAV9 injection and MI induction. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over endogenous ( n =5). ( b , c ) Intracardiac ( b ) and plasmatic ( c ) levels of acyl and des-acyl ghrelin evaluated by EIA 90 days after intracardiac vector injection ( n =4). ( d – g ) Echocardiographic analysis in AAV9-injected and control mice at 2, 7, 30, 60 and 90 days after MI. Left ventricular ejection fraction (LVEF; d ), left ventricular fractional shortening (LVFS; e ), anterior wall thickening (AWTK; f ) and diastolic LV internal diameter (LVID d; g ) of infarcted hearts treated either with AAV9-ghrelin or AAV9-control were measured ( n =12 per group). (h ) Representative M-mode echocardiographic images 90 days after AAV9-control or AAV9-ghrelin injection and MI induction. ( i ) Representative images of whole transverse sections after Azan-trichromic staining of hearts transduced with AAV9-control or AAV9-ghrelin. Fibrotic areas are stained in blue. ( j ) Quantification of infarct size expressed as percentage of LV ( n =12 per group). ( k ) Cardiac expression levels of the indicated genes in AAV9-control and AAV9-ghrelin-treated hearts, 90 days after MI. Values are normalized for GAPDH and expressed as fold over untreated ( n =6). All values are mean±s.e.m. Pairwise comparison was performed with the Student's t -test ( b , c , j ); one-way analysis of variance (ANOVA) and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( a , k ); two-way ANOVA was used in d – g . * P

Techniques Used: Real-time Polymerase Chain Reaction, Injection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Mouse Assay, Staining, Transduction, Expressing

Ghrelin improves muscle functional recovery after femoral artery resection. ( a ) Real-time PCR quantification of ghrelin mRNA in tibialis anterior muscles transduced with 1 × 10 11 vg of AAV. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over endogenous ( n =5). ( b ) Haematoxylin-eosin (HE) staining of tibialis anterior muscles at day 21 after femoral artery resection. Scale bar, 50 μm. ( c ) Quantification of ischaemic lesion in muscles injected with AAV9-ghrelin. Values are expressed as percentage per field ( n =7 per group, four fields counted for each animal). ( d ) Quantification of inflammatory cell infiltration. Values are expressed as percentage of cells per field ( n =7 per group, four fields per animal). ( e ) Expression levels of inflammatory cytokines, analysed by real-time PCR, at day 7 after transduction and surgery. Values are normalized for GAPDH and expressed as fold over untreated ( n =5). ( f , g ) Analysis of apoptosis by TUNEL. Representative images ( f ) and quantification ( g ) of TUNEL-positive nuclei in the muscle areas adjacent to the lesions ( n =5). Red: apoptotic nuclei; blue: 4′-6-diamidino-2-phenylindole (DAPI); green: lectin-stained muscle fibres. Scale bar, 100 μm. ( h ) Ratio between BAX and BCL-2 mRNAs in AAV9-injected muscles, 7 days after femoral artery resection. Values are normalized for GAPDH and expressed as fold over untreated ( n =5). ( i ) Number of fibres with central nuclei per field as a marker of muscle regeneration at 21 days after treatment ( n =7 per group, four fields per animal). ( j ) Fibre size analysis after ischaemia and AAV9 injection. The histograms show the distribution of the fibre cross-sectional areas (μm 2 ); a normal distribution curve is superimposed. Analysis of 20 cross-sections from six animals per group was performed. P
Figure Legend Snippet: Ghrelin improves muscle functional recovery after femoral artery resection. ( a ) Real-time PCR quantification of ghrelin mRNA in tibialis anterior muscles transduced with 1 × 10 11 vg of AAV. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over endogenous ( n =5). ( b ) Haematoxylin-eosin (HE) staining of tibialis anterior muscles at day 21 after femoral artery resection. Scale bar, 50 μm. ( c ) Quantification of ischaemic lesion in muscles injected with AAV9-ghrelin. Values are expressed as percentage per field ( n =7 per group, four fields counted for each animal). ( d ) Quantification of inflammatory cell infiltration. Values are expressed as percentage of cells per field ( n =7 per group, four fields per animal). ( e ) Expression levels of inflammatory cytokines, analysed by real-time PCR, at day 7 after transduction and surgery. Values are normalized for GAPDH and expressed as fold over untreated ( n =5). ( f , g ) Analysis of apoptosis by TUNEL. Representative images ( f ) and quantification ( g ) of TUNEL-positive nuclei in the muscle areas adjacent to the lesions ( n =5). Red: apoptotic nuclei; blue: 4′-6-diamidino-2-phenylindole (DAPI); green: lectin-stained muscle fibres. Scale bar, 100 μm. ( h ) Ratio between BAX and BCL-2 mRNAs in AAV9-injected muscles, 7 days after femoral artery resection. Values are normalized for GAPDH and expressed as fold over untreated ( n =5). ( i ) Number of fibres with central nuclei per field as a marker of muscle regeneration at 21 days after treatment ( n =7 per group, four fields per animal). ( j ) Fibre size analysis after ischaemia and AAV9 injection. The histograms show the distribution of the fibre cross-sectional areas (μm 2 ); a normal distribution curve is superimposed. Analysis of 20 cross-sections from six animals per group was performed. P

Techniques Used: Functional Assay, Real-time Polymerase Chain Reaction, Transduction, Staining, Injection, Expressing, TUNEL Assay, Marker

Reduced apoptosis is paralleled by increased autophagy in ischaemic cardiomyocytes overexpressing ghrelin. ( a , b ) LC3 lipidation (conversion from LC3-I to LC3-II) in the left ventricles of transduced hearts harvested 2 days after MI. Representative western blot ( a ) and densitometric analysis ( b ; n =6). ( c ) Quantification of MAP1LC3A , BECLIN1 and ATG12 mRNA levels in the left ventricles of transduced hearts at 2 days after MI. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over untreated ( n =8). ( d , e ) AMPK phosphorylation in the left ventricles of transduced hearts harvested 2 days after MI. Representative western blot ( d ) and densitometric analysis ( e ) of total AMPK and phosphor (pAMPK; n =6). ( f ) Heart sections of mice injected with AAV9-mRFP-EGFP-LC3 and AAV9-control or AAV9-ghrelin and submitted or not to MI. Autophagosomes appear yellow in the merged image, whereas autolysosomes appear red. Scale bar, 100 μm. ( g ) Quantification of EGFP and mRFP LC3-positive dots per field, using the ImageJ software ( n =6). ( h ) Primary rat neonatal cardiomyocytes transfected with the mRFP-EGFP tandem fluorescent-tagged LC3 plasmid (ptfLC3) and, 48 h later, treated for 4 h with acyl ghrelin, des-acyl ghrelin (both 1 μM) or vehicle in complete or starving medium. Scale bar, 10 μm. ( i ) Quantification of EGFP and mRFP LC3-positive dots per cell, using the ImageJ software ( n =30 cells per group). ( l ) Representative immunofluorescence staining for LC3B (green) in HL-1 cells treated for 4 h with acyl ghrelin, des-acyl ghrelin (both 1 μM) or vehicle in the presence or absence of chloroquine (10 μM) or wortmannin (0.5 μM). Scale bar, 50 μm. ( m ) Quantification of the cells with a high number ( > 30) of LC3B-positive vesicles, expressed as % of the total number of cells ( n =30 cells per group). All values are mean±s.e.m. One-way analysis of variance (ANOVA) and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( b , c , e , g , i , m ). * P
Figure Legend Snippet: Reduced apoptosis is paralleled by increased autophagy in ischaemic cardiomyocytes overexpressing ghrelin. ( a , b ) LC3 lipidation (conversion from LC3-I to LC3-II) in the left ventricles of transduced hearts harvested 2 days after MI. Representative western blot ( a ) and densitometric analysis ( b ; n =6). ( c ) Quantification of MAP1LC3A , BECLIN1 and ATG12 mRNA levels in the left ventricles of transduced hearts at 2 days after MI. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over untreated ( n =8). ( d , e ) AMPK phosphorylation in the left ventricles of transduced hearts harvested 2 days after MI. Representative western blot ( d ) and densitometric analysis ( e ) of total AMPK and phosphor (pAMPK; n =6). ( f ) Heart sections of mice injected with AAV9-mRFP-EGFP-LC3 and AAV9-control or AAV9-ghrelin and submitted or not to MI. Autophagosomes appear yellow in the merged image, whereas autolysosomes appear red. Scale bar, 100 μm. ( g ) Quantification of EGFP and mRFP LC3-positive dots per field, using the ImageJ software ( n =6). ( h ) Primary rat neonatal cardiomyocytes transfected with the mRFP-EGFP tandem fluorescent-tagged LC3 plasmid (ptfLC3) and, 48 h later, treated for 4 h with acyl ghrelin, des-acyl ghrelin (both 1 μM) or vehicle in complete or starving medium. Scale bar, 10 μm. ( i ) Quantification of EGFP and mRFP LC3-positive dots per cell, using the ImageJ software ( n =30 cells per group). ( l ) Representative immunofluorescence staining for LC3B (green) in HL-1 cells treated for 4 h with acyl ghrelin, des-acyl ghrelin (both 1 μM) or vehicle in the presence or absence of chloroquine (10 μM) or wortmannin (0.5 μM). Scale bar, 50 μm. ( m ) Quantification of the cells with a high number ( > 30) of LC3B-positive vesicles, expressed as % of the total number of cells ( n =30 cells per group). All values are mean±s.e.m. One-way analysis of variance (ANOVA) and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( b , c , e , g , i , m ). * P

Techniques Used: Western Blot, Mouse Assay, Injection, Software, Transfection, Plasmid Preparation, Immunofluorescence, Staining

AAV9-ghrelin exerts anti-apoptotic effect on cardiomyocytes exposed to toxic or ischaemic damage in vitro and in vivo . ( a ) TUNEL staining of the infarct border zone in hearts injected with AAV9-control or AAV9-ghrelin 2 days after MI. Nuclei are stained blue with 4′-6-diamidino-2-phenylindole (DAPI) and cardiomyocytes green by an antibody against α-actinin. Red nuclei indicate apoptotic cells. Scale bar, 100 μm. ( b ) Quantification of TUNEL-positive nuclei (% of total) in the peri-infarctual region of AAV9-control and AAV9-ghrelin-treated mice ( n =8 per group). ( c ) Rat neonatal cardiomyocytes, transduced with AAV9-control or AAV9-ghrelin (MOI=5 × 10 4 vg per cell, transduction efficiency > 40%) were either left untreated or treated with (−)-Isoproterenol hydrochloride 10 μM (ISO) or doxorubicin hydrochloride 0.5 μM (DOXO); after 24 or 48 h, respectively, cells were fixed and stained with TUNEL assay. Nuclei are stained blue with DAPI and cardiomyocytes green by an antibody against α-actinin. Red nuclei indicate apoptotic cells. Scale bar, 100 μm. ( d ) Quantification of cardiomyocytes TUNEL-positive nuclei (% of total) after transduction with AAV9-control or AAV9-ghrelin and (−)-Isoproterenol or doxorubicin treatment. Quantification was performed using the ImaJ software ( n =4). ( e , f ) Caspase 3/7 activation analysis in rat neonatal cardiomyocytes ( e ) and HL-1 cells ( f ) transduced with AAV9-ghrelin or AAV9-control and treated with doxorubicin 0.5 μM and 1 μM, respectively, for 20 h ( n =12). All values are mean±s.e.m. Pairwise comparison was performed with the Student's t -test ( b , d ); one-way analysis of variance and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( e , f ). ** P
Figure Legend Snippet: AAV9-ghrelin exerts anti-apoptotic effect on cardiomyocytes exposed to toxic or ischaemic damage in vitro and in vivo . ( a ) TUNEL staining of the infarct border zone in hearts injected with AAV9-control or AAV9-ghrelin 2 days after MI. Nuclei are stained blue with 4′-6-diamidino-2-phenylindole (DAPI) and cardiomyocytes green by an antibody against α-actinin. Red nuclei indicate apoptotic cells. Scale bar, 100 μm. ( b ) Quantification of TUNEL-positive nuclei (% of total) in the peri-infarctual region of AAV9-control and AAV9-ghrelin-treated mice ( n =8 per group). ( c ) Rat neonatal cardiomyocytes, transduced with AAV9-control or AAV9-ghrelin (MOI=5 × 10 4 vg per cell, transduction efficiency > 40%) were either left untreated or treated with (−)-Isoproterenol hydrochloride 10 μM (ISO) or doxorubicin hydrochloride 0.5 μM (DOXO); after 24 or 48 h, respectively, cells were fixed and stained with TUNEL assay. Nuclei are stained blue with DAPI and cardiomyocytes green by an antibody against α-actinin. Red nuclei indicate apoptotic cells. Scale bar, 100 μm. ( d ) Quantification of cardiomyocytes TUNEL-positive nuclei (% of total) after transduction with AAV9-control or AAV9-ghrelin and (−)-Isoproterenol or doxorubicin treatment. Quantification was performed using the ImaJ software ( n =4). ( e , f ) Caspase 3/7 activation analysis in rat neonatal cardiomyocytes ( e ) and HL-1 cells ( f ) transduced with AAV9-ghrelin or AAV9-control and treated with doxorubicin 0.5 μM and 1 μM, respectively, for 20 h ( n =12). All values are mean±s.e.m. Pairwise comparison was performed with the Student's t -test ( b , d ); one-way analysis of variance and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( e , f ). ** P

Techniques Used: In Vitro, In Vivo, TUNEL Assay, Staining, Injection, Mouse Assay, Transduction, Software, Activation Assay

21) Product Images from "The pro-apoptotic Bcl-2 family member Harakiri (HRK) induces cell death in glioblastoma multiforme"

Article Title: The pro-apoptotic Bcl-2 family member Harakiri (HRK) induces cell death in glioblastoma multiforme

Journal: Cell Death Discovery

doi: 10.1038/s41420-019-0144-z

Harakiri overexpression leads to cell death. a Hrk is differentially expressed in four different established cell lines (A172, LN18, U87MG, U373). Values are normalized to the level of housekeeping gene, GAPDH. b Western blot analysis of endogenous HRK expression in A172, LN18, U87MG, U373 cell lines. c Western blot analysis of HRK in the whole cell lysate of GFP- and HRK-overexpressing GBM cell lines. d , e HRK overexpression decreases cell viability ( d ) and increases the activation of caspase 3/7 ( e ). (* denotes p
Figure Legend Snippet: Harakiri overexpression leads to cell death. a Hrk is differentially expressed in four different established cell lines (A172, LN18, U87MG, U373). Values are normalized to the level of housekeeping gene, GAPDH. b Western blot analysis of endogenous HRK expression in A172, LN18, U87MG, U373 cell lines. c Western blot analysis of HRK in the whole cell lysate of GFP- and HRK-overexpressing GBM cell lines. d , e HRK overexpression decreases cell viability ( d ) and increases the activation of caspase 3/7 ( e ). (* denotes p

Techniques Used: Over Expression, Western Blot, Expressing, Activation Assay

Bcl-2 and/or Bcl-xL overexpression inhibits HRK-induced death in GBM cells. a , b Gene expression levels of Bcl-2 and Bcl-xL in A172, LN18, U87MG, and U373 cells detected by qRT-PCR. Values are normalized to the level of housekeeping gene, GAPDH. c – f Cell viability effects of HRK overexpression in GFP, Bcl-2 and/or Bcl-xL overexpressing A172 ( c ), LN18 ( d ), U373 ( e ) and U87MG ( f ) cells after 48 h HRK transduction. g , h Representative fluorescent images of U373 ( g ) and U87MG ( h ) cells transduced with HRK alone (left columns) or together with Bcl-2 and Bcl-xL (right columns) (scale bars:1000 µM) (* denotes p
Figure Legend Snippet: Bcl-2 and/or Bcl-xL overexpression inhibits HRK-induced death in GBM cells. a , b Gene expression levels of Bcl-2 and Bcl-xL in A172, LN18, U87MG, and U373 cells detected by qRT-PCR. Values are normalized to the level of housekeeping gene, GAPDH. c – f Cell viability effects of HRK overexpression in GFP, Bcl-2 and/or Bcl-xL overexpressing A172 ( c ), LN18 ( d ), U373 ( e ) and U87MG ( f ) cells after 48 h HRK transduction. g , h Representative fluorescent images of U373 ( g ) and U87MG ( h ) cells transduced with HRK alone (left columns) or together with Bcl-2 and Bcl-xL (right columns) (scale bars:1000 µM) (* denotes p

Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Transduction

HRK overexpression cooperates with TRAIL to induce cell death in GBM cells. a GBM cells have differential response to TRAIL as measured by cell viability assays of cells in response to TRAIL treatment (0-500 ng/ml) for 24 h. b Cell viability analysis of GFP- or HRK- overexpressing A172, LN18, U87MG, and U373 cells upon 24 h TRAIL treatment (50 ng/ml). c Caspase 3/7 activity analysis of GFP- or HRK- overexpressing A172, LN18, U87MG, U373 GBM cells after 3 h TRAIL treatment (TRAIL concentrations were 20 ng/ml, 20 ng/ml, 50 ng/ml, 200 ng/ml for each cell line respectively). (*, **, *** denote p
Figure Legend Snippet: HRK overexpression cooperates with TRAIL to induce cell death in GBM cells. a GBM cells have differential response to TRAIL as measured by cell viability assays of cells in response to TRAIL treatment (0-500 ng/ml) for 24 h. b Cell viability analysis of GFP- or HRK- overexpressing A172, LN18, U87MG, and U373 cells upon 24 h TRAIL treatment (50 ng/ml). c Caspase 3/7 activity analysis of GFP- or HRK- overexpressing A172, LN18, U87MG, U373 GBM cells after 3 h TRAIL treatment (TRAIL concentrations were 20 ng/ml, 20 ng/ml, 50 ng/ml, 200 ng/ml for each cell line respectively). (*, **, *** denote p

Techniques Used: Over Expression, Activity Assay

22) Product Images from "Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines"

Article Title: Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

Journal: International Journal of Oncology

doi: 10.3892/ijo.2018.4553

Induction of ‘bystander’ senescence in B16 tumour cells. (A) Senescence-associated β-galactosidase activity in B16 cells cultured for 4 days in the medium from DTX-treated cells (SM), IFNγ + TNFα-treated cells or proliferating cell medium (TM). (B) The size and granularity of control and senescent B16 cells was determined by forward and side scatter flow cytometry analysis. (C) Expression of p21 in B16 cells cultured for 4 days in different media (reverse transcription-quantitative polymerase chain reaction). (D) Immunoblotting detection of mouse p21 in B16 cells harvested on day 4 after cultivation in different media. GAPDH was used as a loading control. Data are presented as the mean ± standard deviation. ** P
Figure Legend Snippet: Induction of ‘bystander’ senescence in B16 tumour cells. (A) Senescence-associated β-galactosidase activity in B16 cells cultured for 4 days in the medium from DTX-treated cells (SM), IFNγ + TNFα-treated cells or proliferating cell medium (TM). (B) The size and granularity of control and senescent B16 cells was determined by forward and side scatter flow cytometry analysis. (C) Expression of p21 in B16 cells cultured for 4 days in different media (reverse transcription-quantitative polymerase chain reaction). (D) Immunoblotting detection of mouse p21 in B16 cells harvested on day 4 after cultivation in different media. GAPDH was used as a loading control. Data are presented as the mean ± standard deviation. ** P

Techniques Used: Activity Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

DNA damage response in ‘bystander’ cells treated with conditioned medium from senescent TC-1 and B16 cell lines. Immunofluorescence detection of phosphoSer139 H2A histone family, member X in (A) TC-1 and (B) B16 cells treated with relevant SM and TM medium for 4 days. Scale bar, 20 µ m. Percentage of cells with 1, 2, 3 or more micronuclei in (C) TC-1 and (D) B16 cells treated with SM or TM was quantified. Data are presented as the mean ± standard deviation. ** P
Figure Legend Snippet: DNA damage response in ‘bystander’ cells treated with conditioned medium from senescent TC-1 and B16 cell lines. Immunofluorescence detection of phosphoSer139 H2A histone family, member X in (A) TC-1 and (B) B16 cells treated with relevant SM and TM medium for 4 days. Scale bar, 20 µ m. Percentage of cells with 1, 2, 3 or more micronuclei in (C) TC-1 and (D) B16 cells treated with SM or TM was quantified. Data are presented as the mean ± standard deviation. ** P

Techniques Used: Immunofluorescence, Standard Deviation

DNA damage detection in TC-1 and B16 tumour cell lines. To detect DNA damage, control, DTX- or IFNγ + TNFα-treated (A) TC-1 and (B) B16 cells were stained with phosphoSer139 H2A histone family, member X antibody and mounted with Mowiol containing 4’,6-diamidine-2-phenylindole. Scale bar, 20 µ m. Percentage of cells with 1, 2, 3 or more micronuclei in (C) TC-1 and (D) B16 cells treated with DTX or IFNγ + TNFα was quantified. Data are presented as the mean ± standard deviation. * P
Figure Legend Snippet: DNA damage detection in TC-1 and B16 tumour cell lines. To detect DNA damage, control, DTX- or IFNγ + TNFα-treated (A) TC-1 and (B) B16 cells were stained with phosphoSer139 H2A histone family, member X antibody and mounted with Mowiol containing 4’,6-diamidine-2-phenylindole. Scale bar, 20 µ m. Percentage of cells with 1, 2, 3 or more micronuclei in (C) TC-1 and (D) B16 cells treated with DTX or IFNγ + TNFα was quantified. Data are presented as the mean ± standard deviation. * P

Techniques Used: Staining, Standard Deviation

Secretion of IL-6 and GROα by murine TC-1 and B16 tumour cell lines. Enzyme-linked immunosorbent assay of IL-6 and GROα in supernatants of (A) TC-1 and (B) B16 cells treated with DTX and IFNγ + TNFα. Supernatants were tested in triplicate and the results from three independent experiments are presented as the mean ± standard deviation. *** P
Figure Legend Snippet: Secretion of IL-6 and GROα by murine TC-1 and B16 tumour cell lines. Enzyme-linked immunosorbent assay of IL-6 and GROα in supernatants of (A) TC-1 and (B) B16 cells treated with DTX and IFNγ + TNFα. Supernatants were tested in triplicate and the results from three independent experiments are presented as the mean ± standard deviation. *** P

Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

DTX induces senescence in the B16 cell line. (A) Senescence-associated β-galactosidase activity in B16 cells treated with DTX or IFNγ + TNFα for 4 days. (B) The size and granularity of control or IFNγ + TNFα-treated, senescent B16 cells was determined by forward and side scatter flow cytometry analysis. (C) Autofluorescence of the B16 control cells is presented in light grey, DTX-treated in black and IFNγ + TNFα-treated in grey. (D) Reverse transcription-quantitative polymerase chain reaction quantification of p21 in control, DTX- and IFNγ + TNFα-treated B16 cells. (E) Immunoblotting detection of mouse p21 in control, DTX- and IFNγ + TNFα-treated B16 cells harvested on day 4. GAPDH was used as a loading control. Representative results from at least three independent experiments are presented. Data are presented as the mean ± standard deviation. ** P
Figure Legend Snippet: DTX induces senescence in the B16 cell line. (A) Senescence-associated β-galactosidase activity in B16 cells treated with DTX or IFNγ + TNFα for 4 days. (B) The size and granularity of control or IFNγ + TNFα-treated, senescent B16 cells was determined by forward and side scatter flow cytometry analysis. (C) Autofluorescence of the B16 control cells is presented in light grey, DTX-treated in black and IFNγ + TNFα-treated in grey. (D) Reverse transcription-quantitative polymerase chain reaction quantification of p21 in control, DTX- and IFNγ + TNFα-treated B16 cells. (E) Immunoblotting detection of mouse p21 in control, DTX- and IFNγ + TNFα-treated B16 cells harvested on day 4. GAPDH was used as a loading control. Representative results from at least three independent experiments are presented. Data are presented as the mean ± standard deviation. ** P

Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Standard Deviation

Analysis of TC-1 and B16 cell proliferation during ‘secondary’ induction. (A) TC-1 and (B) B16 cells were seeded in 25 cm 2 cell culture flasks in triplicate and treated with SM and TM. Cell proliferation was determined by counting the cell number on day 4. Data are presented as the mean ± standard deviation. *** P
Figure Legend Snippet: Analysis of TC-1 and B16 cell proliferation during ‘secondary’ induction. (A) TC-1 and (B) B16 cells were seeded in 25 cm 2 cell culture flasks in triplicate and treated with SM and TM. Cell proliferation was determined by counting the cell number on day 4. Data are presented as the mean ± standard deviation. *** P

Techniques Used: Cell Culture, Standard Deviation

Analysis of TC-1 and B16 cell proliferation during ‘primary’ induction. (A) TC-1 and (B) B16 cells were treated with DTX and IFNγ + TNFα. Cell proliferation was determined by counting the cell number on days 4 (TC-1 and B16) and 7 (B16 only). Control cells were passaged on day 4. Data are presented as the mean ± standard deviation. *** P
Figure Legend Snippet: Analysis of TC-1 and B16 cell proliferation during ‘primary’ induction. (A) TC-1 and (B) B16 cells were treated with DTX and IFNγ + TNFα. Cell proliferation was determined by counting the cell number on days 4 (TC-1 and B16) and 7 (B16 only). Control cells were passaged on day 4. Data are presented as the mean ± standard deviation. *** P

Techniques Used: Standard Deviation

23) Product Images from "Discovery of drug-resistant and drug-sensitizing mutations in the oncogenic PI3K isoform p110?"

Article Title: Discovery of drug-resistant and drug-sensitizing mutations in the oncogenic PI3K isoform p110?

Journal: Cancer cell

doi: 10.1016/j.ccr.2008.06.014

Rescue of PI3K-induced growth inhibition in S. cerevisiae by selective PI3K inhibitors
Figure Legend Snippet: Rescue of PI3K-induced growth inhibition in S. cerevisiae by selective PI3K inhibitors

Techniques Used: Inhibition

24) Product Images from "RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions"

Article Title: RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.523

ER stress-induced caspase-8 activation occurs independently of autocrine production of TNF, Fas ligand, or TRAIL. ( a – f ) Ripk1 +/+ MEFs were incubated for 30 min with 10 μ g/ml anti-TNF ( a and b ) or anti-TRAIL ( e and f ) blocking antibodies, or recombinant mouse Fas:Fc chimera ( c and d ), and then stimulated with 1 μ g/ml tunicamycin (Tu) ( a , c , and e ), or with TNF ( b ), anti-Fas antibody ( d ), or TRAIL ( f ) in combination with cycloheximide (CHX). The cells were then lysed and immunoblotted as indicated
Figure Legend Snippet: ER stress-induced caspase-8 activation occurs independently of autocrine production of TNF, Fas ligand, or TRAIL. ( a – f ) Ripk1 +/+ MEFs were incubated for 30 min with 10 μ g/ml anti-TNF ( a and b ) or anti-TRAIL ( e and f ) blocking antibodies, or recombinant mouse Fas:Fc chimera ( c and d ), and then stimulated with 1 μ g/ml tunicamycin (Tu) ( a , c , and e ), or with TNF ( b ), anti-Fas antibody ( d ), or TRAIL ( f ) in combination with cycloheximide (CHX). The cells were then lysed and immunoblotted as indicated

Techniques Used: Activation Assay, Incubation, Blocking Assay, Recombinant

ER stress-induced caspase-8 activation occurs independently of the death receptors TNFR1, FAS, or DR5. ( a – h ) Ripk1 +/+ MEFs were transfected with a control non-silencing siRNA (siNS) or or with siRNA targeting Tnfr1 (siTnfr1), Fas (siFas), Dr5 (siDr5), or Fadd (siFadd), and then exposed to 1 μ g/ml Tu for 12 h ( a , c , e , and g ). As control to test the functionality of repression, the cells were stimulated with TNF in combination with TAK1 inhibitor (TAK1i) ( b ), or with anti-Fas antibody ( d ), or Trail ( f and h ) in combination with cycloheximide (CHX) ( d , f , and h ). The cells were then lysed and immunoblotted as indicated. ( i ) Ripk1 +/+ MEFs were transfected as before, stimulated with 1 μ g/ml Tunicamycin, and then the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of three independent experiments
Figure Legend Snippet: ER stress-induced caspase-8 activation occurs independently of the death receptors TNFR1, FAS, or DR5. ( a – h ) Ripk1 +/+ MEFs were transfected with a control non-silencing siRNA (siNS) or or with siRNA targeting Tnfr1 (siTnfr1), Fas (siFas), Dr5 (siDr5), or Fadd (siFadd), and then exposed to 1 μ g/ml Tu for 12 h ( a , c , e , and g ). As control to test the functionality of repression, the cells were stimulated with TNF in combination with TAK1 inhibitor (TAK1i) ( b ), or with anti-Fas antibody ( d ), or Trail ( f and h ) in combination with cycloheximide (CHX) ( d , f , and h ). The cells were then lysed and immunoblotted as indicated. ( i ) Ripk1 +/+ MEFs were transfected as before, stimulated with 1 μ g/ml Tunicamycin, and then the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of three independent experiments

Techniques Used: Activation Assay, Transfection, Fluorescence

RIPK1 does not regulate JNK or CHOP during ER stress. ( a ) Ripk1 +/+ MEFs were incubated for 30 min with 10 or 20 μ M JNK inhibitor SP 600125 and then exposed to 1 μ g/ml tunicamycin (Tu) for 12 h. The cells were then lysed and immunoblotted as indicated. ( b – d ) Ripk1 +/+ and Ripk1 −/− MEFs were exposed to 1 μ g/ml Tu for the indicated time and cell lysates were either immunoblotted as indicated ( b and d ), or used to measure the expression of CHOP mRNA by RT-qPCR ( c ). Error bars indicate the standard deviation from triplicate samples. The result is representative of two independent experiments
Figure Legend Snippet: RIPK1 does not regulate JNK or CHOP during ER stress. ( a ) Ripk1 +/+ MEFs were incubated for 30 min with 10 or 20 μ M JNK inhibitor SP 600125 and then exposed to 1 μ g/ml tunicamycin (Tu) for 12 h. The cells were then lysed and immunoblotted as indicated. ( b – d ) Ripk1 +/+ and Ripk1 −/− MEFs were exposed to 1 μ g/ml Tu for the indicated time and cell lysates were either immunoblotted as indicated ( b and d ), or used to measure the expression of CHOP mRNA by RT-qPCR ( c ). Error bars indicate the standard deviation from triplicate samples. The result is representative of two independent experiments

Techniques Used: Incubation, Expressing, Quantitative RT-PCR, Standard Deviation

Ripk1 acts upstream of caspase-8 during ER stress-mediated death. ( a ) Ripk1 +/+ and Ripk1 −/− MEFs were exposed to 1 μ g/ml tunicamycin (Tu) for the indicated time, and then lysed and immunoblotted as indicated. ( b and c ) Ripk1 +/+ and Ripk1 −/− MEF transduced with a control shRNA (shCtrl) or with shRNA targeting caspase-8 (shC8) were treated with 1 μ g/ml Tu, and then lysed and immunblotted as indicated. Arrows indicated cleaved fragments of proteins. ( d ) Ripk1 +/+ MEFs were stimulated with 1 μ g/ml Tu for the indicated time, or incubated for 30 min with a TAK1 inhibitor (TAK1i) before stimulation with TNF for 2 h. Caspase-8 immunoprecipitates (IP) and cell lysates (Input 3%) were analyzed by immunoblot (IB) as indicated
Figure Legend Snippet: Ripk1 acts upstream of caspase-8 during ER stress-mediated death. ( a ) Ripk1 +/+ and Ripk1 −/− MEFs were exposed to 1 μ g/ml tunicamycin (Tu) for the indicated time, and then lysed and immunoblotted as indicated. ( b and c ) Ripk1 +/+ and Ripk1 −/− MEF transduced with a control shRNA (shCtrl) or with shRNA targeting caspase-8 (shC8) were treated with 1 μ g/ml Tu, and then lysed and immunblotted as indicated. Arrows indicated cleaved fragments of proteins. ( d ) Ripk1 +/+ MEFs were stimulated with 1 μ g/ml Tu for the indicated time, or incubated for 30 min with a TAK1 inhibitor (TAK1i) before stimulation with TNF for 2 h. Caspase-8 immunoprecipitates (IP) and cell lysates (Input 3%) were analyzed by immunoblot (IB) as indicated

Techniques Used: Transduction, shRNA, Incubation

RIPK1 mediates ER stress-triggered apoptosis independently of its kinase activity. ( a and b ) Ripk1 +/+ and Ripk1 −/− MEFs were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death ( a ) or caspase-3 like activity ( b ) was measured in function of time using the Fluostar Omega fluorescence plate reader.* P
Figure Legend Snippet: RIPK1 mediates ER stress-triggered apoptosis independently of its kinase activity. ( a and b ) Ripk1 +/+ and Ripk1 −/− MEFs were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death ( a ) or caspase-3 like activity ( b ) was measured in function of time using the Fluostar Omega fluorescence plate reader.* P

Techniques Used: Activity Assay, Fluorescence

RIPK1 interacts with the pro-apoptotic receptor IRE1. ( a and b ) Ire1 −/− cells reconstituted with an empty vector (EV) or with a vector coding for hIRE1(hIRE1) were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader ( a ), or cell lysates obtained after 17 h of stimulation were immunoblotted as indicated ( b ). ( c ) HEK293T cells were transiently transfected with plasmids coding for EGFP-hIRE1 and/or Flag-hRIPK1, and RIPK1 was immunprecipitated (IP) using anti-Flag-coated beads. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( d ) HEK293T cells were transiently transfected with plasmids coding for Flag-hIRE1, Flag-hRIPK1 and/or hTNFR1, and IRE1 (upper panels) or RIPK1 (middle panels) were immunoprecipitated with anti-IRE1 or anti-RIPK1 antibodies, respectively. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( e ) Ripk1 +/+ and Ripk1 −/− MEFs were transfected with a control non-silencing siRNA (siNS) or targeting Ire1 (siIre1) and then exposed to 1 μ g/ml Tu for 12 h. The cells were then lysed and immunoblotted as indicated
Figure Legend Snippet: RIPK1 interacts with the pro-apoptotic receptor IRE1. ( a and b ) Ire1 −/− cells reconstituted with an empty vector (EV) or with a vector coding for hIRE1(hIRE1) were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader ( a ), or cell lysates obtained after 17 h of stimulation were immunoblotted as indicated ( b ). ( c ) HEK293T cells were transiently transfected with plasmids coding for EGFP-hIRE1 and/or Flag-hRIPK1, and RIPK1 was immunprecipitated (IP) using anti-Flag-coated beads. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( d ) HEK293T cells were transiently transfected with plasmids coding for Flag-hIRE1, Flag-hRIPK1 and/or hTNFR1, and IRE1 (upper panels) or RIPK1 (middle panels) were immunoprecipitated with anti-IRE1 or anti-RIPK1 antibodies, respectively. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( e ) Ripk1 +/+ and Ripk1 −/− MEFs were transfected with a control non-silencing siRNA (siNS) or targeting Ire1 (siIre1) and then exposed to 1 μ g/ml Tu for 12 h. The cells were then lysed and immunoblotted as indicated

Techniques Used: Plasmid Preparation, Fluorescence, Transfection, Immunoprecipitation

25) Product Images from "Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis"

Article Title: Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0172491

Diagram of the proposed signaling cascade that is induced by Troenan stimulation. PLC: phospholipase C, DAG: diacylglycerol, PKC: protein kinase C, CREB: cAMP response element-binding protein, SOCE: store-operated calcium entry.
Figure Legend Snippet: Diagram of the proposed signaling cascade that is induced by Troenan stimulation. PLC: phospholipase C, DAG: diacylglycerol, PKC: protein kinase C, CREB: cAMP response element-binding protein, SOCE: store-operated calcium entry.

Techniques Used: Planar Chromatography, Binding Assay

Deorphanization of OR51B4. (A) Activation of OR51B4 transfected Hana3A cells by Troenan. Bar chart shows luminescence values upon activation. Luminescence values are normalized to Forskolin activation. OR-transfected cells: Hana3A cells transfected with pCI-vector containing OR51B4. pCI-transfected cells: Hana3A cells transfected with the pCI-vector alone. Control: cells treated with DMSO (0.1%) (B) Molecular receptive field of recombinant expressed OR51B4. Grey inner circle: agonist, white outer circle: inactive substances. N > 3 independent CRE-Luciferase assays.
Figure Legend Snippet: Deorphanization of OR51B4. (A) Activation of OR51B4 transfected Hana3A cells by Troenan. Bar chart shows luminescence values upon activation. Luminescence values are normalized to Forskolin activation. OR-transfected cells: Hana3A cells transfected with pCI-vector containing OR51B4. pCI-transfected cells: Hana3A cells transfected with the pCI-vector alone. Control: cells treated with DMSO (0.1%) (B) Molecular receptive field of recombinant expressed OR51B4. Grey inner circle: agonist, white outer circle: inactive substances. N > 3 independent CRE-Luciferase assays.

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Recombinant, Luciferase

Analysis of the Troenan-induced effect in HCT116 cells containing a doxycycline-sensitive OR51B4-knockdown-sequence. (A) Confirmation of knockdown functionality by qRT-PCR and calcium imaging experiments. M = Marker. Stimulation of HCT116/EV (left) and HCT116/10F1 cells (right) with Troenan (100 μM/ 300 μM). (B) Representative calcium signal of HCT116/EV (above) and HCT116/10F1 (below) cells stimulated with Troenan (300 μM) in calcium imaging analysis. (C) Migration analysis via scratch assay with HCT116/EV and HCT116/10F1 cells with and without doxycycline induction. Stimulation of the cells with Troenan (300 μM) for 48 hours. N = 3 assays with 3 dishes. (D)-(G) Proliferation analysis of HCT116/EV (D, E) and HCT116/10F1 (F, G) cells after treatment with Troenan (300 μM) with and without doxycycline induction. Troenan (300 μM) was applied for 72 hours. N = 20 .
Figure Legend Snippet: Analysis of the Troenan-induced effect in HCT116 cells containing a doxycycline-sensitive OR51B4-knockdown-sequence. (A) Confirmation of knockdown functionality by qRT-PCR and calcium imaging experiments. M = Marker. Stimulation of HCT116/EV (left) and HCT116/10F1 cells (right) with Troenan (100 μM/ 300 μM). (B) Representative calcium signal of HCT116/EV (above) and HCT116/10F1 (below) cells stimulated with Troenan (300 μM) in calcium imaging analysis. (C) Migration analysis via scratch assay with HCT116/EV and HCT116/10F1 cells with and without doxycycline induction. Stimulation of the cells with Troenan (300 μM) for 48 hours. N = 3 assays with 3 dishes. (D)-(G) Proliferation analysis of HCT116/EV (D, E) and HCT116/10F1 (F, G) cells after treatment with Troenan (300 μM) with and without doxycycline induction. Troenan (300 μM) was applied for 72 hours. N = 20 .

Techniques Used: Sequencing, Quantitative RT-PCR, Imaging, Marker, Migration, Wound Healing Assay

Pharmacological analysis of the signaling pathway involved in the activation of OR51B4. Representative calcium imaging traces of HCT116 cells stimulated with Troenan and different specific inhibitors. Grey area represents the duration of the inhibitor applicated. Bar chart showing mean amplitudes of Troenan-induced Ca 2+ signals in HCT116 cells. (A) Localization of Ca 2+ by use of EGTA-Ringer. Investigation of different specific inhibitors of calcium signaling upon Troenan stimulation. ( B ) Gallein (10 μM), (C) U-73522 (10 μM), (D) SQ22.536 (50 μM), (E) H89 (10 μM), (F) RR; Ruthenium red (5 μM), (G) L-cis-diltiazem (150 μM), (H) Mibefradil (10 μM), (I) BTP-2 (25 μM), (J) Thapsigargin (1 μM). N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells. The data are shown as the mean SEM.
Figure Legend Snippet: Pharmacological analysis of the signaling pathway involved in the activation of OR51B4. Representative calcium imaging traces of HCT116 cells stimulated with Troenan and different specific inhibitors. Grey area represents the duration of the inhibitor applicated. Bar chart showing mean amplitudes of Troenan-induced Ca 2+ signals in HCT116 cells. (A) Localization of Ca 2+ by use of EGTA-Ringer. Investigation of different specific inhibitors of calcium signaling upon Troenan stimulation. ( B ) Gallein (10 μM), (C) U-73522 (10 μM), (D) SQ22.536 (50 μM), (E) H89 (10 μM), (F) RR; Ruthenium red (5 μM), (G) L-cis-diltiazem (150 μM), (H) Mibefradil (10 μM), (I) BTP-2 (25 μM), (J) Thapsigargin (1 μM). N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells. The data are shown as the mean SEM.

Techniques Used: Activation Assay, Imaging, Cell Culture

Physiological effects of Troenan stimulation on HCT116 cells. (A) Analyses of cell migration by scratch assay after Troenan stimulation (300 μM) for 48 hours. (B) Bar chart showing statistical analysis of the area overgrown in scratch assay experiments. n = 3 assays. (C) Monitoring of the cell-index equal to the cell proliferation rate of HCT116 cells incubated with Troenan in different concentrations (50, 100, 150 μM). Dynamic real-time monitoring of cell processes in vivo (xCELLigence RTCA-technology). n = 2 in at least 2 independent experiments.
Figure Legend Snippet: Physiological effects of Troenan stimulation on HCT116 cells. (A) Analyses of cell migration by scratch assay after Troenan stimulation (300 μM) for 48 hours. (B) Bar chart showing statistical analysis of the area overgrown in scratch assay experiments. n = 3 assays. (C) Monitoring of the cell-index equal to the cell proliferation rate of HCT116 cells incubated with Troenan in different concentrations (50, 100, 150 μM). Dynamic real-time monitoring of cell processes in vivo (xCELLigence RTCA-technology). n = 2 in at least 2 independent experiments.

Techniques Used: Migration, Wound Healing Assay, Incubation, In Vivo

Impaired actin filament formation and induction of apoptosis upon stimulation of HCT116 cells with Troenan. (A) HCT116 cells treated with Troenan (500 μM) and control cells. (B) and (C) Phalloidin staining of control cells (B) and cells treated with Troenan (300 μM) (C). Scale bar: 10 μm. (D) and (E) Immunocytochemical staining of HCT116 cells with an antibody against caspase-3 after treatment with control (D) or Troenan (300 μM) (E). Cells treated with Troenan (300 μM) for 48 hours. Scale bar: 10 μm. (F) HCT116 cells show decreased serotonin release after application of Troenan (700 μM) for 60 minutes.
Figure Legend Snippet: Impaired actin filament formation and induction of apoptosis upon stimulation of HCT116 cells with Troenan. (A) HCT116 cells treated with Troenan (500 μM) and control cells. (B) and (C) Phalloidin staining of control cells (B) and cells treated with Troenan (300 μM) (C). Scale bar: 10 μm. (D) and (E) Immunocytochemical staining of HCT116 cells with an antibody against caspase-3 after treatment with control (D) or Troenan (300 μM) (E). Cells treated with Troenan (300 μM) for 48 hours. Scale bar: 10 μm. (F) HCT116 cells show decreased serotonin release after application of Troenan (700 μM) for 60 minutes.

Techniques Used: Staining

Western blot analysis of HCT116 cells stimulated with Troenan (300 μM; T) or control (C) for 5 and 25 minutes. (A) Phosphorylation of different isoforms of PKC. (B) Reduced phosphorylation of Stat2, Stat3 and Stat5 upon Troenan stimulation (300 μM). (C) Stimulation of Troenan (300 μM) leads to the time-dependent phosphorylation of p38 MAPK. Reduced phosphorylation was observed for Akt, mTor and Fyn. Troenan stimulation did not affect ERK and SAPK. The total amounts of p38, Akt, ERK, Stat3 and Stat5 and β-actin served as controls. n = 3. (D) Quantification of the mean pixel intensities of the phosphorylated protein kinases Akt, ERK1/2, p38, Stat5 and Stat3. The pixel intensities of duplicates were averaged. Total amounts of the protein kinases were determined and served as controls.
Figure Legend Snippet: Western blot analysis of HCT116 cells stimulated with Troenan (300 μM; T) or control (C) for 5 and 25 minutes. (A) Phosphorylation of different isoforms of PKC. (B) Reduced phosphorylation of Stat2, Stat3 and Stat5 upon Troenan stimulation (300 μM). (C) Stimulation of Troenan (300 μM) leads to the time-dependent phosphorylation of p38 MAPK. Reduced phosphorylation was observed for Akt, mTor and Fyn. Troenan stimulation did not affect ERK and SAPK. The total amounts of p38, Akt, ERK, Stat3 and Stat5 and β-actin served as controls. n = 3. (D) Quantification of the mean pixel intensities of the phosphorylated protein kinases Akt, ERK1/2, p38, Stat5 and Stat3. The pixel intensities of duplicates were averaged. Total amounts of the protein kinases were determined and served as controls.

Techniques Used: Western Blot

Effect of Troenan application on different cancer cell lines. (A) Calcium signals upon Troenan application (300 μM) in the cancer cell lines SW620, Caco-2, HT115, SW982 and SH-EP. N = 3 with n = 6 measurements in 3 cell culture dishes with approximately 200 cells. (B) RNA-Seq data analysis for all cell lines. Bar chart showing the FPKM values in five different cell lines.
Figure Legend Snippet: Effect of Troenan application on different cancer cell lines. (A) Calcium signals upon Troenan application (300 μM) in the cancer cell lines SW620, Caco-2, HT115, SW982 and SH-EP. N = 3 with n = 6 measurements in 3 cell culture dishes with approximately 200 cells. (B) RNA-Seq data analysis for all cell lines. Bar chart showing the FPKM values in five different cell lines.

Techniques Used: Cell Culture, RNA Sequencing Assay

Characterization of Troenan-induced calcium signals in HCT116 cells. (A) Representative image of HCT116 cells stimulated with Troenan (300 μM) in calcium imaging analysis. (B) Number of cells responding to Troenan in different concentrations. (C) Repetitive activation of HCT116 cells upon repetitive Troenan application. (D) Dose-dependent activation of HCT116 cells by Troenan. Troenan was applied at concentrations of 50 μM, 100 μM and 300 μM. Peak amplitudes show the increases in intracellular calcium concentration. To ensure viability of the cells, ATP was applied last, which serves as a positive control. N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells.
Figure Legend Snippet: Characterization of Troenan-induced calcium signals in HCT116 cells. (A) Representative image of HCT116 cells stimulated with Troenan (300 μM) in calcium imaging analysis. (B) Number of cells responding to Troenan in different concentrations. (C) Repetitive activation of HCT116 cells upon repetitive Troenan application. (D) Dose-dependent activation of HCT116 cells by Troenan. Troenan was applied at concentrations of 50 μM, 100 μM and 300 μM. Peak amplitudes show the increases in intracellular calcium concentration. To ensure viability of the cells, ATP was applied last, which serves as a positive control. N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells.

Techniques Used: Imaging, Activation Assay, Concentration Assay, Positive Control, Cell Culture

26) Product Images from "Characterization of the selective in vitro and in vivo binding properties of crenezumab to oligomeric Aβ"

Article Title: Characterization of the selective in vitro and in vivo binding properties of crenezumab to oligomeric Aβ

Journal: Alzheimer's Research & Therapy

doi: 10.1186/s13195-019-0553-5

Crenezumab binding to the hippocampal mossy fibers is Aβ dependent. Representative epifluorescent images of in vivo-dosed crenezumab (80 mg/kg) binding to the mossy fibers ( a ) of PS2APP mice (arrows). Immunostaining for BACE1 shows strong binding in the mossy fibers ( b ) that overlap with crenezumab staining ( c , merge). Scale bar = 50 μm. In vivo-dosed crenezumab (80 mg/kg) staining to the mossy fibers in the PS2APP/BACE1 WT/WT mice ( d ) was nearly completely absent in PS2APP/BACE1 KO/KO ( e ) compared with Ntg/BACE1 WT/WT ( f ) mice. Scale bar, 200 μm. g Significant differences in mossy fiber binding were found between the groups (ANOVA: F 2,8 = 29.16, p
Figure Legend Snippet: Crenezumab binding to the hippocampal mossy fibers is Aβ dependent. Representative epifluorescent images of in vivo-dosed crenezumab (80 mg/kg) binding to the mossy fibers ( a ) of PS2APP mice (arrows). Immunostaining for BACE1 shows strong binding in the mossy fibers ( b ) that overlap with crenezumab staining ( c , merge). Scale bar = 50 μm. In vivo-dosed crenezumab (80 mg/kg) staining to the mossy fibers in the PS2APP/BACE1 WT/WT mice ( d ) was nearly completely absent in PS2APP/BACE1 KO/KO ( e ) compared with Ntg/BACE1 WT/WT ( f ) mice. Scale bar, 200 μm. g Significant differences in mossy fiber binding were found between the groups (ANOVA: F 2,8 = 29.16, p

Techniques Used: Binding Assay, In Vivo, Mouse Assay, Immunostaining, Staining

In vivo-dosed crenezumab binds to (o)ligomeric Aβ, not to (mo)nomeric Aβ, in the hippocampal mossy fiber tract. Plasma and cerebellum PK levels 6 h after the final day of dosing (100 mg/kg daily for 5 d) with an anti-moAβ ( n = 3) or 5 days after a single injection of control IgG (anti-gD 40 mg/kg, n = 4) or crenezumab (80 mg/kg, n = 4) in PS2APP mice. ANOVA found a significant difference in plasma PK levels ( a ) ( F 2,8 = 86.90, p
Figure Legend Snippet: In vivo-dosed crenezumab binds to (o)ligomeric Aβ, not to (mo)nomeric Aβ, in the hippocampal mossy fiber tract. Plasma and cerebellum PK levels 6 h after the final day of dosing (100 mg/kg daily for 5 d) with an anti-moAβ ( n = 3) or 5 days after a single injection of control IgG (anti-gD 40 mg/kg, n = 4) or crenezumab (80 mg/kg, n = 4) in PS2APP mice. ANOVA found a significant difference in plasma PK levels ( a ) ( F 2,8 = 86.90, p

Techniques Used: In Vivo, Injection, Mouse Assay

Crenezumab recognizes Aβ oligomers from in vitro and in vivo sources. Pre-formed (m)onomericAβ 42 , (o)ligomericAβ 42 , or (a)ggregatedAβ 42 were run on native PAGE at 1000, 500, and 250 ng per lane to visualize Aβ 42 banding patterns ( a ). Note that aAβ was too large to enter the gel. Antibodies were incubated with pre-formed Aβ 42 oligomers overnight at 4 °C. To visualize nondenatured oligomers, immunoprecipitated (IP) eluates using were run on native PAGE. Crenezumab recognizes both low molecular weight oligomers between 20 and 50 kDa and high molecular weight (HMW) oligomers between 250 and 700 kDa ( b ). Anti-Aβ IPs from the soluble fraction of PS2APP mouse brain homogenates were run on native PAGE. Crenezumab recognizes HMW oligomers ( c ). 6E10 and 4G8 were used as detection antibodies on all blots
Figure Legend Snippet: Crenezumab recognizes Aβ oligomers from in vitro and in vivo sources. Pre-formed (m)onomericAβ 42 , (o)ligomericAβ 42 , or (a)ggregatedAβ 42 were run on native PAGE at 1000, 500, and 250 ng per lane to visualize Aβ 42 banding patterns ( a ). Note that aAβ was too large to enter the gel. Antibodies were incubated with pre-formed Aβ 42 oligomers overnight at 4 °C. To visualize nondenatured oligomers, immunoprecipitated (IP) eluates using were run on native PAGE. Crenezumab recognizes both low molecular weight oligomers between 20 and 50 kDa and high molecular weight (HMW) oligomers between 250 and 700 kDa ( b ). Anti-Aβ IPs from the soluble fraction of PS2APP mouse brain homogenates were run on native PAGE. Crenezumab recognizes HMW oligomers ( c ). 6E10 and 4G8 were used as detection antibodies on all blots

Techniques Used: In Vitro, In Vivo, Clear Native PAGE, Incubation, Immunoprecipitation, Molecular Weight

In vivo-dosed crenezumab binds in a halo around amyloid plaques and to dystrophic neurites in PS2APP mice. In vivo-dosed crenezumab (200 mg/kg, i.v.) was visualized 7 days postdose with anti-hIgG-Alexa594 antibody (red), and plaques were stained with methoxy-X04 (blue). Representative epifluorescent images of plaque-associated halo of staining by crenezumab alone ( c ) and with plaques ( d ) in the cortex. Note the absence of staining in the control-injected (control IgG, gD) mice around plaques ( a , b ). In the amygdala ( e – g ), confocal z-stacked images show crenezumab binding was prominent around the core of the plaque but in regions not covered by microglia ( e ) (labeled with Iba1, green). This staining pattern was reminiscent of dystrophic neurites and was confirmed by co-staining of crenezumab (80 mg/kg, i.v., red) with markers of dystrophic neurites including BACE1 (green, f ) and LAMP1 (green, g ). Arrowheads indicate example regions of overlap. In vivo-dosed crenezumab ( j , k , red, 120 mg/kg, IP) was localized to regions between methoxy-X04-labeled plaques ( h , k, blue) and GFP-labeled dendrites ( i , k , green) in the dentate gyrus of PS2APP-GFP (line M) mice (2 days postdose). Scale bar, 200 μm ( a – d ) and 50 μm ( e – g )
Figure Legend Snippet: In vivo-dosed crenezumab binds in a halo around amyloid plaques and to dystrophic neurites in PS2APP mice. In vivo-dosed crenezumab (200 mg/kg, i.v.) was visualized 7 days postdose with anti-hIgG-Alexa594 antibody (red), and plaques were stained with methoxy-X04 (blue). Representative epifluorescent images of plaque-associated halo of staining by crenezumab alone ( c ) and with plaques ( d ) in the cortex. Note the absence of staining in the control-injected (control IgG, gD) mice around plaques ( a , b ). In the amygdala ( e – g ), confocal z-stacked images show crenezumab binding was prominent around the core of the plaque but in regions not covered by microglia ( e ) (labeled with Iba1, green). This staining pattern was reminiscent of dystrophic neurites and was confirmed by co-staining of crenezumab (80 mg/kg, i.v., red) with markers of dystrophic neurites including BACE1 (green, f ) and LAMP1 (green, g ). Arrowheads indicate example regions of overlap. In vivo-dosed crenezumab ( j , k , red, 120 mg/kg, IP) was localized to regions between methoxy-X04-labeled plaques ( h , k, blue) and GFP-labeled dendrites ( i , k , green) in the dentate gyrus of PS2APP-GFP (line M) mice (2 days postdose). Scale bar, 200 μm ( a – d ) and 50 μm ( e – g )

Techniques Used: In Vivo, Mouse Assay, Staining, Injection, Binding Assay, Labeling

In vivo-dosed crenezumab binds to the mossy fibers in PS2APP mice. In vivo-dosed crenezumab, but not control IgG (anti-gD IgG4), dose-dependently binds to the mossy fiber axons in the hippocampus of PS2APP mice. Representative epifluorescent images of mossy fiber binding by crenezumab in PS2APP mice ( a ). Quantification of mossy fiber binding integrated density (IntDen) found a significant treatment effect ( b ) (ANOVA: F 4,19 = 50.10, p
Figure Legend Snippet: In vivo-dosed crenezumab binds to the mossy fibers in PS2APP mice. In vivo-dosed crenezumab, but not control IgG (anti-gD IgG4), dose-dependently binds to the mossy fiber axons in the hippocampus of PS2APP mice. Representative epifluorescent images of mossy fiber binding by crenezumab in PS2APP mice ( a ). Quantification of mossy fiber binding integrated density (IntDen) found a significant treatment effect ( b ) (ANOVA: F 4,19 = 50.10, p

Techniques Used: In Vivo, Mouse Assay, Binding Assay

In vivo-dosed crenezumab does not bind to vascular amyloid in PS2APP mice. Representative confocal × 40 images (z-stack maximum projection) of parenchymal amyloid plaques (arrow) and vascular amyloid (arrowhead) stained with methoxy-X04 ( a , c , blue). Note the selective staining of in vivo-dosed crenezumab (200 mg/kg, i.v.) ( b , c , red) to the peri-plaque region and the absence from the vascular amyloid. Scale bar, 100 μm
Figure Legend Snippet: In vivo-dosed crenezumab does not bind to vascular amyloid in PS2APP mice. Representative confocal × 40 images (z-stack maximum projection) of parenchymal amyloid plaques (arrow) and vascular amyloid (arrowhead) stained with methoxy-X04 ( a , c , blue). Note the selective staining of in vivo-dosed crenezumab (200 mg/kg, i.v.) ( b , c , red) to the peri-plaque region and the absence from the vascular amyloid. Scale bar, 100 μm

Techniques Used: In Vivo, Mouse Assay, Staining

27) Product Images from "Repeated lipopolysaccharide exposure causes corticosteroid insensitive airway inflammation via activation of phosphoinositide-3-kinase δ pathway"

Article Title: Repeated lipopolysaccharide exposure causes corticosteroid insensitive airway inflammation via activation of phosphoinositide-3-kinase δ pathway

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2016.07.020

LPS exposure for 3 days reduced HDAC2, Nrf2 levels, and increased Akt phosphorylation. Mice were administered with LPS for 1–3 days, and one day after the last administration, sacrificed. Lung homogenate was separated on SDS-PAGE, and the HDAC2, Nrf2, Akt, p-Akt, and β-actin protein levels were determined by immunoblotting. HDAC2, Nrf2, Akt/p-Akt and β-actin were preliminary detected with predicted band size (A). Upper panel shows a typical immunoblot of HDAC2 (B), Nrf2 (C), Akt, p-Akt (D) and β-actin in the lung homogenates. Lower panel shows the ratios of HDAC2/β-actin (B), Nrf2/β-actin (C) or p-Akt/Akt (D), calculated by measuring band density. Results represent mean±SEM (n=3, # P
Figure Legend Snippet: LPS exposure for 3 days reduced HDAC2, Nrf2 levels, and increased Akt phosphorylation. Mice were administered with LPS for 1–3 days, and one day after the last administration, sacrificed. Lung homogenate was separated on SDS-PAGE, and the HDAC2, Nrf2, Akt, p-Akt, and β-actin protein levels were determined by immunoblotting. HDAC2, Nrf2, Akt/p-Akt and β-actin were preliminary detected with predicted band size (A). Upper panel shows a typical immunoblot of HDAC2 (B), Nrf2 (C), Akt, p-Akt (D) and β-actin in the lung homogenates. Lower panel shows the ratios of HDAC2/β-actin (B), Nrf2/β-actin (C) or p-Akt/Akt (D), calculated by measuring band density. Results represent mean±SEM (n=3, # P

Techniques Used: Mouse Assay, SDS Page

28) Product Images from "Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth"

Article Title: Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth

Journal: The EMBO Journal

doi: 10.15252/embj.201899322

Mechanically stretched human LECs have more VEGFR3‐β1 integrin and less ILK‐β1 integrin interactions LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 min. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (−) mechanical stretch ( n = 6 independent stretch chambers), * P = 0.039. Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 min and used for immunoprecipitation (IP) of HA‐tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates. Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (−) mechanical stretch; normalised to the respective amount of HA‐tagged β1 integrin ( n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers), * P = 0.0007. Data information: Data are presented as means ± SEM, unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.
Figure Legend Snippet: Mechanically stretched human LECs have more VEGFR3‐β1 integrin and less ILK‐β1 integrin interactions LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 min. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (−) mechanical stretch ( n = 6 independent stretch chambers), * P = 0.039. Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 min and used for immunoprecipitation (IP) of HA‐tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates. Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (−) mechanical stretch; normalised to the respective amount of HA‐tagged β1 integrin ( n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers), * P = 0.0007. Data information: Data are presented as means ± SEM, unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.

Techniques Used: Proximity Ligation Assay, Western Blot, Immunoprecipitation, Two Tailed Test

The lymphatic vascular effect of Ilk deletion strictly depends on β1 integrin Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/+ ;Itgb1 ∆/+ mouse embryo (referred to as “control”) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jugular lymph sac/primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 μm, respectively. Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/∆ ;Itgb1 ∆/+ embryo (referred to as “ILK β1 integrin K.O.”), with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jls/pTD. Scale bars: 500 and 100 μm, respectively. LSM images of cross‐sections through the jls/pTD of E13.5 control and ILK β1 integrin K.O. embryos stained for the proliferation marker phospho‐Histone H3. Arrows point to phospho‐Histone H3‐positive LECs. Scale bars: 20 μm. LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p‐Tyr) on stained cross‐sections through the jls/pTD of control and ILK β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1‐stained area. Scale bars: 10 μm. Number of LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. LEC proliferation as determined by the number of phospho‐Histone H3‐positive LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p‐Tyr) per LEC of E13.5 control or ILK β1 integrin K.O. embryos. Data information: Data are presented as means ± SEM, shown as percentage of control embryos with n = 5 embryos per genotype, unpaired two‐tailed Student's t ‐test.
Figure Legend Snippet: The lymphatic vascular effect of Ilk deletion strictly depends on β1 integrin Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/+ ;Itgb1 ∆/+ mouse embryo (referred to as “control”) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jugular lymph sac/primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 μm, respectively. Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/∆ ;Itgb1 ∆/+ embryo (referred to as “ILK β1 integrin K.O.”), with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jls/pTD. Scale bars: 500 and 100 μm, respectively. LSM images of cross‐sections through the jls/pTD of E13.5 control and ILK β1 integrin K.O. embryos stained for the proliferation marker phospho‐Histone H3. Arrows point to phospho‐Histone H3‐positive LECs. Scale bars: 20 μm. LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p‐Tyr) on stained cross‐sections through the jls/pTD of control and ILK β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1‐stained area. Scale bars: 10 μm. Number of LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. LEC proliferation as determined by the number of phospho‐Histone H3‐positive LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p‐Tyr) per LEC of E13.5 control or ILK β1 integrin K.O. embryos. Data information: Data are presented as means ± SEM, shown as percentage of control embryos with n = 5 embryos per genotype, unpaired two‐tailed Student's t ‐test.

Techniques Used: Staining, Marker, Proximity Ligation Assay, Two Tailed Test

ILK controls VEGFR3‐β1 integrin interactions in mouse embryos LSM images of cross‐sections through the jugular lymph sac/primordial thoracic duct (jls/pTD) of an E13.5 control embryo stained for surface VEGFR3 and β1 integrin. Scale bar: 10 μm. LSM images of proximity ligation assay (PLA) dots (arrows) composed of VEGFR3 and β1 integrin on cross‐sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots normalised to Lyve1‐positive area of control or ILK K.O. embryos ( n = 6 embryos per genotype), * P = 0.005. Data information: Data are presented as means ± SEM, shown as percentage of control embryos, unpaired two‐tailed Student's t ‐test.
Figure Legend Snippet: ILK controls VEGFR3‐β1 integrin interactions in mouse embryos LSM images of cross‐sections through the jugular lymph sac/primordial thoracic duct (jls/pTD) of an E13.5 control embryo stained for surface VEGFR3 and β1 integrin. Scale bar: 10 μm. LSM images of proximity ligation assay (PLA) dots (arrows) composed of VEGFR3 and β1 integrin on cross‐sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots normalised to Lyve1‐positive area of control or ILK K.O. embryos ( n = 6 embryos per genotype), * P = 0.005. Data information: Data are presented as means ± SEM, shown as percentage of control embryos, unpaired two‐tailed Student's t ‐test.

Techniques Used: Staining, Proximity Ligation Assay, Two Tailed Test

ILK controls proliferation, VEGFR3 signalling and VEGFR3‐β1 integrin interactions in human LECs Images of adult human LECs after 1 h of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 μm. LEC proliferation as determined by the number of BrdU‐positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 3 independent transfections per siRNA), * P = 0.032 (control versus ILK‐1), * P = 0.005 (control versus ILK‐2), * P = 0.0003 (control versus ILK‐3). VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 4 (control siRNA, ILK‐1 siRNA and ILK‐3 siRNA) or n = 8 (ILK‐2 siRNA) independent transfections per siRNA), * P = 0.0001 (control versus each siRNA). LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK‐1), * P = 0.024 (control versus ILK‐2), * P = 0.001 (control versus ILK‐3). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test.
Figure Legend Snippet: ILK controls proliferation, VEGFR3 signalling and VEGFR3‐β1 integrin interactions in human LECs Images of adult human LECs after 1 h of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 μm. LEC proliferation as determined by the number of BrdU‐positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 3 independent transfections per siRNA), * P = 0.032 (control versus ILK‐1), * P = 0.005 (control versus ILK‐2), * P = 0.0003 (control versus ILK‐3). VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 4 (control siRNA, ILK‐1 siRNA and ILK‐3 siRNA) or n = 8 (ILK‐2 siRNA) independent transfections per siRNA), * P = 0.0001 (control versus each siRNA). LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 μm. Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK‐1), * P = 0.024 (control versus ILK‐2), * P = 0.001 (control versus ILK‐3). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test.

Techniques Used: BrdU Incorporation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Proximity Ligation Assay

Simplified model of mechanosensitive VEGFR3 signalling and ILK‐controlled lymphatic vascular growth In quiescent LECs, VEGFR3 and β1 integrin are physically separated. ILK directly or indirectly interacts with β1 integrin and connects it to the F‐actin cytoskeleton via intracellular proteins, such as α‐parvin, a component of the IPP complex. Upon mechanical stretch, the complex of β1 integrin and ILK (along with the entire IPP complex) transiently disrupts. This releases β1 integrin, resulting in its interaction with VEGFR3, and thus in increased VEGFR3 tyrosine phosphorylation (“P” in yellow circle). As a consequence, LEC proliferation and lymphatic vascular growth are induced. The absence of ILK results in permanent interaction between VEGFR3 and β1 integrin, leading to upregulated VEGFR3 tyrosine phosphorylation (“P” in yellow circle), LEC proliferation and non‐physiologic lymphatic vascular growth.
Figure Legend Snippet: Simplified model of mechanosensitive VEGFR3 signalling and ILK‐controlled lymphatic vascular growth In quiescent LECs, VEGFR3 and β1 integrin are physically separated. ILK directly or indirectly interacts with β1 integrin and connects it to the F‐actin cytoskeleton via intracellular proteins, such as α‐parvin, a component of the IPP complex. Upon mechanical stretch, the complex of β1 integrin and ILK (along with the entire IPP complex) transiently disrupts. This releases β1 integrin, resulting in its interaction with VEGFR3, and thus in increased VEGFR3 tyrosine phosphorylation (“P” in yellow circle). As a consequence, LEC proliferation and lymphatic vascular growth are induced. The absence of ILK results in permanent interaction between VEGFR3 and β1 integrin, leading to upregulated VEGFR3 tyrosine phosphorylation (“P” in yellow circle), LEC proliferation and non‐physiologic lymphatic vascular growth.

Techniques Used:

29) Product Images from "6-Bromoindirubin-3′-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice"

Article Title: 6-Bromoindirubin-3′-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice

Journal: Scientific Reports

doi: 10.1038/s41598-019-54574-4

A pulse of BIO improves skeletal muscle differentiation in-vitro . ( A ) Schematic representation of C2C12 treatment: C2C12 cells were cultured in the presence of growth medium (GM), differentiation medium (DM), BIO (3 μM) or Vehicle (DMSO), for 24 h and then exposed to either GM or DM for 96 h. ( B ) Immunofluorescence analysis of differentiation marker MHC (green) in C2C12 cells. Nuclei are counterstained with DAPI dye (blue). ( C ) The histograms indicate the fusion index and the myotube size of all samples at day 4 of myogenic differentiation. The fusion index was calculated as the ratio of the number of nuclei inside myotubes (MHC positive cells) to the number of total nuclei at day 4 of myogenic differentiation. A myotube was defined by the presence of at least three nuclei within a continuous cell membrane. Data represent means ± SEM relative to GM or DM treated cells Data were analyzed by one-way analysis of variance ANOVA: ( C ) left panel (ns), ( C ) right panel (***P
Figure Legend Snippet: A pulse of BIO improves skeletal muscle differentiation in-vitro . ( A ) Schematic representation of C2C12 treatment: C2C12 cells were cultured in the presence of growth medium (GM), differentiation medium (DM), BIO (3 μM) or Vehicle (DMSO), for 24 h and then exposed to either GM or DM for 96 h. ( B ) Immunofluorescence analysis of differentiation marker MHC (green) in C2C12 cells. Nuclei are counterstained with DAPI dye (blue). ( C ) The histograms indicate the fusion index and the myotube size of all samples at day 4 of myogenic differentiation. The fusion index was calculated as the ratio of the number of nuclei inside myotubes (MHC positive cells) to the number of total nuclei at day 4 of myogenic differentiation. A myotube was defined by the presence of at least three nuclei within a continuous cell membrane. Data represent means ± SEM relative to GM or DM treated cells Data were analyzed by one-way analysis of variance ANOVA: ( C ) left panel (ns), ( C ) right panel (***P

Techniques Used: In Vitro, Cell Culture, Immunofluorescence, Marker

BIO impairs myoblasts cell-cycle progression affecting the expression of cell cycle markers and myomiRs in-vitro . C2C12 cells were cultured in the presence of growth medium (GM), differentiation medium (DM), BIO (3 μM) or Vehicle (DMSO), for 24 h and 48 h. ( A,B ) Cell lysates were subjected to Western blot analysis of GSK3β, Cyclin D1, and GAPDH protein expression. ( C ) qRT-PCR analysis of Cyclin D1 and MyoD in C2C12 treated with BIO (3 μM) or Vehicle (DMSO), for 24 h and 48 h. Data (collected from three independent experiments) represent means ± SEM relative to Vehicle treated cells. Data were analyzed by two-way analysis of variance ANOVA for Cyclin D1 (***P
Figure Legend Snippet: BIO impairs myoblasts cell-cycle progression affecting the expression of cell cycle markers and myomiRs in-vitro . C2C12 cells were cultured in the presence of growth medium (GM), differentiation medium (DM), BIO (3 μM) or Vehicle (DMSO), for 24 h and 48 h. ( A,B ) Cell lysates were subjected to Western blot analysis of GSK3β, Cyclin D1, and GAPDH protein expression. ( C ) qRT-PCR analysis of Cyclin D1 and MyoD in C2C12 treated with BIO (3 μM) or Vehicle (DMSO), for 24 h and 48 h. Data (collected from three independent experiments) represent means ± SEM relative to Vehicle treated cells. Data were analyzed by two-way analysis of variance ANOVA for Cyclin D1 (***P

Techniques Used: Expressing, In Vitro, Cell Culture, Western Blot, Quantitative RT-PCR

GSK3 inhibitors positively affects miR-206 expression. ( A,B ) Schematic representation C2C12 cells transfected with AntagomiR-206 or negative control treated with growth medium (GM), differentiation medium (DM) BIO (3 μM) or Vehicle (DMSO), for 24 h. ( C ) qRT-PCR analysis of miR-206 in C2C12 cells treated as described in panel A. Data (collected from three independent experiments) represent means ± SEM relative to GM treated cells. Data were analyzed by one-way analysis of variance ANOVA (***P
Figure Legend Snippet: GSK3 inhibitors positively affects miR-206 expression. ( A,B ) Schematic representation C2C12 cells transfected with AntagomiR-206 or negative control treated with growth medium (GM), differentiation medium (DM) BIO (3 μM) or Vehicle (DMSO), for 24 h. ( C ) qRT-PCR analysis of miR-206 in C2C12 cells treated as described in panel A. Data (collected from three independent experiments) represent means ± SEM relative to GM treated cells. Data were analyzed by one-way analysis of variance ANOVA (***P

Techniques Used: Expressing, Transfection, Negative Control, Quantitative RT-PCR

Identification of BIO as inhibitor of myoblasts proliferation. ( A ) Schematic representation of the screening cascade: C2C12 cells were probed with the low-molecular weight LOPAC®1280 library, consisting of 1280 pharmacologically active compounds. Identification of BIO as molecule affecting cell proliferation. ( B ) Chemical structure of BIO compound. ( C ) C2C12 cells were cultured in the presence of growth medium (GM), differentiation medium (DM), BIO (3 μM) or Vehicle (DMSO), for 24 h and 48 h. The effect of BIO on both proliferation and viability of C2C12 cells was measured by CellTiter-Glo® Assay and ( D ) by CellTox™ Green Cytotoxicity Assay, respectively. Data were collected from three independent experiments and represent means ± SEM relative to untreated cells (according to one-way analysis of variance ANOVA using the Dunnett’s Multiple Comparison Test). ( E ) C2C12 cells were treated as in C and D panels and cell-cycle distribution was analyzed by flow cytometry. Data (collected from four independent experiments) represent means ± SEM of cells in each phase of the cell cycle compared to a no-treatment group.
Figure Legend Snippet: Identification of BIO as inhibitor of myoblasts proliferation. ( A ) Schematic representation of the screening cascade: C2C12 cells were probed with the low-molecular weight LOPAC®1280 library, consisting of 1280 pharmacologically active compounds. Identification of BIO as molecule affecting cell proliferation. ( B ) Chemical structure of BIO compound. ( C ) C2C12 cells were cultured in the presence of growth medium (GM), differentiation medium (DM), BIO (3 μM) or Vehicle (DMSO), for 24 h and 48 h. The effect of BIO on both proliferation and viability of C2C12 cells was measured by CellTiter-Glo® Assay and ( D ) by CellTox™ Green Cytotoxicity Assay, respectively. Data were collected from three independent experiments and represent means ± SEM relative to untreated cells (according to one-way analysis of variance ANOVA using the Dunnett’s Multiple Comparison Test). ( E ) C2C12 cells were treated as in C and D panels and cell-cycle distribution was analyzed by flow cytometry. Data (collected from four independent experiments) represent means ± SEM of cells in each phase of the cell cycle compared to a no-treatment group.

Techniques Used: Molecular Weight, Cell Culture, Glo Assay, Cytotoxicity Assay, Flow Cytometry, Cytometry

30) Product Images from "MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy"

Article Title: MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0155893

MEK1/2 inhibitor, selumetinib sensitizes GC-resistant ALL cells to dexamethasone. (A) GC-resistant cells exhibit coordinate upregulation of MAPK/ERK pathway components. GSEA plots show relative upregulation of MAPK/ERK cascade components in GC-resistant ALL cells in two independent datasets [ 25 , 26 ]. Relative expression of pathway components is visualized by the heat map. FDR- false discovery rate. (B) ALL cell lines with active MAPK/ERK pathway (SEMK2, 697, CCRF-CEM) and ALL cells with undetectable expression of MAPK/ERK pathway (RS4;11) were incubated for 4h with MEK1/2 inhibitor, selumetinib (SEL, 200 nM). Thereafter, phosphorylation status of ERK1/2 and its substrate p90RSK were assessed by immunoblotting. (C) ALL cell lines were incubated with DEX (0.05 μg/ml 2 μg/ml and 30 μg/ml), SEL (200 nM) or combination of DEX+SEL for 72h and cell death was assessed by annexinV/PI staining followed by flow cytometry analysis. * p
Figure Legend Snippet: MEK1/2 inhibitor, selumetinib sensitizes GC-resistant ALL cells to dexamethasone. (A) GC-resistant cells exhibit coordinate upregulation of MAPK/ERK pathway components. GSEA plots show relative upregulation of MAPK/ERK cascade components in GC-resistant ALL cells in two independent datasets [ 25 , 26 ]. Relative expression of pathway components is visualized by the heat map. FDR- false discovery rate. (B) ALL cell lines with active MAPK/ERK pathway (SEMK2, 697, CCRF-CEM) and ALL cells with undetectable expression of MAPK/ERK pathway (RS4;11) were incubated for 4h with MEK1/2 inhibitor, selumetinib (SEL, 200 nM). Thereafter, phosphorylation status of ERK1/2 and its substrate p90RSK were assessed by immunoblotting. (C) ALL cell lines were incubated with DEX (0.05 μg/ml 2 μg/ml and 30 μg/ml), SEL (200 nM) or combination of DEX+SEL for 72h and cell death was assessed by annexinV/PI staining followed by flow cytometry analysis. * p

Techniques Used: Expressing, Incubation, Staining, Flow Cytometry, Cytometry

Selumetinib sensitized primary ALL blast do DEX through modulation of mTORC1 activity. (A) Primary blast were isolated from peripheral blood of ALL patients and incubated with DEX (0.05 μg/ml), SEL (200 nM) or DEX+SEL. Cell death was assessed by annexinV/PI staining and flow cytometry analysis. (B) Primary blasts from patients indicated with asterisks (panel A) were incubated as described above and phosphorylation status of 4E-BP1 and LC3 processing were assessed in total cell lysates by immunoblotting. Densitometric analyses of LC3II/I are indicated below the blots.
Figure Legend Snippet: Selumetinib sensitized primary ALL blast do DEX through modulation of mTORC1 activity. (A) Primary blast were isolated from peripheral blood of ALL patients and incubated with DEX (0.05 μg/ml), SEL (200 nM) or DEX+SEL. Cell death was assessed by annexinV/PI staining and flow cytometry analysis. (B) Primary blasts from patients indicated with asterisks (panel A) were incubated as described above and phosphorylation status of 4E-BP1 and LC3 processing were assessed in total cell lysates by immunoblotting. Densitometric analyses of LC3II/I are indicated below the blots.

Techniques Used: Activity Assay, Isolation, Incubation, Staining, Flow Cytometry, Cytometry

Overexpression of a constitutively active MEK1 mutant (MEK-Q56P) in GC-sensitive RS4;11 cells induces resistance to DEX. (A) RS4;11 cells were retrovirally transduced with MEK-Q56P or empty control. Cells were lysed and ERK1/2 phoshorylation status was assessed by immunoblotting. (B-C) Control cells and MEK-Q56P—transduced cells were incubated with DEX (0.05, 2 or 30 μg/ml) for 72h. Thereafter, cell death was assessed by annexinV/PI staining and flow cytometry analysis. Absolute, averaged numbers of apoptotic cells in two independent experiments are indicated in (C). Error bars represent SD. P value was calculated using Student’s t-test.
Figure Legend Snippet: Overexpression of a constitutively active MEK1 mutant (MEK-Q56P) in GC-sensitive RS4;11 cells induces resistance to DEX. (A) RS4;11 cells were retrovirally transduced with MEK-Q56P or empty control. Cells were lysed and ERK1/2 phoshorylation status was assessed by immunoblotting. (B-C) Control cells and MEK-Q56P—transduced cells were incubated with DEX (0.05, 2 or 30 μg/ml) for 72h. Thereafter, cell death was assessed by annexinV/PI staining and flow cytometry analysis. Absolute, averaged numbers of apoptotic cells in two independent experiments are indicated in (C). Error bars represent SD. P value was calculated using Student’s t-test.

Techniques Used: Over Expression, Mutagenesis, Transduction, Incubation, Staining, Flow Cytometry, Cytometry

MEK1/2 inhibitor, selumetinib, intensifies DEX induced LC3 conversion, MDC staining and GFP-LC3 relocalization in GC-resistant SEMK2 ALL cells. (A) GC-sensitive (RS4;11) and GC-resistant (SEMK2) cells were incubated with DEX (0.05 μg/mL) in the presence or absence of MEK1/2 inhibitor, selumetinib (SEL, 200 nM) for 24h. When indicated, cells were pretreated for 3 h with 50 μM or 100 μM of chloroquine (CQ). Thereafter, LC3 processing was assessed by immunoblotting. Densitometric analyses of LC3II/I are indicated below the blots. (B) SEMK2 and RS4;11 cells were cultured as described above for 24h, stained with MDC (50 μM) and analyzed by fluorescence microscopy. (C) SEMK2 cells were stably transduced with GFP-LC3 and incubated with DEX, SEL or combination of DEX+SEL for 24h. GFP-LC3 relocalization from diffuse cytoplasmic in control cells to a massive dotty pattern in DEX+SEL treated cells indicates LC3 recruitment to autophagosome membranes. In the lower panel, the percentage of cells with GFP-LC3 dots was quantified by counting the number of cells with > 3 dots and divided by a total number of GFP positive cells in 5 random non-overlapping fields. Pictures were taken at 630 × magnification. P value was calculated using Student’s t-test. (D) Induction of autophagy markers by the DEX and SEL co-treatment involves mTOR suppression. SEMK2 and RS4;11 cells were incubated with DEX in the presence or absence of SEL and lysed. 4E-BP1 phoshorylation status was assessed by immunoblotting. (E) GC-resistant (SEMK2) and—sensitive (RS4;11) cells were incubated with mTOR inhibitor rapamycin (100nM) in the presence or absence of DEX (0.05 μg/mL) for 24h. Thereafter, LC3 processing was assessed by immunoblotting and quantified. Densitometric analyses of LC3II/I are indicated below the blots. (F) Cells were treated as in (E) for 72h. Thereafter, cell numbers were assessed by counting 6 independent fields in Burker’s chamber. Data represent two independent experiments. P-values were calculated using 2-sided Student’s t-test.
Figure Legend Snippet: MEK1/2 inhibitor, selumetinib, intensifies DEX induced LC3 conversion, MDC staining and GFP-LC3 relocalization in GC-resistant SEMK2 ALL cells. (A) GC-sensitive (RS4;11) and GC-resistant (SEMK2) cells were incubated with DEX (0.05 μg/mL) in the presence or absence of MEK1/2 inhibitor, selumetinib (SEL, 200 nM) for 24h. When indicated, cells were pretreated for 3 h with 50 μM or 100 μM of chloroquine (CQ). Thereafter, LC3 processing was assessed by immunoblotting. Densitometric analyses of LC3II/I are indicated below the blots. (B) SEMK2 and RS4;11 cells were cultured as described above for 24h, stained with MDC (50 μM) and analyzed by fluorescence microscopy. (C) SEMK2 cells were stably transduced with GFP-LC3 and incubated with DEX, SEL or combination of DEX+SEL for 24h. GFP-LC3 relocalization from diffuse cytoplasmic in control cells to a massive dotty pattern in DEX+SEL treated cells indicates LC3 recruitment to autophagosome membranes. In the lower panel, the percentage of cells with GFP-LC3 dots was quantified by counting the number of cells with > 3 dots and divided by a total number of GFP positive cells in 5 random non-overlapping fields. Pictures were taken at 630 × magnification. P value was calculated using Student’s t-test. (D) Induction of autophagy markers by the DEX and SEL co-treatment involves mTOR suppression. SEMK2 and RS4;11 cells were incubated with DEX in the presence or absence of SEL and lysed. 4E-BP1 phoshorylation status was assessed by immunoblotting. (E) GC-resistant (SEMK2) and—sensitive (RS4;11) cells were incubated with mTOR inhibitor rapamycin (100nM) in the presence or absence of DEX (0.05 μg/mL) for 24h. Thereafter, LC3 processing was assessed by immunoblotting and quantified. Densitometric analyses of LC3II/I are indicated below the blots. (F) Cells were treated as in (E) for 72h. Thereafter, cell numbers were assessed by counting 6 independent fields in Burker’s chamber. Data represent two independent experiments. P-values were calculated using 2-sided Student’s t-test.

Techniques Used: Staining, Incubation, Cell Culture, Fluorescence, Microscopy, Stable Transfection, Transduction

31) Product Images from "Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation"

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004741

ASYN biochemical state. A. Native Gels. Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. B. STED microscopy. Selected mutants were imaged in order to characterize the fine structure of the inclusions. C. Thioflavin S staining. H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. D-E . Triton X-100 solubility assay and quantification. H4 cells show that all mutants form detergent insoluble species. Student's t test (*p
Figure Legend Snippet: ASYN biochemical state. A. Native Gels. Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. B. STED microscopy. Selected mutants were imaged in order to characterize the fine structure of the inclusions. C. Thioflavin S staining. H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. D-E . Triton X-100 solubility assay and quantification. H4 cells show that all mutants form detergent insoluble species. Student's t test (*p

Techniques Used: Clear Native PAGE, Transfection, Bimolecular Fluorescence Complementation Assay, Construct, Microscopy, Staining, Expressing, Incubation, Solubility

ASYN mutation effects in the inclusion formation. A. Constructs used in the aggregation model.  This model consists of co-expressing SynT together with synphilin-1.  B. Inclusion pattern in H4 cells.  Different SynT mutants resulted in the formation of distinct inclusion formation in human H4 cells. Scale bar: 10 µm.  C. Inclusion quantification. > 50 cells were scored per experiment and classified in different groups according to the pattern of inclusions. Representative cells were drawn to show type of inclusions present in each categories. Lysine mutants (E35K, E57K) increase the percentage of cells with inclusions and the number of inclusions per cell, whereas A30P and proline mutants reduce percentage of cells with inclusions and also the number of inclusions per cell.  D-E. Levels of ASYN.  Immunoblot analysis of the expression levels of ASYN. Student's  t  test (*p
Figure Legend Snippet: ASYN mutation effects in the inclusion formation. A. Constructs used in the aggregation model. This model consists of co-expressing SynT together with synphilin-1. B. Inclusion pattern in H4 cells. Different SynT mutants resulted in the formation of distinct inclusion formation in human H4 cells. Scale bar: 10 µm. C. Inclusion quantification. > 50 cells were scored per experiment and classified in different groups according to the pattern of inclusions. Representative cells were drawn to show type of inclusions present in each categories. Lysine mutants (E35K, E57K) increase the percentage of cells with inclusions and the number of inclusions per cell, whereas A30P and proline mutants reduce percentage of cells with inclusions and also the number of inclusions per cell. D-E. Levels of ASYN. Immunoblot analysis of the expression levels of ASYN. Student's t test (*p

Techniques Used: Mutagenesis, Construct, Expressing

ASYN bPCA. A. Schematic representation of the ASYN bPCA constructs. Non-bioluminescent halves of humanized Gaussia luciferase (hGLuc) were fused to ASYN monomers. B-C . Intact cells (intracellular) and medium (extracellular) from H4 cells co-transfected with S1 and S2 were assayed for luciferase activity 48 hours post-transfection. Intracellular ( B ) and extracellular ( C ) TP displayed a 3-fold increase in luciferase activity compared to WT. n = 12. Student's t test (*p
Figure Legend Snippet: ASYN bPCA. A. Schematic representation of the ASYN bPCA constructs. Non-bioluminescent halves of humanized Gaussia luciferase (hGLuc) were fused to ASYN monomers. B-C . Intact cells (intracellular) and medium (extracellular) from H4 cells co-transfected with S1 and S2 were assayed for luciferase activity 48 hours post-transfection. Intracellular ( B ) and extracellular ( C ) TP displayed a 3-fold increase in luciferase activity compared to WT. n = 12. Student's t test (*p

Techniques Used: Construct, Luciferase, Transfection, Activity Assay

ASYN secretion is inversely correlated with toxicity. A. Secretion of ASYN. B. Toxicity measurements. Medium from H4 cells were collected to determine the secretion and the percentage cytotoxicity for each mutant. To measure the release of ASYN, an ELISA assay was performed. Using the same media we also measured the release of lactate dehydrogenase as a measure of cytotoxicity. We observed that these values were inversely correlated with those obtained in the release/secretion experiments. A decrease trend particularly for TP and Y125F detected in terms of secretion, was higher in toxicity. n = 3. C. Correlation between Secretion and Toxicity. The graph shows the inverse trend in secretion and toxicity.
Figure Legend Snippet: ASYN secretion is inversely correlated with toxicity. A. Secretion of ASYN. B. Toxicity measurements. Medium from H4 cells were collected to determine the secretion and the percentage cytotoxicity for each mutant. To measure the release of ASYN, an ELISA assay was performed. Using the same media we also measured the release of lactate dehydrogenase as a measure of cytotoxicity. We observed that these values were inversely correlated with those obtained in the release/secretion experiments. A decrease trend particularly for TP and Y125F detected in terms of secretion, was higher in toxicity. n = 3. C. Correlation between Secretion and Toxicity. The graph shows the inverse trend in secretion and toxicity.

Techniques Used: Mutagenesis, Enzyme-linked Immunosorbent Assay

ASYN partially co-localizes with endosomes/lysosomes. A. Immunocytochemistry analysis of H4 cells expressing selected ASYN mutants. Partial co-localization of ASYN and LAMP1 suggests interplay between lysosomal degradation and ASYN inclusion formation. B. E57K and Y125F inclusions co-localize with lysosomal marker LAMP-1. We detected the presence of endosomes/lysosomes surrounding the aggregates in E57K and Y125F. This indicates that, maybe this could be the preferential via for degradation for these mutations. Scale bar: 10 µm.
Figure Legend Snippet: ASYN partially co-localizes with endosomes/lysosomes. A. Immunocytochemistry analysis of H4 cells expressing selected ASYN mutants. Partial co-localization of ASYN and LAMP1 suggests interplay between lysosomal degradation and ASYN inclusion formation. B. E57K and Y125F inclusions co-localize with lysosomal marker LAMP-1. We detected the presence of endosomes/lysosomes surrounding the aggregates in E57K and Y125F. This indicates that, maybe this could be the preferential via for degradation for these mutations. Scale bar: 10 µm.

Techniques Used: Immunocytochemistry, Expressing, Marker

32) Product Images from "Critical role for the catalytic activity of phospholipase C-?1 in epidermal growth factor-induced cell migration"

Article Title: Critical role for the catalytic activity of phospholipase C-?1 in epidermal growth factor-induced cell migration

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2010.07.098

EGF-induced intracellular calcium rise requires the catalytic activity of PLC-γ1. SCC4 cells cultured on glass coverslips were transfected with the full-length (FL) and X-domain truncated (ΔX) PLC-γ1-FLAG-tagged fusion constructs
Figure Legend Snippet: EGF-induced intracellular calcium rise requires the catalytic activity of PLC-γ1. SCC4 cells cultured on glass coverslips were transfected with the full-length (FL) and X-domain truncated (ΔX) PLC-γ1-FLAG-tagged fusion constructs

Techniques Used: Activity Assay, Planar Chromatography, Cell Culture, Transfection, Construct

Lipase activity of the full-length and X-domain truncated PLC-γ1-FLAG-tagged fusion constructs. SCC4 cells were transfected with the full-length (FL) and X-domain truncated (ΔX) PLC-γ1-FLAG-tagged fusion constructs as indicated.
Figure Legend Snippet: Lipase activity of the full-length and X-domain truncated PLC-γ1-FLAG-tagged fusion constructs. SCC4 cells were transfected with the full-length (FL) and X-domain truncated (ΔX) PLC-γ1-FLAG-tagged fusion constructs as indicated.

Techniques Used: Activity Assay, Planar Chromatography, Construct, Transfection

Schematic diagram of the full-length and truncated PLC-γ1-FLAG-tagged fusion constructs. A) The constructs of the full-length PLC-γ1 (FL) and PLC-γ1 lacking the X domain (ΔX). (B) SCC4 cells were transfected with DNA constructs
Figure Legend Snippet: Schematic diagram of the full-length and truncated PLC-γ1-FLAG-tagged fusion constructs. A) The constructs of the full-length PLC-γ1 (FL) and PLC-γ1 lacking the X domain (ΔX). (B) SCC4 cells were transfected with DNA constructs

Techniques Used: Planar Chromatography, Construct, Transfection

EGF-induced cell migration requires the catalytic activity of PLC-γ1, IP 3 and intracellular calcium. SCC4 cells were transfected with the full-length (FL) and X-domain truncated (ΔX) PLC-γ1-FLAG-tagged fusion constructs as indicated.
Figure Legend Snippet: EGF-induced cell migration requires the catalytic activity of PLC-γ1, IP 3 and intracellular calcium. SCC4 cells were transfected with the full-length (FL) and X-domain truncated (ΔX) PLC-γ1-FLAG-tagged fusion constructs as indicated.

Techniques Used: Migration, Activity Assay, Planar Chromatography, Transfection, Construct

33) Product Images from "The pro-apoptotic Bcl-2 family member Harakiri (HRK) induces cell death in glioblastoma multiforme"

Article Title: The pro-apoptotic Bcl-2 family member Harakiri (HRK) induces cell death in glioblastoma multiforme

Journal: Cell Death Discovery

doi: 10.1038/s41420-019-0144-z

HRK expression is partly required for TRAIL response and TRAIL sensitization by secondary agents. a , b Viability analysis of U87MG cells ( a ) and LN18 cells ( b ) upon differential doses of TRAIL and MS-275 (5 μM) combination. c qRT-PCR analysis of Hrk expression in four established GBM cell lines (A172, LN18, U87MG, U373) upon MS-275 treatment (5 μM). d qRT-PCR analysis of Bcl-2 family member genes when treated with MS-275 (5 μM). e qRT-PCR analysis of Hrk expression in shControl and shHRK transduced U87MG cells. Values are normalized to the level of housekeeping gene, GAPDH. f Long-term cell growth analysis of shControl and shHRK U87MG cells (t0: time of cell seeding, t1: time of media change). g Cell viability analysis of shControl and shHRK transduced U87MG cells upon TRAIL treatment (75 ng/ml). h Cell viability analysis of shControl and shHRK transduced U87MG cells upon TRAIL (75 ng/ml) and MS-275 (5 μM) combination. (*, **, *** denote p
Figure Legend Snippet: HRK expression is partly required for TRAIL response and TRAIL sensitization by secondary agents. a , b Viability analysis of U87MG cells ( a ) and LN18 cells ( b ) upon differential doses of TRAIL and MS-275 (5 μM) combination. c qRT-PCR analysis of Hrk expression in four established GBM cell lines (A172, LN18, U87MG, U373) upon MS-275 treatment (5 μM). d qRT-PCR analysis of Bcl-2 family member genes when treated with MS-275 (5 μM). e qRT-PCR analysis of Hrk expression in shControl and shHRK transduced U87MG cells. Values are normalized to the level of housekeeping gene, GAPDH. f Long-term cell growth analysis of shControl and shHRK U87MG cells (t0: time of cell seeding, t1: time of media change). g Cell viability analysis of shControl and shHRK transduced U87MG cells upon TRAIL treatment (75 ng/ml). h Cell viability analysis of shControl and shHRK transduced U87MG cells upon TRAIL (75 ng/ml) and MS-275 (5 μM) combination. (*, **, *** denote p

Techniques Used: Expressing, Mass Spectrometry, Quantitative RT-PCR

HRK is differentially expressed in different GBM cell subpopulations and cooperates with TRAIL in primary GBM cell lines. a , b HRK overexpression decreases the viability of primary GBM cell lines, GBM8 ( a ) and MGG152 ( b ), in a time-dependent manner. c TRAIL response of an isogenic GBM cell line (GBM8) pair selected for TS (TRAIL sensitive) and TR (TRAIL resistance) subpopulations that were exposed with differential doses of TRAIL (0, 10, 50, 250, 400 ng/mL) were assessed. d qRT-PCR analysis of apoptotic members in GBM8-TS and GBM8-TR subpopulations was performed. e , f HRK overexpression decreases cell viability and cooperates with TRAIL in TRAIL-resistant ( e ) and TRAIL-sensitive ( f ) subpopulation of a primary GBM cells. (n.s., *, *** denote non-significant, p
Figure Legend Snippet: HRK is differentially expressed in different GBM cell subpopulations and cooperates with TRAIL in primary GBM cell lines. a , b HRK overexpression decreases the viability of primary GBM cell lines, GBM8 ( a ) and MGG152 ( b ), in a time-dependent manner. c TRAIL response of an isogenic GBM cell line (GBM8) pair selected for TS (TRAIL sensitive) and TR (TRAIL resistance) subpopulations that were exposed with differential doses of TRAIL (0, 10, 50, 250, 400 ng/mL) were assessed. d qRT-PCR analysis of apoptotic members in GBM8-TS and GBM8-TR subpopulations was performed. e , f HRK overexpression decreases cell viability and cooperates with TRAIL in TRAIL-resistant ( e ) and TRAIL-sensitive ( f ) subpopulation of a primary GBM cells. (n.s., *, *** denote non-significant, p

Techniques Used: Over Expression, Quantitative RT-PCR

Harakiri overexpression leads to cell death. a Hrk is differentially expressed in four different established cell lines (A172, LN18, U87MG, U373). Values are normalized to the level of housekeeping gene, GAPDH. b Western blot analysis of endogenous HRK expression in A172, LN18, U87MG, U373 cell lines. c Western blot analysis of HRK in the whole cell lysate of GFP- and HRK-overexpressing GBM cell lines. d , e HRK overexpression decreases cell viability ( d ) and increases the activation of caspase 3/7 ( e ). (* denotes p
Figure Legend Snippet: Harakiri overexpression leads to cell death. a Hrk is differentially expressed in four different established cell lines (A172, LN18, U87MG, U373). Values are normalized to the level of housekeeping gene, GAPDH. b Western blot analysis of endogenous HRK expression in A172, LN18, U87MG, U373 cell lines. c Western blot analysis of HRK in the whole cell lysate of GFP- and HRK-overexpressing GBM cell lines. d , e HRK overexpression decreases cell viability ( d ) and increases the activation of caspase 3/7 ( e ). (* denotes p

Techniques Used: Over Expression, Western Blot, Expressing, Activation Assay

Bcl-2 and/or Bcl-xL overexpression inhibits HRK-induced death in GBM cells. a , b Gene expression levels of Bcl-2 and Bcl-xL in A172, LN18, U87MG, and U373 cells detected by qRT-PCR. Values are normalized to the level of housekeeping gene, GAPDH. c – f Cell viability effects of HRK overexpression in GFP, Bcl-2 and/or Bcl-xL overexpressing A172 ( c ), LN18 ( d ), U373 ( e ) and U87MG ( f ) cells after 48 h HRK transduction. g , h Representative fluorescent images of U373 ( g ) and U87MG ( h ) cells transduced with HRK alone (left columns) or together with Bcl-2 and Bcl-xL (right columns) (scale bars:1000 µM) (* denotes p
Figure Legend Snippet: Bcl-2 and/or Bcl-xL overexpression inhibits HRK-induced death in GBM cells. a , b Gene expression levels of Bcl-2 and Bcl-xL in A172, LN18, U87MG, and U373 cells detected by qRT-PCR. Values are normalized to the level of housekeeping gene, GAPDH. c – f Cell viability effects of HRK overexpression in GFP, Bcl-2 and/or Bcl-xL overexpressing A172 ( c ), LN18 ( d ), U373 ( e ) and U87MG ( f ) cells after 48 h HRK transduction. g , h Representative fluorescent images of U373 ( g ) and U87MG ( h ) cells transduced with HRK alone (left columns) or together with Bcl-2 and Bcl-xL (right columns) (scale bars:1000 µM) (* denotes p

Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Transduction

HRK overexpression cooperates with TRAIL to induce cell death in GBM cells. a GBM cells have differential response to TRAIL as measured by cell viability assays of cells in response to TRAIL treatment (0-500 ng/ml) for 24 h. b Cell viability analysis of GFP- or HRK- overexpressing A172, LN18, U87MG, and U373 cells upon 24 h TRAIL treatment (50 ng/ml). c Caspase 3/7 activity analysis of GFP- or HRK- overexpressing A172, LN18, U87MG, U373 GBM cells after 3 h TRAIL treatment (TRAIL concentrations were 20 ng/ml, 20 ng/ml, 50 ng/ml, 200 ng/ml for each cell line respectively). (*, **, *** denote p
Figure Legend Snippet: HRK overexpression cooperates with TRAIL to induce cell death in GBM cells. a GBM cells have differential response to TRAIL as measured by cell viability assays of cells in response to TRAIL treatment (0-500 ng/ml) for 24 h. b Cell viability analysis of GFP- or HRK- overexpressing A172, LN18, U87MG, and U373 cells upon 24 h TRAIL treatment (50 ng/ml). c Caspase 3/7 activity analysis of GFP- or HRK- overexpressing A172, LN18, U87MG, U373 GBM cells after 3 h TRAIL treatment (TRAIL concentrations were 20 ng/ml, 20 ng/ml, 50 ng/ml, 200 ng/ml for each cell line respectively). (*, **, *** denote p

Techniques Used: Over Expression, Activity Assay

34) Product Images from "p62/SQSTM1/Sequestosome-1 is an N-recognin of the N-end rule pathway which modulates autophagosome biogenesis"

Article Title: p62/SQSTM1/Sequestosome-1 is an N-recognin of the N-end rule pathway which modulates autophagosome biogenesis

Journal: Nature Communications

doi: 10.1038/s41467-017-00085-7

XIE62-1004 and XIE2008 accelerate autophagic clearance of mutant huntingtin protein aggregates (mHTT). a Stimulated degradation of GFP-HDQ103 induced by XIE compounds. HeLa cells transiently expressing GFP-HDQ103 were treated with XIE62-1004 (1004), XIE2008 or rapamycin for 18 h and fractionated into soluble and insoluble proteins in 1% Triton X100, followed by immunoblotting analysis. b Inhibition of inclusion body formation by XIE62-1004. HeLa cells expressing GFP-HDQ103 were treated with 10 μM XIE62-1004 for 18 h and analyzed by immunofluorescent analysis of GFP-HDQ103 and immunostaining of p62. c Inhibition of HDQ103 aggregate formation by XIE62-1004. HeLa cells transiently expressing GFP-HDQ25 or GFP-HDQ103 were treated with 10 μM XIE62-1004 or 2 μM rapamycin for 18 h, followed by filter trap analysis. d Facilitated autophagic clearance of HDQ103 aggregates by XIE compounds. Wild-type and ATG5 −/− MEFs transiently expressing GFP-HDQ103 were treated with 10 μM XIE62-1004 (1004), 10 μM XIE2008 (2008) or 2 μM rapamycin for 18 h, followed by soluble and insoluble fractionation and immunoblotting analyses. e Inhibition of HDQ74-GFP inclusion body formation by XIE compounds. Inducible PC12 cells stably expressing EGFP-HDQ74 (mutant Htt) were treated with 1 μg/ml doxycycline for 8 h followed by stimulation with XIE62-1004 (1004), XIE2008, or rapamycin for 18 h and subjected to fluorescence analysis of GFP. Average percentage of cells positive for HDQ74-GFP puncta was calculated by counting 100 cells per experimental condition in each experiment. Data represent the mean (±S.D.) of three independent experiments. Statistical significance was calculated using a one-way ANOVA test (**** P ≤ 0.0001). f Enhanced autophagic degradation of GFP-HDQ74 in XIE62-1004 stimulated PC12 cells. Inducible PC12 cells stably expressing EGFP-HDQ23 or EGFP-HDQ74 were treated with 10 μM XIE62-1004 or 2 μM rapamycin for 18 h following induction with 1 μg/ml doxycycline for 8 h and fractionated into soluble and insoluble proteins
Figure Legend Snippet: XIE62-1004 and XIE2008 accelerate autophagic clearance of mutant huntingtin protein aggregates (mHTT). a Stimulated degradation of GFP-HDQ103 induced by XIE compounds. HeLa cells transiently expressing GFP-HDQ103 were treated with XIE62-1004 (1004), XIE2008 or rapamycin for 18 h and fractionated into soluble and insoluble proteins in 1% Triton X100, followed by immunoblotting analysis. b Inhibition of inclusion body formation by XIE62-1004. HeLa cells expressing GFP-HDQ103 were treated with 10 μM XIE62-1004 for 18 h and analyzed by immunofluorescent analysis of GFP-HDQ103 and immunostaining of p62. c Inhibition of HDQ103 aggregate formation by XIE62-1004. HeLa cells transiently expressing GFP-HDQ25 or GFP-HDQ103 were treated with 10 μM XIE62-1004 or 2 μM rapamycin for 18 h, followed by filter trap analysis. d Facilitated autophagic clearance of HDQ103 aggregates by XIE compounds. Wild-type and ATG5 −/− MEFs transiently expressing GFP-HDQ103 were treated with 10 μM XIE62-1004 (1004), 10 μM XIE2008 (2008) or 2 μM rapamycin for 18 h, followed by soluble and insoluble fractionation and immunoblotting analyses. e Inhibition of HDQ74-GFP inclusion body formation by XIE compounds. Inducible PC12 cells stably expressing EGFP-HDQ74 (mutant Htt) were treated with 1 μg/ml doxycycline for 8 h followed by stimulation with XIE62-1004 (1004), XIE2008, or rapamycin for 18 h and subjected to fluorescence analysis of GFP. Average percentage of cells positive for HDQ74-GFP puncta was calculated by counting 100 cells per experimental condition in each experiment. Data represent the mean (±S.D.) of three independent experiments. Statistical significance was calculated using a one-way ANOVA test (**** P ≤ 0.0001). f Enhanced autophagic degradation of GFP-HDQ74 in XIE62-1004 stimulated PC12 cells. Inducible PC12 cells stably expressing EGFP-HDQ23 or EGFP-HDQ74 were treated with 10 μM XIE62-1004 or 2 μM rapamycin for 18 h following induction with 1 μg/ml doxycycline for 8 h and fractionated into soluble and insoluble proteins

Techniques Used: Mutagenesis, Expressing, Inhibition, Immunostaining, Fractionation, Stable Transfection, Fluorescence

The binding of Nt-Arg to p62 ZZ domain facilitates disulfide bond-linked aggregation of p62 and p62–LC3 interaction. a In vitro oligomerization assay using myc/His tagged p62 deletion mutants (Supplementary Fig. 2a ), followed by non-reducing SDS-PAGE and immunoblotting using a mixture of antibodies to p62 and Myc. b In vitro oligomerization assay using myc/His tagged wild type p62 and mutants carrying point mutations in ZZ domain. c In vitro p62 oligomerization assay using p62 D69A and D129A mutants in comparison with wild-type p62. d R-nsP4 pulldown assay using constructs used in c . e In vitro oligomerization assay of p62 ectopically expressed in HEK293 cells treated with 50 mM Arg-Ala in the presence of 50 mM β-mercaptoethanol (β-ME). f R-nsP4 peptide binding assay of p62 Cys mutants. Each Cys/Ala p62 mutant with myc/His tag was expressed in HEK293 cells, and 50 μg of total protein was used for pulldown. g In vitro oligomerization assay of p62 Cys mutants used in f . Cell lysates with ectopically expressed wild-type p62 and Cys mutants that can bind Nt-Arg were incubated with 20 mM RIFS tetrapeptide for 1.5 h at room temperature. h ELISA measuring the interaction of p62 ZZ domain mutants with LC3. Cell lysates overexpressing p62 proteins were incubated with Arg-Ala at different concentrations to allow p62 binding to LC3-GST linked to glutathione coated plates. Shown are the means (±S.D.) of three independent experiments, each performed in triplicate. A one-way ANOVA was performed to determine statistical significance (** P
Figure Legend Snippet: The binding of Nt-Arg to p62 ZZ domain facilitates disulfide bond-linked aggregation of p62 and p62–LC3 interaction. a In vitro oligomerization assay using myc/His tagged p62 deletion mutants (Supplementary Fig. 2a ), followed by non-reducing SDS-PAGE and immunoblotting using a mixture of antibodies to p62 and Myc. b In vitro oligomerization assay using myc/His tagged wild type p62 and mutants carrying point mutations in ZZ domain. c In vitro p62 oligomerization assay using p62 D69A and D129A mutants in comparison with wild-type p62. d R-nsP4 pulldown assay using constructs used in c . e In vitro oligomerization assay of p62 ectopically expressed in HEK293 cells treated with 50 mM Arg-Ala in the presence of 50 mM β-mercaptoethanol (β-ME). f R-nsP4 peptide binding assay of p62 Cys mutants. Each Cys/Ala p62 mutant with myc/His tag was expressed in HEK293 cells, and 50 μg of total protein was used for pulldown. g In vitro oligomerization assay of p62 Cys mutants used in f . Cell lysates with ectopically expressed wild-type p62 and Cys mutants that can bind Nt-Arg were incubated with 20 mM RIFS tetrapeptide for 1.5 h at room temperature. h ELISA measuring the interaction of p62 ZZ domain mutants with LC3. Cell lysates overexpressing p62 proteins were incubated with Arg-Ala at different concentrations to allow p62 binding to LC3-GST linked to glutathione coated plates. Shown are the means (±S.D.) of three independent experiments, each performed in triplicate. A one-way ANOVA was performed to determine statistical significance (** P

Techniques Used: Binding Assay, In Vitro, SDS Page, Construct, Mutagenesis, Incubation, Enzyme-linked Immunosorbent Assay

ZZ ligands induce self-polymerization and autophagy targeting of p62. a In vitro p62 oligomerization assay using HEK293 cell extracts expressing myc/His-tagged p62. After 2 h incubation of cell extracts with XIE62-1004 at room temperature, forms of p62 was detected by immunoblotting analysis using anti-Myc antibody following non-reducing SDS-PAGE. b In vitro filter trap assay of p62 using myc/His-tagged p62 expressed using the TnT lysate system. c In vitro oligomerization assay using myc/His tagged p62 deletion mutants, Forms of p62 were detected as described in a . d In vitro p62 oligomerization assay using myc/His-tagged p62 wild type and ZZ point mutants. e Similar to d except that in vitro p62 oligomerization assay was performed in the presence or absence of 50 mM β-mercaptoethanol. f In vivo p62 oligomerization assay using 1% Triton X-100 insoluble p62 (wild type and D129A mutant) expressed in HeLa cells treated with 5 μM XIE compounds for 24 h. g In vivo filter trap assay of 1% Triton X-100 insoluble p62. HeLa cells were treated with 10 μM XIE62-1004, 5 μM MG132 or 25 mM hydroxychloroquine for 16 h. h In vivo p62 puncta formation analysis employing immunocytochemistry. HeLa cells treated with XIE compounds for 12 h were stained for p62. Scale bar, 10 μm. i Quantification of h . Data are representative of three independent experiments, and values are expressed as the average number of p62 puncta per cell with the indicated S.D. Statistical significance was calculated using a one-way ANOVA test (* P
Figure Legend Snippet: ZZ ligands induce self-polymerization and autophagy targeting of p62. a In vitro p62 oligomerization assay using HEK293 cell extracts expressing myc/His-tagged p62. After 2 h incubation of cell extracts with XIE62-1004 at room temperature, forms of p62 was detected by immunoblotting analysis using anti-Myc antibody following non-reducing SDS-PAGE. b In vitro filter trap assay of p62 using myc/His-tagged p62 expressed using the TnT lysate system. c In vitro oligomerization assay using myc/His tagged p62 deletion mutants, Forms of p62 were detected as described in a . d In vitro p62 oligomerization assay using myc/His-tagged p62 wild type and ZZ point mutants. e Similar to d except that in vitro p62 oligomerization assay was performed in the presence or absence of 50 mM β-mercaptoethanol. f In vivo p62 oligomerization assay using 1% Triton X-100 insoluble p62 (wild type and D129A mutant) expressed in HeLa cells treated with 5 μM XIE compounds for 24 h. g In vivo filter trap assay of 1% Triton X-100 insoluble p62. HeLa cells were treated with 10 μM XIE62-1004, 5 μM MG132 or 25 mM hydroxychloroquine for 16 h. h In vivo p62 puncta formation analysis employing immunocytochemistry. HeLa cells treated with XIE compounds for 12 h were stained for p62. Scale bar, 10 μm. i Quantification of h . Data are representative of three independent experiments, and values are expressed as the average number of p62 puncta per cell with the indicated S.D. Statistical significance was calculated using a one-way ANOVA test (* P

Techniques Used: In Vitro, Expressing, Incubation, SDS Page, TRAP Assay, In Vivo, Mutagenesis, Immunocytochemistry, Staining

Development of small molecule ligands to p62 ZZ domain using 3D-modeling of p62 and virtual screening. a A 3D-model representing the structure of p62 that shows the predicted binding pocket present in ZZ domain. b The chemical structures of XIE62-1004 and XIE2008. c Docking model of p62 with XIE62-1004 and XIE2008. d The chemical structure of biotinylated XIE2008. e Pulldown assay using biotinylated XIE2008 and myc/His tagged ZZ point mutants expressed in HEK293 cells. 75 μg of total protein was used in pulldown assay, and p62 was detected by immunoblotting analysis using anti-Myc antibody. f Similar to e except that biotinylated XIE2008 pulldown assay was performed using myc/His tagged p62 deletion mutants. g Pulldown assay using biotinylated XIE2008 and 93-residue p62 ZZ -GST containing intact ZZ domain
Figure Legend Snippet: Development of small molecule ligands to p62 ZZ domain using 3D-modeling of p62 and virtual screening. a A 3D-model representing the structure of p62 that shows the predicted binding pocket present in ZZ domain. b The chemical structures of XIE62-1004 and XIE2008. c Docking model of p62 with XIE62-1004 and XIE2008. d The chemical structure of biotinylated XIE2008. e Pulldown assay using biotinylated XIE2008 and myc/His tagged ZZ point mutants expressed in HEK293 cells. 75 μg of total protein was used in pulldown assay, and p62 was detected by immunoblotting analysis using anti-Myc antibody. f Similar to e except that biotinylated XIE2008 pulldown assay was performed using myc/His tagged p62 deletion mutants. g Pulldown assay using biotinylated XIE2008 and 93-residue p62 ZZ -GST containing intact ZZ domain

Techniques Used: Binding Assay

35) Product Images from "STING-dependent sensing of self-DNA drives silica-induced lung inflammation"

Article Title: STING-dependent sensing of self-DNA drives silica-induced lung inflammation

Journal: Nature Communications

doi: 10.1038/s41467-018-07425-1

Circulating DNA and CXCL10 in the airways of silicosis patients, STING, and type I IFN pathway activation in ILD patient lungs. a , b Presence of ( a ) dsDNA in the plasma and ( b ) CXCL10 in the sputum of patients with silicosis (ILO 5–12, see Table 1 ), as compared with healthy individuals (ILO 0). c – f Human lung tissue samples of ILD patients treated or not with cortisone (see Table 2 ). c Confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 50 µm. Bars, 20 µm for zoomed regions. d Immunoblots of phosphorylated-STING (p-STING), STING, TBK1, phospho-TBK1, IRF3, phospho-IRF3, and β-actin. STING dimers quantification. e CXCL10 levels. f Correlation between STING dimers and epithelial damage scored on human histological tissue sections for necrotic cells, desquamation or denudation, and flattening of the epithelial barrier (indicated by arrows). Control lung tissue (patient G) and fibrotic area (patient D). Bars, 100 µm. g – n Human PBMCs were stimulated with silica microparticles (250 µg/mL) for 18 h, transfected with c-di-AMP (6 µg/mL) or unstimulated. g Extracellular dsDNA in cell supernatant, h IFNβ transcript, i correlation between IFNβ transcripts and released dsDNA. ( j ) CXCL10. ( k ) Confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 5 µm. l Immunoblots of phospho-STING, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3, and β-actin. m Flow cytometry annexin V/PI analysis gated on singlet cells. n Correlations between dead cells and extracellular dsDNA or IFNβ transcripts. * p
Figure Legend Snippet: Circulating DNA and CXCL10 in the airways of silicosis patients, STING, and type I IFN pathway activation in ILD patient lungs. a , b Presence of ( a ) dsDNA in the plasma and ( b ) CXCL10 in the sputum of patients with silicosis (ILO 5–12, see Table 1 ), as compared with healthy individuals (ILO 0). c – f Human lung tissue samples of ILD patients treated or not with cortisone (see Table 2 ). c Confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 50 µm. Bars, 20 µm for zoomed regions. d Immunoblots of phosphorylated-STING (p-STING), STING, TBK1, phospho-TBK1, IRF3, phospho-IRF3, and β-actin. STING dimers quantification. e CXCL10 levels. f Correlation between STING dimers and epithelial damage scored on human histological tissue sections for necrotic cells, desquamation or denudation, and flattening of the epithelial barrier (indicated by arrows). Control lung tissue (patient G) and fibrotic area (patient D). Bars, 100 µm. g – n Human PBMCs were stimulated with silica microparticles (250 µg/mL) for 18 h, transfected with c-di-AMP (6 µg/mL) or unstimulated. g Extracellular dsDNA in cell supernatant, h IFNβ transcript, i correlation between IFNβ transcripts and released dsDNA. ( j ) CXCL10. ( k ) Confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 5 µm. l Immunoblots of phospho-STING, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3, and β-actin. m Flow cytometry annexin V/PI analysis gated on singlet cells. n Correlations between dead cells and extracellular dsDNA or IFNβ transcripts. * p

Techniques Used: Activation Assay, Western Blot, Transfection, Flow Cytometry, Cytometry

36) Product Images from "CCDC88A, a prognostic factor for human pancreatic cancers, promotes the motility and invasiveness of pancreatic cancer cells"

Article Title: CCDC88A, a prognostic factor for human pancreatic cancers, promotes the motility and invasiveness of pancreatic cancer cells

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-016-0466-0

Roles of CCDC88A in the formation of cell protrusions. a . Confocal Z stack images of S2-013 cells that were transiently transfected with scrambled control-siRNA (Scr) or CCDC88A -siRNA (siCCDC88A). The transfected cells were incubated on fibronectin, and were subsequently stained with anti-CCDC88A antibody ( green ) and phalloidin ( red ). The lower and right panels in the confocal Z stack show a vertical cross-section ( yellow lines ) through the cells. Arrows, peripheral actin structures in cell protrusions of control-siRNA transfected cells. Blue, nuclear DAPI staining. Bars, 10 μm. b . Quantification of the data shown in Fig. 5a. Columns , mean; bars , SD. * p
Figure Legend Snippet: Roles of CCDC88A in the formation of cell protrusions. a . Confocal Z stack images of S2-013 cells that were transiently transfected with scrambled control-siRNA (Scr) or CCDC88A -siRNA (siCCDC88A). The transfected cells were incubated on fibronectin, and were subsequently stained with anti-CCDC88A antibody ( green ) and phalloidin ( red ). The lower and right panels in the confocal Z stack show a vertical cross-section ( yellow lines ) through the cells. Arrows, peripheral actin structures in cell protrusions of control-siRNA transfected cells. Blue, nuclear DAPI staining. Bars, 10 μm. b . Quantification of the data shown in Fig. 5a. Columns , mean; bars , SD. * p

Techniques Used: Transfection, Incubation, Staining

Roles of CCDC88A in regulating the activation of Akt. a . Scr or siCCDC88A oligos were transfected into S2-013 cells. After 48 h, the cells were incubated on fibronectin for 5 h, and whole cell lysates were prepared for western blot analysis using anti-CCDC88A, anti-Akt and anti-phosphorylated Akt antibodies. Data are representative of three independent experiments. b . Confocal immunofluorescence microscopic images of S2-013 cells that were transiently transfected with scrambled control-siRNA or CCDC88A -siRNA. After 48 h, the cells were incubated on fibronectin for 5 h and were then stained with anti-phosphorylated Akt antibody ( green ), anti-CCDC88A antibody ( red ) and phalloidin ( violet ; actin filaments ). Blue, nuclear DAPI staining. Bars, 10 μm. c . S2-013 cells were pretreated with or without 5 μM of the Akt inhibitor triciribine for 2 h, following which the cells were plated on migration ( left panel ) and Matrigel invasion ( right panel ) chambers. Migrated cells in four fields per group were counted. Data are representative of three independent experiments. Columns , mean; bars , SD. d . S2-013 cells were pretreated with or without 100 ng/mL IGF-1 for 10 min, and whole cell lysates were prepared for western blot analysis using anti-CCDC88A, anti-phosphorylated CCDC88A, anti-PI3K, anti-phosphorylated PI3K, anti-Akt, and anti-phosphorylated Akt antibodies. Data are representative of three independent experiments. e . S2-013 cells were pretreated with or without 100 ng/mL IGF-1 for 10 min and the cells were then plated on migration ( left panel ) and Matrigel invasion ( right panel ) chambers. Migrated cells in four fields per group were counted. Data are representative of three independent experiments. Columns , mean; bars , SD. f . Confocal immunofluorescence microscopic images of S2-013 cells that were cultured on fibronectin with or without IGF-1 stimulation following which the cells were stained with anti-phosphorylated Akt antibody ( green ), anti-CCDC88A antibody ( red ) and phalloidin ( violet ; actin ). Arrows, phosphorylated Akt accumulated in cell protrusions; arrowheads, CCDC88A accumulated in cell protrusions. Blue, nuclear DAPI staining. Bars, 10 μm
Figure Legend Snippet: Roles of CCDC88A in regulating the activation of Akt. a . Scr or siCCDC88A oligos were transfected into S2-013 cells. After 48 h, the cells were incubated on fibronectin for 5 h, and whole cell lysates were prepared for western blot analysis using anti-CCDC88A, anti-Akt and anti-phosphorylated Akt antibodies. Data are representative of three independent experiments. b . Confocal immunofluorescence microscopic images of S2-013 cells that were transiently transfected with scrambled control-siRNA or CCDC88A -siRNA. After 48 h, the cells were incubated on fibronectin for 5 h and were then stained with anti-phosphorylated Akt antibody ( green ), anti-CCDC88A antibody ( red ) and phalloidin ( violet ; actin filaments ). Blue, nuclear DAPI staining. Bars, 10 μm. c . S2-013 cells were pretreated with or without 5 μM of the Akt inhibitor triciribine for 2 h, following which the cells were plated on migration ( left panel ) and Matrigel invasion ( right panel ) chambers. Migrated cells in four fields per group were counted. Data are representative of three independent experiments. Columns , mean; bars , SD. d . S2-013 cells were pretreated with or without 100 ng/mL IGF-1 for 10 min, and whole cell lysates were prepared for western blot analysis using anti-CCDC88A, anti-phosphorylated CCDC88A, anti-PI3K, anti-phosphorylated PI3K, anti-Akt, and anti-phosphorylated Akt antibodies. Data are representative of three independent experiments. e . S2-013 cells were pretreated with or without 100 ng/mL IGF-1 for 10 min and the cells were then plated on migration ( left panel ) and Matrigel invasion ( right panel ) chambers. Migrated cells in four fields per group were counted. Data are representative of three independent experiments. Columns , mean; bars , SD. f . Confocal immunofluorescence microscopic images of S2-013 cells that were cultured on fibronectin with or without IGF-1 stimulation following which the cells were stained with anti-phosphorylated Akt antibody ( green ), anti-CCDC88A antibody ( red ) and phalloidin ( violet ; actin ). Arrows, phosphorylated Akt accumulated in cell protrusions; arrowheads, CCDC88A accumulated in cell protrusions. Blue, nuclear DAPI staining. Bars, 10 μm

Techniques Used: Activation Assay, Transfection, Incubation, Western Blot, Immunofluorescence, Staining, Migration, Cell Culture

Association of CCDC88A and AMPK1 with cell migration and invasion. a. Effects of suppression of CCDC88A on the expression of selected phosphoproteins in S2-013 cells. Cell extracts obtained from fibronectin-stimulated scrambled control-siRNA transfected S2-013 cells or CCDC88A -siRNA transfected S2-013 cells were probed on human phosphoprotein arrays. b . Confocal immunofluorescence microscopic images of S2-013 cells that were cultured on fibronectin and were then labeled with anti-AMPK1 antibody ( green ), anti-CCDC88A antibody ( red ) and phalloidin (violet; actin filaments). Arrows, AMPK1 localized in cell protrusions. Blue, nuclear DAPI staining. Bar, 10 μm. c . Western blot analysis of AMPK1 following transient transfection of S2-013 and PANC-1 cells with a single mixture containing four different siRNA oligonucleotides targeting AMPK1 (siAMPK1) or negative scrambled control (Scr). Western blotting was performed using an anti-AMPK1 antibody. d . Confocal immunofluorescence microscopic images. A myc-tagged CCDC88A-rescue construct was transfected into S2-013 and PANC-1 cells that had been transfected with both CCDC88A -siRNA and AMPK1 -siRNA. 48 h later, the cells were incubated on fibronectin. Cells were stained with anti-myc antibody ( violet ), anti-AMPK1 antibody ( green ), and phalloidin ( red ). Blue, DAPI staining. Bars, 10 μm. e . siRNA oligonucleotides targeting AMPK1 or Scr were transiently transfected into S2-013 and PANC-1 cells. After 48 h, migration and two-chamber invasion assays were performed. Migrating cells in four fields per group were scored ( lower panel ). Data are representative of three independent experiments. Columns , mean; bars , SD. * p
Figure Legend Snippet: Association of CCDC88A and AMPK1 with cell migration and invasion. a. Effects of suppression of CCDC88A on the expression of selected phosphoproteins in S2-013 cells. Cell extracts obtained from fibronectin-stimulated scrambled control-siRNA transfected S2-013 cells or CCDC88A -siRNA transfected S2-013 cells were probed on human phosphoprotein arrays. b . Confocal immunofluorescence microscopic images of S2-013 cells that were cultured on fibronectin and were then labeled with anti-AMPK1 antibody ( green ), anti-CCDC88A antibody ( red ) and phalloidin (violet; actin filaments). Arrows, AMPK1 localized in cell protrusions. Blue, nuclear DAPI staining. Bar, 10 μm. c . Western blot analysis of AMPK1 following transient transfection of S2-013 and PANC-1 cells with a single mixture containing four different siRNA oligonucleotides targeting AMPK1 (siAMPK1) or negative scrambled control (Scr). Western blotting was performed using an anti-AMPK1 antibody. d . Confocal immunofluorescence microscopic images. A myc-tagged CCDC88A-rescue construct was transfected into S2-013 and PANC-1 cells that had been transfected with both CCDC88A -siRNA and AMPK1 -siRNA. 48 h later, the cells were incubated on fibronectin. Cells were stained with anti-myc antibody ( violet ), anti-AMPK1 antibody ( green ), and phalloidin ( red ). Blue, DAPI staining. Bars, 10 μm. e . siRNA oligonucleotides targeting AMPK1 or Scr were transiently transfected into S2-013 and PANC-1 cells. After 48 h, migration and two-chamber invasion assays were performed. Migrating cells in four fields per group were scored ( lower panel ). Data are representative of three independent experiments. Columns , mean; bars , SD. * p

Techniques Used: Migration, Expressing, Transfection, Immunofluorescence, Cell Culture, Labeling, Staining, Western Blot, Construct, Incubation

Subcellular localization of CCDC88A in PDAC cells. a . Confocal immunofluorescence microscopic images of S2-013 and PANC-1 cells that were cultured with or without fibronectin and were then labeled with anti-CCDC88A antibody ( green ) and phalloidin ( red; actin filaments ). Arrows, CCDC88A localized in cell protrusions. Blue, nuclear DAPI staining. Bars, 10 μm. b . Confocal Z stack images show nuclear DAPI staining ( blue ), abundant cytoplasmic CCDC88A and the accumulation of CCDC88A ( green ) in membrane protrusions of fibronectin-stimulated S2-013 cells. Arrows, CCDC88A localized in cell protrusions. The lower and right panels in the confocal Z stack show a vertical cross-section ( yellow lines ) through the cells. Bar, 10 μm
Figure Legend Snippet: Subcellular localization of CCDC88A in PDAC cells. a . Confocal immunofluorescence microscopic images of S2-013 and PANC-1 cells that were cultured with or without fibronectin and were then labeled with anti-CCDC88A antibody ( green ) and phalloidin ( red; actin filaments ). Arrows, CCDC88A localized in cell protrusions. Blue, nuclear DAPI staining. Bars, 10 μm. b . Confocal Z stack images show nuclear DAPI staining ( blue ), abundant cytoplasmic CCDC88A and the accumulation of CCDC88A ( green ) in membrane protrusions of fibronectin-stimulated S2-013 cells. Arrows, CCDC88A localized in cell protrusions. The lower and right panels in the confocal Z stack show a vertical cross-section ( yellow lines ) through the cells. Bar, 10 μm

Techniques Used: Immunofluorescence, Cell Culture, Labeling, Staining

Co-localization of CCDC88A with actin-filaments in cell protrusions. a . Immunoprecipitation (IP) of CCDC88A from S2-013 cells cultured on fibronectin. Proteins within the immunoprecipitates were examined by western blotting. The blots were probed with antibodies against CCDC88A and actin. Mouse IgG isotype control antibody was used as an isotype control. b . Confocal immunofluorescence microscopic images show nuclear DAPI staining ( blue ), abundant cytoplasmic CCDC88A, and the accumulation of CCDC88A ( green ) in membrane protrusions of fibronectin-stimulated S2-013 cells. Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Bar, 10 μm. c . Confocal immunofluorescence microscopic images of S2-013 and PANC-1 cells that were pretreated with 100 μM Cytochalasin D for 12 h and were then incubated on fibronectin. Cells were stained with anti-CCDC88A antibody ( green ). Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Blue, DAPI staining. Bars, 10 μm. d . Confocal immunofluorescence microscopic images. CCDC88A -siRNA transfected S2-013 and PANC-1 cells, which were transiently transfected with a myc-tagged CCDC88A-rescue construct, were pretreated with 100 μM Cytochalasin D for 12 h, and were subsequently incubated on fibronectin. Cells were stained with anti-myc antibody ( green ). Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Blue, DAPI staining. Bars, 10 μm
Figure Legend Snippet: Co-localization of CCDC88A with actin-filaments in cell protrusions. a . Immunoprecipitation (IP) of CCDC88A from S2-013 cells cultured on fibronectin. Proteins within the immunoprecipitates were examined by western blotting. The blots were probed with antibodies against CCDC88A and actin. Mouse IgG isotype control antibody was used as an isotype control. b . Confocal immunofluorescence microscopic images show nuclear DAPI staining ( blue ), abundant cytoplasmic CCDC88A, and the accumulation of CCDC88A ( green ) in membrane protrusions of fibronectin-stimulated S2-013 cells. Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Bar, 10 μm. c . Confocal immunofluorescence microscopic images of S2-013 and PANC-1 cells that were pretreated with 100 μM Cytochalasin D for 12 h and were then incubated on fibronectin. Cells were stained with anti-CCDC88A antibody ( green ). Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Blue, DAPI staining. Bars, 10 μm. d . Confocal immunofluorescence microscopic images. CCDC88A -siRNA transfected S2-013 and PANC-1 cells, which were transiently transfected with a myc-tagged CCDC88A-rescue construct, were pretreated with 100 μM Cytochalasin D for 12 h, and were subsequently incubated on fibronectin. Cells were stained with anti-myc antibody ( green ). Actin filaments were labeled with phalloidin ( red ). Arrows, CCDC88A that was colocalized with actin-filaments in cell protrusions. Blue, DAPI staining. Bars, 10 μm

Techniques Used: Immunoprecipitation, Cell Culture, Western Blot, Immunofluorescence, Staining, Labeling, Incubation, Transfection, Construct

37) Product Images from "Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation"

Article Title: Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

Journal: Molecular Systems Biology

doi: 10.15252/msb.20145880

Proteomic analysis of BCR signalosomes A The Venn diagram shows the overlap between the proteins that were enriched in BCR signalosomes at 5 and 15 min. B A network view of proteins present in BCR signalosome. Proteins are color-coded based on their association with BCR signalosomes at 5 or 15 min after BCR cross-linking. C Validation of the dynamic association of proteins with BCR signalosomes. A20 cells were stimulated with biotinylated α-IgG F(ab′)2 for the indicated time points, the signalosomes were isolated by streptavidin pull-downs, and enrichment of the indicated proteins was analyzed by immunoblotting. D, E ANKRD13A associates with BCR signalosomes. A20 cells were transiently transfected with GFP-tagged ANKRD13A WT and the ANRKD13A ΔUIM mutant (D), or ANKRD13A WT and ANKRD13A UIM3/4 mutant (E). BCR signalosomes were isolated as described in (C), and the enrichment of ANKRD13A was probed using α-GFP antibody. The expression of GFP-tagged ANKRD13A WT, ANRKD13A ΔUIM, and ANKRD13A UIM3/4 mutants in the input material was verified.
Figure Legend Snippet: Proteomic analysis of BCR signalosomes A The Venn diagram shows the overlap between the proteins that were enriched in BCR signalosomes at 5 and 15 min. B A network view of proteins present in BCR signalosome. Proteins are color-coded based on their association with BCR signalosomes at 5 or 15 min after BCR cross-linking. C Validation of the dynamic association of proteins with BCR signalosomes. A20 cells were stimulated with biotinylated α-IgG F(ab′)2 for the indicated time points, the signalosomes were isolated by streptavidin pull-downs, and enrichment of the indicated proteins was analyzed by immunoblotting. D, E ANKRD13A associates with BCR signalosomes. A20 cells were transiently transfected with GFP-tagged ANKRD13A WT and the ANRKD13A ΔUIM mutant (D), or ANKRD13A WT and ANKRD13A UIM3/4 mutant (E). BCR signalosomes were isolated as described in (C), and the enrichment of ANKRD13A was probed using α-GFP antibody. The expression of GFP-tagged ANKRD13A WT, ANRKD13A ΔUIM, and ANKRD13A UIM3/4 mutants in the input material was verified.

Techniques Used: Isolation, Transfection, Mutagenesis, Expressing

38) Product Images from "Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth"

Article Title: Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth

Journal: The EMBO Journal

doi: 10.15252/embj.201899322

Mechanically stretched human LECs have more VEGFR3‐β1 integrin and less ILK‐β1 integrin interactions A–D LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 min. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 μm. E Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (−) mechanical stretch ( n = 6 independent stretch chambers), * P = 0.039. F Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 min and used for immunoprecipitation (IP) of HA‐tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates. G Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (−) mechanical stretch; normalised to the respective amount of HA‐tagged β1 integrin ( n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers), * P = 0.0007. Data information: Data are presented as means ± SEM, unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.
Figure Legend Snippet: Mechanically stretched human LECs have more VEGFR3‐β1 integrin and less ILK‐β1 integrin interactions A–D LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 min. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 μm. E Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (−) mechanical stretch ( n = 6 independent stretch chambers), * P = 0.039. F Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 min and used for immunoprecipitation (IP) of HA‐tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates. G Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (−) mechanical stretch; normalised to the respective amount of HA‐tagged β1 integrin ( n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers), * P = 0.0007. Data information: Data are presented as means ± SEM, unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.

Techniques Used: Proximity Ligation Assay, Western Blot, Immunoprecipitation, Two Tailed Test

ILK regulates α‐parvin expression in adult human LECs ILK and α‐parvin protein bands in Western blots of lysates from human LECs transfected with control siRNA or ILK siRNAs (ILK1‐3). The GAPDH protein band served as a loading control. ILK protein expression normalised to GAPDH protein expression of siRNA‐transfected human LECs ( n = 9 (control and ILK‐3), n = 10 (ILK‐1) and n = 8 (ILK‐2) independent transfections), * P = 0.0001 (control versus ILK‐1 and control versus ILK‐3), * P = 0.017 (control versus ILK‐2). α‐parvin protein expression normalised to GAPDH protein expression of transfected human LEC ( n = 9 (control, ILK‐2 and ILK‐3) and n = 10 (ILK‐1) independent transfections per siRNA), * P = 0.0002 (control versus ILK‐1), P = 0.075 (control versus ILK‐2), * P = 0.0001 (control versus ILK‐3). ILK and α‐parvin protein bands in Western blots of lysates from human LECs transfected with control siRNA or α‐parvin siRNAs (PARVA1‐3). The GAPDH protein band served as a loading control. α‐parvin protein expression normalised to GAPDH protein expression of transfected human LECs ( n = 16 (control and PARVA‐1), n = 11 (PARVA‐2) and n = 14 (PARVA‐3) independent transfections per siRNA), * P = 0.0001. ILK protein expression normalised to GAPDH protein expression of transfected human LECs ( n = 16 (control), n = 15 (PARVA‐1), n = 12 (PARVA‐2) and n = 14 (PARVA‐3) independent transfections per siRNA). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test. Kruskal–Wallis test with Dunn's multiple comparisons test was additionally performed as a non‐parametric test for (B) with * P = 0.011 (control versus ILK‐1) and * P = 0.0003 (control versus ILK‐3), (C) with * P = 0.006 (control versus ILK‐1) and * P = 0.0003 (control versus ILK‐3), (E) with * P = 0.013 (control versus ILK‐1) and * P = 0.0001 (control versus ILK‐2 and control versus ILK‐3) and (F). Source data are available online for this figure.
Figure Legend Snippet: ILK regulates α‐parvin expression in adult human LECs ILK and α‐parvin protein bands in Western blots of lysates from human LECs transfected with control siRNA or ILK siRNAs (ILK1‐3). The GAPDH protein band served as a loading control. ILK protein expression normalised to GAPDH protein expression of siRNA‐transfected human LECs ( n = 9 (control and ILK‐3), n = 10 (ILK‐1) and n = 8 (ILK‐2) independent transfections), * P = 0.0001 (control versus ILK‐1 and control versus ILK‐3), * P = 0.017 (control versus ILK‐2). α‐parvin protein expression normalised to GAPDH protein expression of transfected human LEC ( n = 9 (control, ILK‐2 and ILK‐3) and n = 10 (ILK‐1) independent transfections per siRNA), * P = 0.0002 (control versus ILK‐1), P = 0.075 (control versus ILK‐2), * P = 0.0001 (control versus ILK‐3). ILK and α‐parvin protein bands in Western blots of lysates from human LECs transfected with control siRNA or α‐parvin siRNAs (PARVA1‐3). The GAPDH protein band served as a loading control. α‐parvin protein expression normalised to GAPDH protein expression of transfected human LECs ( n = 16 (control and PARVA‐1), n = 11 (PARVA‐2) and n = 14 (PARVA‐3) independent transfections per siRNA), * P = 0.0001. ILK protein expression normalised to GAPDH protein expression of transfected human LECs ( n = 16 (control), n = 15 (PARVA‐1), n = 12 (PARVA‐2) and n = 14 (PARVA‐3) independent transfections per siRNA). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test. Kruskal–Wallis test with Dunn's multiple comparisons test was additionally performed as a non‐parametric test for (B) with * P = 0.011 (control versus ILK‐1) and * P = 0.0003 (control versus ILK‐3), (C) with * P = 0.006 (control versus ILK‐1) and * P = 0.0003 (control versus ILK‐3), (E) with * P = 0.013 (control versus ILK‐1) and * P = 0.0001 (control versus ILK‐2 and control versus ILK‐3) and (F). Source data are available online for this figure.

Techniques Used: Expressing, Western Blot, Transfection

The lymphatic vascular effect of Ilk deletion strictly depends on β1 integrin A, B Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/+ ;Itgb1 ∆/+ mouse embryo (referred to as “control”) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jugular lymph sac/primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 μm, respectively. C, D Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/∆ ;Itgb1 ∆/+ embryo (referred to as “ILK β1 integrin K.O.”), with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jls/pTD. Scale bars: 500 and 100 μm, respectively. E–H LSM images of cross‐sections through the jls/pTD of E13.5 control and ILK β1 integrin K.O. embryos stained for the proliferation marker phospho‐Histone H3. Arrows point to phospho‐Histone H3‐positive LECs. Scale bars: 20 μm. I–L LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p‐Tyr) on stained cross‐sections through the jls/pTD of control and ILK β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1‐stained area. Scale bars: 10 μm. M Number of LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. N LEC proliferation as determined by the number of phospho‐Histone H3‐positive LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. O Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p‐Tyr) per LEC of E13.5 control or ILK β1 integrin K.O. embryos. Data information: Data are presented as means ± SEM, shown as percentage of control embryos with n = 5 embryos per genotype, unpaired two‐tailed Student's t ‐test.
Figure Legend Snippet: The lymphatic vascular effect of Ilk deletion strictly depends on β1 integrin A, B Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/+ ;Itgb1 ∆/+ mouse embryo (referred to as “control”) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jugular lymph sac/primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 μm, respectively. C, D Bright‐field image of an E13.5 Flk1‐Cre;Ilk ∆/∆ ;Itgb1 ∆/+ embryo (referred to as “ILK β1 integrin K.O.”), with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross‐section through its jls/pTD. Scale bars: 500 and 100 μm, respectively. E–H LSM images of cross‐sections through the jls/pTD of E13.5 control and ILK β1 integrin K.O. embryos stained for the proliferation marker phospho‐Histone H3. Arrows point to phospho‐Histone H3‐positive LECs. Scale bars: 20 μm. I–L LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p‐Tyr) on stained cross‐sections through the jls/pTD of control and ILK β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1‐stained area. Scale bars: 10 μm. M Number of LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. N LEC proliferation as determined by the number of phospho‐Histone H3‐positive LECs per jls/pTD section in E13.5 control or ILK β1 integrin K.O. embryos. O Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p‐Tyr) per LEC of E13.5 control or ILK β1 integrin K.O. embryos. Data information: Data are presented as means ± SEM, shown as percentage of control embryos with n = 5 embryos per genotype, unpaired two‐tailed Student's t ‐test.

Techniques Used: Staining, Marker, Proximity Ligation Assay, Two Tailed Test

ILK controls proliferation, VEGFR3 signalling and VEGFR3‐β1 integrin interactions in human LECs A, B Images of adult human LECs after 1 h of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 μm. C LEC proliferation as determined by the number of BrdU‐positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 3 independent transfections per siRNA), * P = 0.032 (control versus ILK‐1), * P = 0.005 (control versus ILK‐2), * P = 0.0003 (control versus ILK‐3). D VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 4 (control siRNA, ILK‐1 siRNA and ILK‐3 siRNA) or n = 8 (ILK‐2 siRNA) independent transfections per siRNA), * P = 0.0001 (control versus each siRNA). E, F LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 μm. G Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK‐1), * P = 0.024 (control versus ILK‐2), * P = 0.001 (control versus ILK‐3). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test.
Figure Legend Snippet: ILK controls proliferation, VEGFR3 signalling and VEGFR3‐β1 integrin interactions in human LECs A, B Images of adult human LECs after 1 h of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 μm. C LEC proliferation as determined by the number of BrdU‐positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 3 independent transfections per siRNA), * P = 0.032 (control versus ILK‐1), * P = 0.005 (control versus ILK‐2), * P = 0.0003 (control versus ILK‐3). D VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF‐C Cys156Ser ( n = 4 (control siRNA, ILK‐1 siRNA and ILK‐3 siRNA) or n = 8 (ILK‐2 siRNA) independent transfections per siRNA), * P = 0.0001 (control versus each siRNA). E, F LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 μm. G Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK‐1), * P = 0.024 (control versus ILK‐2), * P = 0.001 (control versus ILK‐3). Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one‐way ANOVA with Dunnett's multiple comparisons test.

Techniques Used: BrdU Incorporation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Proximity Ligation Assay

Simplified model of mechanosensitive VEGFR3 signalling and ILK‐controlled lymphatic vascular growth In quiescent LECs, VEGFR3 and β1 integrin are physically separated. ILK directly or indirectly interacts with β1 integrin and connects it to the F‐actin cytoskeleton via intracellular proteins, such as α‐parvin, a component of the IPP complex. Upon mechanical stretch, the complex of β1 integrin and ILK (along with the entire IPP complex) transiently disrupts. This releases β1 integrin, resulting in its interaction with VEGFR3, and thus in increased VEGFR3 tyrosine phosphorylation (“P” in yellow circle). As a consequence, LEC proliferation and lymphatic vascular growth are induced. The absence of ILK results in permanent interaction between VEGFR3 and β1 integrin, leading to upregulated VEGFR3 tyrosine phosphorylation (“P” in yellow circle), LEC proliferation and non‐physiologic lymphatic vascular growth.
Figure Legend Snippet: Simplified model of mechanosensitive VEGFR3 signalling and ILK‐controlled lymphatic vascular growth In quiescent LECs, VEGFR3 and β1 integrin are physically separated. ILK directly or indirectly interacts with β1 integrin and connects it to the F‐actin cytoskeleton via intracellular proteins, such as α‐parvin, a component of the IPP complex. Upon mechanical stretch, the complex of β1 integrin and ILK (along with the entire IPP complex) transiently disrupts. This releases β1 integrin, resulting in its interaction with VEGFR3, and thus in increased VEGFR3 tyrosine phosphorylation (“P” in yellow circle). As a consequence, LEC proliferation and lymphatic vascular growth are induced. The absence of ILK results in permanent interaction between VEGFR3 and β1 integrin, leading to upregulated VEGFR3 tyrosine phosphorylation (“P” in yellow circle), LEC proliferation and non‐physiologic lymphatic vascular growth.

Techniques Used:

39) Product Images from "cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production"

Article Title: cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2010.138

cIAP1 repression induces RIP1 kinase activity. ( a ) L929 cells were pre-treated with 10 μ M Nec-1 and 1 μ M BV6 for 2 h and stimulated with TNF for 4 h. ( b ) RIP3 levels in L929 were reduced by using RNAi. After 72 h, cells were pre-treated with BV6 and stimulated with TNF for 6 h. Results are representative of at least three independent experiments. Error bars indicate standard deviations. * P
Figure Legend Snippet: cIAP1 repression induces RIP1 kinase activity. ( a ) L929 cells were pre-treated with 10 μ M Nec-1 and 1 μ M BV6 for 2 h and stimulated with TNF for 4 h. ( b ) RIP3 levels in L929 were reduced by using RNAi. After 72 h, cells were pre-treated with BV6 and stimulated with TNF for 6 h. Results are representative of at least three independent experiments. Error bars indicate standard deviations. * P

Techniques Used: Activity Assay

cIAP1 and TAK1 regulate the formation of the necrosome complex. ( a ) TAK1 levels in L929 were repressed by using RNAi. After 72 h, cells were pre-treated with Nec-1 and triggered with TNF. ( b ) L929 cells were pre-treated with Nec-1 (10 μ M) and TAK1 kinase inhibitor 5Z-7 (1 μ M) for 1 h, and then stimulated with TNF (10 000 IU/ml) for 2 h. ( c ) CYLD levels were reduced in L929 cells as described above, and then stimulated with TNF for 4 or 6 h. Cell death (% Sytox positivity) was analyzed by flow cytometry. Knockdown efficiency was checked by western blot. Results are representative of at least three independent experiments. Error bars indicate standard deviations. * P
Figure Legend Snippet: cIAP1 and TAK1 regulate the formation of the necrosome complex. ( a ) TAK1 levels in L929 were repressed by using RNAi. After 72 h, cells were pre-treated with Nec-1 and triggered with TNF. ( b ) L929 cells were pre-treated with Nec-1 (10 μ M) and TAK1 kinase inhibitor 5Z-7 (1 μ M) for 1 h, and then stimulated with TNF (10 000 IU/ml) for 2 h. ( c ) CYLD levels were reduced in L929 cells as described above, and then stimulated with TNF for 4 or 6 h. Cell death (% Sytox positivity) was analyzed by flow cytometry. Knockdown efficiency was checked by western blot. Results are representative of at least three independent experiments. Error bars indicate standard deviations. * P

Techniques Used: Flow Cytometry, Cytometry, Western Blot

cIAP1 and TAK1 depletion augments RIP1/RIP3-dependent ROS generation induced by TNF. ( a ) L929 cells were pre-treated with BV6 and Nec-1, and then stimulated with TNF (10 000 IU/ml). ( b ) RIP3 levels in L929 cells were reduced for 72 h using RNAi, and then treated with BV6 and TNF. Protein levels of ( c ) cIAP1 or XIAP and ( d ) TAK1 or CYLD were reduced by using RNAi, followed by TNF stimulation. ( e ) L929 cells were pre-treated with BV6 and 100 μ M BHA. ( f ) p22phox (encoded by the Cyba gene) and Ndufb8 levels were repressed using RNAi, followed by BV6 treatment and TNF stimulation. Knockdown efficiency was checked by western blot or RT-PCR. Cell death (% Sytox positivity) and ROS generation (ΔMFI (DHR123)) were analyzed simultaneously by flow cytometry. Results are representative of at least two independent experiments. Error bars indicate standard deviations
Figure Legend Snippet: cIAP1 and TAK1 depletion augments RIP1/RIP3-dependent ROS generation induced by TNF. ( a ) L929 cells were pre-treated with BV6 and Nec-1, and then stimulated with TNF (10 000 IU/ml). ( b ) RIP3 levels in L929 cells were reduced for 72 h using RNAi, and then treated with BV6 and TNF. Protein levels of ( c ) cIAP1 or XIAP and ( d ) TAK1 or CYLD were reduced by using RNAi, followed by TNF stimulation. ( e ) L929 cells were pre-treated with BV6 and 100 μ M BHA. ( f ) p22phox (encoded by the Cyba gene) and Ndufb8 levels were repressed using RNAi, followed by BV6 treatment and TNF stimulation. Knockdown efficiency was checked by western blot or RT-PCR. Cell death (% Sytox positivity) and ROS generation (ΔMFI (DHR123)) were analyzed simultaneously by flow cytometry. Results are representative of at least two independent experiments. Error bars indicate standard deviations

Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry

BV6 treatment sensitizes cells to necrosis induced by TNF, but not to necrosis induced by anti-Fas, poly(I:C), or H 2 O 2 . ( a ) L929 cells were treated with 1 μ M BV6 for the indicated durations. Cells were lysed and cIAP1/2 and XIAP were immunoblotted. ( b ) L929 cells were pre-treated with IFN β , zVAD-fmk, or medium in the presence of BV6, and then stimulated with the mentioned triggers for the indicated times. Cell death (% Sytox positivity) was analyzed by flow cytometry. Data are representative of three independent experiments. Error bars represent standard deviation. ( c ) L929 cells were pre-treated with BV6 and stimulated with a serial dilution of TNF. After 20 h, cell viability was determined by an MTT assay. Error bars represent standard deviations of triplicates. ( d ) L929 cells were pre-treated with BV6 and stimulated with TNF. Cell lysates were made, and processed caspase-3 was checked by western blot. Stimulation with anti-Fas was included as a positive control for apoptosis. Cell death was determined by flow cytometry. ( e ) FADD−/− Jurkat cells were pre-treated with BV6 and stimulated with TNF for 24 h. Cell death (PI positivity) was analyzed by flow cytometry. Levels of cIAP1, cIAP2 and XIAP were checked on western blot. * P
Figure Legend Snippet: BV6 treatment sensitizes cells to necrosis induced by TNF, but not to necrosis induced by anti-Fas, poly(I:C), or H 2 O 2 . ( a ) L929 cells were treated with 1 μ M BV6 for the indicated durations. Cells were lysed and cIAP1/2 and XIAP were immunoblotted. ( b ) L929 cells were pre-treated with IFN β , zVAD-fmk, or medium in the presence of BV6, and then stimulated with the mentioned triggers for the indicated times. Cell death (% Sytox positivity) was analyzed by flow cytometry. Data are representative of three independent experiments. Error bars represent standard deviation. ( c ) L929 cells were pre-treated with BV6 and stimulated with a serial dilution of TNF. After 20 h, cell viability was determined by an MTT assay. Error bars represent standard deviations of triplicates. ( d ) L929 cells were pre-treated with BV6 and stimulated with TNF. Cell lysates were made, and processed caspase-3 was checked by western blot. Stimulation with anti-Fas was included as a positive control for apoptosis. Cell death was determined by flow cytometry. ( e ) FADD−/− Jurkat cells were pre-treated with BV6 and stimulated with TNF for 24 h. Cell death (PI positivity) was analyzed by flow cytometry. Levels of cIAP1, cIAP2 and XIAP were checked on western blot. * P

Techniques Used: Flow Cytometry, Cytometry, Standard Deviation, Serial Dilution, MTT Assay, Western Blot, Positive Control

Loss of cIAP1 sensitizes L929 cells to TNF-induced necrosis. ( a ) Protein levels of cIAP1, cIAP2, and XIAP were repressed by using RNAi. Knockdown efficiency was checked by western blot. Detection of cIAP2 is below detection limit. ( b ) mRNA levels of cIAP2 were tested using RT-PCR. cDNA from macrophages was included as a positive control. ( c ) L929 cells with repressed levels of cIAP1, cIAP2, or XIAP were stimulated with TNF (10 000 IU/ml) for 2, 4, or 6 h. Cell death (% Sytox positivity) was analyzed by flow cytometry. Results are representative of at least three independent experiments. Error bars indicate standard deviation. *** P
Figure Legend Snippet: Loss of cIAP1 sensitizes L929 cells to TNF-induced necrosis. ( a ) Protein levels of cIAP1, cIAP2, and XIAP were repressed by using RNAi. Knockdown efficiency was checked by western blot. Detection of cIAP2 is below detection limit. ( b ) mRNA levels of cIAP2 were tested using RT-PCR. cDNA from macrophages was included as a positive control. ( c ) L929 cells with repressed levels of cIAP1, cIAP2, or XIAP were stimulated with TNF (10 000 IU/ml) for 2, 4, or 6 h. Cell death (% Sytox positivity) was analyzed by flow cytometry. Results are representative of at least three independent experiments. Error bars indicate standard deviation. *** P

Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control, Flow Cytometry, Cytometry, Standard Deviation

40) Product Images from "Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-β"

Article Title: Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-β

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201308031

Activation of latent TGF-β by the FBG domain requires cell adhesion and the α11β1 integrin. (A) Adhesion of NMuMG and BL41-(CAGA) 9 -Luc cells to an increasing quantity of recombinant FBG domain. (B) Luciferase activity of BL41-(CAGA) 9 -Luc reporter cells cultured for 24 h on coated FBG domain or treated with 5 ng/ml TGF-β1. (C) Adhesion analysis of HT-1080 fibrosarcoma cells transiently expressing a scrambled shRNA or one of two shRNAs (#1 and #2) targeting different sequences of the ITGA11 mRNA and cultured for 30 min onto coated recombinant FBG domain or other TNX fragments (111 pmol/cm 2 ). Results represent the percentage of cell adhesion relative to the scramble shRNA condition for each recombinant protein analyzed. (D) Solid-phase assay of the interaction between the inserted domain of α11 integrin chain in fusion with GST (1 µM) and 5 µg/ml of native or denatured (Denat.) type I collagen or the different TNX derivatives (111 nM). FL, full length. (E) Immunoblotting analysis of phospho-Smad2 and the α11 integrin chain in HT-1080 cells transiently transfected as in C and seeded for 3 h onto uncoated (−) or 222 pmol/cm 2 FBG-coated (+) dishes. (F) Firefly luciferase activity of HT-1080 cells transiently cotransfected with the Smad-responsive (CAGA) 9 -Luc reporter construct and a scrambled or an ITGA11 -targeting shRNA and cultured for 24 h in noncoated (N-C) or 222 pmol/cm 2 FBG-coated wells. *, P
Figure Legend Snippet: Activation of latent TGF-β by the FBG domain requires cell adhesion and the α11β1 integrin. (A) Adhesion of NMuMG and BL41-(CAGA) 9 -Luc cells to an increasing quantity of recombinant FBG domain. (B) Luciferase activity of BL41-(CAGA) 9 -Luc reporter cells cultured for 24 h on coated FBG domain or treated with 5 ng/ml TGF-β1. (C) Adhesion analysis of HT-1080 fibrosarcoma cells transiently expressing a scrambled shRNA or one of two shRNAs (#1 and #2) targeting different sequences of the ITGA11 mRNA and cultured for 30 min onto coated recombinant FBG domain or other TNX fragments (111 pmol/cm 2 ). Results represent the percentage of cell adhesion relative to the scramble shRNA condition for each recombinant protein analyzed. (D) Solid-phase assay of the interaction between the inserted domain of α11 integrin chain in fusion with GST (1 µM) and 5 µg/ml of native or denatured (Denat.) type I collagen or the different TNX derivatives (111 nM). FL, full length. (E) Immunoblotting analysis of phospho-Smad2 and the α11 integrin chain in HT-1080 cells transiently transfected as in C and seeded for 3 h onto uncoated (−) or 222 pmol/cm 2 FBG-coated (+) dishes. (F) Firefly luciferase activity of HT-1080 cells transiently cotransfected with the Smad-responsive (CAGA) 9 -Luc reporter construct and a scrambled or an ITGA11 -targeting shRNA and cultured for 24 h in noncoated (N-C) or 222 pmol/cm 2 FBG-coated wells. *, P

Techniques Used: Activation Assay, Recombinant, Luciferase, Activity Assay, Cell Culture, Expressing, shRNA, Transfection, Construct

The FBG domain of TNX interacts physically with the SLC in vitro and in vivo. (A) Quantitative (ELISA) analysis of mature human TGF-β1 associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF fragment, subjected (+) or not subjected (−) to conditions activating latent TGF-β (heat and acid treatments). (B) Levels of mature human TGF-β1 and of its LAP(β1) propeptide associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF protein were determined by immunoblotting. The monoclonal anti-TNX antibody recognizes the 10th FNIII domain. (C) Levels of phospho-Smad2 (P-Smad2) in NMuMG cells cultured for 3 h on noncoated dishes (N-C) or dishes coated with the different recombinant TNX variants (111 pmol/cm 2 ) or stimulated with 5 ng/ml of soluble TGF-β1. Ratio of phospho-Smad2 to total Smad2/3 levels is indicated below. (D) Coimmunoprecipitation of the mature TGF-β1 entity and of its LAP(β1) propeptide with the purified recombinant FBG domain. Immunoprecipitations were performed with anti-FBG (α-FBG), anti-–human TGF-β1 (α-TGF-β1), or anti–human LAP(β1) antibodies or with control IgG. (E) Quantitative detection (ELISA) of mature TGF-β1 associated with the FBG domain of bovine TNX immunoprecipitated from FBS with either anti-FBG domain (α-FBG) antibody or control IgG. Samples were subjected (+) or not subjected (−) to activating conditions before the ELISA. An FBS fraction corresponding to 2.5% of the total volume used for the immunoprecipitation was also subjected to ELISA. *, P
Figure Legend Snippet: The FBG domain of TNX interacts physically with the SLC in vitro and in vivo. (A) Quantitative (ELISA) analysis of mature human TGF-β1 associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF fragment, subjected (+) or not subjected (−) to conditions activating latent TGF-β (heat and acid treatments). (B) Levels of mature human TGF-β1 and of its LAP(β1) propeptide associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF protein were determined by immunoblotting. The monoclonal anti-TNX antibody recognizes the 10th FNIII domain. (C) Levels of phospho-Smad2 (P-Smad2) in NMuMG cells cultured for 3 h on noncoated dishes (N-C) or dishes coated with the different recombinant TNX variants (111 pmol/cm 2 ) or stimulated with 5 ng/ml of soluble TGF-β1. Ratio of phospho-Smad2 to total Smad2/3 levels is indicated below. (D) Coimmunoprecipitation of the mature TGF-β1 entity and of its LAP(β1) propeptide with the purified recombinant FBG domain. Immunoprecipitations were performed with anti-FBG (α-FBG), anti-–human TGF-β1 (α-TGF-β1), or anti–human LAP(β1) antibodies or with control IgG. (E) Quantitative detection (ELISA) of mature TGF-β1 associated with the FBG domain of bovine TNX immunoprecipitated from FBS with either anti-FBG domain (α-FBG) antibody or control IgG. Samples were subjected (+) or not subjected (−) to activating conditions before the ELISA. An FBS fraction corresponding to 2.5% of the total volume used for the immunoprecipitation was also subjected to ELISA. *, P

Techniques Used: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Cell Culture, Immunoprecipitation

41) Product Images from "Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis"

Article Title: Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0172491

Analysis of the Troenan-induced effect in HCT116 cells containing a doxycycline-sensitive OR51B4-knockdown-sequence. (A) Confirmation of knockdown functionality by qRT-PCR and calcium imaging experiments. M = Marker. Stimulation of HCT116/EV (left) and HCT116/10F1 cells (right) with Troenan (100 μM/ 300 μM). (B) Representative calcium signal of HCT116/EV (above) and HCT116/10F1 (below) cells stimulated with Troenan (300 μM) in calcium imaging analysis. (C) Migration analysis via scratch assay with HCT116/EV and HCT116/10F1 cells with and without doxycycline induction. Stimulation of the cells with Troenan (300 μM) for 48 hours. N = 3 assays with 3 dishes. (D)-(G) Proliferation analysis of HCT116/EV (D, E) and HCT116/10F1 (F, G) cells after treatment with Troenan (300 μM) with and without doxycycline induction. Troenan (300 μM) was applied for 72 hours. N = 20 .
Figure Legend Snippet: Analysis of the Troenan-induced effect in HCT116 cells containing a doxycycline-sensitive OR51B4-knockdown-sequence. (A) Confirmation of knockdown functionality by qRT-PCR and calcium imaging experiments. M = Marker. Stimulation of HCT116/EV (left) and HCT116/10F1 cells (right) with Troenan (100 μM/ 300 μM). (B) Representative calcium signal of HCT116/EV (above) and HCT116/10F1 (below) cells stimulated with Troenan (300 μM) in calcium imaging analysis. (C) Migration analysis via scratch assay with HCT116/EV and HCT116/10F1 cells with and without doxycycline induction. Stimulation of the cells with Troenan (300 μM) for 48 hours. N = 3 assays with 3 dishes. (D)-(G) Proliferation analysis of HCT116/EV (D, E) and HCT116/10F1 (F, G) cells after treatment with Troenan (300 μM) with and without doxycycline induction. Troenan (300 μM) was applied for 72 hours. N = 20 .

Techniques Used: Sequencing, Quantitative RT-PCR, Imaging, Marker, Migration, Wound Healing Assay

Pharmacological analysis of the signaling pathway involved in the activation of OR51B4. Representative calcium imaging traces of HCT116 cells stimulated with Troenan and different specific inhibitors. Grey area represents the duration of the inhibitor applicated. Bar chart showing mean amplitudes of Troenan-induced Ca 2+ signals in HCT116 cells. (A) Localization of Ca 2+ by use of EGTA-Ringer. Investigation of different specific inhibitors of calcium signaling upon Troenan stimulation. ( B ) Gallein (10 μM), (C) U-73522 (10 μM), (D) SQ22.536 (50 μM), (E) H89 (10 μM), (F) RR; Ruthenium red (5 μM), (G) L-cis-diltiazem (150 μM), (H) Mibefradil (10 μM), (I) BTP-2 (25 μM), (J) Thapsigargin (1 μM). N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells. The data are shown as the mean SEM.
Figure Legend Snippet: Pharmacological analysis of the signaling pathway involved in the activation of OR51B4. Representative calcium imaging traces of HCT116 cells stimulated with Troenan and different specific inhibitors. Grey area represents the duration of the inhibitor applicated. Bar chart showing mean amplitudes of Troenan-induced Ca 2+ signals in HCT116 cells. (A) Localization of Ca 2+ by use of EGTA-Ringer. Investigation of different specific inhibitors of calcium signaling upon Troenan stimulation. ( B ) Gallein (10 μM), (C) U-73522 (10 μM), (D) SQ22.536 (50 μM), (E) H89 (10 μM), (F) RR; Ruthenium red (5 μM), (G) L-cis-diltiazem (150 μM), (H) Mibefradil (10 μM), (I) BTP-2 (25 μM), (J) Thapsigargin (1 μM). N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells. The data are shown as the mean SEM.

Techniques Used: Activation Assay, Imaging, Cell Culture

Transcript abundance of potential effector channels in HCT116 cells determined by RNA-Seq. (A) Bar chart showing the FPKM values of different possible effector channels. Voltage-dependent L- and T-Type channels: CACNA1S, CACNA1C, CACNA1D, CACNA1F, CACNA2D1, CACNA2D2; CACNA1G, CACNA1H, CACNA1I; CACNB1, CACNB3. Cyclic nucleotide-gated ion channels: CNGA1, CNGA2, CNGA3, CNGA4, CNGB1, CNGB3. Voltage-dependent Ca 2+ channel: CATSPER1. Transient receptor potential channels: TRPCI, TRPC6, TRPV1, TRPM8, TRPC6, TRPV2, TRPM7, TRPM8. Calcium release-activated calcium channels: ORAI1, ORAI2, ORAI3. (B) Transcript expression of PLC isoforms in the colon cancer cell line HCT116. Bar chart showing FPKM values of different PLCs in the colon cancer cell line HCT116.
Figure Legend Snippet: Transcript abundance of potential effector channels in HCT116 cells determined by RNA-Seq. (A) Bar chart showing the FPKM values of different possible effector channels. Voltage-dependent L- and T-Type channels: CACNA1S, CACNA1C, CACNA1D, CACNA1F, CACNA2D1, CACNA2D2; CACNA1G, CACNA1H, CACNA1I; CACNB1, CACNB3. Cyclic nucleotide-gated ion channels: CNGA1, CNGA2, CNGA3, CNGA4, CNGB1, CNGB3. Voltage-dependent Ca 2+ channel: CATSPER1. Transient receptor potential channels: TRPCI, TRPC6, TRPV1, TRPM8, TRPC6, TRPV2, TRPM7, TRPM8. Calcium release-activated calcium channels: ORAI1, ORAI2, ORAI3. (B) Transcript expression of PLC isoforms in the colon cancer cell line HCT116. Bar chart showing FPKM values of different PLCs in the colon cancer cell line HCT116.

Techniques Used: RNA Sequencing Assay, Expressing, Planar Chromatography

Expression of OR51B4 in HCT116 cells and colon cancer tissues. (A) Bar chart displays the FPKM values of the most highly expressed ORs in HCT116 cells. (B) Immunocytochemical staining of OR51B4 in HCT116 cells with a specific OR51B4-antibody. Left: HCT116 cells. Right: Negative control: HCT116 cells stained with second antibody alone. Bottom left: Hana3A cells transfected with OR51B4 plasmid. Bottom right: untransfected Hana3A cells. Scale bar: 10 μm. (C) The most highly expressed ORs in NGS analyses, validated by RT-PCR. + = +RT, cDNA;— = -RT, RNA; g = genomic DNA as a control; M = marker. (D) OR51B4 expressed in human colon cancer tissues: RT-PCR analysis shows OR51B4 expression in human colon cancer tissues. Expression in A: Colon tissue B: Colorectal cancer tissue C, D, E: Colon carcinoma tissues. M = marker.
Figure Legend Snippet: Expression of OR51B4 in HCT116 cells and colon cancer tissues. (A) Bar chart displays the FPKM values of the most highly expressed ORs in HCT116 cells. (B) Immunocytochemical staining of OR51B4 in HCT116 cells with a specific OR51B4-antibody. Left: HCT116 cells. Right: Negative control: HCT116 cells stained with second antibody alone. Bottom left: Hana3A cells transfected with OR51B4 plasmid. Bottom right: untransfected Hana3A cells. Scale bar: 10 μm. (C) The most highly expressed ORs in NGS analyses, validated by RT-PCR. + = +RT, cDNA;— = -RT, RNA; g = genomic DNA as a control; M = marker. (D) OR51B4 expressed in human colon cancer tissues: RT-PCR analysis shows OR51B4 expression in human colon cancer tissues. Expression in A: Colon tissue B: Colorectal cancer tissue C, D, E: Colon carcinoma tissues. M = marker.

Techniques Used: Expressing, Staining, Negative Control, Transfection, Plasmid Preparation, Next-Generation Sequencing, Reverse Transcription Polymerase Chain Reaction, Marker

Physiological effects of Troenan stimulation on HCT116 cells. (A) Analyses of cell migration by scratch assay after Troenan stimulation (300 μM) for 48 hours. (B) Bar chart showing statistical analysis of the area overgrown in scratch assay experiments. n = 3 assays. (C) Monitoring of the cell-index equal to the cell proliferation rate of HCT116 cells incubated with Troenan in different concentrations (50, 100, 150 μM). Dynamic real-time monitoring of cell processes in vivo (xCELLigence RTCA-technology). n = 2 in at least 2 independent experiments.
Figure Legend Snippet: Physiological effects of Troenan stimulation on HCT116 cells. (A) Analyses of cell migration by scratch assay after Troenan stimulation (300 μM) for 48 hours. (B) Bar chart showing statistical analysis of the area overgrown in scratch assay experiments. n = 3 assays. (C) Monitoring of the cell-index equal to the cell proliferation rate of HCT116 cells incubated with Troenan in different concentrations (50, 100, 150 μM). Dynamic real-time monitoring of cell processes in vivo (xCELLigence RTCA-technology). n = 2 in at least 2 independent experiments.

Techniques Used: Migration, Wound Healing Assay, Incubation, In Vivo

Impaired actin filament formation and induction of apoptosis upon stimulation of HCT116 cells with Troenan. (A) HCT116 cells treated with Troenan (500 μM) and control cells. (B) and (C) Phalloidin staining of control cells (B) and cells treated with Troenan (300 μM) (C). Scale bar: 10 μm. (D) and (E) Immunocytochemical staining of HCT116 cells with an antibody against caspase-3 after treatment with control (D) or Troenan (300 μM) (E). Cells treated with Troenan (300 μM) for 48 hours. Scale bar: 10 μm. (F) HCT116 cells show decreased serotonin release after application of Troenan (700 μM) for 60 minutes.
Figure Legend Snippet: Impaired actin filament formation and induction of apoptosis upon stimulation of HCT116 cells with Troenan. (A) HCT116 cells treated with Troenan (500 μM) and control cells. (B) and (C) Phalloidin staining of control cells (B) and cells treated with Troenan (300 μM) (C). Scale bar: 10 μm. (D) and (E) Immunocytochemical staining of HCT116 cells with an antibody against caspase-3 after treatment with control (D) or Troenan (300 μM) (E). Cells treated with Troenan (300 μM) for 48 hours. Scale bar: 10 μm. (F) HCT116 cells show decreased serotonin release after application of Troenan (700 μM) for 60 minutes.

Techniques Used: Staining

Western blot analysis of HCT116 cells stimulated with Troenan (300 μM; T) or control (C) for 5 and 25 minutes. (A) Phosphorylation of different isoforms of PKC. (B) Reduced phosphorylation of Stat2, Stat3 and Stat5 upon Troenan stimulation (300 μM). (C) Stimulation of Troenan (300 μM) leads to the time-dependent phosphorylation of p38 MAPK. Reduced phosphorylation was observed for Akt, mTor and Fyn. Troenan stimulation did not affect ERK and SAPK. The total amounts of p38, Akt, ERK, Stat3 and Stat5 and β-actin served as controls. n = 3. (D) Quantification of the mean pixel intensities of the phosphorylated protein kinases Akt, ERK1/2, p38, Stat5 and Stat3. The pixel intensities of duplicates were averaged. Total amounts of the protein kinases were determined and served as controls.
Figure Legend Snippet: Western blot analysis of HCT116 cells stimulated with Troenan (300 μM; T) or control (C) for 5 and 25 minutes. (A) Phosphorylation of different isoforms of PKC. (B) Reduced phosphorylation of Stat2, Stat3 and Stat5 upon Troenan stimulation (300 μM). (C) Stimulation of Troenan (300 μM) leads to the time-dependent phosphorylation of p38 MAPK. Reduced phosphorylation was observed for Akt, mTor and Fyn. Troenan stimulation did not affect ERK and SAPK. The total amounts of p38, Akt, ERK, Stat3 and Stat5 and β-actin served as controls. n = 3. (D) Quantification of the mean pixel intensities of the phosphorylated protein kinases Akt, ERK1/2, p38, Stat5 and Stat3. The pixel intensities of duplicates were averaged. Total amounts of the protein kinases were determined and served as controls.

Techniques Used: Western Blot

Characterization of Troenan-induced calcium signals in HCT116 cells. (A) Representative image of HCT116 cells stimulated with Troenan (300 μM) in calcium imaging analysis. (B) Number of cells responding to Troenan in different concentrations. (C) Repetitive activation of HCT116 cells upon repetitive Troenan application. (D) Dose-dependent activation of HCT116 cells by Troenan. Troenan was applied at concentrations of 50 μM, 100 μM and 300 μM. Peak amplitudes show the increases in intracellular calcium concentration. To ensure viability of the cells, ATP was applied last, which serves as a positive control. N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells.
Figure Legend Snippet: Characterization of Troenan-induced calcium signals in HCT116 cells. (A) Representative image of HCT116 cells stimulated with Troenan (300 μM) in calcium imaging analysis. (B) Number of cells responding to Troenan in different concentrations. (C) Repetitive activation of HCT116 cells upon repetitive Troenan application. (D) Dose-dependent activation of HCT116 cells by Troenan. Troenan was applied at concentrations of 50 μM, 100 μM and 300 μM. Peak amplitudes show the increases in intracellular calcium concentration. To ensure viability of the cells, ATP was applied last, which serves as a positive control. N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells.

Techniques Used: Imaging, Activation Assay, Concentration Assay, Positive Control, Cell Culture

42) Product Images from "Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma"

Article Title: Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma

Journal: The Journal of Pathology

doi: 10.1002/path.4222

Interactions between TGM2 and integrin– α 5 β 1 in cervical SCC cells. (A) Flow-cytometric quantification of cell-surface TGM2 expressed by SW756 and CaSki, comparing cells treated with OSM for 48 h (OSM) with control cells treated with vehicle only (C). Negative control cells were stained with an isotype control IgG. (B) Immunofluorescence for TGM2 (green) and integrin– α 5 (red) in SW756 cells treated with vehicle (control) or OSM (48 h) for 48 h, showing focal co-localization (yellow); scale bars = 10 µm. (C) Immunoprecipitation showing physical interaction between TGM2 and integrin– α 5 β 1 in SW756 and CaSki. Cells cultured on fibronectin-coated plates and treated with vehicle (C) or OSM (OSM) for 48 h were immunoprecipitated with anti-integrin– α 5 β 1 antibodies or with IgG isotype-matched control antibodies. The immunoprecipitated (IP) and unbound (UNB) fractions, together with whole-cell lysates, were then analysed by western blotting, using antibodies against TGM2 (top row), integrin– α 5 (ITGA5; middle row) and integrin– β 1 (ITGB1; bottom row). The anti-integrin– α 5 β 1 antibody precipitated TGM2 in both cell lines (cf lanes 3 and 4 versus 7 and 8 for each panel), with a stronger band for SW756 than for CaSki. Increased levels of precipitated protein were seen in OSM-treated cells (cf lanes 4 versus 3 in each panel). (D) Western blot showing levels of Y397-phosphorylated Fak (P-Fak) and total Fak in SW756 and CaSki cells cultured on fibronectin-coated plates and treated with OSM or vehicle (C) for 48 h
Figure Legend Snippet: Interactions between TGM2 and integrin– α 5 β 1 in cervical SCC cells. (A) Flow-cytometric quantification of cell-surface TGM2 expressed by SW756 and CaSki, comparing cells treated with OSM for 48 h (OSM) with control cells treated with vehicle only (C). Negative control cells were stained with an isotype control IgG. (B) Immunofluorescence for TGM2 (green) and integrin– α 5 (red) in SW756 cells treated with vehicle (control) or OSM (48 h) for 48 h, showing focal co-localization (yellow); scale bars = 10 µm. (C) Immunoprecipitation showing physical interaction between TGM2 and integrin– α 5 β 1 in SW756 and CaSki. Cells cultured on fibronectin-coated plates and treated with vehicle (C) or OSM (OSM) for 48 h were immunoprecipitated with anti-integrin– α 5 β 1 antibodies or with IgG isotype-matched control antibodies. The immunoprecipitated (IP) and unbound (UNB) fractions, together with whole-cell lysates, were then analysed by western blotting, using antibodies against TGM2 (top row), integrin– α 5 (ITGA5; middle row) and integrin– β 1 (ITGB1; bottom row). The anti-integrin– α 5 β 1 antibody precipitated TGM2 in both cell lines (cf lanes 3 and 4 versus 7 and 8 for each panel), with a stronger band for SW756 than for CaSki. Increased levels of precipitated protein were seen in OSM-treated cells (cf lanes 4 versus 3 in each panel). (D) Western blot showing levels of Y397-phosphorylated Fak (P-Fak) and total Fak in SW756 and CaSki cells cultured on fibronectin-coated plates and treated with OSM or vehicle (C) for 48 h

Techniques Used: Flow Cytometry, Negative Control, Staining, Immunofluorescence, Immunoprecipitation, Cell Culture, Western Blot

43) Product Images from "Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity"

Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity

Journal: Pancreas

doi: 10.1097/MPA.0000000000000270

Prdx1 associates with phospho-p38 MAPK in cell protrusions. A, Immunoprecipitation of Prdx1 from S2-013 cells cultured on fibronectin. Immunoprecipitates were examined on Western blots probed with antibodies against phospho-ASK1, phospho-p38 MAPK, and phospho-JNK. Rabbit immunoglobulin G was used as an isotype control. B, Immunocytochemical staining in S2-013 cells cultured on fibronectin; anti-Prdx1 (green) and anti–phospho-p38 MAPK (red) antibodies were used to label endogenous proteins. Arrows, Prdx1 colocalized with phospho-p38 MAPK in cell protrusions. Blue, DAPI staining (bar, 10 μm). C, Western blotting of total cell lysates probing for phospho-ASK1, phospho-p38 MAPK, and phospho-JNK in scrambled control and Prdx1 RNAi S2-013 cells cultured on fibronectin.
Figure Legend Snippet: Prdx1 associates with phospho-p38 MAPK in cell protrusions. A, Immunoprecipitation of Prdx1 from S2-013 cells cultured on fibronectin. Immunoprecipitates were examined on Western blots probed with antibodies against phospho-ASK1, phospho-p38 MAPK, and phospho-JNK. Rabbit immunoglobulin G was used as an isotype control. B, Immunocytochemical staining in S2-013 cells cultured on fibronectin; anti-Prdx1 (green) and anti–phospho-p38 MAPK (red) antibodies were used to label endogenous proteins. Arrows, Prdx1 colocalized with phospho-p38 MAPK in cell protrusions. Blue, DAPI staining (bar, 10 μm). C, Western blotting of total cell lysates probing for phospho-ASK1, phospho-p38 MAPK, and phospho-JNK in scrambled control and Prdx1 RNAi S2-013 cells cultured on fibronectin.

Techniques Used: Immunoprecipitation, Cell Culture, Western Blot, Staining

44) Product Images from "Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes"

Article Title: Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes

Journal: Oncotarget

doi:

Comparative analysis of the kinetics of DNA demethylation of the APM genes induced by IFNγ or 5AC TC-1/A9 cells were cultured in the presence of either IFNγ or 5AC. For the indicated time periods, DNA samples were isolated, bisulphite treated and subjected to MSP analysis of the TAP-1, TAP-2 , LMP-2 LMP-7 promoter sequences. U = unmethylated primer, M = methylated. In untreated cells, the core CpG island was highly methylated, and demethylation was detected within 2 hours after the IFNγ treatment, while nearly complete demethylation was evident by 6 hours (A). After 5AC treatment, strong demethylation was evident by 24 hours (A). The amount of 1 μg of RNA was reverse transcribed to cDNA and the PCR products were quantified. Upregulation of APM genes in TC-1/A9 cells after the treatment with IFNγ after 2 hours (A) and with 5AC after 48 hour (B). * denote significant changes (P
Figure Legend Snippet: Comparative analysis of the kinetics of DNA demethylation of the APM genes induced by IFNγ or 5AC TC-1/A9 cells were cultured in the presence of either IFNγ or 5AC. For the indicated time periods, DNA samples were isolated, bisulphite treated and subjected to MSP analysis of the TAP-1, TAP-2 , LMP-2 LMP-7 promoter sequences. U = unmethylated primer, M = methylated. In untreated cells, the core CpG island was highly methylated, and demethylation was detected within 2 hours after the IFNγ treatment, while nearly complete demethylation was evident by 6 hours (A). After 5AC treatment, strong demethylation was evident by 24 hours (A). The amount of 1 μg of RNA was reverse transcribed to cDNA and the PCR products were quantified. Upregulation of APM genes in TC-1/A9 cells after the treatment with IFNγ after 2 hours (A) and with 5AC after 48 hour (B). * denote significant changes (P

Techniques Used: Cell Culture, Isolation, Methylation, Polymerase Chain Reaction

IFNγ stimulates DNA demethylation of the APM gene promoter regions DNA from tumour cell lines cultured in the absence or presence of IFNγ were bisulphite treated and subjected to MSP analysis of the TAP-1/LMP-2, TAP-2 and LMP-7 promoter sequences. Higher proportion of DNA methylation, as compared to TC-1 cells and DNA demethylation induced by IFNγ, is documented in TC-1/A9 cells (A). Similar results were obtained in TRAMP-C2 cells (B), while no effects were noticed in IFNγ-insensitive RVP-3 tumour cells (C). U = unmethylated primer, M = methylated. Experiments were repeated three times with similar results.
Figure Legend Snippet: IFNγ stimulates DNA demethylation of the APM gene promoter regions DNA from tumour cell lines cultured in the absence or presence of IFNγ were bisulphite treated and subjected to MSP analysis of the TAP-1/LMP-2, TAP-2 and LMP-7 promoter sequences. Higher proportion of DNA methylation, as compared to TC-1 cells and DNA demethylation induced by IFNγ, is documented in TC-1/A9 cells (A). Similar results were obtained in TRAMP-C2 cells (B), while no effects were noticed in IFNγ-insensitive RVP-3 tumour cells (C). U = unmethylated primer, M = methylated. Experiments were repeated three times with similar results.

Techniques Used: Cell Culture, DNA Methylation Assay, Methylation

IFNγ-induced DNA demethylation of the TAP-2 and TAP-1/LMP-2 promoters in TC-1/A9 cells analysed by bisulphite sequencing DNA isolated from treated and control untreated TC-1/A9 cells was subjected to bisulphite conversion and cloned. Sequences from 11 clones from each sample are presented. After treatment with IFNγ, strong DNA demethylation of both the TAP-2 and TAP-1/LMP-2 gene promoter regions was observed. For LMP-7, we did not see any dramatic changes in bisulphite sequencing analysis targeting cytosines located at the positions -502 upstream to +130 downstream from the LMP-7 transcription start site. White and black circles indicate unmethylated and methylated CpGs, respectively. Rhombuses indicate the CpG islands that were investigated with bisulphite sequencing. White colour marks the CpG islands investigated with MSP. TS: transcription start.
Figure Legend Snippet: IFNγ-induced DNA demethylation of the TAP-2 and TAP-1/LMP-2 promoters in TC-1/A9 cells analysed by bisulphite sequencing DNA isolated from treated and control untreated TC-1/A9 cells was subjected to bisulphite conversion and cloned. Sequences from 11 clones from each sample are presented. After treatment with IFNγ, strong DNA demethylation of both the TAP-2 and TAP-1/LMP-2 gene promoter regions was observed. For LMP-7, we did not see any dramatic changes in bisulphite sequencing analysis targeting cytosines located at the positions -502 upstream to +130 downstream from the LMP-7 transcription start site. White and black circles indicate unmethylated and methylated CpGs, respectively. Rhombuses indicate the CpG islands that were investigated with bisulphite sequencing. White colour marks the CpG islands investigated with MSP. TS: transcription start.

Techniques Used: Bisulfite Sequencing, Isolation, Clone Assay, Methylation

Histone H3 acetylation levels in the APM regulatory gene sequences in TC-1/A9 cells are lower than those in TC-1 cells, but can be increased by IFNγ ChIP analysis of chromatin from the TAP-1/LMP-2, TAP-2, and LMP-7 promoter sequences isolated from control and treated TC-1/A9 cells demonstrates an increase in acetylated histone H3 (H3K18) after IFNγ treatment. Results were normalized to the levels of the relative input in TC-1 cells. Experiments were repeated five times with similar results. * denotes significant changes (P
Figure Legend Snippet: Histone H3 acetylation levels in the APM regulatory gene sequences in TC-1/A9 cells are lower than those in TC-1 cells, but can be increased by IFNγ ChIP analysis of chromatin from the TAP-1/LMP-2, TAP-2, and LMP-7 promoter sequences isolated from control and treated TC-1/A9 cells demonstrates an increase in acetylated histone H3 (H3K18) after IFNγ treatment. Results were normalized to the levels of the relative input in TC-1 cells. Experiments were repeated five times with similar results. * denotes significant changes (P

Techniques Used: Chromatin Immunoprecipitation, Isolation

JAK/STAT inhibitors abrogated IFNγ-induced DNA demethylation of the APM gene promoters in TC-1/A9 cells Inhibitor of Janus kinases (JAK inhibitor 1), as well as STAT1 phosphorylation inhibitor fludarabine, blocked the IFNγ induction of the MHC class I cell-surface expression (A), as well as APM gene activation (B), and caused impaired demethylation of the corresponding gene promoter regions (C). Both inhibitors blocked IFNγ-induced STAT1 phosphorylation (D). The effect of fludarabine was much weaker, as compared to Janus kinase inhibitor 1. * denotes significant changes (P
Figure Legend Snippet: JAK/STAT inhibitors abrogated IFNγ-induced DNA demethylation of the APM gene promoters in TC-1/A9 cells Inhibitor of Janus kinases (JAK inhibitor 1), as well as STAT1 phosphorylation inhibitor fludarabine, blocked the IFNγ induction of the MHC class I cell-surface expression (A), as well as APM gene activation (B), and caused impaired demethylation of the corresponding gene promoter regions (C). Both inhibitors blocked IFNγ-induced STAT1 phosphorylation (D). The effect of fludarabine was much weaker, as compared to Janus kinase inhibitor 1. * denotes significant changes (P

Techniques Used: Expressing, Activation Assay

IFNγ upregulation of the cell-surface MHC class I expression cells is associated with APM gene expression in experimental tumour cells MHC class I expression (H-2D b and H-2K b together) was determined by the FACS analysis of control tumour cells and after the treatment with IFNγ. Representative results are presented. (A) Upregulation of APM genes in TC-1/A9 and TRAMP-C2 tumours after treatment with IFNγ. (B) Expression levels of selected APM genes in TC-1/A9 and TRAMP-C2 control tumour cells and after the treatment with IFNγ. As a negative control, MHC class I-deficient RVP-3 cell line that did not respond to the IFNγ treatment was used. TC-1 cells, a MHC class I-positive parental cell line to the TC-1/A9 cells, that also displayed higher APM expression levels compared to TC-1/A9 cells, served as a MHC class I-positive control. *denote significant changes (P
Figure Legend Snippet: IFNγ upregulation of the cell-surface MHC class I expression cells is associated with APM gene expression in experimental tumour cells MHC class I expression (H-2D b and H-2K b together) was determined by the FACS analysis of control tumour cells and after the treatment with IFNγ. Representative results are presented. (A) Upregulation of APM genes in TC-1/A9 and TRAMP-C2 tumours after treatment with IFNγ. (B) Expression levels of selected APM genes in TC-1/A9 and TRAMP-C2 control tumour cells and after the treatment with IFNγ. As a negative control, MHC class I-deficient RVP-3 cell line that did not respond to the IFNγ treatment was used. TC-1 cells, a MHC class I-positive parental cell line to the TC-1/A9 cells, that also displayed higher APM expression levels compared to TC-1/A9 cells, served as a MHC class I-positive control. *denote significant changes (P

Techniques Used: Expressing, FACS, Negative Control, Positive Control

45) Product Images from "Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease"

Article Title: Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017953

Elevated levels of α-Syn, p-Tau and p-GSK-3β in striata of α-Syn A53T mutant mice. Striata from wild type non-transgenic mice and A53T α-Syn mutant transgenic mice, 8 months of age, were dissected and homogenized in RIPA buffer, and analyzed by Western blots, as described under “ Materials and Methods ”. After exposure to initial antibodies, blots were stripped and probed for other proteins. The blots show representative gels while the bar graphs are composites summarized from all animals ( n = 4–9). (A) α-Syn and p-α-Syn were expressed relative to β-actin used as a loading control. (B). p-Tau was probed using antibodies specific for pSer202, pSer262 and pSer396/404, and expressed relative to total Tau used as loading control. (C). p-GSK-3β levels were probed using antibodies which recognize phosphorylation at Tyr216, and expressed relative to total GSK-3β. (D) PP1, PP2A and PP2B levels were probed using specific antibodies and normalized to β-actin. Asterisks (*) indicate values significantly different from wild-type animals ( P
Figure Legend Snippet: Elevated levels of α-Syn, p-Tau and p-GSK-3β in striata of α-Syn A53T mutant mice. Striata from wild type non-transgenic mice and A53T α-Syn mutant transgenic mice, 8 months of age, were dissected and homogenized in RIPA buffer, and analyzed by Western blots, as described under “ Materials and Methods ”. After exposure to initial antibodies, blots were stripped and probed for other proteins. The blots show representative gels while the bar graphs are composites summarized from all animals ( n = 4–9). (A) α-Syn and p-α-Syn were expressed relative to β-actin used as a loading control. (B). p-Tau was probed using antibodies specific for pSer202, pSer262 and pSer396/404, and expressed relative to total Tau used as loading control. (C). p-GSK-3β levels were probed using antibodies which recognize phosphorylation at Tyr216, and expressed relative to total GSK-3β. (D) PP1, PP2A and PP2B levels were probed using specific antibodies and normalized to β-actin. Asterisks (*) indicate values significantly different from wild-type animals ( P

Techniques Used: Mutagenesis, Mouse Assay, Transgenic Assay, Western Blot

46) Product Images from "RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions"

Article Title: RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.523

ER stress-induced caspase-8 activation occurs independently of autocrine production of TNF, Fas ligand, or TRAIL. ( a – f ) Ripk1 +/+ MEFs were incubated for 30 min with 10 μ g/ml anti-TNF ( a and b ) or anti-TRAIL ( e and f ) blocking antibodies, or recombinant mouse Fas:Fc chimera ( c and d ), and then stimulated with 1 μ g/ml tunicamycin (Tu) ( a , c , and e ), or with TNF ( b ), anti-Fas antibody ( d ), or TRAIL ( f ) in combination with cycloheximide (CHX). The cells were then lysed and immunoblotted as indicated
Figure Legend Snippet: ER stress-induced caspase-8 activation occurs independently of autocrine production of TNF, Fas ligand, or TRAIL. ( a – f ) Ripk1 +/+ MEFs were incubated for 30 min with 10 μ g/ml anti-TNF ( a and b ) or anti-TRAIL ( e and f ) blocking antibodies, or recombinant mouse Fas:Fc chimera ( c and d ), and then stimulated with 1 μ g/ml tunicamycin (Tu) ( a , c , and e ), or with TNF ( b ), anti-Fas antibody ( d ), or TRAIL ( f ) in combination with cycloheximide (CHX). The cells were then lysed and immunoblotted as indicated

Techniques Used: Activation Assay, Incubation, Blocking Assay, Recombinant

ER stress-induced caspase-8 activation occurs independently of the death receptors TNFR1, FAS, or DR5. ( a – h ) Ripk1 +/+ MEFs were transfected with a control non-silencing siRNA (siNS) or or with siRNA targeting Tnfr1 (siTnfr1), Fas (siFas), Dr5 (siDr5), or Fadd (siFadd), and then exposed to 1 μ g/ml Tu for 12 h ( a , c , e , and g ). As control to test the functionality of repression, the cells were stimulated with TNF in combination with TAK1 inhibitor (TAK1i) ( b ), or with anti-Fas antibody ( d ), or Trail ( f and h ) in combination with cycloheximide (CHX) ( d , f , and h ). The cells were then lysed and immunoblotted as indicated. ( i ) Ripk1 +/+ MEFs were transfected as before, stimulated with 1 μ g/ml Tunicamycin, and then the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of three independent experiments
Figure Legend Snippet: ER stress-induced caspase-8 activation occurs independently of the death receptors TNFR1, FAS, or DR5. ( a – h ) Ripk1 +/+ MEFs were transfected with a control non-silencing siRNA (siNS) or or with siRNA targeting Tnfr1 (siTnfr1), Fas (siFas), Dr5 (siDr5), or Fadd (siFadd), and then exposed to 1 μ g/ml Tu for 12 h ( a , c , e , and g ). As control to test the functionality of repression, the cells were stimulated with TNF in combination with TAK1 inhibitor (TAK1i) ( b ), or with anti-Fas antibody ( d ), or Trail ( f and h ) in combination with cycloheximide (CHX) ( d , f , and h ). The cells were then lysed and immunoblotted as indicated. ( i ) Ripk1 +/+ MEFs were transfected as before, stimulated with 1 μ g/ml Tunicamycin, and then the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of three independent experiments

Techniques Used: Activation Assay, Transfection, Fluorescence

RIPK1 does not regulate JNK or CHOP during ER stress. ( a ) Ripk1 +/+ MEFs were incubated for 30 min with 10 or 20 μ M JNK inhibitor SP 600125 and then exposed to 1 μ g/ml tunicamycin (Tu) for 12 h. The cells were then lysed and immunoblotted as indicated. ( b – d ) Ripk1 +/+ and Ripk1 −/− MEFs were exposed to 1 μ g/ml Tu for the indicated time and cell lysates were either immunoblotted as indicated ( b and d ), or used to measure the expression of CHOP mRNA by RT-qPCR ( c ). Error bars indicate the standard deviation from triplicate samples. The result is representative of two independent experiments
Figure Legend Snippet: RIPK1 does not regulate JNK or CHOP during ER stress. ( a ) Ripk1 +/+ MEFs were incubated for 30 min with 10 or 20 μ M JNK inhibitor SP 600125 and then exposed to 1 μ g/ml tunicamycin (Tu) for 12 h. The cells were then lysed and immunoblotted as indicated. ( b – d ) Ripk1 +/+ and Ripk1 −/− MEFs were exposed to 1 μ g/ml Tu for the indicated time and cell lysates were either immunoblotted as indicated ( b and d ), or used to measure the expression of CHOP mRNA by RT-qPCR ( c ). Error bars indicate the standard deviation from triplicate samples. The result is representative of two independent experiments

Techniques Used: Incubation, Expressing, Quantitative RT-PCR, Standard Deviation

Ripk1 acts upstream of caspase-8 during ER stress-mediated death. ( a ) Ripk1 +/+ and Ripk1 −/− MEFs were exposed to 1 μ g/ml tunicamycin (Tu) for the indicated time, and then lysed and immunoblotted as indicated. ( b and c ) Ripk1 +/+ and Ripk1 −/− MEF transduced with a control shRNA (shCtrl) or with shRNA targeting caspase-8 (shC8) were treated with 1 μ g/ml Tu, and then lysed and immunblotted as indicated. Arrows indicated cleaved fragments of proteins. ( d ) Ripk1 +/+ MEFs were stimulated with 1 μ g/ml Tu for the indicated time, or incubated for 30 min with a TAK1 inhibitor (TAK1i) before stimulation with TNF for 2 h. Caspase-8 immunoprecipitates (IP) and cell lysates (Input 3%) were analyzed by immunoblot (IB) as indicated
Figure Legend Snippet: Ripk1 acts upstream of caspase-8 during ER stress-mediated death. ( a ) Ripk1 +/+ and Ripk1 −/− MEFs were exposed to 1 μ g/ml tunicamycin (Tu) for the indicated time, and then lysed and immunoblotted as indicated. ( b and c ) Ripk1 +/+ and Ripk1 −/− MEF transduced with a control shRNA (shCtrl) or with shRNA targeting caspase-8 (shC8) were treated with 1 μ g/ml Tu, and then lysed and immunblotted as indicated. Arrows indicated cleaved fragments of proteins. ( d ) Ripk1 +/+ MEFs were stimulated with 1 μ g/ml Tu for the indicated time, or incubated for 30 min with a TAK1 inhibitor (TAK1i) before stimulation with TNF for 2 h. Caspase-8 immunoprecipitates (IP) and cell lysates (Input 3%) were analyzed by immunoblot (IB) as indicated

Techniques Used: Transduction, shRNA, Incubation

RIPK1 mediates ER stress-triggered apoptosis independently of its kinase activity. ( a and b ) Ripk1 +/+ and Ripk1 −/− MEFs were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death ( a ) or caspase-3 like activity ( b ) was measured in function of time using the Fluostar Omega fluorescence plate reader.* P
Figure Legend Snippet: RIPK1 mediates ER stress-triggered apoptosis independently of its kinase activity. ( a and b ) Ripk1 +/+ and Ripk1 −/− MEFs were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death ( a ) or caspase-3 like activity ( b ) was measured in function of time using the Fluostar Omega fluorescence plate reader.* P

Techniques Used: Activity Assay, Fluorescence

RIPK1 interacts with the pro-apoptotic receptor IRE1. ( a and b ) Ire1 −/− cells reconstituted with an empty vector (EV) or with a vector coding for hIRE1(hIRE1) were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader ( a ), or cell lysates obtained after 17 h of stimulation were immunoblotted as indicated ( b ). ( c ) HEK293T cells were transiently transfected with plasmids coding for EGFP-hIRE1 and/or Flag-hRIPK1, and RIPK1 was immunprecipitated (IP) using anti-Flag-coated beads. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( d ) HEK293T cells were transiently transfected with plasmids coding for Flag-hIRE1, Flag-hRIPK1 and/or hTNFR1, and IRE1 (upper panels) or RIPK1 (middle panels) were immunoprecipitated with anti-IRE1 or anti-RIPK1 antibodies, respectively. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( e ) Ripk1 +/+ and Ripk1 −/− MEFs were transfected with a control non-silencing siRNA (siNS) or targeting Ire1 (siIre1) and then exposed to 1 μ g/ml Tu for 12 h. The cells were then lysed and immunoblotted as indicated
Figure Legend Snippet: RIPK1 interacts with the pro-apoptotic receptor IRE1. ( a and b ) Ire1 −/− cells reconstituted with an empty vector (EV) or with a vector coding for hIRE1(hIRE1) were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader ( a ), or cell lysates obtained after 17 h of stimulation were immunoblotted as indicated ( b ). ( c ) HEK293T cells were transiently transfected with plasmids coding for EGFP-hIRE1 and/or Flag-hRIPK1, and RIPK1 was immunprecipitated (IP) using anti-Flag-coated beads. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( d ) HEK293T cells were transiently transfected with plasmids coding for Flag-hIRE1, Flag-hRIPK1 and/or hTNFR1, and IRE1 (upper panels) or RIPK1 (middle panels) were immunoprecipitated with anti-IRE1 or anti-RIPK1 antibodies, respectively. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( e ) Ripk1 +/+ and Ripk1 −/− MEFs were transfected with a control non-silencing siRNA (siNS) or targeting Ire1 (siIre1) and then exposed to 1 μ g/ml Tu for 12 h. The cells were then lysed and immunoblotted as indicated

Techniques Used: Plasmid Preparation, Fluorescence, Transfection, Immunoprecipitation

47) Product Images from "PLIN2 is a Key Regulator of the Unfolded Protein Response and Endoplasmic Reticulum Stress Resolution in Pancreatic β Cells"

Article Title: PLIN2 is a Key Regulator of the Unfolded Protein Response and Endoplasmic Reticulum Stress Resolution in Pancreatic β Cells

Journal: Scientific Reports

doi: 10.1038/srep40855

Downregulation of PLIN2 protects against ER stress in MIN6 cells and mouse pancreatic islets. ( A , B ) MIN6 cells and ( C , D ) pancreatic islets of wild-type (WT) and Plin2 −/− mice were treated with vehicle or 5 μg/ml tunicamycin (TM) for 6 hours. ( A , C ) Immunoblotting and quantification for p-PERK, PERK, p-eIF2α, eIF2α, and nuclear ATF6 protein. β-ACTIN and P84 were used as loading controls for total and nuclear proteins respectively. ( B , D ) Semi-quantitative RT-PCR and quantification results for spliced Xbp1 (s Xbp1 ) and unspliced Xbp1 (u Xbp1 ) mRNA. Rn18s was used as a loading control. Quantification was normalized to scramble-shRNA vehicle or WT vehicle. * p
Figure Legend Snippet: Downregulation of PLIN2 protects against ER stress in MIN6 cells and mouse pancreatic islets. ( A , B ) MIN6 cells and ( C , D ) pancreatic islets of wild-type (WT) and Plin2 −/− mice were treated with vehicle or 5 μg/ml tunicamycin (TM) for 6 hours. ( A , C ) Immunoblotting and quantification for p-PERK, PERK, p-eIF2α, eIF2α, and nuclear ATF6 protein. β-ACTIN and P84 were used as loading controls for total and nuclear proteins respectively. ( B , D ) Semi-quantitative RT-PCR and quantification results for spliced Xbp1 (s Xbp1 ) and unspliced Xbp1 (u Xbp1 ) mRNA. Rn18s was used as a loading control. Quantification was normalized to scramble-shRNA vehicle or WT vehicle. * p

Techniques Used: Mouse Assay, Quantitative RT-PCR, shRNA

ER stress upregulates PLIN2 expression in MIN6 cells and mouse pancreatic islets. ( A , B ) MIN6 cells and ( C , D ) isolated C57BL/6 mouse pancreatic islets were treated with vehicle or 5 μg/ml tunicamycin (TM) for 6 hours (n = 4–6). ( A , C ) Quantitative RT-PCR and ( B , D ) immunoblotting and quantification for PLIN2 expression. GAPDH was used as a loading control. ( E,F ) MIN6 cells treated with vehicle or 5 μM thapsigargin (THAPS) for 6 hours (n = 4–6). ( E ) Quantitative RT-PCR and ( F ) immunoblotting and quantification for PLIN2 expression. GAPDH was used as a loading control. All immunoblotting quantification was normalized to vehicle. * p
Figure Legend Snippet: ER stress upregulates PLIN2 expression in MIN6 cells and mouse pancreatic islets. ( A , B ) MIN6 cells and ( C , D ) isolated C57BL/6 mouse pancreatic islets were treated with vehicle or 5 μg/ml tunicamycin (TM) for 6 hours (n = 4–6). ( A , C ) Quantitative RT-PCR and ( B , D ) immunoblotting and quantification for PLIN2 expression. GAPDH was used as a loading control. ( E,F ) MIN6 cells treated with vehicle or 5 μM thapsigargin (THAPS) for 6 hours (n = 4–6). ( E ) Quantitative RT-PCR and ( F ) immunoblotting and quantification for PLIN2 expression. GAPDH was used as a loading control. All immunoblotting quantification was normalized to vehicle. * p

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

PLIN2 accumulation enhances the ER stress of β cells. ( A , B ) MIN6 cells were transduced with control or Plin2 -retrovirus. PLIN2 overexpression was examined by ( A ) quantitative RT-PCR (n = 4) and ( B ) immunoblotting and quantification. GAPDH was used as a loading control. ( C ) Quantitative RT-PCR. Control and Plin2 -overexpression MIN6 cells treated with vehicle or 5 μg/ml tunicamycin (TM) for 6 hours (n = 4–6). ( D , E ) Total RNA and protein were collected from control wild-type (WT) β cells and Akita β cells (n = 5–6). PLIN2 expression was examined by ( D ) quantitative RT-PCR and ( E ) immunoblotting and quantification. GAPDH was used as a loading control. ( F , G ) Control and Plin2 -knockdown Akita β cells were treated with vehicle or 5 μg/ml TM for 6 hours (n = 4–6). ( F ) Immunoblotting and quantification. GAPDH was used as loading control. ( G ) Quantitative RT-PCR. All immunoblotting quantification was normalized to vehicle/control. * p
Figure Legend Snippet: PLIN2 accumulation enhances the ER stress of β cells. ( A , B ) MIN6 cells were transduced with control or Plin2 -retrovirus. PLIN2 overexpression was examined by ( A ) quantitative RT-PCR (n = 4) and ( B ) immunoblotting and quantification. GAPDH was used as a loading control. ( C ) Quantitative RT-PCR. Control and Plin2 -overexpression MIN6 cells treated with vehicle or 5 μg/ml tunicamycin (TM) for 6 hours (n = 4–6). ( D , E ) Total RNA and protein were collected from control wild-type (WT) β cells and Akita β cells (n = 5–6). PLIN2 expression was examined by ( D ) quantitative RT-PCR and ( E ) immunoblotting and quantification. GAPDH was used as a loading control. ( F , G ) Control and Plin2 -knockdown Akita β cells were treated with vehicle or 5 μg/ml TM for 6 hours (n = 4–6). ( F ) Immunoblotting and quantification. GAPDH was used as loading control. ( G ) Quantitative RT-PCR. All immunoblotting quantification was normalized to vehicle/control. * p

Techniques Used: Transduction, Over Expression, Quantitative RT-PCR, Expressing

PLIN2 regulates lipid homeostasis and NEFA-induced ER stress in pancreatic β cells. ( A , B ) MIN6 cells were treated with vehicle, 0.4 mM oleic acid (OA), or 0.4 mM palmitic acid (PA) for 16 hours (n = 4–6). ( A ) Quantitative RT-PCR. ( B ) Immunoblotting and quantification. GAPDH was used as a loading control. ( C , D ) C57BL/6 mice were fed with normal chow or 40% high fat diet (HFD) for 12 weeks (n = 6). ( C ) Plasma non-esterified fatty acids (NEFA). ( D ) Immunoblotting and quantification for PLIN2 protein in pancreatic islets. β-ACTIN was used as a loading control. ( E , F ) MIN6 cells were transduced with scramble-shRNA or Plin2 -shRNA lentivirus. Plin2 -knockdown was examined by ( E ) quantitative RT-PCR (n = 4) and ( F ) immunoblotting. β-ACTIN was used as a loading control. ( G , H ) MIN6 cells were treated the same as panels (A,B) (n = 5). ( G ) Quantification of triglycerides (TG). TG levels were normalized to total protein. * p
Figure Legend Snippet: PLIN2 regulates lipid homeostasis and NEFA-induced ER stress in pancreatic β cells. ( A , B ) MIN6 cells were treated with vehicle, 0.4 mM oleic acid (OA), or 0.4 mM palmitic acid (PA) for 16 hours (n = 4–6). ( A ) Quantitative RT-PCR. ( B ) Immunoblotting and quantification. GAPDH was used as a loading control. ( C , D ) C57BL/6 mice were fed with normal chow or 40% high fat diet (HFD) for 12 weeks (n = 6). ( C ) Plasma non-esterified fatty acids (NEFA). ( D ) Immunoblotting and quantification for PLIN2 protein in pancreatic islets. β-ACTIN was used as a loading control. ( E , F ) MIN6 cells were transduced with scramble-shRNA or Plin2 -shRNA lentivirus. Plin2 -knockdown was examined by ( E ) quantitative RT-PCR (n = 4) and ( F ) immunoblotting. β-ACTIN was used as a loading control. ( G , H ) MIN6 cells were treated the same as panels (A,B) (n = 5). ( G ) Quantification of triglycerides (TG). TG levels were normalized to total protein. * p

Techniques Used: Quantitative RT-PCR, Mouse Assay, Transduction, shRNA

PLIN2 modulates autophagic flux in pancreatic β cells. ( A ) Immunoblotting and quantification. Control and Plin2 -knockdown MIN6 cells were treated with vehicle or 5 μg/ml tunicamycin (TM) for 6 hours. GAPDH was used as loading control. ( B ) Control and Plin2 -knockdown MIN6 cells were transfected with LC3-GFP construct and subquently treated with vehicle or 0.1 mM chloroquine (CQ) for 6 hours. Nuclei were stained with DAPI (blue). The number of LC3-GFP punctae (green dots) was quantified. Total of 50 GFP-positive cells were calculated each group. ( C ) Immunoblotting and quantification. Wild-type control and Akita β cells were treated with vehicle or 5 μg/ml TM for 6 hours. GAPDH was used as loading control. ( D ) Immunoblotting and quantification. Cells were treated with vehicle or 0.1 mM CQ for 6 hours. GAPDH was used as loading control. All immunoblotting quantification was normalized to scramble-shRNA vehicle. * p
Figure Legend Snippet: PLIN2 modulates autophagic flux in pancreatic β cells. ( A ) Immunoblotting and quantification. Control and Plin2 -knockdown MIN6 cells were treated with vehicle or 5 μg/ml tunicamycin (TM) for 6 hours. GAPDH was used as loading control. ( B ) Control and Plin2 -knockdown MIN6 cells were transfected with LC3-GFP construct and subquently treated with vehicle or 0.1 mM chloroquine (CQ) for 6 hours. Nuclei were stained with DAPI (blue). The number of LC3-GFP punctae (green dots) was quantified. Total of 50 GFP-positive cells were calculated each group. ( C ) Immunoblotting and quantification. Wild-type control and Akita β cells were treated with vehicle or 5 μg/ml TM for 6 hours. GAPDH was used as loading control. ( D ) Immunoblotting and quantification. Cells were treated with vehicle or 0.1 mM CQ for 6 hours. GAPDH was used as loading control. All immunoblotting quantification was normalized to scramble-shRNA vehicle. * p

Techniques Used: Transfection, Construct, Staining, shRNA

48) Product Images from "Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain"

Article Title: Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18091968

Effect of IRCR201 on c-Met receptor internalization and degradation. ( a – c ) U87MG, A549, and MKN45 were treated with 100 nM IRCR201 or human IgG for the indicated durations and lysed. The level of total c-Met was measured by ELISA-based assay as described in the “Materials and Methods” section; ( d – f ) After c-Met-positive cell lines were incubated with 100 nM of IRCR201 or human IgG for 30 min at 4 °C, the cell lines were incubated at 37 °C to induce internalization and degradation of the c-Met/IRCR201 complex. The residual levels of cell surface c-Met were detected by rabbit anti-c-Met antibody-conjugated Alexa Fluor ® 488 in which the epitope of IRCR201 does not overlap. The c-Met levels on the cell surface were measured based on the mean fluorescence intensity (MFI) and normalized to a value of MFI at 0 min; ( g ) MKN45 cells were treated with 100 nM IRCR201 at 37 °C. Thereafter, IRCR201 was allowed to internalize for up to 120 min at 37 °C. MKN45 cells were then stained with anti-human IgG-Alexa Fluor ® 647 to recognize IRCR201 and anti-LAMP1-Alexa Fluor ® 488 to detect lysosomes. The fluorescence images are visualized by confocal microscopy.
Figure Legend Snippet: Effect of IRCR201 on c-Met receptor internalization and degradation. ( a – c ) U87MG, A549, and MKN45 were treated with 100 nM IRCR201 or human IgG for the indicated durations and lysed. The level of total c-Met was measured by ELISA-based assay as described in the “Materials and Methods” section; ( d – f ) After c-Met-positive cell lines were incubated with 100 nM of IRCR201 or human IgG for 30 min at 4 °C, the cell lines were incubated at 37 °C to induce internalization and degradation of the c-Met/IRCR201 complex. The residual levels of cell surface c-Met were detected by rabbit anti-c-Met antibody-conjugated Alexa Fluor ® 488 in which the epitope of IRCR201 does not overlap. The c-Met levels on the cell surface were measured based on the mean fluorescence intensity (MFI) and normalized to a value of MFI at 0 min; ( g ) MKN45 cells were treated with 100 nM IRCR201 at 37 °C. Thereafter, IRCR201 was allowed to internalize for up to 120 min at 37 °C. MKN45 cells were then stained with anti-human IgG-Alexa Fluor ® 647 to recognize IRCR201 and anti-LAMP1-Alexa Fluor ® 488 to detect lysosomes. The fluorescence images are visualized by confocal microscopy.

Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation, Fluorescence, Staining, Confocal Microscopy

Immunohistological staining of Ki-67, apoptotic cells, c-Met, phospho-c-Met, phospho-Akt, phospho-Erk1/2, and platelet endothelial cell adhesion molecule 1 (PECAM1) in an MKN45 xenograft mouse tumor model. ( a – g ) All the immunohistochemistry (IHC) images were obtained at the same magnification. Sections were counterstained with hematoxylin (blue); ( a – d ) Immunohistochemistry analysis of MKN45 tumors in mice at 48 h post-treatment with 10 mg/kg IRCR201. The control group was intravenously injected with PBS (vehicle); ( a ) Immunohistological staining of MKN45 tumor section for Ki-67 (brown); ( b ) Immunohistochemistry analysis of apoptotic cells (brown) using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay; ( c , d ) IHC expression images of total c-Met (brown) and phospho-c-Met (brown) in the MKN45 tumor section at 48-h post-treatment at 10 mg/kg; ( e – g ) Immunohistochemistry analysis of phospho-Akt, phospho-Erk1/2, and PECAM1 on 15 mg/kg IRCR201-treated MKN45 tissue sections; ( e , f ) IHC expression images of phospho-Akt (brown) and phospho-Erk1/2 (brown) in the MKN45 tumor sections; ( g ) Immunohistological analysis of PECAM1-positive blood vessels (brown) in the 15 mg/kg IRCR201-treated MKN45 xenograft tumor section.
Figure Legend Snippet: Immunohistological staining of Ki-67, apoptotic cells, c-Met, phospho-c-Met, phospho-Akt, phospho-Erk1/2, and platelet endothelial cell adhesion molecule 1 (PECAM1) in an MKN45 xenograft mouse tumor model. ( a – g ) All the immunohistochemistry (IHC) images were obtained at the same magnification. Sections were counterstained with hematoxylin (blue); ( a – d ) Immunohistochemistry analysis of MKN45 tumors in mice at 48 h post-treatment with 10 mg/kg IRCR201. The control group was intravenously injected with PBS (vehicle); ( a ) Immunohistological staining of MKN45 tumor section for Ki-67 (brown); ( b ) Immunohistochemistry analysis of apoptotic cells (brown) using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay; ( c , d ) IHC expression images of total c-Met (brown) and phospho-c-Met (brown) in the MKN45 tumor section at 48-h post-treatment at 10 mg/kg; ( e – g ) Immunohistochemistry analysis of phospho-Akt, phospho-Erk1/2, and PECAM1 on 15 mg/kg IRCR201-treated MKN45 tissue sections; ( e , f ) IHC expression images of phospho-Akt (brown) and phospho-Erk1/2 (brown) in the MKN45 tumor sections; ( g ) Immunohistological analysis of PECAM1-positive blood vessels (brown) in the 15 mg/kg IRCR201-treated MKN45 xenograft tumor section.

Techniques Used: Staining, Immunohistochemistry, Mouse Assay, Injection, TUNEL Assay, Expressing

Epitope mapping of IRCR201. ( a , b ) The reactivity of IRCR201 against a panel of overlapping peptides representing the c-Met extracellular domain was determined by peptide array. A peptide library spanning amino acids 1–932 of the c-Met extracellular domain was synthesized (JPT Peptide Technologies GmbH, Berlin, Germany). The library was prepared as overlapping linear peptides covalently bound to a cellulose membrane. The results show dot blots of specific epitope sequences for IRCR201 (yellow frame). The quantified levels of dot intensity were determined by Multi Gauge V3.0 program; ( c ) Binding pattern analysis using domain proteins of c-Met; ( d ) Hepatocyte growth factor (HGF)/c-Met competitive ELISA. After the HGF (2.5 μg/mL) was pre-treated with the human c-Met protein-immobilized plates, IRCR201 or huOA5D5.v2 was added to confirm whether HGF and each antibody were competitively bound. IPT: immunoglobulin–plexin–transcription; PSI: plexin-semaphorin-integrin domain.
Figure Legend Snippet: Epitope mapping of IRCR201. ( a , b ) The reactivity of IRCR201 against a panel of overlapping peptides representing the c-Met extracellular domain was determined by peptide array. A peptide library spanning amino acids 1–932 of the c-Met extracellular domain was synthesized (JPT Peptide Technologies GmbH, Berlin, Germany). The library was prepared as overlapping linear peptides covalently bound to a cellulose membrane. The results show dot blots of specific epitope sequences for IRCR201 (yellow frame). The quantified levels of dot intensity were determined by Multi Gauge V3.0 program; ( c ) Binding pattern analysis using domain proteins of c-Met; ( d ) Hepatocyte growth factor (HGF)/c-Met competitive ELISA. After the HGF (2.5 μg/mL) was pre-treated with the human c-Met protein-immobilized plates, IRCR201 or huOA5D5.v2 was added to confirm whether HGF and each antibody were competitively bound. IPT: immunoglobulin–plexin–transcription; PSI: plexin-semaphorin-integrin domain.

Techniques Used: Peptide Microarray, Synthesized, Binding Assay, Competitive ELISA

IRCR201 suppresses c-Met signaling pathway via the degradation of c-Met. ( a , b ) A549 cells were pre-treated with dimethyl sulfoxide (DMSO) or MG132 (5 μM)/lactacystin (5 μM) mixture for 2 h and incubated with 100 nM anti-c-Met antibodies (IRCR201 or 5D5) or phosphate-buffered saline (PBS). After 4 h of incubation, the c-Met degradation pattern was measured by Western blot analysis. The band intensities of c-Met were quantified by a Multi Gauge V3.0 program and normalized by the intensity of corresponding β-actin band; ( c , d ) A549 cells were pre-treated with DMSO or 100 nM concanamycin A for 2 h and subsequently treated with 100 nM anti-c-Met antibodies (IRCR201 or 5D5) or PBS for 4 h. The c-Met degradation pattern was evaluated by immunoblot analysis and analyzed by Multi Gauge V3.0 program; ( e ) Western blot analysis of c-Met, phospho-c-Met, Akt, phospho-Akt, Erk1/2, and phospho-Erk1/2 after 24-h treatment with IRCR201 (100 nM) or PBS in U87MG and MKN45; ( f ) Western blot analysis of c-Met, phospho-c-Met, Akt, phospho-Akt, Erk1/2, and phospho-Erk1/2 after 24-h treatment with IRCR201 (100 nM) or PBS in A549 in the presence or absence of HGF (50 ng/mL).
Figure Legend Snippet: IRCR201 suppresses c-Met signaling pathway via the degradation of c-Met. ( a , b ) A549 cells were pre-treated with dimethyl sulfoxide (DMSO) or MG132 (5 μM)/lactacystin (5 μM) mixture for 2 h and incubated with 100 nM anti-c-Met antibodies (IRCR201 or 5D5) or phosphate-buffered saline (PBS). After 4 h of incubation, the c-Met degradation pattern was measured by Western blot analysis. The band intensities of c-Met were quantified by a Multi Gauge V3.0 program and normalized by the intensity of corresponding β-actin band; ( c , d ) A549 cells were pre-treated with DMSO or 100 nM concanamycin A for 2 h and subsequently treated with 100 nM anti-c-Met antibodies (IRCR201 or 5D5) or PBS for 4 h. The c-Met degradation pattern was evaluated by immunoblot analysis and analyzed by Multi Gauge V3.0 program; ( e ) Western blot analysis of c-Met, phospho-c-Met, Akt, phospho-Akt, Erk1/2, and phospho-Erk1/2 after 24-h treatment with IRCR201 (100 nM) or PBS in U87MG and MKN45; ( f ) Western blot analysis of c-Met, phospho-c-Met, Akt, phospho-Akt, Erk1/2, and phospho-Erk1/2 after 24-h treatment with IRCR201 (100 nM) or PBS in A549 in the presence or absence of HGF (50 ng/mL).

Techniques Used: Incubation, Western Blot

Binding properties of IRCR201. ( a ) Binding patterns of IRCR201 to the human c-Met extracellular domain (ECD)-fragment crystallizable region (Fc) and the human RON (recepteur d’origine nantais) ECD-Fc were measured by enzyme-linked immunosorbent assay (ELISA). IRCR201 binds to the human c-Met ECD-Fc with specificity and selectivity; ( b – d ) Surface plasmon resonance (SPR) sensorgrams binding with varying concentrations of IRCR201 to human c-Met, mouse c-Met, or bovine serum albumin (BSA) immobilized onto a CM5 Biacore TM sensor chip; ( e ) Cross-reactivity analysis of IRCR201 to human and mouse c-Met; ( f ) Analysis of c-Met expression in various types of cancer cell lines; ( g ) Binding analysis of IRCR201 to the cell surface c-Met through a flow cytometer (FACSAria™ III).
Figure Legend Snippet: Binding properties of IRCR201. ( a ) Binding patterns of IRCR201 to the human c-Met extracellular domain (ECD)-fragment crystallizable region (Fc) and the human RON (recepteur d’origine nantais) ECD-Fc were measured by enzyme-linked immunosorbent assay (ELISA). IRCR201 binds to the human c-Met ECD-Fc with specificity and selectivity; ( b – d ) Surface plasmon resonance (SPR) sensorgrams binding with varying concentrations of IRCR201 to human c-Met, mouse c-Met, or bovine serum albumin (BSA) immobilized onto a CM5 Biacore TM sensor chip; ( e ) Cross-reactivity analysis of IRCR201 to human and mouse c-Met; ( f ) Analysis of c-Met expression in various types of cancer cell lines; ( g ) Binding analysis of IRCR201 to the cell surface c-Met through a flow cytometer (FACSAria™ III).

Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Chromatin Immunoprecipitation, Expressing, Flow Cytometry, Cytometry

In vivo potency of IRCR201 in c-Met-expressing tumor xenograft models. ( a ) IRCR201 was dosed intravenously twice a week at 3 mg/kg in an A549 non-small cell lung cancer (NSCLC) xenograft model, as compared with the PBS-treated group or huOA5D5.v2-treated group; ( b ) IRCR201 was intravenously injected at 3 or 15 mg/kg twice a week in an MKN45 gastric cancer xenograft model compared with the PBS-treated group or huOA5D5.v2-treated group. Six mice per group were used for the A549 and MKN45 studies. Asterisks (*) indicate p -values versus PBS-treated group (vehicle group) according to one-tailed two-sample t -test. p -Value
Figure Legend Snippet: In vivo potency of IRCR201 in c-Met-expressing tumor xenograft models. ( a ) IRCR201 was dosed intravenously twice a week at 3 mg/kg in an A549 non-small cell lung cancer (NSCLC) xenograft model, as compared with the PBS-treated group or huOA5D5.v2-treated group; ( b ) IRCR201 was intravenously injected at 3 or 15 mg/kg twice a week in an MKN45 gastric cancer xenograft model compared with the PBS-treated group or huOA5D5.v2-treated group. Six mice per group were used for the A549 and MKN45 studies. Asterisks (*) indicate p -values versus PBS-treated group (vehicle group) according to one-tailed two-sample t -test. p -Value

Techniques Used: In Vivo, Expressing, Injection, Mouse Assay, One-tailed Test

Docking of IRCR201 to human c-Met. ( a ) A three-dimensional (3D) model of IRCR201 variable domains was generated in a single-chain variable fragment (scFv) format by Rosetta computational homology modeling. The complementarity determining regions (CDRs) of the V H and V L domains are shown in blue and red, respectively. The framework regions of the V H and V L domains are represented in dark grey and light grey, respectively. The CDR sequences of IRCR201 are represented according to the Kabat numbering scheme; ( b , c ) IRCR201 was docked to c-Met (PDB accession: 1SHY) using the ZDOCK docking program. Epitope was mapped on to the 3D structure of c-Met (25–567), incorporating the Sema domain and plexin-semaphorin-integrin (PSI) domain. The figures were drawn with PyMOL (DeLano Scientific LLC, Palo Alto, CA, USA). The V H and V L domains of IRCR201 are shown in blue and red, respectively. Yellow = IRCR201 binding site (SAPPFVQ). Light grey = Sema domain. Dark grey = PSI domain. Green = serine protease homology domain (SPHD) of HGF.
Figure Legend Snippet: Docking of IRCR201 to human c-Met. ( a ) A three-dimensional (3D) model of IRCR201 variable domains was generated in a single-chain variable fragment (scFv) format by Rosetta computational homology modeling. The complementarity determining regions (CDRs) of the V H and V L domains are shown in blue and red, respectively. The framework regions of the V H and V L domains are represented in dark grey and light grey, respectively. The CDR sequences of IRCR201 are represented according to the Kabat numbering scheme; ( b , c ) IRCR201 was docked to c-Met (PDB accession: 1SHY) using the ZDOCK docking program. Epitope was mapped on to the 3D structure of c-Met (25–567), incorporating the Sema domain and plexin-semaphorin-integrin (PSI) domain. The figures were drawn with PyMOL (DeLano Scientific LLC, Palo Alto, CA, USA). The V H and V L domains of IRCR201 are shown in blue and red, respectively. Yellow = IRCR201 binding site (SAPPFVQ). Light grey = Sema domain. Dark grey = PSI domain. Green = serine protease homology domain (SPHD) of HGF.

Techniques Used: Generated, Binding Assay

In vitro potency of IRCR201. All results are shown as the mean ± standard error of mean (SEM) from triplicate treatments. ( a – d ) Inhibitory effect of IRCR201 on cancer cell proliferation. MCF7, U87MG, MKN45, and A549 cells were treated with IRCR201, huOA5D5.v2, or human IgG control for 72 h. Cell proliferation was measured using CellTiter Glo ® (Promega); ( e ) HGF-induced growth inhibition by IRCR201. The inhibitory effect of IRCR201 and huOA5D5.v2 on cell growth was examined under the condition of the addition of 50 ng/mL HGF in A549, an HGF-dependent cell. After 72 h of antibody treatment, the number of cells was measured with CellTiter Glo ® (Promega); ( f – i ) Apoptosis assay. MCF7, U87MG, MKN45, and A549 cells were treated with IRCR201 or human IgG for 24 h. Apoptosis activity was detected with caspase-3/7 activity.
Figure Legend Snippet: In vitro potency of IRCR201. All results are shown as the mean ± standard error of mean (SEM) from triplicate treatments. ( a – d ) Inhibitory effect of IRCR201 on cancer cell proliferation. MCF7, U87MG, MKN45, and A549 cells were treated with IRCR201, huOA5D5.v2, or human IgG control for 72 h. Cell proliferation was measured using CellTiter Glo ® (Promega); ( e ) HGF-induced growth inhibition by IRCR201. The inhibitory effect of IRCR201 and huOA5D5.v2 on cell growth was examined under the condition of the addition of 50 ng/mL HGF in A549, an HGF-dependent cell. After 72 h of antibody treatment, the number of cells was measured with CellTiter Glo ® (Promega); ( f – i ) Apoptosis assay. MCF7, U87MG, MKN45, and A549 cells were treated with IRCR201 or human IgG for 24 h. Apoptosis activity was detected with caspase-3/7 activity.

Techniques Used: In Vitro, Inhibition, Apoptosis Assay, Activity Assay

49) Product Images from "Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease"

Article Title: Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017953

Postmortem PD brains show increased levels of α-Syn, p-GSK-3β and p-Tau at phosphorylation sites Ser202, Ser262 and Ser396/404 similar to striatum in the 8 month-old A53T mice. Postmortem control and PD brain lysates were prepared and immunoblots probed for (A) α-synuclein and p-α-synuclein, (B) phospho-specific Tau epitopes at Ser202, Ser262 and Ser396/404 and (C) p-GSK-3β. (D) PP1, PP2A and PP2B levels were probed using specific antibodies and normalized to β-actin. Representative immunoblots from postmortem samples are shown. β-actin levels were measured as loading controls. Increases in the level of α-synuclein and are normalized to β-actin and data are expressed as a percent increase relative to amounts present in control, non-diseased brains (100%). Phospho-specific Tau and p-GSK-3β are expressed as a percent increase with respect to non-diseased controls (100%) after normalization to total Tau and GSK-3β, respectively. Values are mean ± SEM. Asterisks (*) indicate values significantly different from age-matched control postmortem striata (p
Figure Legend Snippet: Postmortem PD brains show increased levels of α-Syn, p-GSK-3β and p-Tau at phosphorylation sites Ser202, Ser262 and Ser396/404 similar to striatum in the 8 month-old A53T mice. Postmortem control and PD brain lysates were prepared and immunoblots probed for (A) α-synuclein and p-α-synuclein, (B) phospho-specific Tau epitopes at Ser202, Ser262 and Ser396/404 and (C) p-GSK-3β. (D) PP1, PP2A and PP2B levels were probed using specific antibodies and normalized to β-actin. Representative immunoblots from postmortem samples are shown. β-actin levels were measured as loading controls. Increases in the level of α-synuclein and are normalized to β-actin and data are expressed as a percent increase relative to amounts present in control, non-diseased brains (100%). Phospho-specific Tau and p-GSK-3β are expressed as a percent increase with respect to non-diseased controls (100%) after normalization to total Tau and GSK-3β, respectively. Values are mean ± SEM. Asterisks (*) indicate values significantly different from age-matched control postmortem striata (p

Techniques Used: Mouse Assay, Western Blot

Binding of striatal proteins from A53T α-Syn mutant and wild-type non-Tg mice to cytoskeleton. Lysates from striata of mutant and non-Tg mice [ n = 4 per group] were extracted in PIPES buffer and SDS buffer to obtain cytoskeleton-free and cytoskeleton-bound fractions, as described in Methods. (A) α-Syn and p-GSK-3β were normalized to β-actin and GSK-3β, respectively, while (B) pSer396/404 was normalized to total Tau. Results were expressed as percent of control non-Tg mice. Blots show representative gels while the bar graphs are composites summarized from all animals. *, P
Figure Legend Snippet: Binding of striatal proteins from A53T α-Syn mutant and wild-type non-Tg mice to cytoskeleton. Lysates from striata of mutant and non-Tg mice [ n = 4 per group] were extracted in PIPES buffer and SDS buffer to obtain cytoskeleton-free and cytoskeleton-bound fractions, as described in Methods. (A) α-Syn and p-GSK-3β were normalized to β-actin and GSK-3β, respectively, while (B) pSer396/404 was normalized to total Tau. Results were expressed as percent of control non-Tg mice. Blots show representative gels while the bar graphs are composites summarized from all animals. *, P

Techniques Used: Binding Assay, Mutagenesis, Mouse Assay

Elevated levels of α-Syn, p-Tau and p-GSK-3β in striata of α-Syn A53T mutant mice. Striata from wild type non-transgenic mice and A53T α-Syn mutant transgenic mice, 8 months of age, were dissected and homogenized in RIPA buffer, and analyzed by Western blots, as described under “ Materials and Methods ”. After exposure to initial antibodies, blots were stripped and probed for other proteins. The blots show representative gels while the bar graphs are composites summarized from all animals ( n = 4–9). (A) α-Syn and p-α-Syn were expressed relative to β-actin used as a loading control. (B). p-Tau was probed using antibodies specific for pSer202, pSer262 and pSer396/404, and expressed relative to total Tau used as loading control. (C). p-GSK-3β levels were probed using antibodies which recognize phosphorylation at Tyr216, and expressed relative to total GSK-3β. (D) PP1, PP2A and PP2B levels were probed using specific antibodies and normalized to β-actin. Asterisks (*) indicate values significantly different from wild-type animals ( P
Figure Legend Snippet: Elevated levels of α-Syn, p-Tau and p-GSK-3β in striata of α-Syn A53T mutant mice. Striata from wild type non-transgenic mice and A53T α-Syn mutant transgenic mice, 8 months of age, were dissected and homogenized in RIPA buffer, and analyzed by Western blots, as described under “ Materials and Methods ”. After exposure to initial antibodies, blots were stripped and probed for other proteins. The blots show representative gels while the bar graphs are composites summarized from all animals ( n = 4–9). (A) α-Syn and p-α-Syn were expressed relative to β-actin used as a loading control. (B). p-Tau was probed using antibodies specific for pSer202, pSer262 and pSer396/404, and expressed relative to total Tau used as loading control. (C). p-GSK-3β levels were probed using antibodies which recognize phosphorylation at Tyr216, and expressed relative to total GSK-3β. (D) PP1, PP2A and PP2B levels were probed using specific antibodies and normalized to β-actin. Asterisks (*) indicate values significantly different from wild-type animals ( P

Techniques Used: Mutagenesis, Mouse Assay, Transgenic Assay, Western Blot

50) Product Images from "RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions"

Article Title: RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.523

RIPK1 interacts with the pro-apoptotic receptor IRE1. ( a and b ) Ire1 −/− cells reconstituted with an empty vector (EV) or with a vector coding for hIRE1(hIRE1) were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader ( a ), or cell lysates obtained after 17 h of stimulation were immunoblotted as indicated ( b ). ( c ) HEK293T cells were transiently transfected with plasmids coding for EGFP-hIRE1 and/or Flag-hRIPK1, and RIPK1 was immunprecipitated (IP) using anti-Flag-coated beads. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( d ) HEK293T cells were transiently transfected with plasmids coding for Flag-hIRE1, Flag-hRIPK1 and/or hTNFR1, and IRE1 (upper panels) or RIPK1 (middle panels) were immunoprecipitated with anti-IRE1 or anti-RIPK1 antibodies, respectively. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( e ) Ripk1 +/+ and Ripk1 −/− MEFs were transfected with a control non-silencing siRNA (siNS) or targeting Ire1 (siIre1) and then exposed to 1 μ g/ml Tu for 12 h. The cells were then lysed and immunoblotted as indicated
Figure Legend Snippet: RIPK1 interacts with the pro-apoptotic receptor IRE1. ( a and b ) Ire1 −/− cells reconstituted with an empty vector (EV) or with a vector coding for hIRE1(hIRE1) were stimulated with 1 μ g/ml tunicamycin (Tu), and the percentage of cell death was measured in function of time using the Fluostar Omega fluorescence plate reader ( a ), or cell lysates obtained after 17 h of stimulation were immunoblotted as indicated ( b ). ( c ) HEK293T cells were transiently transfected with plasmids coding for EGFP-hIRE1 and/or Flag-hRIPK1, and RIPK1 was immunprecipitated (IP) using anti-Flag-coated beads. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( d ) HEK293T cells were transiently transfected with plasmids coding for Flag-hIRE1, Flag-hRIPK1 and/or hTNFR1, and IRE1 (upper panels) or RIPK1 (middle panels) were immunoprecipitated with anti-IRE1 or anti-RIPK1 antibodies, respectively. Cell lysates and immunoprecipitates were analyzed by immunoblot as indicated. ( e ) Ripk1 +/+ and Ripk1 −/− MEFs were transfected with a control non-silencing siRNA (siNS) or targeting Ire1 (siIre1) and then exposed to 1 μ g/ml Tu for 12 h. The cells were then lysed and immunoblotted as indicated

Techniques Used: Plasmid Preparation, Fluorescence, Transfection, Immunoprecipitation

51) Product Images from "The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability"

Article Title: The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkh172

Expression profiling of Pol II LS mutants. ( A ) The amino acid sequence of the last CTD repeat is shown for the mutants produced in this study. LS* wt MCS consists of the wild-type 52 repeats plus an additional seven amino acids resulting from the introduction of a multiple cloning site in the DNA sequence (MCS). Green boxes signify regions containing the CKII consensus recognition sequence S/TxxD/E. LS*49+52 consists of a total of 50 repeats, whereby the last repeat corresponds to the sequence of CTD52. Variations of this mutant were produced where one or both potential sites for phosphorylation by CKII are mutated (S→A; labelled red). Similarly, mutants LS*49+NS and LS*49+ATM contain a total of 50 repeats, where the last repeat is either a scrambled CTD52 (NS) or consists of repeat 50 of the wild-type sequence plus the c-Abl interaction domain of ATM. In addition, a mutant truncated to the 50th repeat (LS*49+50) was also produced. ( B ) Cell lines were cultivated in the absence of Tc for 24 h before the addition of α-amanitin and harvesting a further 24 h later. The induction of the recombinant large subunits was analysed by western blot using HA-specific antibodies (3F10). Samples were prepared using Laemmli buffer. The same blot was stripped and re-probed using CTD-specific antibodies (8WG16). The same samples were also screened using antibodies directed against CKII-phosphorylated CTD (DEEP).
Figure Legend Snippet: Expression profiling of Pol II LS mutants. ( A ) The amino acid sequence of the last CTD repeat is shown for the mutants produced in this study. LS* wt MCS consists of the wild-type 52 repeats plus an additional seven amino acids resulting from the introduction of a multiple cloning site in the DNA sequence (MCS). Green boxes signify regions containing the CKII consensus recognition sequence S/TxxD/E. LS*49+52 consists of a total of 50 repeats, whereby the last repeat corresponds to the sequence of CTD52. Variations of this mutant were produced where one or both potential sites for phosphorylation by CKII are mutated (S→A; labelled red). Similarly, mutants LS*49+NS and LS*49+ATM contain a total of 50 repeats, where the last repeat is either a scrambled CTD52 (NS) or consists of repeat 50 of the wild-type sequence plus the c-Abl interaction domain of ATM. In addition, a mutant truncated to the 50th repeat (LS*49+50) was also produced. ( B ) Cell lines were cultivated in the absence of Tc for 24 h before the addition of α-amanitin and harvesting a further 24 h later. The induction of the recombinant large subunits was analysed by western blot using HA-specific antibodies (3F10). Samples were prepared using Laemmli buffer. The same blot was stripped and re-probed using CTD-specific antibodies (8WG16). The same samples were also screened using antibodies directed against CKII-phosphorylated CTD (DEEP).

Techniques Used: Expressing, Sequencing, Produced, Clone Assay, Mutagenesis, Recombinant, Western Blot

52) Product Images from "Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-?"

Article Title: Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-?

Journal: BMC Cancer

doi: 10.1186/1471-2407-12-450

Schematic representation of antibody epitope mapping studies. Mammalian expression plasmids expressing full-length HER2 (p219D) or partially deleted HER2 were transfected into Huh7 cells. Anti-HER2 antibody reactivity was tested by indirect immunofluorescence (Figure 4 ) and immunoprecipitation-western blotting (Additional file 3 ) techniques. The boxes named as I, II, III, IV represent four subdomains of ECD. Grey box: transmembrane domain, TK: tyrosine kinase domain, CT: carboxyl-terminal domain. Tzm: Trastuzumab.+: antibody reactivity, -: no antibody reactivity. Stars (*) near plasmid names indicate that immunofluorescence data were confirmed with immunoprecipitation-western blot assay.
Figure Legend Snippet: Schematic representation of antibody epitope mapping studies. Mammalian expression plasmids expressing full-length HER2 (p219D) or partially deleted HER2 were transfected into Huh7 cells. Anti-HER2 antibody reactivity was tested by indirect immunofluorescence (Figure 4 ) and immunoprecipitation-western blotting (Additional file 3 ) techniques. The boxes named as I, II, III, IV represent four subdomains of ECD. Grey box: transmembrane domain, TK: tyrosine kinase domain, CT: carboxyl-terminal domain. Tzm: Trastuzumab.+: antibody reactivity, -: no antibody reactivity. Stars (*) near plasmid names indicate that immunofluorescence data were confirmed with immunoprecipitation-western blot assay.

Techniques Used: Expressing, Transfection, Immunofluorescence, Immunoprecipitation, Western Blot, Plasmid Preparation

Epitope mapping of anti-HER2 antibodies using indirect immunofluorescence assay. Huh7 cells were transfected in 6-well plates using a set of mammalian expression plasmids encoding full-length or N-terminally truncated HER2 protein. Transfected cells were cultivated for 48 h, and subjected to indirect immunofluorescence assay. Transfected cells that were recognized by specific antibodies displayed strong green fluorescence. The letters from (a) to (k) indicate the plasmids in the following order: pDEST26 (empty vector), p219D, pCC2001, pCC2005, pCC2004, pCC2003, pCC2002, pCC2008, pCC2007, pCC2006, pCC2012 (see Figure 3 for description of HER2-expression plasmids). (−) No primary antibody, Tzm: Trastuzumab, CB11: a monoclonal antibody recognizing an epitope at intracellular domain of HER2 that was used as a positive control for expression of transfected plasmids.
Figure Legend Snippet: Epitope mapping of anti-HER2 antibodies using indirect immunofluorescence assay. Huh7 cells were transfected in 6-well plates using a set of mammalian expression plasmids encoding full-length or N-terminally truncated HER2 protein. Transfected cells were cultivated for 48 h, and subjected to indirect immunofluorescence assay. Transfected cells that were recognized by specific antibodies displayed strong green fluorescence. The letters from (a) to (k) indicate the plasmids in the following order: pDEST26 (empty vector), p219D, pCC2001, pCC2005, pCC2004, pCC2003, pCC2002, pCC2008, pCC2007, pCC2006, pCC2012 (see Figure 3 for description of HER2-expression plasmids). (−) No primary antibody, Tzm: Trastuzumab, CB11: a monoclonal antibody recognizing an epitope at intracellular domain of HER2 that was used as a positive control for expression of transfected plasmids.

Techniques Used: Immunofluorescence, Transfection, Expressing, Fluorescence, Plasmid Preparation, Positive Control

Synergy and antagonism between anti-HER2 antibodies and tumor necrosis factor-α. Anti-Her2 antibodies and TNF-α acted synergistically in SK-BR-3 cells (top), but antagonistically in BT-474 cells (bottom). Growth effects observed under antibody (white columns), TNF-α (striped columns), and antibody + 1000 TNF-α (black columns) were obtained as follows. Cells were plated onto 96-well plates and incubated at 37°C with 5% CO 2 . After 2 h of pre-incubation, antibodies (5 μg/ml) were added, and cells were incubated for 6 days to study the effects of antibodies alone (white columns). The effects TNF-α (1000 U/ml) alone (striped columns) were studied by adding this cytokine into cell culture medium after 4 h of pre-incubation. For combined treatments, antibodies (5 μg/ml) and TNF-α (1000 U/ml) were added after 2 h and 4 h of pre-incubations, respectively (black columns). Cells amount was determined by Sulforhodamine B assay after 6 days of treatment. Growth level under a drug condition was defined as the growth under that condition normalized by growth under no drug condition. Expected growth level under no interaction (gray columns) was calculated by multiplying the growth levels under each individual drug. The observed growth level for each combination was divided by the expected growth level to find an interaction score according to Bliss Independence Model for drug interactions (displayed under each antibody label). The combination of TNF-α with isotype control antibody (IgG1) or Trastuzumab (Tzm) provided scores near 1, meaning no interaction or independence in both cell lines. New anti-HER2 antibodies provided scores less than 0.57 in SK-BR-3 cells, meaning synergy. In contrast their interaction scores were more than 1.40 in BT-474 cells, meaning antagonism.
Figure Legend Snippet: Synergy and antagonism between anti-HER2 antibodies and tumor necrosis factor-α. Anti-Her2 antibodies and TNF-α acted synergistically in SK-BR-3 cells (top), but antagonistically in BT-474 cells (bottom). Growth effects observed under antibody (white columns), TNF-α (striped columns), and antibody + 1000 TNF-α (black columns) were obtained as follows. Cells were plated onto 96-well plates and incubated at 37°C with 5% CO 2 . After 2 h of pre-incubation, antibodies (5 μg/ml) were added, and cells were incubated for 6 days to study the effects of antibodies alone (white columns). The effects TNF-α (1000 U/ml) alone (striped columns) were studied by adding this cytokine into cell culture medium after 4 h of pre-incubation. For combined treatments, antibodies (5 μg/ml) and TNF-α (1000 U/ml) were added after 2 h and 4 h of pre-incubations, respectively (black columns). Cells amount was determined by Sulforhodamine B assay after 6 days of treatment. Growth level under a drug condition was defined as the growth under that condition normalized by growth under no drug condition. Expected growth level under no interaction (gray columns) was calculated by multiplying the growth levels under each individual drug. The observed growth level for each combination was divided by the expected growth level to find an interaction score according to Bliss Independence Model for drug interactions (displayed under each antibody label). The combination of TNF-α with isotype control antibody (IgG1) or Trastuzumab (Tzm) provided scores near 1, meaning no interaction or independence in both cell lines. New anti-HER2 antibodies provided scores less than 0.57 in SK-BR-3 cells, meaning synergy. In contrast their interaction scores were more than 1.40 in BT-474 cells, meaning antagonism.

Techniques Used: Incubation, Cell Culture, Sulforhodamine B Assay

53) Product Images from "Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease"

Article Title: Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017953

Triton X-100 solubilization of striatal lysates from A53T α-Syn mutant and age-matched control animals. Striatal lysates from A53T α-Syn mutant mice and control, non-Tg mice [4 animals per group] were extracted with Triton X-100 as described in Methods. Proteins in Triton X-100-soluble and Triton X-100-insoluble fractions were measured by Western blots. Blots show representative gels while the bar graphs are composites summarized from all animals, expressed as percent of control, non-Tg mice. (A) β-actin was added as a loading control. (B) p-GSK-3β was normalized to GSK-3β from within each fraction on the blot. (C) pSer202, pSer262 and PSer396/404 were all normalized to total Tau in each fraction, and β-actin was added as a loading control. *,  P
Figure Legend Snippet: Triton X-100 solubilization of striatal lysates from A53T α-Syn mutant and age-matched control animals. Striatal lysates from A53T α-Syn mutant mice and control, non-Tg mice [4 animals per group] were extracted with Triton X-100 as described in Methods. Proteins in Triton X-100-soluble and Triton X-100-insoluble fractions were measured by Western blots. Blots show representative gels while the bar graphs are composites summarized from all animals, expressed as percent of control, non-Tg mice. (A) β-actin was added as a loading control. (B) p-GSK-3β was normalized to GSK-3β from within each fraction on the blot. (C) pSer202, pSer262 and PSer396/404 were all normalized to total Tau in each fraction, and β-actin was added as a loading control. *, P

Techniques Used: Mutagenesis, Mouse Assay, Western Blot

54) Product Images from "Ablation of CCAAT/Enhancer-Binding Protein Delta (C/EBPD): Increased Plaque Burden in a Murine Alzheimer’s Disease Model"

Article Title: Ablation of CCAAT/Enhancer-Binding Protein Delta (C/EBPD): Increased Plaque Burden in a Murine Alzheimer’s Disease Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0134228

Western blot detection of Abeta in brain extracts from APP/PS1 and APP/PS1 x C/EBPD (-/-) mice. Abeta protein loads in PBS-soluble ( A ) and formic acid-soluble ( C ) brain extracts detected using the 6E10 antibody at 9 months of age. Synthetic Abeta1-42 peptide served as additional size marker. ( B and D ) Densitometric quantification of monomeric Abeta band intensities from ( A ) and ( C ), respectively (**p
Figure Legend Snippet: Western blot detection of Abeta in brain extracts from APP/PS1 and APP/PS1 x C/EBPD (-/-) mice. Abeta protein loads in PBS-soluble ( A ) and formic acid-soluble ( C ) brain extracts detected using the 6E10 antibody at 9 months of age. Synthetic Abeta1-42 peptide served as additional size marker. ( B and D ) Densitometric quantification of monomeric Abeta band intensities from ( A ) and ( C ), respectively (**p

Techniques Used: Western Blot, Mouse Assay, Marker

55) Product Images from "Yin Yang 1 Intronic Binding Sequences and Splicing Elicit Intron-Mediated Enhancement of Ubiquitin C Gene Expression"

Article Title: Yin Yang 1 Intronic Binding Sequences and Splicing Elicit Intron-Mediated Enhancement of Ubiquitin C Gene Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065932

Effects of ectopic expression of YY1 on both reporter and endogenous target gene expression. ( A ) Immunoblotting of proteins from HeLa cells transfected with YY1 expression vector (lanes 2, 4, 6) or control empty vector (lanes 1, 3, 5), at 48 h post-transfection. Nuclear (Nuc, 10 µg), cytosol (Cyt, 20 µg) and total (20 µg) extracts were obtained as reported under “Materials and Methods”. Arrows mark the YY1 and actin bands (upper and lower panel, respectively). Molecular weight standards (kDa) are indicated on the left. Actin was employed as the endogenous internal control. A representative blot is shown. Experiments were repeated three times with similar results. ( B ) EMSA performed with 32 P-labeled ODN IIa, containing a YY1 binding sequence, as the probe and HeLa nuclear extracts of cells transfected with control (-, lane 1) or YY1 expression vector (+, lane 2). The parentheses indicate the major nucleoprotein complexes. A representative image of three different EMSA is shown. Quantification of DNA-protein complexes was performed in a Molecular Imager and results are reported in the histogram as mean counts (±SE) of three different experiments (***, p
Figure Legend Snippet: Effects of ectopic expression of YY1 on both reporter and endogenous target gene expression. ( A ) Immunoblotting of proteins from HeLa cells transfected with YY1 expression vector (lanes 2, 4, 6) or control empty vector (lanes 1, 3, 5), at 48 h post-transfection. Nuclear (Nuc, 10 µg), cytosol (Cyt, 20 µg) and total (20 µg) extracts were obtained as reported under “Materials and Methods”. Arrows mark the YY1 and actin bands (upper and lower panel, respectively). Molecular weight standards (kDa) are indicated on the left. Actin was employed as the endogenous internal control. A representative blot is shown. Experiments were repeated three times with similar results. ( B ) EMSA performed with 32 P-labeled ODN IIa, containing a YY1 binding sequence, as the probe and HeLa nuclear extracts of cells transfected with control (-, lane 1) or YY1 expression vector (+, lane 2). The parentheses indicate the major nucleoprotein complexes. A representative image of three different EMSA is shown. Quantification of DNA-protein complexes was performed in a Molecular Imager and results are reported in the histogram as mean counts (±SE) of three different experiments (***, p

Techniques Used: Expressing, Transfection, Plasmid Preparation, Molecular Weight, Labeling, Binding Assay, Sequencing

56) Product Images from "TUSC3 Loss Alters the ER Stress Response and Accelerates Prostate Cancer Growth in vivo"

Article Title: TUSC3 Loss Alters the ER Stress Response and Accelerates Prostate Cancer Growth in vivo

Journal: Scientific Reports

doi: 10.1038/srep03739

TUSC3 loss leads to increased viability, N-glycosylation and Akt signaling. (a) shTUSC3 and control cells were serum starved for 36 hours (−) and serum was added for 30 minutes before lysis (+). PI3K/Akt and MAPK signaling pathway were evaluated by immunoblotting. Increased downstream activation of Akt can be observed in serum starved TUSC3 silenced PC3 cells as well as in DU145 cells after stimulation with serum. ER stress and CHOP are induced by prolonged serum starvation (lane 5 and 6) in PC3 cells. Loss of TUSC3 decreases CHOP levels in PC3 cells (lane 7 and 8). (b) Increased N-glycosylation in shTUSC3 cells. Lectin blotting using Concanavalin A and Phytohaemagglutinin-L lectins on isolated cell surface proteins was performed in cell membrane fractions of PC3 and DU145 cells following 72 h serum starvation. Control for protein loading was performed by amido black staining ( Supplementary Figure S2C ). (c) DU145 and PC3 prostate cancer cell lines were serum starved for 72 hours before lysis. Silencing of TUSC3 (sh) leads to sustained phosphorylation of Akt and decreased expression of CHOP in both cell lines. (d) Viability of TUSC3 silenced (shTUSC3) and control (scrambled shRNA) prostate cancer cells was assessed with the CellTiter-Blue® Assay after treatment with 5 μM tunicamycin or DMSO for 72 hours in full medium. Experiments were performed in triplicates and results are representative of several independent experiments. * p = 0.01.
Figure Legend Snippet: TUSC3 loss leads to increased viability, N-glycosylation and Akt signaling. (a) shTUSC3 and control cells were serum starved for 36 hours (−) and serum was added for 30 minutes before lysis (+). PI3K/Akt and MAPK signaling pathway were evaluated by immunoblotting. Increased downstream activation of Akt can be observed in serum starved TUSC3 silenced PC3 cells as well as in DU145 cells after stimulation with serum. ER stress and CHOP are induced by prolonged serum starvation (lane 5 and 6) in PC3 cells. Loss of TUSC3 decreases CHOP levels in PC3 cells (lane 7 and 8). (b) Increased N-glycosylation in shTUSC3 cells. Lectin blotting using Concanavalin A and Phytohaemagglutinin-L lectins on isolated cell surface proteins was performed in cell membrane fractions of PC3 and DU145 cells following 72 h serum starvation. Control for protein loading was performed by amido black staining ( Supplementary Figure S2C ). (c) DU145 and PC3 prostate cancer cell lines were serum starved for 72 hours before lysis. Silencing of TUSC3 (sh) leads to sustained phosphorylation of Akt and decreased expression of CHOP in both cell lines. (d) Viability of TUSC3 silenced (shTUSC3) and control (scrambled shRNA) prostate cancer cells was assessed with the CellTiter-Blue® Assay after treatment with 5 μM tunicamycin or DMSO for 72 hours in full medium. Experiments were performed in triplicates and results are representative of several independent experiments. * p = 0.01.

Techniques Used: Lysis, Activation Assay, Isolation, Staining, Expressing, shRNA, CtB Assay

57) Product Images from "Luminal breast cancer metastasis is dependent on estrogen signaling"

Article Title: Luminal breast cancer metastasis is dependent on estrogen signaling

Journal: Clinical & experimental metastasis

doi: 10.1007/s10585-012-9466-4

Luminal breast cancer metastasis does not require TGF-β signaling. a Conditioned medium of MCF-7-5624A-GF cells ( red circles ) produced much higher amounts of TGF-β2 than parental MCF-7 cells ( blue squares ), while production of TGF-β1
Figure Legend Snippet: Luminal breast cancer metastasis does not require TGF-β signaling. a Conditioned medium of MCF-7-5624A-GF cells ( red circles ) produced much higher amounts of TGF-β2 than parental MCF-7 cells ( blue squares ), while production of TGF-β1

Techniques Used: Produced

58) Product Images from "Reversible interconversion and maintenance of mammary epithelial cell characteristics by the ligand-regulated EGFR system"

Article Title: Reversible interconversion and maintenance of mammary epithelial cell characteristics by the ligand-regulated EGFR system

Journal: Scientific Reports

doi: 10.1038/srep20209

Ligand-switching reversibly interconverted distinct characteristics of mammary epithelial cells. ( a ) Representative projected Z-stack images of the acinus-like structures formed by E-cells and A-cells after a 2-week culture on the reconstituted basement membrane. Cells were visualized by fluorescent phalloidin staining. Yellow arrowheads indicate cell clusters judged as non-acinus. Scale bar: 100 μm. ( b ) Quantification of acinus-formation efficiency of sequentially generated E-cells and A-cells. The criteria for evaluating the acinus size are shown in Supplementary Fig. 3a . ( c ) Flow cytometric analysis of CD24 (X-axis) and CD44 (Y-axis) expression in sequentially generated E-cells and A-cells. ( d ) Western blot analysis of EMT related factors and EGFR in HMT-3522 S1 cells generated by the ligand-switching between EGF (10 ng/mL) and AREG (20 ng/mL). S1-EGF and S1-AREG indicate EGF- and AREG-cultured parental cells, respectively. S1-AREG cells were further cultured in the presence of EGF, generating S1-EGF (2nd). ( e ) Immunofluorescent images of S1-EGF, S1-AREG and S1-EGF (2nd) cells stained with anti-E-cadherin antibody (green) and anti-EGFR antibody (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar: 40 μm. ( f ) FACS analysis of the expression of CD44 and CD24 in S1-EGF, S1-AREG and S1-EGF (2nd) cells.
Figure Legend Snippet: Ligand-switching reversibly interconverted distinct characteristics of mammary epithelial cells. ( a ) Representative projected Z-stack images of the acinus-like structures formed by E-cells and A-cells after a 2-week culture on the reconstituted basement membrane. Cells were visualized by fluorescent phalloidin staining. Yellow arrowheads indicate cell clusters judged as non-acinus. Scale bar: 100 μm. ( b ) Quantification of acinus-formation efficiency of sequentially generated E-cells and A-cells. The criteria for evaluating the acinus size are shown in Supplementary Fig. 3a . ( c ) Flow cytometric analysis of CD24 (X-axis) and CD44 (Y-axis) expression in sequentially generated E-cells and A-cells. ( d ) Western blot analysis of EMT related factors and EGFR in HMT-3522 S1 cells generated by the ligand-switching between EGF (10 ng/mL) and AREG (20 ng/mL). S1-EGF and S1-AREG indicate EGF- and AREG-cultured parental cells, respectively. S1-AREG cells were further cultured in the presence of EGF, generating S1-EGF (2nd). ( e ) Immunofluorescent images of S1-EGF, S1-AREG and S1-EGF (2nd) cells stained with anti-E-cadherin antibody (green) and anti-EGFR antibody (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar: 40 μm. ( f ) FACS analysis of the expression of CD44 and CD24 in S1-EGF, S1-AREG and S1-EGF (2nd) cells.

Techniques Used: Staining, Generated, Flow Cytometry, Expressing, Western Blot, HMT Assay, Cell Culture, FACS

EGFR was responsible for EGF- and AREG-induced phenotypic conversion. ( a ) Quantification of EGFR mRNA by RT-qPCR (left) and EGFR protein in whole cell lysates by western blot analysis (right). The mRNA and protein levels of EGFR in A-cells were expressed relative to that of E-cells. *p
Figure Legend Snippet: EGFR was responsible for EGF- and AREG-induced phenotypic conversion. ( a ) Quantification of EGFR mRNA by RT-qPCR (left) and EGFR protein in whole cell lysates by western blot analysis (right). The mRNA and protein levels of EGFR in A-cells were expressed relative to that of E-cells. *p

Techniques Used: Quantitative RT-PCR, Western Blot

Dose-changing of EGF and AREG induces phenotypic conversion. ( a ) Phase-contrast images of cells cultured in the presence of different concentrations of EGF. Scale bar: 50 μm. ( b ) Western blot analysis of EMT-related factors and EGFR in cells cultured as in ( a ). ( c ) Immunofluorescent images of cells cultured as in ( a ). Cells were stained with antibodies against E-cadherin (green) and EGFR (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar: 50 μm. ( d ) Quantification of acinus-formation efficiency. Cells shown in ( a ) were cultured for 2 weeks on a reconstituted basement membrane and their acinus size was evaluated. ( e ) Phase contrast images of cells cultured in the presence of different concentration of AREG. Scale bar: 50 μm. ( f ) Western blot analysis of EMT-related factors and EGFR expression in cells cultured as in ( e ). ( g ) Immunofluorescent images of cells cultured as in ( e ). Cells were stained as in ( c ). Scale bar: 50 μm. ( h ) Quantification of acinus-formation efficiency for cells shown in ( e ). Cells were processed as in ( d ).
Figure Legend Snippet: Dose-changing of EGF and AREG induces phenotypic conversion. ( a ) Phase-contrast images of cells cultured in the presence of different concentrations of EGF. Scale bar: 50 μm. ( b ) Western blot analysis of EMT-related factors and EGFR in cells cultured as in ( a ). ( c ) Immunofluorescent images of cells cultured as in ( a ). Cells were stained with antibodies against E-cadherin (green) and EGFR (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar: 50 μm. ( d ) Quantification of acinus-formation efficiency. Cells shown in ( a ) were cultured for 2 weeks on a reconstituted basement membrane and their acinus size was evaluated. ( e ) Phase contrast images of cells cultured in the presence of different concentration of AREG. Scale bar: 50 μm. ( f ) Western blot analysis of EMT-related factors and EGFR expression in cells cultured as in ( e ). ( g ) Immunofluorescent images of cells cultured as in ( e ). Cells were stained as in ( c ). Scale bar: 50 μm. ( h ) Quantification of acinus-formation efficiency for cells shown in ( e ). Cells were processed as in ( d ).

Techniques Used: Cell Culture, Western Blot, Staining, Concentration Assay, Expressing

Ligand-switching between EGF and AREG altered signal strength of EGFR and ERK. ( a ) Western blot time course analysis of the phosphorylation of EGFR and ERK1/2 by 10 ng/mL EGF or 20 ng/mL AREG in E-cells and A-cells. ( b ) Schematic representation of the quantitative measurement of EGFR or ERK activation (Modified from Andreu-Perez et al. Fig. 1b ). ( c ) Quantification of pERK phosphorylation. Data were obtained from 3 independent experiments, one of which is shown in ( a ). The band intensities were normalized to that of pERK in E-cells treated with EGF for 10 min. ( d ) Quantification of the integrated signal strength of ERK, which was calculated from the area under the curve (0 to 60 min) shown in ( c ). The values were normalized to that of pERK in E-cells treated with EGF. ( e ) Western blot analysis of the phosphorylation of ERK1/2 and FRA1 by EGF or AREG in A-cells over a time course. ( f ) Quantification of pERK phosphorylation. Data were obtained from 3 independent experiments, one of which is shown in ( e ). ( g ) Quantification of the integrated signal strength of ERK, which was calculated from the area under the curve (0 to 310 min) shown in ( f ). The values were normalized to that of pERK in A-cells treated with AREG. ( h ) Phase-contrast and immunofluorescent images of A-cells treated with EGFR inhibitors. AREG-depleted A-cells were treated with EGF or AREG, allowing the immediate activation of EGFR signaling. After the 2 h incubation, a control antibody (10 μg/mL) or a neutralizing antibody against EGFR (10 μg/mL) was administered. Cells were further cultured for 2 days and stained with an anti-E-cadherin antibody (green). Nuclei were stained with Hoechst 33342 (blue). See also Supplementary Fig. 8d . Scale bar: upper panel, 100 μm; lower panel, 50 μm.
Figure Legend Snippet: Ligand-switching between EGF and AREG altered signal strength of EGFR and ERK. ( a ) Western blot time course analysis of the phosphorylation of EGFR and ERK1/2 by 10 ng/mL EGF or 20 ng/mL AREG in E-cells and A-cells. ( b ) Schematic representation of the quantitative measurement of EGFR or ERK activation (Modified from Andreu-Perez et al. Fig. 1b ). ( c ) Quantification of pERK phosphorylation. Data were obtained from 3 independent experiments, one of which is shown in ( a ). The band intensities were normalized to that of pERK in E-cells treated with EGF for 10 min. ( d ) Quantification of the integrated signal strength of ERK, which was calculated from the area under the curve (0 to 60 min) shown in ( c ). The values were normalized to that of pERK in E-cells treated with EGF. ( e ) Western blot analysis of the phosphorylation of ERK1/2 and FRA1 by EGF or AREG in A-cells over a time course. ( f ) Quantification of pERK phosphorylation. Data were obtained from 3 independent experiments, one of which is shown in ( e ). ( g ) Quantification of the integrated signal strength of ERK, which was calculated from the area under the curve (0 to 310 min) shown in ( f ). The values were normalized to that of pERK in A-cells treated with AREG. ( h ) Phase-contrast and immunofluorescent images of A-cells treated with EGFR inhibitors. AREG-depleted A-cells were treated with EGF or AREG, allowing the immediate activation of EGFR signaling. After the 2 h incubation, a control antibody (10 μg/mL) or a neutralizing antibody against EGFR (10 μg/mL) was administered. Cells were further cultured for 2 days and stained with an anti-E-cadherin antibody (green). Nuclei were stained with Hoechst 33342 (blue). See also Supplementary Fig. 8d . Scale bar: upper panel, 100 μm; lower panel, 50 μm.

Techniques Used: Western Blot, Activation Assay, Modification, Incubation, Cell Culture, Staining

Manipulation of signal strength led to phenotypic conversion. ( a ) Phase-contrast images of E-cells infected with a lentivirus vector encoding dominant negative MEK1 (DN-MEK1). Enforced expression of DN-MEK1 increased cell-cell contact in EGF medium. Scale bar: 50 μm. ( b ) Western blot analysis of EMT-related factors, MEK1 and ERK1/2 in cells cultured as in ( a ). ( c ) Flow cytometric analysis of DN-MEK1-expressing cells cultured as in ( a ). ( d ) Quantification of acinus-formation efficiency. Cells shown in ( a ) were cultured for 2 weeks on a reconstituted basement membrane. ( e ) Phase-contrast images of A-cells infected with the lentivirus vector encoding constitutive active MEK1 (CA-MEK1). Enforced expression of CA-MEK1 decreased cell-cell contact in AREG medium. Scale bar: 50 μm. ( f ) Western blot analysis of EMT-related factors MEK1 and ERK1/2 in cells cultured as in ( e ). ( g ) Flow cytometric analysis of CA-MEK1-expressing cells cultured as in ( e ). ( h ) Quantification of acinus-formation efficiency of cells shown in ( e ). Cells were processed as in ( d ).
Figure Legend Snippet: Manipulation of signal strength led to phenotypic conversion. ( a ) Phase-contrast images of E-cells infected with a lentivirus vector encoding dominant negative MEK1 (DN-MEK1). Enforced expression of DN-MEK1 increased cell-cell contact in EGF medium. Scale bar: 50 μm. ( b ) Western blot analysis of EMT-related factors, MEK1 and ERK1/2 in cells cultured as in ( a ). ( c ) Flow cytometric analysis of DN-MEK1-expressing cells cultured as in ( a ). ( d ) Quantification of acinus-formation efficiency. Cells shown in ( a ) were cultured for 2 weeks on a reconstituted basement membrane. ( e ) Phase-contrast images of A-cells infected with the lentivirus vector encoding constitutive active MEK1 (CA-MEK1). Enforced expression of CA-MEK1 decreased cell-cell contact in AREG medium. Scale bar: 50 μm. ( f ) Western blot analysis of EMT-related factors MEK1 and ERK1/2 in cells cultured as in ( e ). ( g ) Flow cytometric analysis of CA-MEK1-expressing cells cultured as in ( e ). ( h ) Quantification of acinus-formation efficiency of cells shown in ( e ). Cells were processed as in ( d ).

Techniques Used: Infection, Plasmid Preparation, Dominant Negative Mutation, Expressing, Western Blot, Cell Culture, Flow Cytometry

59) Product Images from "Internalized Tau sensitizes cells to stress by promoting formation and stability of stress granules"

Article Title: Internalized Tau sensitizes cells to stress by promoting formation and stability of stress granules

Journal: Scientific Reports

doi: 10.1038/srep30498

Tau localization to stress granules requires TIA-1. ( A ) TIA-1 shRNA knockdown efficiency determined by qPCR following shRNA plasmid transfection of HEK293T cells. Levels of TIA-1 mRNA were normalized to GAPDH mRNA levels (n = 2). ( B ) TIA-1 was transiently knocked down in HEK293T cells (middle and right images) that were then exposed to Tau-GLuc-conditioned media and stained with Tau-5 and stress granule marker antibodies. The middle image shows typical cells with mostly cytosolic, non-punctate staining of Tau. The image on the right shows an example of a cell with SGs costaining with Tau and eIF3η. ( C ) Quantitative analysis of Tau-positive stress granule formation. SGs were present in the majority of cells exposed to Tau media when TIA-1 is normally expressed but were significantly decreased when TIA-1 was knocked down. Similar results were obtained with both TIA-1 and eIF3η staining (n = 3). ( D ) Resazurin-based cell viability assay showed that TIA-1 knockdown had no effect on cell viability per se but was able to improve viability in cells exposed to Tau-GLuc-conditioned media (n = 3). Scalebar = 10 μm; average +/− SEM is shown; ***p
Figure Legend Snippet: Tau localization to stress granules requires TIA-1. ( A ) TIA-1 shRNA knockdown efficiency determined by qPCR following shRNA plasmid transfection of HEK293T cells. Levels of TIA-1 mRNA were normalized to GAPDH mRNA levels (n = 2). ( B ) TIA-1 was transiently knocked down in HEK293T cells (middle and right images) that were then exposed to Tau-GLuc-conditioned media and stained with Tau-5 and stress granule marker antibodies. The middle image shows typical cells with mostly cytosolic, non-punctate staining of Tau. The image on the right shows an example of a cell with SGs costaining with Tau and eIF3η. ( C ) Quantitative analysis of Tau-positive stress granule formation. SGs were present in the majority of cells exposed to Tau media when TIA-1 is normally expressed but were significantly decreased when TIA-1 was knocked down. Similar results were obtained with both TIA-1 and eIF3η staining (n = 3). ( D ) Resazurin-based cell viability assay showed that TIA-1 knockdown had no effect on cell viability per se but was able to improve viability in cells exposed to Tau-GLuc-conditioned media (n = 3). Scalebar = 10 μm; average +/− SEM is shown; ***p

Techniques Used: shRNA, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Staining, Marker, Viability Assay

Transfected and internalized extracellular Tau differ in their ability to associate with stress granules. ( A ) Expression of non-tagged Tau, Tau-GLuc1/2 and TauE14-GLuc1/2 in HEK293T cells as detected by Western blot. The blot picture was cropped from a larger original image, maintaining all the stained bands. ( B ) HEK293T cells transiently transfected with the above-mentioned constructs and stained with Tau-5 (green) and TIA-1 (red) antibodies. Arsenite (0.5 mM for 30 min) was used as a positive control for induction of stress granules. ( C ) Quantitative analysis of stress granule formation. Stress granule-positive cells were counted among the Tau-transfected cells. Arsenite treatment promoted stress granule-formation in all cells while only some Tau-transfected, and more efficiently TauE14-transfected, cells contained stress-granules (n = 3). ( D ) Resazurin-based cell viability assay with HEK293T cells transiently transfected with the Tau constructs. Salubrinal and arsenite were used as positive controls for stress granule induction (n = 4). ( E ) LDH release assay in HEK293T cells transfected with the Tau constructs and treated with salubrinal and arsenite as positive controls (n = 4). Relative LDH release was calculated from the ratio of LDH in the media and total LDH (from media and the cells). ( F ) Protein-fragment complementation assay (PCA) in HEK293T cells transiently transfected with TIA-1-GLuc1, Tau-GLuc2 and TauE14-GLuc2 (n = 3). ( G ) PCA-based coplating experiment to study cell-to-cell transfer of Tau. Batches of HEK293T cells were separately transfected with a single PCA construct (TIA-1-GLuc1, Tau-GLuc2, TauE14-GLuc2 or GSK3β-GLuc2 as a control), followed by replating the cells in various combinations 24 h post-transfection (n = 3). Scalebar = 10 μm; average +/−SEM is shown; ***p
Figure Legend Snippet: Transfected and internalized extracellular Tau differ in their ability to associate with stress granules. ( A ) Expression of non-tagged Tau, Tau-GLuc1/2 and TauE14-GLuc1/2 in HEK293T cells as detected by Western blot. The blot picture was cropped from a larger original image, maintaining all the stained bands. ( B ) HEK293T cells transiently transfected with the above-mentioned constructs and stained with Tau-5 (green) and TIA-1 (red) antibodies. Arsenite (0.5 mM for 30 min) was used as a positive control for induction of stress granules. ( C ) Quantitative analysis of stress granule formation. Stress granule-positive cells were counted among the Tau-transfected cells. Arsenite treatment promoted stress granule-formation in all cells while only some Tau-transfected, and more efficiently TauE14-transfected, cells contained stress-granules (n = 3). ( D ) Resazurin-based cell viability assay with HEK293T cells transiently transfected with the Tau constructs. Salubrinal and arsenite were used as positive controls for stress granule induction (n = 4). ( E ) LDH release assay in HEK293T cells transfected with the Tau constructs and treated with salubrinal and arsenite as positive controls (n = 4). Relative LDH release was calculated from the ratio of LDH in the media and total LDH (from media and the cells). ( F ) Protein-fragment complementation assay (PCA) in HEK293T cells transiently transfected with TIA-1-GLuc1, Tau-GLuc2 and TauE14-GLuc2 (n = 3). ( G ) PCA-based coplating experiment to study cell-to-cell transfer of Tau. Batches of HEK293T cells were separately transfected with a single PCA construct (TIA-1-GLuc1, Tau-GLuc2, TauE14-GLuc2 or GSK3β-GLuc2 as a control), followed by replating the cells in various combinations 24 h post-transfection (n = 3). Scalebar = 10 μm; average +/−SEM is shown; ***p

Techniques Used: Transfection, Expressing, Western Blot, Staining, Construct, Positive Control, Viability Assay, Lactate Dehydrogenase Assay, Protein-Fragment Complementation Assay

60) Product Images from "WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4. WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4"

Article Title: WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4. WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4

Journal: Cancer Medicine

doi: 10.1002/cam4.1837

Association of p27 with peripheral rearrangements of the actin cytoskeleton. A, siRNA oligonucleotides targeting p27 (sip27) or scrambled control siRNAs (Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐p27 antibody. B, MTT assays of S2‐013 and PANC‐1 cells transiently transfected with scrambled control siRNA, ACTN4 siRNA, or p27 siRNA were performed to evaluate cell viability. Data are representative of three independent experiments and are the means ±SD. ABS on Y ‐axis means absorbance at 490 nm and at 630 nm as reference measured with a microplate reader. C, Confocal immunofluorescence microscopic images. Scr‐transfected S2‐013 cells and sip27‐transfected S2‐013 cells were incubated on fibronectin and subsequently stained with anti‐p27 antibody (green) and phalloidin (red). Arrows, peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. D, Quantification of the data shown in B. The values represent the number of cells with fibronectin‐stimulated cell protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P
Figure Legend Snippet: Association of p27 with peripheral rearrangements of the actin cytoskeleton. A, siRNA oligonucleotides targeting p27 (sip27) or scrambled control siRNAs (Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐p27 antibody. B, MTT assays of S2‐013 and PANC‐1 cells transiently transfected with scrambled control siRNA, ACTN4 siRNA, or p27 siRNA were performed to evaluate cell viability. Data are representative of three independent experiments and are the means ±SD. ABS on Y ‐axis means absorbance at 490 nm and at 630 nm as reference measured with a microplate reader. C, Confocal immunofluorescence microscopic images. Scr‐transfected S2‐013 cells and sip27‐transfected S2‐013 cells were incubated on fibronectin and subsequently stained with anti‐p27 antibody (green) and phalloidin (red). Arrows, peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. D, Quantification of the data shown in B. The values represent the number of cells with fibronectin‐stimulated cell protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P

Techniques Used: Transfection, Western Blot, MTT Assay, Immunofluorescence, Incubation, Staining, Derivative Assay

Roles of WAVE2 and ACTN4 in the motility and invasiveness of PDAC cells. A and B, siACTN4 oligonucleotides or Scr oligonucleotides were transiently transfected into S2‐013 and PANC‐1 cells. Motility (A) and two‐chamber invasion (B) assays were performed. Migrating cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P
Figure Legend Snippet: Roles of WAVE2 and ACTN4 in the motility and invasiveness of PDAC cells. A and B, siACTN4 oligonucleotides or Scr oligonucleotides were transiently transfected into S2‐013 and PANC‐1 cells. Motility (A) and two‐chamber invasion (B) assays were performed. Migrating cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P

Techniques Used: Transfection, Derivative Assay

Effects of WAVE2 and ACTN4 on p27 activity. A, Confocal immunofluorescence microscopic images. S2‐013 and HPNE cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐phosphorylated p27 (red) antibodies. Actin filaments were labeled with phalloidin (violet). Arrows, phosphorylated p27 in the nucleus. Blue, DAPI staining. Scale bar, 10 μm. B, S2‐013 and HPNE cells were incubated on fibronectin and fractionated into cytosolic (c) and nuclear (n) fractions. Western blotting of the fractions was performed using anti‐phosphorylated p27 and anti‐p27 antibodies. C, Scr‐transfected S2‐013 cells and siWAVE2‐transfected S2‐013 cells were incubated on fibronectin. Western blotting was performed using anti‐WAVE2, anti‐phosphorylated p27, and anti‐p27 antibodies. D, A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with scrambled control siRNA or WAVE2 siRNA with or without ACTN4 siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies
Figure Legend Snippet: Effects of WAVE2 and ACTN4 on p27 activity. A, Confocal immunofluorescence microscopic images. S2‐013 and HPNE cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐phosphorylated p27 (red) antibodies. Actin filaments were labeled with phalloidin (violet). Arrows, phosphorylated p27 in the nucleus. Blue, DAPI staining. Scale bar, 10 μm. B, S2‐013 and HPNE cells were incubated on fibronectin and fractionated into cytosolic (c) and nuclear (n) fractions. Western blotting of the fractions was performed using anti‐phosphorylated p27 and anti‐p27 antibodies. C, Scr‐transfected S2‐013 cells and siWAVE2‐transfected S2‐013 cells were incubated on fibronectin. Western blotting was performed using anti‐WAVE2, anti‐phosphorylated p27, and anti‐p27 antibodies. D, A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with scrambled control siRNA or WAVE2 siRNA with or without ACTN4 siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies

Techniques Used: Activity Assay, Immunofluorescence, Cell Culture, Labeling, Staining, Incubation, Western Blot, Transfection, Construct

Roles of WAVE2 in the translocation of ACTN4 to actin filaments in cell protrusions. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2, ACTN4, and actin. Rabbit IgG isotype control antibody was used as the control. B, Confocal immunofluorescence microscopic images. Oligonucleotides (siRNAs targeting WAVE2 (siWAVE2) or scrambled siRNAs (Scr) as the negative control) were transiently transfected into S2‐013 cells. Transfected cells were incubated on fibronectin and were subsequently stained with anti‐WAVE2 antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, ACTN4 bound to peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. C, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with WAVE2 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, exogenous WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 µm. D, Oligonucleotides (siRNAs targeting ACTN4 (siACTN4) or Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐ACTN4 antibody. E, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Blue, DAPI staining. Scale bars, 10 µm
Figure Legend Snippet: Roles of WAVE2 in the translocation of ACTN4 to actin filaments in cell protrusions. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2, ACTN4, and actin. Rabbit IgG isotype control antibody was used as the control. B, Confocal immunofluorescence microscopic images. Oligonucleotides (siRNAs targeting WAVE2 (siWAVE2) or scrambled siRNAs (Scr) as the negative control) were transiently transfected into S2‐013 cells. Transfected cells were incubated on fibronectin and were subsequently stained with anti‐WAVE2 antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, ACTN4 bound to peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. C, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with WAVE2 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Arrows, exogenous WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 µm. D, Oligonucleotides (siRNAs targeting ACTN4 (siACTN4) or Scr) were transiently transfected into S2‐013 cells. Western blotting was performed using anti‐ACTN4 antibody. E, Confocal immunofluorescence microscopic images. A myc‐tagged WAVE2 rescue construct was transfected into S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green), anti‐ACTN4 antibody (red), and phalloidin (violet). Blue, DAPI staining. Scale bars, 10 µm

Techniques Used: Translocation Assay, Immunoprecipitation, Cell Culture, Western Blot, Immunofluorescence, Negative Control, Transfection, Incubation, Staining, Construct

Association of WAVE2 with ACTN4. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Rabbit IgG isotype control antibody was used as the control. Proteins within immunoprecipitates were examined on Western blots probed with anti‐WAVE2 antibody. B, Proteins in immunoprecipitates were examined with silver staining. Rabbit IgG isotype control antibody was used as the control. A 100‐kDa band is indicated by the arrow. C, The percent coverage for ACTN4 is represented by the identified peptides in the total protein sequence (accession number NP_004915). D, Immunoprecipitation of WAVE2 or ACTN4 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2 and ACTN4. Rabbit IgG isotype control antibody for WAVE2 and mouse IgG isotype control antibody for ACTN4 was used as controls. E, Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐ACTN4 (red) antibodies. Arrows, WAVE2 co‐localized with ACTN4 in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm
Figure Legend Snippet: Association of WAVE2 with ACTN4. A, Immunoprecipitation of WAVE2 from S2‐013 cells cultured on fibronectin. Rabbit IgG isotype control antibody was used as the control. Proteins within immunoprecipitates were examined on Western blots probed with anti‐WAVE2 antibody. B, Proteins in immunoprecipitates were examined with silver staining. Rabbit IgG isotype control antibody was used as the control. A 100‐kDa band is indicated by the arrow. C, The percent coverage for ACTN4 is represented by the identified peptides in the total protein sequence (accession number NP_004915). D, Immunoprecipitation of WAVE2 or ACTN4 from S2‐013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2 and ACTN4. Rabbit IgG isotype control antibody for WAVE2 and mouse IgG isotype control antibody for ACTN4 was used as controls. E, Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 (green) and anti‐ACTN4 (red) antibodies. Arrows, WAVE2 co‐localized with ACTN4 in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm

Techniques Used: Immunoprecipitation, Cell Culture, Western Blot, Silver Staining, Sequencing, Immunofluorescence, Labeling, Staining

Roles of WAVE2 in the motility and invasiveness of PDAC cells. A, siRNA oligonucleotides targeting WAVE2 (siWAVE2) or negative control scrambled siRNAs (Scr) were transiently transfected into S2‐013 and PANC‐1 cells. Western blotting was performed using anti‐WAVE2 antibody. B, MTT assays of S2‐013 and PANC‐1 cells transiently transfected with scrambled control siRNA or WAVE2 siRNA were performed to evaluate cell viability. Data are representative of three independent experiments and are the means ±SD. ABS on Y ‐axis means absorbance at 490 nm and at 630 nm as reference measured with a microplate reader. C and D, Scr or siWAVE2 was transiently transfected into S2‐013 and PANC‐1 cells. Motility (C) and two‐chamber invasion (D) assays were performed. Migrating cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , standard deviation (SD). * P
Figure Legend Snippet: Roles of WAVE2 in the motility and invasiveness of PDAC cells. A, siRNA oligonucleotides targeting WAVE2 (siWAVE2) or negative control scrambled siRNAs (Scr) were transiently transfected into S2‐013 and PANC‐1 cells. Western blotting was performed using anti‐WAVE2 antibody. B, MTT assays of S2‐013 and PANC‐1 cells transiently transfected with scrambled control siRNA or WAVE2 siRNA were performed to evaluate cell viability. Data are representative of three independent experiments and are the means ±SD. ABS on Y ‐axis means absorbance at 490 nm and at 630 nm as reference measured with a microplate reader. C and D, Scr or siWAVE2 was transiently transfected into S2‐013 and PANC‐1 cells. Motility (C) and two‐chamber invasion (D) assays were performed. Migrating cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , standard deviation (SD). * P

Techniques Used: Negative Control, Transfection, Western Blot, MTT Assay, Derivative Assay, Standard Deviation

Subcellular localization of WAVE2 in PDAC cells grown on fibronectin. Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 antibody (green). Actin filaments were labeled with phalloidin (red). Arrows, WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 μm
Figure Legend Snippet: Subcellular localization of WAVE2 in PDAC cells grown on fibronectin. Confocal immunofluorescence microscopic images. S2‐013 cells were cultured on fibronectin and then labeled with anti‐WAVE2 antibody (green). Actin filaments were labeled with phalloidin (red). Arrows, WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 μm

Techniques Used: Immunofluorescence, Cell Culture, Labeling, Staining

Signaling pathway molecules associated with WAVE2 and ACTN4. A, Human phosphoprotein arrays showing the differential phosphorylation of proteins between WAVE2 rescue construct‐transfected S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA, and WAVE2 rescue construct‐transfected S2‐013 cells that had been transfected with WAVE2 siRNA alone. Data are representative of three independent experiments. B, Densitometric analysis of the results of A. The level of phosphorylated p27 in WAVE2 rescue construct‐transfected S2‐013 cells that had been transfected with WAVE2 siRNA alone was compared to that in WAVE2 rescue construct‐transfected S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA. Data are derived from three independent experiments. Columns , mean; bars , SD. * P
Figure Legend Snippet: Signaling pathway molecules associated with WAVE2 and ACTN4. A, Human phosphoprotein arrays showing the differential phosphorylation of proteins between WAVE2 rescue construct‐transfected S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA, and WAVE2 rescue construct‐transfected S2‐013 cells that had been transfected with WAVE2 siRNA alone. Data are representative of three independent experiments. B, Densitometric analysis of the results of A. The level of phosphorylated p27 in WAVE2 rescue construct‐transfected S2‐013 cells that had been transfected with WAVE2 siRNA alone was compared to that in WAVE2 rescue construct‐transfected S2‐013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA. Data are derived from three independent experiments. Columns , mean; bars , SD. * P

Techniques Used: Construct, Transfection, Derivative Assay

Association of WAVE2 with p27 in the regulation of the motility and invasiveness of PDAC cells. A and B, siRNA oligonucleotides targeting p27 (sip27) or scrambled control siRNAs (Scr) were transiently transfected into S2‐013 and PANC‐1 cells. Motility (A) and two‐chamber invasion (B) assays were performed. Migrating cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P
Figure Legend Snippet: Association of WAVE2 with p27 in the regulation of the motility and invasiveness of PDAC cells. A and B, siRNA oligonucleotides targeting p27 (sip27) or scrambled control siRNAs (Scr) were transiently transfected into S2‐013 and PANC‐1 cells. Motility (A) and two‐chamber invasion (B) assays were performed. Migrating cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P

Techniques Used: Transfection, Derivative Assay

Roles of WAVE2 and ACTN4 in forming cell protrusions. A, Confocal immunofluorescence microscopic images showing phalloidin‐labeled peripheral actin structures (red) and DAPI‐labeled nuclei (blue) in scrambled control siRNA‐transfected S2‐013 and PANC‐1 cells, and WAVE2 siRNA‐transfected S2‐013 and PANC‐1 cells grown on fibronectin. Arrows, peripheral actin structures in cell protrusions. Scale bars, 10 µm. B, Quantification of data shown in A; the values represent the number of cells with protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P
Figure Legend Snippet: Roles of WAVE2 and ACTN4 in forming cell protrusions. A, Confocal immunofluorescence microscopic images showing phalloidin‐labeled peripheral actin structures (red) and DAPI‐labeled nuclei (blue) in scrambled control siRNA‐transfected S2‐013 and PANC‐1 cells, and WAVE2 siRNA‐transfected S2‐013 and PANC‐1 cells grown on fibronectin. Arrows, peripheral actin structures in cell protrusions. Scale bars, 10 µm. B, Quantification of data shown in A; the values represent the number of cells with protrusions in which the levels of peripheral actin structures were increased. All cells in four fields per group were scored. Data were derived from three independent experiments. Columns , mean; bars , SD. * P

Techniques Used: Immunofluorescence, Labeling, Transfection, Derivative Assay

61) Product Images from "Bi-directional signaling by membrane-bound KitL induces proliferation and coordinates thymic endothelial cell and thymocyte expansion"

Article Title: Bi-directional signaling by membrane-bound KitL induces proliferation and coordinates thymic endothelial cell and thymocyte expansion

Journal: Nature Communications

doi: 10.1038/s41467-018-07024-0

Membrane-bound Kit ligand acts as a c-Kit receptor to induce cell proliferation. a Flow cytometry plots of pRRL-Venus-transduced (left plot; NIH-Venus) and pRRL–c-Kit-Venus-transduced NIH3T3 cells (right plot; NIH-Kit) stained with anti–c-Kit antibody. Data are representative of three experiments. b Flow cytometry plot of NIH3T3 cells stained with anti-SCF antibody or control IgG, as indicated. Data are representative of three experiments. c Ki67 expression in NIH3T3 co-cultured together with NIH-Venus (left plot) or NIH-Kit (right plot) detected by intracellular flow cytometry. Gating shows Ki67 high cells. d Percentage of Ki67 high cells in cultures from c ( N = 7, two experiments). Bars show mean ± s.e.m. P -value is shown (Student’s t -test). e Mean fluorescence intensity (MFI) of Ki67 signal in samples from d . Bars show mean ± s.e.m., with NIH-Venus mean = 100. P -value is shown (Student’s t -test). f Percentage of Ki67 + cells in cultures from NIH3T3 treated with 200 ng/ml Kit-Fc ( N = 10; two experiments) or IgG-Fc ( N = 10, two experiments), as indicated, for 24 h prior to analysis by intracellular flow cytometry. Bars show mean ± s.e.m., with NIH-Venus mean = 100. P - value is shown (Student’s t -test). g Mean fluorescence intensity (MFI) of Ki67 signal in NIH3T3 cells from f . Bars show mean ± s.e.m., with NIH-Venus mean = 100. P -value is shown (Student’s t -test). h Representative flow cytometric analysis of Ki67 expression in IgG-Fc (left panel) and Kit-Fc (right panel) treated NIH3T3 cells from f . i Percentage of NIH3T3 cells incorporating BrdU after 8 h treatment with 200 ng/ml IgG-Fc or Kit-Fc ( N = 8; two experiments) as indicated. BrdU was added to the culture medium 2 h before analysis. Bars show mean ± s.e.m. P -value is shown (Student’s t -test)
Figure Legend Snippet: Membrane-bound Kit ligand acts as a c-Kit receptor to induce cell proliferation. a Flow cytometry plots of pRRL-Venus-transduced (left plot; NIH-Venus) and pRRL–c-Kit-Venus-transduced NIH3T3 cells (right plot; NIH-Kit) stained with anti–c-Kit antibody. Data are representative of three experiments. b Flow cytometry plot of NIH3T3 cells stained with anti-SCF antibody or control IgG, as indicated. Data are representative of three experiments. c Ki67 expression in NIH3T3 co-cultured together with NIH-Venus (left plot) or NIH-Kit (right plot) detected by intracellular flow cytometry. Gating shows Ki67 high cells. d Percentage of Ki67 high cells in cultures from c ( N = 7, two experiments). Bars show mean ± s.e.m. P -value is shown (Student’s t -test). e Mean fluorescence intensity (MFI) of Ki67 signal in samples from d . Bars show mean ± s.e.m., with NIH-Venus mean = 100. P -value is shown (Student’s t -test). f Percentage of Ki67 + cells in cultures from NIH3T3 treated with 200 ng/ml Kit-Fc ( N = 10; two experiments) or IgG-Fc ( N = 10, two experiments), as indicated, for 24 h prior to analysis by intracellular flow cytometry. Bars show mean ± s.e.m., with NIH-Venus mean = 100. P - value is shown (Student’s t -test). g Mean fluorescence intensity (MFI) of Ki67 signal in NIH3T3 cells from f . Bars show mean ± s.e.m., with NIH-Venus mean = 100. P -value is shown (Student’s t -test). h Representative flow cytometric analysis of Ki67 expression in IgG-Fc (left panel) and Kit-Fc (right panel) treated NIH3T3 cells from f . i Percentage of NIH3T3 cells incorporating BrdU after 8 h treatment with 200 ng/ml IgG-Fc or Kit-Fc ( N = 8; two experiments) as indicated. BrdU was added to the culture medium 2 h before analysis. Bars show mean ± s.e.m. P -value is shown (Student’s t -test)

Techniques Used: Flow Cytometry, Cytometry, Staining, Expressing, Cell Culture, Fluorescence

mKitL signaling induces CREB and Rps6 phosphorylation. a ELISA quantification of CREB S133 phosphorylation in NIH3T3 treated with 200 ng/ml IgG-Fc ( N = 3, two experiments) or Kit-Fc ( N = 3, two experiments) at the indicated time points. Bars show mean ± s.e.m. P- value is shown (Student’s t -test). b ELISA quantification of Rps6 S235/S236 phosphorylation in NIH3T3 treated with IgG-Fc ( N = 3, two experiments) or Kit-Fc ( N = 3, two experiments) at the indicated time points. Values represent the mean of the ratio between Rps6 S235/S236 and Histone H3 at the indicated time points, quantified by ImageJ analysis of Western blots. Bars show mean ± s.e.m. P -value is shown (Student’s t -test). c ELISA quantification of Erk1 T202/Y204 + Erk2 T185/Y187 phosphorylation in NIH3T3 treated with IgG-Fc ( N = 3, two experiments) or Kit-Fc ( N = 3, two experiments) at the indicated time points, quantified by ImageJ analysis of western blots. Bars show mean ± s.e.m. No statistically significant differences were observed. d ELISA quantification of p38α T180/Y182 phosphorylation in NIH3T3 treated with IgG-Fc ( N = 3, two experiments) or Kit-Fc ( N = 3, two experiments) at the indicated time points, quantified by ImageJ analysis of western blots. Bars show mean ± s.e.m. No statistically significant differences were observed. e Immunofluorescence analysis of NIH3T3 cells treated as in i showing CREB phospho-S133 (green) and DNA (blue, visualized with DAPI). Scale bars: 75 μm. Data are representative of three experiments. f Quantification of CREB S133 phosphorylation in NIH3T3 cells pre-treated with goat-IgG (g-IgG) or goat anti-KitL antibody (α-KitL) for 1 h, and treated IgG-Fc ( N = 4, two experiments) or Kit-Fc ( N = 6, two experiments) for 10 min. Values represent the mean of the ratio between of CREB phospho-S133 and Histone H3, quantified by ImageJ analysis of western blots. Bars show mean ± s.e.m. P -value is shown (Student’s t -test)
Figure Legend Snippet: mKitL signaling induces CREB and Rps6 phosphorylation. a ELISA quantification of CREB S133 phosphorylation in NIH3T3 treated with 200 ng/ml IgG-Fc ( N = 3, two experiments) or Kit-Fc ( N = 3, two experiments) at the indicated time points. Bars show mean ± s.e.m. P- value is shown (Student’s t -test). b ELISA quantification of Rps6 S235/S236 phosphorylation in NIH3T3 treated with IgG-Fc ( N = 3, two experiments) or Kit-Fc ( N = 3, two experiments) at the indicated time points. Values represent the mean of the ratio between Rps6 S235/S236 and Histone H3 at the indicated time points, quantified by ImageJ analysis of Western blots. Bars show mean ± s.e.m. P -value is shown (Student’s t -test). c ELISA quantification of Erk1 T202/Y204 + Erk2 T185/Y187 phosphorylation in NIH3T3 treated with IgG-Fc ( N = 3, two experiments) or Kit-Fc ( N = 3, two experiments) at the indicated time points, quantified by ImageJ analysis of western blots. Bars show mean ± s.e.m. No statistically significant differences were observed. d ELISA quantification of p38α T180/Y182 phosphorylation in NIH3T3 treated with IgG-Fc ( N = 3, two experiments) or Kit-Fc ( N = 3, two experiments) at the indicated time points, quantified by ImageJ analysis of western blots. Bars show mean ± s.e.m. No statistically significant differences were observed. e Immunofluorescence analysis of NIH3T3 cells treated as in i showing CREB phospho-S133 (green) and DNA (blue, visualized with DAPI). Scale bars: 75 μm. Data are representative of three experiments. f Quantification of CREB S133 phosphorylation in NIH3T3 cells pre-treated with goat-IgG (g-IgG) or goat anti-KitL antibody (α-KitL) for 1 h, and treated IgG-Fc ( N = 4, two experiments) or Kit-Fc ( N = 6, two experiments) for 10 min. Values represent the mean of the ratio between of CREB phospho-S133 and Histone H3, quantified by ImageJ analysis of western blots. Bars show mean ± s.e.m. P -value is shown (Student’s t -test)

Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence

CAML is required for mKitL signal transduction and proliferation induction. a Expression of mKitL in NIH3T3 cells transduced with lentivirus expressing CAML shRNA (NIH-CAML) and Control shRNA (NIH-Con), as indicated. Protein levels were quantified by western blotting, and mKitL normalized to Histone H3 ( N = 4, three experiments). Bars show mean ± s.e.m. P -value is shown (Student’s t -test). b Flow cytometry analysis of surface mKitL expression in cells from a , using an anti-KitL antibody or corresponding isotype control (Isotype). Data are representative of two experiments. c Quantification of phospho-Akt, -CREB and –Erk in NIH-CAML and NIH-Con cells gown in medium with 2% FBS. Protein levels were quantified by western blotting, and phospho-protein levels normalized to the corresponding total protein ( N = 6, three experiments). Bars show mean ± s.e.m. P -value is shown (Student’s t -test); n.s. not significant. d Quantification of CAML input protein (left), and CAML (centre) and KitL protein immuno-precipitated by control IgG (IgG) and anti-CAML antibody (α-CAML) as indicated. Proteins were quantified by western blotting ( N = 3). CAML input was normalized to β-tubulin. Bars show mean ± s.e.m. P -value is shown (Student’s t -test). e Quantification of CREB phopho-Ser133 in NIH-CAML and NIH-Con cells, incubated in 0.5% FBS medium for 24 h and stimulated with 200 ng/ml IgG-Fc or Kit-Fc for 8 h, as indicated ( N = 6 in two experiments, except IgG-Fc-treated NIH-Con ( N = 7, two experiments)). P -values are shown (Student’s t -test). f MFI of Ki67 expression in NIH-CAML cells grown in 2% FBS medium and stimulated with 200 ng/ml IgG-Fc or Kit-Fc for 8 h, measured by intracellular flow cytometry ( N = 5, two experiments). Bars show mean ± s.e.m. P -value is shown (Student’s t -test); n.s. not significant. g Proliferation curve of NIH-Con (left) and NIH-CAML cells (right) grown in 2% FBS medium with 200 ng/ml IgG-Fc or Kit-Fc. Medium was changed every 48 h. Cells were counted at 0, 3, and 7 days ( N = 6 for each time point, two experiments). Bars show mean ± s.e.m. P -values are shown (Student’s t -test); n.s. not significant
Figure Legend Snippet: CAML is required for mKitL signal transduction and proliferation induction. a Expression of mKitL in NIH3T3 cells transduced with lentivirus expressing CAML shRNA (NIH-CAML) and Control shRNA (NIH-Con), as indicated. Protein levels were quantified by western blotting, and mKitL normalized to Histone H3 ( N = 4, three experiments). Bars show mean ± s.e.m. P -value is shown (Student’s t -test). b Flow cytometry analysis of surface mKitL expression in cells from a , using an anti-KitL antibody or corresponding isotype control (Isotype). Data are representative of two experiments. c Quantification of phospho-Akt, -CREB and –Erk in NIH-CAML and NIH-Con cells gown in medium with 2% FBS. Protein levels were quantified by western blotting, and phospho-protein levels normalized to the corresponding total protein ( N = 6, three experiments). Bars show mean ± s.e.m. P -value is shown (Student’s t -test); n.s. not significant. d Quantification of CAML input protein (left), and CAML (centre) and KitL protein immuno-precipitated by control IgG (IgG) and anti-CAML antibody (α-CAML) as indicated. Proteins were quantified by western blotting ( N = 3). CAML input was normalized to β-tubulin. Bars show mean ± s.e.m. P -value is shown (Student’s t -test). e Quantification of CREB phopho-Ser133 in NIH-CAML and NIH-Con cells, incubated in 0.5% FBS medium for 24 h and stimulated with 200 ng/ml IgG-Fc or Kit-Fc for 8 h, as indicated ( N = 6 in two experiments, except IgG-Fc-treated NIH-Con ( N = 7, two experiments)). P -values are shown (Student’s t -test). f MFI of Ki67 expression in NIH-CAML cells grown in 2% FBS medium and stimulated with 200 ng/ml IgG-Fc or Kit-Fc for 8 h, measured by intracellular flow cytometry ( N = 5, two experiments). Bars show mean ± s.e.m. P -value is shown (Student’s t -test); n.s. not significant. g Proliferation curve of NIH-Con (left) and NIH-CAML cells (right) grown in 2% FBS medium with 200 ng/ml IgG-Fc or Kit-Fc. Medium was changed every 48 h. Cells were counted at 0, 3, and 7 days ( N = 6 for each time point, two experiments). Bars show mean ± s.e.m. P -values are shown (Student’s t -test); n.s. not significant

Techniques Used: Transduction, Expressing, shRNA, Western Blot, Flow Cytometry, Cytometry, Incubation

Activation of the Akt/mTOR pathway by reverse mKitL signaling. a Intracellular flow cytometry analysis of Akt S473, mTOR S2448, and Rps6 S235/S236 phosphorylation in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells as indicated ( N = 7, two experiments). Values are normalized MFI (NIH-Venus mean = 100). Bars show mean ± s.e.m. P -value is shown (Student’s t -test). b Representative intracellular flow cytometry plots measuring Rps6 S235/S236 phosphorylation in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells, as indicated. Gating shows cells with high phospho-Rps6 levels. Data are representative of seven experiments. c Percentage of phospho-Rps6 positive cells in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells in b ( N = 7, two experiments). Bars show mean percentage of S235/S236 positive cells ± s.e.m normalized to the average value for NIH-Venus ( = 100). P -value is shown (Student’s t -test). d CREB phopho-Ser133 MFI (left) and percentage CREB phopho-Ser133 positive cells (right) in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells, as indicated ( N = 8, two experiments). Bars show mean ± s.e.m. P- value is shown (Student’s t -test). e Representative intracellular flow cytometry plots measuring Ki67 in NIH3T3 cells co-cultured with NIH-Kit cells and treated for 9 h with DMSO, Akt inhibitor (Akti1/2) or CREB inhibitor (666–15), as indicated. Gating shows the Ki67 high population. f Percentage of Ki67 high cells (left) and Ki67 MFI (right) in NIH3T3 cells co-cultured with NIH-Kit cells and treated for 3, 6, or 9 h (as indicated) with DMSO ( n = 13, two experiments), Akt inhibitor (Akti1/2), or CREB inhibitor (666–15), as indicated ( N = 6, two experiments, except the Akti1/2 3 h time point where N = 9, two experiments). Bars show mean ± s.e.m. P- value is shown (Student’s t -test)
Figure Legend Snippet: Activation of the Akt/mTOR pathway by reverse mKitL signaling. a Intracellular flow cytometry analysis of Akt S473, mTOR S2448, and Rps6 S235/S236 phosphorylation in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells as indicated ( N = 7, two experiments). Values are normalized MFI (NIH-Venus mean = 100). Bars show mean ± s.e.m. P -value is shown (Student’s t -test). b Representative intracellular flow cytometry plots measuring Rps6 S235/S236 phosphorylation in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells, as indicated. Gating shows cells with high phospho-Rps6 levels. Data are representative of seven experiments. c Percentage of phospho-Rps6 positive cells in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells in b ( N = 7, two experiments). Bars show mean percentage of S235/S236 positive cells ± s.e.m normalized to the average value for NIH-Venus ( = 100). P -value is shown (Student’s t -test). d CREB phopho-Ser133 MFI (left) and percentage CREB phopho-Ser133 positive cells (right) in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells, as indicated ( N = 8, two experiments). Bars show mean ± s.e.m. P- value is shown (Student’s t -test). e Representative intracellular flow cytometry plots measuring Ki67 in NIH3T3 cells co-cultured with NIH-Kit cells and treated for 9 h with DMSO, Akt inhibitor (Akti1/2) or CREB inhibitor (666–15), as indicated. Gating shows the Ki67 high population. f Percentage of Ki67 high cells (left) and Ki67 MFI (right) in NIH3T3 cells co-cultured with NIH-Kit cells and treated for 3, 6, or 9 h (as indicated) with DMSO ( n = 13, two experiments), Akt inhibitor (Akti1/2), or CREB inhibitor (666–15), as indicated ( N = 6, two experiments, except the Akti1/2 3 h time point where N = 9, two experiments). Bars show mean ± s.e.m. P- value is shown (Student’s t -test)

Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Cell Culture

62) Product Images from "Vascular endothelial growth factor expression and inhibition in uveal melanoma cell lines"

Article Title: Vascular endothelial growth factor expression and inhibition in uveal melanoma cell lines

Journal: ecancermedicalscience

doi: 10.3332/ecancer.2013.336

Figure 3: Phosphorylated VEGF-R2 expression in three UM cell lines following bevacizumab treatment as determined by western blot. Expression of phosphorylated-VEGF-R2 before and after bevacizumab treatment is expressed as a percentage of the control. The OD of the western blots was quantified using ImageJ software (NIH). * P
Figure Legend Snippet: Figure 3: Phosphorylated VEGF-R2 expression in three UM cell lines following bevacizumab treatment as determined by western blot. Expression of phosphorylated-VEGF-R2 before and after bevacizumab treatment is expressed as a percentage of the control. The OD of the western blots was quantified using ImageJ software (NIH). * P

Techniques Used: Expressing, Western Blot, Software

Figure 2: MMP-9 and CCL3 protein secretion by UM cell lines: secretion of CCL3 and MMP-9 by three UM cell lines following treatment with 100 μg/mL of bevacizumab. Results are shown as percentage of control. *P
Figure Legend Snippet: Figure 2: MMP-9 and CCL3 protein secretion by UM cell lines: secretion of CCL3 and MMP-9 by three UM cell lines following treatment with 100 μg/mL of bevacizumab. Results are shown as percentage of control. *P

Techniques Used:

Figure 1: VEGF-A secretion by three UM cell lines before and after bevacizumab treatment: secretion of VEGF-A by the three UM cell lines (92.1, OCM-1, and UW-1) in conditioned media before and after the administration of 100 μg/mL of bevacizumab, as determined using a sandwich ELISA. Normal media with and without bevacizumab were used as controls. *P
Figure Legend Snippet: Figure 1: VEGF-A secretion by three UM cell lines before and after bevacizumab treatment: secretion of VEGF-A by the three UM cell lines (92.1, OCM-1, and UW-1) in conditioned media before and after the administration of 100 μg/mL of bevacizumab, as determined using a sandwich ELISA. Normal media with and without bevacizumab were used as controls. *P

Techniques Used: Sandwich ELISA

63) Product Images from "Peucedanum japonicum Thunb. ethanol extract suppresses RANKL-mediated osteoclastogenesis"

Article Title: Peucedanum japonicum Thunb. ethanol extract suppresses RANKL-mediated osteoclastogenesis

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2017.4480

Effects of PEE on RANKL-induced intracellular signaling. Bone marrow-derived macrophages were treated with or without PEE (25 µg/ml) in the presence of RANKL and M-CSF for the indicated durations (0, 1, 2, 3 and 4 days). Whole cell extracts were subjected to western blot analysis. (A) Phospho-CREB, (B) NFATc1 and (C) c-fos were detected with the specific antibodies. β-actin was used as a loading control. PEE, Peucedanum japonicum Thunb. ethanol extract; RANKL, receptor activator of nuclear factor κB ligand; M-CSF, macrophage colony-stimulating factor; CREB, cAMP response element-binding protein; NFATc1, nuclear factor of activated T cells, cytoplasmic 1.
Figure Legend Snippet: Effects of PEE on RANKL-induced intracellular signaling. Bone marrow-derived macrophages were treated with or without PEE (25 µg/ml) in the presence of RANKL and M-CSF for the indicated durations (0, 1, 2, 3 and 4 days). Whole cell extracts were subjected to western blot analysis. (A) Phospho-CREB, (B) NFATc1 and (C) c-fos were detected with the specific antibodies. β-actin was used as a loading control. PEE, Peucedanum japonicum Thunb. ethanol extract; RANKL, receptor activator of nuclear factor κB ligand; M-CSF, macrophage colony-stimulating factor; CREB, cAMP response element-binding protein; NFATc1, nuclear factor of activated T cells, cytoplasmic 1.

Techniques Used: Derivative Assay, Western Blot, Binding Assay

64) Product Images from "Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation"

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004741

ASYN biochemical state. A. Native Gels. Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. B. STED microscopy. Selected mutants were imaged in order to characterize the fine structure of the inclusions. C. Thioflavin S staining. H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. D-E . Triton X-100 solubility assay and quantification. H4 cells show that all mutants form detergent insoluble species. Student's t test (*p
Figure Legend Snippet: ASYN biochemical state. A. Native Gels. Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. B. STED microscopy. Selected mutants were imaged in order to characterize the fine structure of the inclusions. C. Thioflavin S staining. H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. D-E . Triton X-100 solubility assay and quantification. H4 cells show that all mutants form detergent insoluble species. Student's t test (*p

Techniques Used: Clear Native PAGE, Transfection, Bimolecular Fluorescence Complementation Assay, Construct, Microscopy, Staining, Expressing, Incubation, Solubility

65) Product Images from "Characterization of adipose-derived stem cells from subcutaneous and visceral adipose tissues and their function in breast cancer cells"

Article Title: Characterization of adipose-derived stem cells from subcutaneous and visceral adipose tissues and their function in breast cancer cells

Journal: Oncotarget

doi:

ASCs interact with cancer cells directly and indirectly A. The cytokine/chemokine array assay. The factors were measured in the supernatants of visceral ASCs (ASCvis) and subcutaneous ASCs (ASCsub) cultured for 3 days by using a human cytokine antibody array. The six most prominent chemokines/chemokines are demonstrated. The results, relative to the positive control provided by the array, are based on three independent experiments with ASCs obtained from three different donors and presented as mean ± SEM. B. Evaluation of membrane protrusion of ASCs toward MCF-7, MDA-MB-231 and MCF-10A cells. The results are based on three independent experiments with ASCs from three different donors and shown as mean ± SEM. C. Representatives of ASCs homing to breast cancer cell lines. Red arrows indicate membrane protrusions of ASCs toward breast cancer cells. Normal mammary epithelial MCF-10A cells served as negative control. Scale bar: 250 μm. D. Immunofluorescence staining of the migration front between ASCs and MCF-7 or MDA-MB-231 cells. Both cell types were stained for p-FAK, phalloidin, acetylated α-tubulin and DNA. White arrows depict the connections stabilized by acetylated α-tubulin between ASCs and breast cancer cells. Scale bar: 25 μm.
Figure Legend Snippet: ASCs interact with cancer cells directly and indirectly A. The cytokine/chemokine array assay. The factors were measured in the supernatants of visceral ASCs (ASCvis) and subcutaneous ASCs (ASCsub) cultured for 3 days by using a human cytokine antibody array. The six most prominent chemokines/chemokines are demonstrated. The results, relative to the positive control provided by the array, are based on three independent experiments with ASCs obtained from three different donors and presented as mean ± SEM. B. Evaluation of membrane protrusion of ASCs toward MCF-7, MDA-MB-231 and MCF-10A cells. The results are based on three independent experiments with ASCs from three different donors and shown as mean ± SEM. C. Representatives of ASCs homing to breast cancer cell lines. Red arrows indicate membrane protrusions of ASCs toward breast cancer cells. Normal mammary epithelial MCF-10A cells served as negative control. Scale bar: 250 μm. D. Immunofluorescence staining of the migration front between ASCs and MCF-7 or MDA-MB-231 cells. Both cell types were stained for p-FAK, phalloidin, acetylated α-tubulin and DNA. White arrows depict the connections stabilized by acetylated α-tubulin between ASCs and breast cancer cells. Scale bar: 25 μm.

Techniques Used: Cell Culture, Ab Array, Positive Control, Multiple Displacement Amplification, Negative Control, Immunofluorescence, Staining, Migration

66) Product Images from "Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer"

Article Title: Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.56

Phenothiazines disrupt autophagy. ( a ) H82, H69, U-1810 and A549 cells were treated with TFP at the indicated concentrations for 6, 24 or 48 h; WCL was used for immunoblotting with antibodies specific for p62, LC3B (which detect both LC3-I and LC3-II) and GAPDH (loading control). ( b ) H592, U-1285, H125, H157 cells were treated with TFP at the indicated concentrations for 72 h; WCL was used for immunoblotting with antibodies as in ( a ). ( c ) H82 and U-1810 cells were treated with 10 μ M of the indicated phenothiazines for 24 h; WCL was used for immunoblotting with antibodies specific for LC3B and GAPDH (loading control). ( d ) H82 and U-1810 cells were treated with TFP at the indicated concentrations in the absence or presence of E-64d (30 μ M) for 24 h; WCL was used for immunoblotting as in ( a ). ( e ) H82 and U-1810 cells were treated with TFP for 24 h followed by 24 h with either TFP, cycloheximide (CHX, 100 μ g/ml) or TFP+CHX. WCL was used for immunoblotting as in ( a ). Data shown are representative of three independent experiments
Figure Legend Snippet: Phenothiazines disrupt autophagy. ( a ) H82, H69, U-1810 and A549 cells were treated with TFP at the indicated concentrations for 6, 24 or 48 h; WCL was used for immunoblotting with antibodies specific for p62, LC3B (which detect both LC3-I and LC3-II) and GAPDH (loading control). ( b ) H592, U-1285, H125, H157 cells were treated with TFP at the indicated concentrations for 72 h; WCL was used for immunoblotting with antibodies as in ( a ). ( c ) H82 and U-1810 cells were treated with 10 μ M of the indicated phenothiazines for 24 h; WCL was used for immunoblotting with antibodies specific for LC3B and GAPDH (loading control). ( d ) H82 and U-1810 cells were treated with TFP at the indicated concentrations in the absence or presence of E-64d (30 μ M) for 24 h; WCL was used for immunoblotting as in ( a ). ( e ) H82 and U-1810 cells were treated with TFP for 24 h followed by 24 h with either TFP, cycloheximide (CHX, 100 μ g/ml) or TFP+CHX. WCL was used for immunoblotting as in ( a ). Data shown are representative of three independent experiments

Techniques Used:

TFP induces cell death distinct from classical apoptosis. ( a ) SCLC (H82, H69) and NSCLC (U-1810, A549) cells were treated with TFP at the indicated concentrations for 6, 24 or 48 h; WCL was used for immunoblotting with an antibody recognizing full-length and cleaved PARP (113 and 89 kDa respectively). GAPDH antibody was used to confirm equal loading. ( b ) H592, U-1285, H125, H157 cells were treated with TFP at the indicated concentrations for 72 h; immunoblotting was performed as in ( a ). ( c ) H82 and U-1810 cells were pre-treated (1 h) with z-VAD-fmk (20 μ M) and thereafter exposed to TFP at the indicated concentrations for 72 h; cell viability was measured by MTT. For ( a and b ), data shown are representative of three independent experiments. For ( c ), data depict mean±S.D. compiled from three independent experiments. NS, nonsignificant
Figure Legend Snippet: TFP induces cell death distinct from classical apoptosis. ( a ) SCLC (H82, H69) and NSCLC (U-1810, A549) cells were treated with TFP at the indicated concentrations for 6, 24 or 48 h; WCL was used for immunoblotting with an antibody recognizing full-length and cleaved PARP (113 and 89 kDa respectively). GAPDH antibody was used to confirm equal loading. ( b ) H592, U-1285, H125, H157 cells were treated with TFP at the indicated concentrations for 72 h; immunoblotting was performed as in ( a ). ( c ) H82 and U-1810 cells were pre-treated (1 h) with z-VAD-fmk (20 μ M) and thereafter exposed to TFP at the indicated concentrations for 72 h; cell viability was measured by MTT. For ( a and b ), data shown are representative of three independent experiments. For ( c ), data depict mean±S.D. compiled from three independent experiments. NS, nonsignificant

Techniques Used: MTT Assay

67) Product Images from "AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia"

Article Title: AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia

Journal: Nature Communications

doi: 10.1038/ncomms8388

Ghrelin preserves cardiac function and reduces infarct size after myocardial infarction (MI). ( a ) Real-time PCR quantification of ghrelin mRNA in total ventricular RNA extracted 2 or 90 days after AAV9 injection and MI induction. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over endogenous ( n =5). ( b , c ) Intracardiac ( b ) and plasmatic ( c ) levels of acyl and des-acyl ghrelin evaluated by EIA 90 days after intracardiac vector injection ( n =4). ( d – g ) Echocardiographic analysis in AAV9-injected and control mice at 2, 7, 30, 60 and 90 days after MI. Left ventricular ejection fraction (LVEF; d ), left ventricular fractional shortening (LVFS; e ), anterior wall thickening (AWTK; f ) and diastolic LV internal diameter (LVID d; g ) of infarcted hearts treated either with AAV9-ghrelin or AAV9-control were measured ( n =12 per group). (h ) Representative M-mode echocardiographic images 90 days after AAV9-control or AAV9-ghrelin injection and MI induction. ( i ) Representative images of whole transverse sections after Azan-trichromic staining of hearts transduced with AAV9-control or AAV9-ghrelin. Fibrotic areas are stained in blue. ( j ) Quantification of infarct size expressed as percentage of LV ( n =12 per group). ( k ) Cardiac expression levels of the indicated genes in AAV9-control and AAV9-ghrelin-treated hearts, 90 days after MI. Values are normalized for GAPDH and expressed as fold over untreated ( n =6). All values are mean±s.e.m. Pairwise comparison was performed with the Student's t -test ( b , c , j ); one-way analysis of variance (ANOVA) and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( a , k ); two-way ANOVA was used in d – g . * P
Figure Legend Snippet: Ghrelin preserves cardiac function and reduces infarct size after myocardial infarction (MI). ( a ) Real-time PCR quantification of ghrelin mRNA in total ventricular RNA extracted 2 or 90 days after AAV9 injection and MI induction. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over endogenous ( n =5). ( b , c ) Intracardiac ( b ) and plasmatic ( c ) levels of acyl and des-acyl ghrelin evaluated by EIA 90 days after intracardiac vector injection ( n =4). ( d – g ) Echocardiographic analysis in AAV9-injected and control mice at 2, 7, 30, 60 and 90 days after MI. Left ventricular ejection fraction (LVEF; d ), left ventricular fractional shortening (LVFS; e ), anterior wall thickening (AWTK; f ) and diastolic LV internal diameter (LVID d; g ) of infarcted hearts treated either with AAV9-ghrelin or AAV9-control were measured ( n =12 per group). (h ) Representative M-mode echocardiographic images 90 days after AAV9-control or AAV9-ghrelin injection and MI induction. ( i ) Representative images of whole transverse sections after Azan-trichromic staining of hearts transduced with AAV9-control or AAV9-ghrelin. Fibrotic areas are stained in blue. ( j ) Quantification of infarct size expressed as percentage of LV ( n =12 per group). ( k ) Cardiac expression levels of the indicated genes in AAV9-control and AAV9-ghrelin-treated hearts, 90 days after MI. Values are normalized for GAPDH and expressed as fold over untreated ( n =6). All values are mean±s.e.m. Pairwise comparison was performed with the Student's t -test ( b , c , j ); one-way analysis of variance (ANOVA) and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( a , k ); two-way ANOVA was used in d – g . * P

Techniques Used: Real-time Polymerase Chain Reaction, Injection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Mouse Assay, Staining, Transduction, Expressing

Ghrelin improves muscle functional recovery after femoral artery resection. ( a ) Real-time PCR quantification of ghrelin mRNA in tibialis anterior muscles transduced with 1 × 10 11 vg of AAV. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over endogenous ( n =5). ( b ) Haematoxylin-eosin (HE) staining of tibialis anterior muscles at day 21 after femoral artery resection. Scale bar, 50 μm. ( c ) Quantification of ischaemic lesion in muscles injected with AAV9-ghrelin. Values are expressed as percentage per field ( n =7 per group, four fields counted for each animal). ( d ) Quantification of inflammatory cell infiltration. Values are expressed as percentage of cells per field ( n =7 per group, four fields per animal). ( e ) Expression levels of inflammatory cytokines, analysed by real-time PCR, at day 7 after transduction and surgery. Values are normalized for GAPDH and expressed as fold over untreated ( n =5). ( f , g ) Analysis of apoptosis by TUNEL. Representative images ( f ) and quantification ( g ) of TUNEL-positive nuclei in the muscle areas adjacent to the lesions ( n =5). Red: apoptotic nuclei; blue: 4′-6-diamidino-2-phenylindole (DAPI); green: lectin-stained muscle fibres. Scale bar, 100 μm. ( h ) Ratio between BAX and BCL-2 mRNAs in AAV9-injected muscles, 7 days after femoral artery resection. Values are normalized for GAPDH and expressed as fold over untreated ( n =5). ( i ) Number of fibres with central nuclei per field as a marker of muscle regeneration at 21 days after treatment ( n =7 per group, four fields per animal). ( j ) Fibre size analysis after ischaemia and AAV9 injection. The histograms show the distribution of the fibre cross-sectional areas (μm 2 ); a normal distribution curve is superimposed. Analysis of 20 cross-sections from six animals per group was performed. P
Figure Legend Snippet: Ghrelin improves muscle functional recovery after femoral artery resection. ( a ) Real-time PCR quantification of ghrelin mRNA in tibialis anterior muscles transduced with 1 × 10 11 vg of AAV. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over endogenous ( n =5). ( b ) Haematoxylin-eosin (HE) staining of tibialis anterior muscles at day 21 after femoral artery resection. Scale bar, 50 μm. ( c ) Quantification of ischaemic lesion in muscles injected with AAV9-ghrelin. Values are expressed as percentage per field ( n =7 per group, four fields counted for each animal). ( d ) Quantification of inflammatory cell infiltration. Values are expressed as percentage of cells per field ( n =7 per group, four fields per animal). ( e ) Expression levels of inflammatory cytokines, analysed by real-time PCR, at day 7 after transduction and surgery. Values are normalized for GAPDH and expressed as fold over untreated ( n =5). ( f , g ) Analysis of apoptosis by TUNEL. Representative images ( f ) and quantification ( g ) of TUNEL-positive nuclei in the muscle areas adjacent to the lesions ( n =5). Red: apoptotic nuclei; blue: 4′-6-diamidino-2-phenylindole (DAPI); green: lectin-stained muscle fibres. Scale bar, 100 μm. ( h ) Ratio between BAX and BCL-2 mRNAs in AAV9-injected muscles, 7 days after femoral artery resection. Values are normalized for GAPDH and expressed as fold over untreated ( n =5). ( i ) Number of fibres with central nuclei per field as a marker of muscle regeneration at 21 days after treatment ( n =7 per group, four fields per animal). ( j ) Fibre size analysis after ischaemia and AAV9 injection. The histograms show the distribution of the fibre cross-sectional areas (μm 2 ); a normal distribution curve is superimposed. Analysis of 20 cross-sections from six animals per group was performed. P

Techniques Used: Functional Assay, Real-time Polymerase Chain Reaction, Transduction, Staining, Injection, Expressing, TUNEL Assay, Marker

Reduced apoptosis is paralleled by increased autophagy in ischaemic cardiomyocytes overexpressing ghrelin. ( a , b ) LC3 lipidation (conversion from LC3-I to LC3-II) in the left ventricles of transduced hearts harvested 2 days after MI. Representative western blot ( a ) and densitometric analysis ( b ; n =6). ( c ) Quantification of MAP1LC3A , BECLIN1 and ATG12 mRNA levels in the left ventricles of transduced hearts at 2 days after MI. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over untreated ( n =8). ( d , e ) AMPK phosphorylation in the left ventricles of transduced hearts harvested 2 days after MI. Representative western blot ( d ) and densitometric analysis ( e ) of total AMPK and phosphor (pAMPK; n =6). ( f ) Heart sections of mice injected with AAV9-mRFP-EGFP-LC3 and AAV9-control or AAV9-ghrelin and submitted or not to MI. Autophagosomes appear yellow in the merged image, whereas autolysosomes appear red. Scale bar, 100 μm. ( g ) Quantification of EGFP and mRFP LC3-positive dots per field, using the ImageJ software ( n =6). ( h ) Primary rat neonatal cardiomyocytes transfected with the mRFP-EGFP tandem fluorescent-tagged LC3 plasmid (ptfLC3) and, 48 h later, treated for 4 h with acyl ghrelin, des-acyl ghrelin (both 1 μM) or vehicle in complete or starving medium. Scale bar, 10 μm. ( i ) Quantification of EGFP and mRFP LC3-positive dots per cell, using the ImageJ software ( n =30 cells per group). ( l ) Representative immunofluorescence staining for LC3B (green) in HL-1 cells treated for 4 h with acyl ghrelin, des-acyl ghrelin (both 1 μM) or vehicle in the presence or absence of chloroquine (10 μM) or wortmannin (0.5 μM). Scale bar, 50 μm. ( m ) Quantification of the cells with a high number ( > 30) of LC3B-positive vesicles, expressed as % of the total number of cells ( n =30 cells per group). All values are mean±s.e.m. One-way analysis of variance (ANOVA) and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( b , c , e , g , i , m ). * P
Figure Legend Snippet: Reduced apoptosis is paralleled by increased autophagy in ischaemic cardiomyocytes overexpressing ghrelin. ( a , b ) LC3 lipidation (conversion from LC3-I to LC3-II) in the left ventricles of transduced hearts harvested 2 days after MI. Representative western blot ( a ) and densitometric analysis ( b ; n =6). ( c ) Quantification of MAP1LC3A , BECLIN1 and ATG12 mRNA levels in the left ventricles of transduced hearts at 2 days after MI. Values are normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold over untreated ( n =8). ( d , e ) AMPK phosphorylation in the left ventricles of transduced hearts harvested 2 days after MI. Representative western blot ( d ) and densitometric analysis ( e ) of total AMPK and phosphor (pAMPK; n =6). ( f ) Heart sections of mice injected with AAV9-mRFP-EGFP-LC3 and AAV9-control or AAV9-ghrelin and submitted or not to MI. Autophagosomes appear yellow in the merged image, whereas autolysosomes appear red. Scale bar, 100 μm. ( g ) Quantification of EGFP and mRFP LC3-positive dots per field, using the ImageJ software ( n =6). ( h ) Primary rat neonatal cardiomyocytes transfected with the mRFP-EGFP tandem fluorescent-tagged LC3 plasmid (ptfLC3) and, 48 h later, treated for 4 h with acyl ghrelin, des-acyl ghrelin (both 1 μM) or vehicle in complete or starving medium. Scale bar, 10 μm. ( i ) Quantification of EGFP and mRFP LC3-positive dots per cell, using the ImageJ software ( n =30 cells per group). ( l ) Representative immunofluorescence staining for LC3B (green) in HL-1 cells treated for 4 h with acyl ghrelin, des-acyl ghrelin (both 1 μM) or vehicle in the presence or absence of chloroquine (10 μM) or wortmannin (0.5 μM). Scale bar, 50 μm. ( m ) Quantification of the cells with a high number ( > 30) of LC3B-positive vesicles, expressed as % of the total number of cells ( n =30 cells per group). All values are mean±s.e.m. One-way analysis of variance (ANOVA) and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( b , c , e , g , i , m ). * P

Techniques Used: Western Blot, Mouse Assay, Injection, Software, Transfection, Plasmid Preparation, Immunofluorescence, Staining

AAV9-ghrelin exerts anti-apoptotic effect on cardiomyocytes exposed to toxic or ischaemic damage in vitro and in vivo . ( a ) TUNEL staining of the infarct border zone in hearts injected with AAV9-control or AAV9-ghrelin 2 days after MI. Nuclei are stained blue with 4′-6-diamidino-2-phenylindole (DAPI) and cardiomyocytes green by an antibody against α-actinin. Red nuclei indicate apoptotic cells. Scale bar, 100 μm. ( b ) Quantification of TUNEL-positive nuclei (% of total) in the peri-infarctual region of AAV9-control and AAV9-ghrelin-treated mice ( n =8 per group). ( c ) Rat neonatal cardiomyocytes, transduced with AAV9-control or AAV9-ghrelin (MOI=5 × 10 4 vg per cell, transduction efficiency > 40%) were either left untreated or treated with (−)-Isoproterenol hydrochloride 10 μM (ISO) or doxorubicin hydrochloride 0.5 μM (DOXO); after 24 or 48 h, respectively, cells were fixed and stained with TUNEL assay. Nuclei are stained blue with DAPI and cardiomyocytes green by an antibody against α-actinin. Red nuclei indicate apoptotic cells. Scale bar, 100 μm. ( d ) Quantification of cardiomyocytes TUNEL-positive nuclei (% of total) after transduction with AAV9-control or AAV9-ghrelin and (−)-Isoproterenol or doxorubicin treatment. Quantification was performed using the ImaJ software ( n =4). ( e , f ) Caspase 3/7 activation analysis in rat neonatal cardiomyocytes ( e ) and HL-1 cells ( f ) transduced with AAV9-ghrelin or AAV9-control and treated with doxorubicin 0.5 μM and 1 μM, respectively, for 20 h ( n =12). All values are mean±s.e.m. Pairwise comparison was performed with the Student's t -test ( b , d ); one-way analysis of variance and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( e , f ). ** P
Figure Legend Snippet: AAV9-ghrelin exerts anti-apoptotic effect on cardiomyocytes exposed to toxic or ischaemic damage in vitro and in vivo . ( a ) TUNEL staining of the infarct border zone in hearts injected with AAV9-control or AAV9-ghrelin 2 days after MI. Nuclei are stained blue with 4′-6-diamidino-2-phenylindole (DAPI) and cardiomyocytes green by an antibody against α-actinin. Red nuclei indicate apoptotic cells. Scale bar, 100 μm. ( b ) Quantification of TUNEL-positive nuclei (% of total) in the peri-infarctual region of AAV9-control and AAV9-ghrelin-treated mice ( n =8 per group). ( c ) Rat neonatal cardiomyocytes, transduced with AAV9-control or AAV9-ghrelin (MOI=5 × 10 4 vg per cell, transduction efficiency > 40%) were either left untreated or treated with (−)-Isoproterenol hydrochloride 10 μM (ISO) or doxorubicin hydrochloride 0.5 μM (DOXO); after 24 or 48 h, respectively, cells were fixed and stained with TUNEL assay. Nuclei are stained blue with DAPI and cardiomyocytes green by an antibody against α-actinin. Red nuclei indicate apoptotic cells. Scale bar, 100 μm. ( d ) Quantification of cardiomyocytes TUNEL-positive nuclei (% of total) after transduction with AAV9-control or AAV9-ghrelin and (−)-Isoproterenol or doxorubicin treatment. Quantification was performed using the ImaJ software ( n =4). ( e , f ) Caspase 3/7 activation analysis in rat neonatal cardiomyocytes ( e ) and HL-1 cells ( f ) transduced with AAV9-ghrelin or AAV9-control and treated with doxorubicin 0.5 μM and 1 μM, respectively, for 20 h ( n =12). All values are mean±s.e.m. Pairwise comparison was performed with the Student's t -test ( b , d ); one-way analysis of variance and Bonferroni/Dunn's post hoc test were used to compare multiple groups ( e , f ). ** P

Techniques Used: In Vitro, In Vivo, TUNEL Assay, Staining, Injection, Mouse Assay, Transduction, Software, Activation Assay

68) Product Images from "STING-dependent sensing of self-DNA drives silica-induced lung inflammation"

Article Title: STING-dependent sensing of self-DNA drives silica-induced lung inflammation

Journal: Nature Communications

doi: 10.1038/s41467-018-07425-1

Silica induces macrophage mitochondrial stress and necrosis, dsDNA leakage, and STING pathway activation. a , b WT and STING −/− bone marrow-derived macrophages were unstimulated or stimulated with silica (250 µg/mL) for 18 h. a Brightfield confocal microscopy showing intracellular silica microparticles and MitoTracker (green) labeled mitochondria co-localizing with superoxide production detected by MitoSOX staining (red). Images are representative of five slides from n = 3 cell cultures. Bars, 5 µm. b Colocalization analysis of MitoTracker versus MitoSOX staining from the slides shown in a . Overlap coefficient and Pearson correlation coefficient were determined using ImageJ. c Flow cytometry Annexin V/PI staining of pre-gated singlets (SSC-A/SSC-H) and CD11b + F4/80 + CD11c – cells. d Concentration of extracellular dsDNA in the culture supernatant. e Immunoblots of caspase 3, cleaved caspase 3, gasdermin D, MLKL, and phospho-MLKL in WT and STING −/− macrophages, with β-actin as a reference. f Confocal images of DNA Draq5 (cyan) and β-actin (red) staining in WT macrophages unstimulated or stimulated with silica (250 µg/mL) for 18 h. g Immunoblots of STING/IRF3 axis, including phospho-STING, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3, and β-actin as a reference, in WT macrophages stimulated as in a – b or transfected with c-di-AMP (6 µg/mL; cDN) for 18 h as a positive control. h Confocal images of DNA dye Draq5 (cyan) and STING-specific antibody (red) in WT BMDMs stimulated as in f . Bars, 20 µm. *** p
Figure Legend Snippet: Silica induces macrophage mitochondrial stress and necrosis, dsDNA leakage, and STING pathway activation. a , b WT and STING −/− bone marrow-derived macrophages were unstimulated or stimulated with silica (250 µg/mL) for 18 h. a Brightfield confocal microscopy showing intracellular silica microparticles and MitoTracker (green) labeled mitochondria co-localizing with superoxide production detected by MitoSOX staining (red). Images are representative of five slides from n = 3 cell cultures. Bars, 5 µm. b Colocalization analysis of MitoTracker versus MitoSOX staining from the slides shown in a . Overlap coefficient and Pearson correlation coefficient were determined using ImageJ. c Flow cytometry Annexin V/PI staining of pre-gated singlets (SSC-A/SSC-H) and CD11b + F4/80 + CD11c – cells. d Concentration of extracellular dsDNA in the culture supernatant. e Immunoblots of caspase 3, cleaved caspase 3, gasdermin D, MLKL, and phospho-MLKL in WT and STING −/− macrophages, with β-actin as a reference. f Confocal images of DNA Draq5 (cyan) and β-actin (red) staining in WT macrophages unstimulated or stimulated with silica (250 µg/mL) for 18 h. g Immunoblots of STING/IRF3 axis, including phospho-STING, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3, and β-actin as a reference, in WT macrophages stimulated as in a – b or transfected with c-di-AMP (6 µg/mL; cDN) for 18 h as a positive control. h Confocal images of DNA dye Draq5 (cyan) and STING-specific antibody (red) in WT BMDMs stimulated as in f . Bars, 20 µm. *** p

Techniques Used: Activation Assay, Derivative Assay, Confocal Microscopy, Labeling, Staining, Flow Cytometry, Cytometry, Concentration Assay, Western Blot, Transfection, Positive Control

69) Product Images from "ZRF1 is a novel S6 kinase substrate that drives the senescence programme"

Article Title: ZRF1 is a novel S6 kinase substrate that drives the senescence programme

Journal: The EMBO Journal

doi: 10.15252/embj.201694966

Validation of analogue‐specific S6K1 mutation Myc‐WT‐S6K1 and myc‐AS‐S6K1 were transfected in HEK293 cells with HA‐RpS6WT or HA‐RpS65A. In vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation using an anti‐HA antibody, the thiophosphorylation of RpS6 was revealed by Western blot using an anti‐thiophosphate ester antibody. Expression level of myc‐WT‐S6K1 and myc‐AS‐S6K1 or total S6K1 was revealed by Western blot on total extracts using the indicated antibody. HEK293 or U2OS cells stably expressing myc‐WT‐S6K1 (WT) or myc‐AS‐S6K1 (AS) were transfected with tagged forms of S6K1 substrates (CAD, eIF4B, eEF2K, SKAR, IRS1, RpS6, PDCD4) or PRAS40. In vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation, the thiophosphorylation was revealed by Western blot using an anti‐thiophosphate ester antibody. * indicates unspecific band.
Figure Legend Snippet: Validation of analogue‐specific S6K1 mutation Myc‐WT‐S6K1 and myc‐AS‐S6K1 were transfected in HEK293 cells with HA‐RpS6WT or HA‐RpS65A. In vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation using an anti‐HA antibody, the thiophosphorylation of RpS6 was revealed by Western blot using an anti‐thiophosphate ester antibody. Expression level of myc‐WT‐S6K1 and myc‐AS‐S6K1 or total S6K1 was revealed by Western blot on total extracts using the indicated antibody. HEK293 or U2OS cells stably expressing myc‐WT‐S6K1 (WT) or myc‐AS‐S6K1 (AS) were transfected with tagged forms of S6K1 substrates (CAD, eIF4B, eEF2K, SKAR, IRS1, RpS6, PDCD4) or PRAS40. In vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation, the thiophosphorylation was revealed by Western blot using an anti‐thiophosphate ester antibody. * indicates unspecific band.

Techniques Used: Mutagenesis, Transfection, In Vivo, Kinase Assay, Immunoprecipitation, Western Blot, Expressing, Stable Transfection

Analogue‐specific S6K1 mutation to screen for direct in vivo substrates HEK293 cells stably expressing myc‐WT‐S6K1 (WT) or myc‐AS‐S6K1 (AS), or U2OS transduced with adeno‐myc‐WT‐S6K1 (WT) or adeno‐myc‐AS‐S6K1 (AS), were transfected with tagged forms of candidates (lamin A, ZRF1, CUX1, NPM1). In vivo kinase assay were performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation, the thiophosphorylation was revealed by Western blot using an anti‐thiophosphate ester antibody. * indicates unspecific band. HEK293 cells stably expressing myc‐WT‐S6K1 (WT) or myc‐AS‐S6K1 (AS) were transfected with Flag‐tagged forms of ZRF1 or a mutant of ZRF1, ZRF1 S47A . In vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation using an anti‐Flag antibody, the thiophosphorylation was revealed by Western blot using an anti‐thiophosphate ester antibody. HEK293 was transfected with Flag‐ZRF1WT or Flag‐ZRF1S47A mutant plasmids. Twenty‐four hours post‐transfection, cells were starved overnight and treated for 3 h with Torin 1 (100 nM). After immunoprecipitation with anti‐Flag antibody, an in vitro kinase assay was performed with a recombinant active S6K1. ZRF1 phosphorylation was analysed by immunoblotting.
Figure Legend Snippet: Analogue‐specific S6K1 mutation to screen for direct in vivo substrates HEK293 cells stably expressing myc‐WT‐S6K1 (WT) or myc‐AS‐S6K1 (AS), or U2OS transduced with adeno‐myc‐WT‐S6K1 (WT) or adeno‐myc‐AS‐S6K1 (AS), were transfected with tagged forms of candidates (lamin A, ZRF1, CUX1, NPM1). In vivo kinase assay were performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation, the thiophosphorylation was revealed by Western blot using an anti‐thiophosphate ester antibody. * indicates unspecific band. HEK293 cells stably expressing myc‐WT‐S6K1 (WT) or myc‐AS‐S6K1 (AS) were transfected with Flag‐tagged forms of ZRF1 or a mutant of ZRF1, ZRF1 S47A . In vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation using an anti‐Flag antibody, the thiophosphorylation was revealed by Western blot using an anti‐thiophosphate ester antibody. HEK293 was transfected with Flag‐ZRF1WT or Flag‐ZRF1S47A mutant plasmids. Twenty‐four hours post‐transfection, cells were starved overnight and treated for 3 h with Torin 1 (100 nM). After immunoprecipitation with anti‐Flag antibody, an in vitro kinase assay was performed with a recombinant active S6K1. ZRF1 phosphorylation was analysed by immunoblotting.

Techniques Used: Mutagenesis, In Vivo, Stable Transfection, Expressing, Transduction, Transfection, Kinase Assay, Immunoprecipitation, Western Blot, In Vitro, Recombinant

70) Product Images from "Phospholipase C-?1 is Required for the Epidermal Growth Factor Receptor-induced Squamous Cell Carcinoma Cell Mitogenesis"

Article Title: Phospholipase C-?1 is Required for the Epidermal Growth Factor Receptor-induced Squamous Cell Carcinoma Cell Mitogenesis

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2010.05.103

EGF-induced SCC cell mitogenesis was blocked by knockdown of PLC-γ1 Cultured SCC4 ( a ) and SCC12B2 ( b ) cells were treated with PLC-γ1 siRNA (100 nM) for 72 hours and then with EGF (200 ng/ml) for 24 hours. Cells were harvested and total cell lysates were isolated. The protein levels for PLC-γ1, β1 and δ1 were determined by western analysis. Cell mitogenesis was determined by [ 3 H]-thymidine incorporation assay. The results of [ 3 H]-thymidine incorporation are expressed as percentages of the control values. Data are mean ± SD of triplicates within a single representative experiments, *p
Figure Legend Snippet: EGF-induced SCC cell mitogenesis was blocked by knockdown of PLC-γ1 Cultured SCC4 ( a ) and SCC12B2 ( b ) cells were treated with PLC-γ1 siRNA (100 nM) for 72 hours and then with EGF (200 ng/ml) for 24 hours. Cells were harvested and total cell lysates were isolated. The protein levels for PLC-γ1, β1 and δ1 were determined by western analysis. Cell mitogenesis was determined by [ 3 H]-thymidine incorporation assay. The results of [ 3 H]-thymidine incorporation are expressed as percentages of the control values. Data are mean ± SD of triplicates within a single representative experiments, *p

Techniques Used: Planar Chromatography, Cell Culture, Isolation, Western Blot, Thymidine Incorporation Assay

Elevated expression of PLC-γ1 in human SCC Cultured normal human keratinocytes, SCC4 and SCC12B2 cells were grown to 90% confluence and total protein was isolated. Protein levels for PLC-γ1 and involucrin were determined by western analysis ( a ). Sections of paraffin-embedded human skin SCC from 14 different patients were processed for hematoxylin and eosin (H E) staining and immunohistochemical staining with antibody for PLC-γ1. The figure shows a representative field of sections stained red and blue with H E ( b ) or immunostained brown with PLC-γ1 antibody and counterstained blue with hematoxylin ( c ).
Figure Legend Snippet: Elevated expression of PLC-γ1 in human SCC Cultured normal human keratinocytes, SCC4 and SCC12B2 cells were grown to 90% confluence and total protein was isolated. Protein levels for PLC-γ1 and involucrin were determined by western analysis ( a ). Sections of paraffin-embedded human skin SCC from 14 different patients were processed for hematoxylin and eosin (H E) staining and immunohistochemical staining with antibody for PLC-γ1. The figure shows a representative field of sections stained red and blue with H E ( b ) or immunostained brown with PLC-γ1 antibody and counterstained blue with hematoxylin ( c ).

Techniques Used: Expressing, Planar Chromatography, Cell Culture, Isolation, Western Blot, Staining, Immunohistochemistry

71) Product Images from "Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation"

Article Title: Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation

Journal: Nature Communications

doi: 10.1038/s41467-019-09690-0

Phospho-Ser25 prevents RIPK1 kinase activation in complex I. MEFs ( a – d ), BMDMs ( e ) or MDFs ( f ) of the indicated genotypes were pretreated for 30 min with the indicated compounds before stimulation with 1 µg/ml FLAG-hTNF for the indicated duration. ( a , c – f ) TNFR1 complex I was then FLAG-immunoprecipitated and the IPs were then treated with USP2 when indicated. b The lysates were then split in two to isolate in parallel TNFR1 complex I by FLAG immunoprecipitation and pS25 RIPK1. The IPs were respectively subsequently treated with USP2 and USP2 + PPase. Protein levels were determined by immunoblot. Immunoblots are representative of two ( a – f ) independent experiments
Figure Legend Snippet: Phospho-Ser25 prevents RIPK1 kinase activation in complex I. MEFs ( a – d ), BMDMs ( e ) or MDFs ( f ) of the indicated genotypes were pretreated for 30 min with the indicated compounds before stimulation with 1 µg/ml FLAG-hTNF for the indicated duration. ( a , c – f ) TNFR1 complex I was then FLAG-immunoprecipitated and the IPs were then treated with USP2 when indicated. b The lysates were then split in two to isolate in parallel TNFR1 complex I by FLAG immunoprecipitation and pS25 RIPK1. The IPs were respectively subsequently treated with USP2 and USP2 + PPase. Protein levels were determined by immunoblot. Immunoblots are representative of two ( a – f ) independent experiments

Techniques Used: Activation Assay, Immunoprecipitation, Western Blot

Defective phospho-Ser25 can drive multi-organ inflammation. a Shpn +/+ or Shpn cpdm/cpdm MDFs were pretreated with the indicated compounds for 30 min before stimulation with 1 µg/ml hTNF for the indicated duration. pS25 RIPK1 was then immunoprecipitated and treated with USP2 and λ phosphatase (PPase) post-IP when indicated. b , c Shpn cpdm/cpdm ; Ripk1 +/+ or Shpn cpdm/cpdm ; Ripk1 S25D/S25D MDFs were pretreated with the indicated inhibitors for 30 min prior to 1 ng/ml hTNF stimulation. Cell death and protein activation were determined in function of time respectively by SytoxGreen positivity ( b ) and immunoblot ( c ). Cell death data are presented as mean ± SEM of three independent experiments ( n = 3). Statistical significance for the cell death assays was determined using two-way ANOVA followed by a Tukey post-hoc test. d Representative picture of 8-weeks-old Shpn +/+ Ripk1 S25D/S25D , Shpn cpdm/cpdm Ripk1 +/+ , and Shpn cpdm/cpdm Ripk1 S25D/S25D mice, and section of their skin and spleen stained for H E, TUNEL or cleaved caspase-3. Scale bar represents 40 µm. e Spleen weight from age-matched 8–12-weeks-old mice ( Shpn cpdm/cpdm Ripk1 +/+ n = 19, Shpn +/+ Ripk1 S25D/S25D n = 7 , Shpn cpdm/cpdm Ripk1 +/S25D n = 6 , Shpn cpdm/cpdm Ripk1 S25D/S25D n = 6 , Shpn +/+ Ripk1 +/+ n = 6). f Lactate dehydrogenase (LDH) ( Shpn cpdm/cpdm Ripk1 +/+ n = 12, Shpn +/+ Ripk1 S25D/S25D n = 8 , Shpn cpdm/cpdm Ripk1 S25D/S25D n = 10 , Shpn +/+ Ripk1 +/+ n = 5), g IL-6 ( Shpn cpdm/cpdm Ripk1 +/+ n = 7, Shpn +/+ Ripk1 S25D/S25D n = 6 , Shpn cpdm/cpdm Ripk1 S25D/S25D n = 6 , Shpn +/+ ; Ripk1 +/+ n = 7), and h MCP-1 ( Shpn cpdm/cpdm ; Ripk1 +/+ n = 8, Shpn +/+ ; Ripk1 S25D/S25D n = 7 , Shpn cpdm/cpdm ; Ripk1 S25D/S25D n = 6 , Shpn +/+ ; Ripk1 +/+ n = 7) levels were determined in the serum of 8–12-weeks-old mice. Spleen weight, LDH, IL-6, and MCP-1 are presented as mean ± SEM from n samples from individual mice. Statistical significance was determined by one-way ANOVA followed by a Tukey post-hoc test. Significance between samples is indicated in the figures as follows: ∗∗ p
Figure Legend Snippet: Defective phospho-Ser25 can drive multi-organ inflammation. a Shpn +/+ or Shpn cpdm/cpdm MDFs were pretreated with the indicated compounds for 30 min before stimulation with 1 µg/ml hTNF for the indicated duration. pS25 RIPK1 was then immunoprecipitated and treated with USP2 and λ phosphatase (PPase) post-IP when indicated. b , c Shpn cpdm/cpdm ; Ripk1 +/+ or Shpn cpdm/cpdm ; Ripk1 S25D/S25D MDFs were pretreated with the indicated inhibitors for 30 min prior to 1 ng/ml hTNF stimulation. Cell death and protein activation were determined in function of time respectively by SytoxGreen positivity ( b ) and immunoblot ( c ). Cell death data are presented as mean ± SEM of three independent experiments ( n = 3). Statistical significance for the cell death assays was determined using two-way ANOVA followed by a Tukey post-hoc test. d Representative picture of 8-weeks-old Shpn +/+ Ripk1 S25D/S25D , Shpn cpdm/cpdm Ripk1 +/+ , and Shpn cpdm/cpdm Ripk1 S25D/S25D mice, and section of their skin and spleen stained for H E, TUNEL or cleaved caspase-3. Scale bar represents 40 µm. e Spleen weight from age-matched 8–12-weeks-old mice ( Shpn cpdm/cpdm Ripk1 +/+ n = 19, Shpn +/+ Ripk1 S25D/S25D n = 7 , Shpn cpdm/cpdm Ripk1 +/S25D n = 6 , Shpn cpdm/cpdm Ripk1 S25D/S25D n = 6 , Shpn +/+ Ripk1 +/+ n = 6). f Lactate dehydrogenase (LDH) ( Shpn cpdm/cpdm Ripk1 +/+ n = 12, Shpn +/+ Ripk1 S25D/S25D n = 8 , Shpn cpdm/cpdm Ripk1 S25D/S25D n = 10 , Shpn +/+ Ripk1 +/+ n = 5), g IL-6 ( Shpn cpdm/cpdm Ripk1 +/+ n = 7, Shpn +/+ Ripk1 S25D/S25D n = 6 , Shpn cpdm/cpdm Ripk1 S25D/S25D n = 6 , Shpn +/+ ; Ripk1 +/+ n = 7), and h MCP-1 ( Shpn cpdm/cpdm ; Ripk1 +/+ n = 8, Shpn +/+ ; Ripk1 S25D/S25D n = 7 , Shpn cpdm/cpdm ; Ripk1 S25D/S25D n = 6 , Shpn +/+ ; Ripk1 +/+ n = 7) levels were determined in the serum of 8–12-weeks-old mice. Spleen weight, LDH, IL-6, and MCP-1 are presented as mean ± SEM from n samples from individual mice. Statistical significance was determined by one-way ANOVA followed by a Tukey post-hoc test. Significance between samples is indicated in the figures as follows: ∗∗ p

Techniques Used: Immunoprecipitation, Activation Assay, Mouse Assay, Staining, TUNEL Assay

Phospho-Ser25 regulates the immune response against Yersinia. a Wild-type MEFs were pretreated with the indicated compounds for 30 min before stimulation with 1 µg/ml hTNF for the indicated duration. pS25 RIPK1 was then immunoprecipitated and treated with USP2 and λ phosphatase (PPase) post-IP when indicated. b – f Ripk1 +/+ and Ripk1 S25D/S25D BMDMs ( b , e – f ) or MEFs ( c , d ) were pretreated with the indicated compounds for 30 min before stimulation with hTNF (10 pg/ml for BMDMs and 100 pg/ml for MEFs) ( b – d ) or 50 ng/ml LPS for 6 h ( e ) or 4 h ( f ). Activation of cytosolic proteins was monitored by immunoblot ( d ) and cell death was measured in function of time ( b – c , e – f ). ( g ) Ripk1 +/+ and Ripk1 S25D/S25D BMDMs were infected with wild-type Y. enterocolitica ( Ye ) or with the YopP-negative mutant Ye.ΔP in presence or absence of Nec-1s. Cell death was quantified by SytoxGreen staining 4 h post-infection. h – i Ripk1 +/+ , Ripk1 S25D/S25D and Ripk1 K45A/K45A BMDMs were pretreated, or not, for 1 h with 5 µM IKK inhibitor BMS-345541 (IKKi), then infected with wild-type Y. pseudotuberculosis ( Yp ) or the YopJ-negative mutant Yp.ΔJ . Cell death was measured by LDH 6 h post-infection ( h ) and cytosolic RIPK1 activation was monitored by immunoblotting for pS166 RIPK1 ( i ). Cell death data are presented as mean ± SEM three independent experiments ( c ). BMDMs results were obtained with cells isolated from three ( n = 3) ( b , e – h ) different mice of each genotype. Statistical significance for the cell death assays was determined using two-way ANOVA followed by a Tukey ( b , c , h ) or Sidak ( g ) post-hoc test. J BM chimeras were generated by reconstituting lethally irradiated congenic hosts with BM from either Ripk1 +/+ , Ripk1 S25D/S25D , or Ripk1 K45A/K45A mice. k – m BM chimeric mice were infected with 1–2 10 8 CFUs Y. pseudotuberculosis by oral gavage. Liver ( k ) ( Ripk1 +/+ n = 16, Ripk1 S25D/S25D n = 13, Ripk1 K45A/K45A n = 14) and spleen ( l ) ( Ripk1 +/+ n = 16, Ripk1 S25D/S25D n = 13, Ripk1 K45A/K45A n = 14) bacterial burdens were measured on day 5 post-infection and survival was recorded during two weeks ( m ) ( Ripk1 +/+ n = 6, Ripk1 S25D/S25D n = 6, Ripk1 K45A/K45A n = 6). Statistical significance for the bacterial burdens was determined using a Mann–Whitney test. Significance between samples is indicated in the figures as follows: ∗∗ p
Figure Legend Snippet: Phospho-Ser25 regulates the immune response against Yersinia. a Wild-type MEFs were pretreated with the indicated compounds for 30 min before stimulation with 1 µg/ml hTNF for the indicated duration. pS25 RIPK1 was then immunoprecipitated and treated with USP2 and λ phosphatase (PPase) post-IP when indicated. b – f Ripk1 +/+ and Ripk1 S25D/S25D BMDMs ( b , e – f ) or MEFs ( c , d ) were pretreated with the indicated compounds for 30 min before stimulation with hTNF (10 pg/ml for BMDMs and 100 pg/ml for MEFs) ( b – d ) or 50 ng/ml LPS for 6 h ( e ) or 4 h ( f ). Activation of cytosolic proteins was monitored by immunoblot ( d ) and cell death was measured in function of time ( b – c , e – f ). ( g ) Ripk1 +/+ and Ripk1 S25D/S25D BMDMs were infected with wild-type Y. enterocolitica ( Ye ) or with the YopP-negative mutant Ye.ΔP in presence or absence of Nec-1s. Cell death was quantified by SytoxGreen staining 4 h post-infection. h – i Ripk1 +/+ , Ripk1 S25D/S25D and Ripk1 K45A/K45A BMDMs were pretreated, or not, for 1 h with 5 µM IKK inhibitor BMS-345541 (IKKi), then infected with wild-type Y. pseudotuberculosis ( Yp ) or the YopJ-negative mutant Yp.ΔJ . Cell death was measured by LDH 6 h post-infection ( h ) and cytosolic RIPK1 activation was monitored by immunoblotting for pS166 RIPK1 ( i ). Cell death data are presented as mean ± SEM three independent experiments ( c ). BMDMs results were obtained with cells isolated from three ( n = 3) ( b , e – h ) different mice of each genotype. Statistical significance for the cell death assays was determined using two-way ANOVA followed by a Tukey ( b , c , h ) or Sidak ( g ) post-hoc test. J BM chimeras were generated by reconstituting lethally irradiated congenic hosts with BM from either Ripk1 +/+ , Ripk1 S25D/S25D , or Ripk1 K45A/K45A mice. k – m BM chimeric mice were infected with 1–2 10 8 CFUs Y. pseudotuberculosis by oral gavage. Liver ( k ) ( Ripk1 +/+ n = 16, Ripk1 S25D/S25D n = 13, Ripk1 K45A/K45A n = 14) and spleen ( l ) ( Ripk1 +/+ n = 16, Ripk1 S25D/S25D n = 13, Ripk1 K45A/K45A n = 14) bacterial burdens were measured on day 5 post-infection and survival was recorded during two weeks ( m ) ( Ripk1 +/+ n = 6, Ripk1 S25D/S25D n = 6, Ripk1 K45A/K45A n = 6). Statistical significance for the bacterial burdens was determined using a Mann–Whitney test. Significance between samples is indicated in the figures as follows: ∗∗ p

Techniques Used: Immunoprecipitation, Activation Assay, Infection, Mutagenesis, Staining, Isolation, Mouse Assay, Generated, Irradiation, MANN-WHITNEY

72) Product Images from "A human microprotein that interacts with the mRNA decapping complex"

Article Title: A human microprotein that interacts with the mRNA decapping complex

Journal: Nature chemical biology

doi: 10.1038/nchembio.2249

The LOC550643/LINC01420 gene encodes the NoBody peptide in a short open reading frame (sORF) ( a ) K562 and HEK293T cellular peptides were enriched and subjected to multidimensional LC-MS proteomics. Peptide mass spectra were searched against a custom protein database obtained from the 3-frame translation of RNA-Seq data from these cell lines. Annotated peptides were removed by BLAST search to afford a list of non-annotated peptides. This workflow led to the discovery of a tryptic peptide (underlined sequence) derived from a polypeptide translated from a sORF (black) in the LOC550643 RNA transcript (gray). The polypeptide is hereafter referred to as NoBody. ( b ) Transfection of an expression construct corresponding to the annotated full-length LOC550643 cDNA sequence (gray), with an epitope tag (red) at the C-terminus of the putative short ORF (black) into HEK293T cells resulted in expression of NoBody (red anti-FLAG immunofluorescence image superimposed on differential interference contrast (DIC) image). Scale bar, 20 µm. ( c ) ClustalW2 alignment of full-length NoBody polypeptide sequence from a variety of mammals. Amino acid identity is indicated by asterisks.
Figure Legend Snippet: The LOC550643/LINC01420 gene encodes the NoBody peptide in a short open reading frame (sORF) ( a ) K562 and HEK293T cellular peptides were enriched and subjected to multidimensional LC-MS proteomics. Peptide mass spectra were searched against a custom protein database obtained from the 3-frame translation of RNA-Seq data from these cell lines. Annotated peptides were removed by BLAST search to afford a list of non-annotated peptides. This workflow led to the discovery of a tryptic peptide (underlined sequence) derived from a polypeptide translated from a sORF (black) in the LOC550643 RNA transcript (gray). The polypeptide is hereafter referred to as NoBody. ( b ) Transfection of an expression construct corresponding to the annotated full-length LOC550643 cDNA sequence (gray), with an epitope tag (red) at the C-terminus of the putative short ORF (black) into HEK293T cells resulted in expression of NoBody (red anti-FLAG immunofluorescence image superimposed on differential interference contrast (DIC) image). Scale bar, 20 µm. ( c ) ClustalW2 alignment of full-length NoBody polypeptide sequence from a variety of mammals. Amino acid identity is indicated by asterisks.

Techniques Used: Liquid Chromatography with Mass Spectroscopy, RNA Sequencing Assay, Sequencing, Derivative Assay, Transfection, Expressing, Construct, Immunofluorescence

73) Product Images from "Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-β"

Article Title: Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-β

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201308031

The FBG domain of TNX interacts physically with the SLC in vitro and in vivo. (A) Quantitative (ELISA) analysis of mature human TGF-β1 associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF fragment, subjected (+) or not subjected (−) to conditions activating latent TGF-β (heat and acid treatments). (B) Levels of mature human TGF-β1 and of its LAP(β1) propeptide associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF protein were determined by immunoblotting. The monoclonal anti-TNX antibody recognizes the 10th FNIII domain. (C) Levels of phospho-Smad2 (P-Smad2) in NMuMG cells cultured for 3 h on noncoated dishes (N-C) or dishes coated with the different recombinant TNX variants (111 pmol/cm 2 ) or stimulated with 5 ng/ml of soluble TGF-β1. Ratio of phospho-Smad2 to total Smad2/3 levels is indicated below. (D) Coimmunoprecipitation of the mature TGF-β1 entity and of its LAP(β1) propeptide with the purified recombinant FBG domain. Immunoprecipitations were performed with anti-FBG (α-FBG), anti-–human TGF-β1 (α-TGF-β1), or anti–human LAP(β1) antibodies or with control IgG. (E) Quantitative detection (ELISA) of mature TGF-β1 associated with the FBG domain of bovine TNX immunoprecipitated from FBS with either anti-FBG domain (α-FBG) antibody or control IgG. Samples were subjected (+) or not subjected (−) to activating conditions before the ELISA. An FBS fraction corresponding to 2.5% of the total volume used for the immunoprecipitation was also subjected to ELISA. *, P
Figure Legend Snippet: The FBG domain of TNX interacts physically with the SLC in vitro and in vivo. (A) Quantitative (ELISA) analysis of mature human TGF-β1 associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF fragment, subjected (+) or not subjected (−) to conditions activating latent TGF-β (heat and acid treatments). (B) Levels of mature human TGF-β1 and of its LAP(β1) propeptide associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF protein were determined by immunoblotting. The monoclonal anti-TNX antibody recognizes the 10th FNIII domain. (C) Levels of phospho-Smad2 (P-Smad2) in NMuMG cells cultured for 3 h on noncoated dishes (N-C) or dishes coated with the different recombinant TNX variants (111 pmol/cm 2 ) or stimulated with 5 ng/ml of soluble TGF-β1. Ratio of phospho-Smad2 to total Smad2/3 levels is indicated below. (D) Coimmunoprecipitation of the mature TGF-β1 entity and of its LAP(β1) propeptide with the purified recombinant FBG domain. Immunoprecipitations were performed with anti-FBG (α-FBG), anti-–human TGF-β1 (α-TGF-β1), or anti–human LAP(β1) antibodies or with control IgG. (E) Quantitative detection (ELISA) of mature TGF-β1 associated with the FBG domain of bovine TNX immunoprecipitated from FBS with either anti-FBG domain (α-FBG) antibody or control IgG. Samples were subjected (+) or not subjected (−) to activating conditions before the ELISA. An FBS fraction corresponding to 2.5% of the total volume used for the immunoprecipitation was also subjected to ELISA. *, P

Techniques Used: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Cell Culture, Immunoprecipitation

74) Product Images from "Hyperphosphorylated Tau in an ?-synuclein overexpressing transgenic model of Parkinson's disease"

Article Title: Hyperphosphorylated Tau in an ?-synuclein overexpressing transgenic model of Parkinson's disease

Journal: The European journal of neuroscience

doi: 10.1111/j.1460-9568.2011.07660.x

Triton X-100 extraction of striatal lysates from PDGF-α-Syn overexpressing transgenic mice Striata from 11 month old transgenic mice and age-matched litter mates were extracted in Triton X-100 and soluble and insoluble fractions were isolated as described under Methods.  [A]  α-Syn, p-GSK-3β and  [B]  p-Tau levels were all expressed relative to GAPDH. All values are expressed as percent change relative to changes observed in WT control animals. Results are from 3–4 animals per group; [*,  P
Figure Legend Snippet: Triton X-100 extraction of striatal lysates from PDGF-α-Syn overexpressing transgenic mice Striata from 11 month old transgenic mice and age-matched litter mates were extracted in Triton X-100 and soluble and insoluble fractions were isolated as described under Methods. [A] α-Syn, p-GSK-3β and [B] p-Tau levels were all expressed relative to GAPDH. All values are expressed as percent change relative to changes observed in WT control animals. Results are from 3–4 animals per group; [*, P

Techniques Used: Transgenic Assay, Mouse Assay, Isolation

75) Product Images from ""

Article Title:

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E07-06-0607

Suppression of glc7-td by prk1 Δ. (A) Actin staining of glc7-ntd (YMC499), glc7-td (YMC500), and prk1 Δ glc7-td (YMC501) cells. The strains were grown at 25°C to early log phase and incubated at 37°C for 6 h followed by fixation
Figure Legend Snippet: Suppression of glc7-td by prk1 Δ. (A) Actin staining of glc7-ntd (YMC499), glc7-td (YMC500), and prk1 Δ glc7-td (YMC501) cells. The strains were grown at 25°C to early log phase and incubated at 37°C for 6 h followed by fixation

Techniques Used: Staining, Incubation

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Transduction:

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Centrifugation:

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Nucleic Acid Electrophoresis:

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Bradford Assay:

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Cytometry:

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Electrophoresis:

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Transplantation Assay:

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Incubation:

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Cell Culture:

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Cell Fractionation:

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Modification:

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Article Snippet: Immunoblotting Total protein was extracted from ventricular tissue or harvested CFs by homogenizing in a lysis buffer containing 50 mmol/L HEPES, 5 mmol/L EDTA, 100 mmol/L NaCl, 1% Triton X-100, and 1x protease inhibitor cocktail (Roche, Mannheim, Germany) at pH 7.4. .. Protein concentrations were determined using a modified Bradford assay, and equal amounts of protein were separated by 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes.

Article Title: Geometry sensing by dendritic cells dictates spatial organization and PGE2-induced dissolution of podosomes
Article Snippet: .. The following chemicals were used: fibronectin (Roche, Mannheim, Germany), gelatin, laminin and poly-l -lysine (PLL) (Sigma), polytetrafluoroethylene (Teflon), polystyrene (PS), polyethylene naphthalate (PEN), and impact modified poly(methyl methacrylate) (PMMA) (Goodfellow, Bad Nauheim, Germany). .. Hydrogels are p-slides from Nexterion (Schott, Mainz, Germany).

Western Blot:

Article Title: Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus
Article Snippet: Paragraph title: Western blots. ... 3T3 cells were treated with 10 μM Purv, 10 μM MeO-Ros, 0.3 μM Flavo, or vehicle (DMSO) for 6 h. Cells were lysed in a solution containing 10 mM Tris (pH 7.6), 150 mM NaCl, 2 mM MgCl2 , 1% Triton X-100, 15% glycerol, 0.1% of a saturated solution of phenylmethanesulfonyl fluoride in isopropanol, and protease inhibitor cocktail tablets (Roche).

Flow Cytometry:

Article Title: Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
Article Snippet: The integrin-specific antibodies used for flow cytometry, in situ immunohistochemistry, colocalization, binding, and infection competition analyses were the following: MAbs anti-α2 (clone P1E6 [mouse]), anti-α3 (clones P1B5 [mouse] and ASC-1 [mouse]), anti-α4 (clones P4C2 [mouse] and PS/2 [rat]), and anti-α5 (clone P1D6 [mouse]), all from Merck Millipore; anti-α6 MAbs (clones GoH3 [rat] and MP 4F10 [mouse]) from Abcam; MAb anti-αV (clone 272-17E6 [mouse]) from Thermo Fisher Scientific; MAb anti-β1 (clone P5D2 [mouse]) and PAb anti-β1 (AF1778 [goat]), both from R & D Systems; MAb anti-β3 (clone MHF4 [mouse]) from Abnova; MAb anti-β4 (clone 422325 [mouse]) from R & D Systems; and PAb anti-β5 (H00003693D01P [rabbit]) from Abnova. .. The integrin-recognizing ligands used in binding and infection competition analyses were vitronectin (R & D Systems), fibronectin (Roche), and laminin 511 (Biolamina).

Immunohistochemistry:

Article Title: Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
Article Snippet: The integrin-specific antibodies used for flow cytometry, in situ immunohistochemistry, colocalization, binding, and infection competition analyses were the following: MAbs anti-α2 (clone P1E6 [mouse]), anti-α3 (clones P1B5 [mouse] and ASC-1 [mouse]), anti-α4 (clones P4C2 [mouse] and PS/2 [rat]), and anti-α5 (clone P1D6 [mouse]), all from Merck Millipore; anti-α6 MAbs (clones GoH3 [rat] and MP 4F10 [mouse]) from Abcam; MAb anti-αV (clone 272-17E6 [mouse]) from Thermo Fisher Scientific; MAb anti-β1 (clone P5D2 [mouse]) and PAb anti-β1 (AF1778 [goat]), both from R & D Systems; MAb anti-β3 (clone MHF4 [mouse]) from Abnova; MAb anti-β4 (clone 422325 [mouse]) from R & D Systems; and PAb anti-β5 (H00003693D01P [rabbit]) from Abnova. .. The integrin-recognizing ligands used in binding and infection competition analyses were vitronectin (R & D Systems), fibronectin (Roche), and laminin 511 (Biolamina).

Protease Inhibitor:

Article Title: Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus
Article Snippet: .. 3T3 cells were treated with 10 μM Purv, 10 μM MeO-Ros, 0.3 μM Flavo, or vehicle (DMSO) for 6 h. Cells were lysed in a solution containing 10 mM Tris (pH 7.6), 150 mM NaCl, 2 mM MgCl2 , 1% Triton X-100, 15% glycerol, 0.1% of a saturated solution of phenylmethanesulfonyl fluoride in isopropanol, and protease inhibitor cocktail tablets (Roche). .. The cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immobilized on nitrocellulose filters.

Article Title: A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli
Article Snippet: .. The cells were then washed with ice-cold isotonic saline and lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM KCl, 0.5% Triton X-100 with Roche mini-complete protease inhibitor) by repeated vortexing at maximal setting and keeping the cells on ice for 10 min. Lysing cells with Triton X-100 at isotonic salt concentrations has been shown to be a rapid and effective way to isolate purified nuclei . .. The lysates were centrifuged for 10 min at 5,000 g, the extra-nuclear faction was transferred to a new tube and the nuclear pellets were washed with lysis buffer.

Article Title: Angiogenic Sprouting Requires the Fine Tuning of Endothelial Cell Cohesion by the Raf-1/Rok-? Complex
Article Snippet: .. Immunoprecipitation and Immunoblotting Cells were washed once with ice-cold PBS and lysed in IP-buffer (20 mM Tris/acetate [pH7.0], 0.27 M sucrose, 1% Triton X-100, 1 mM DTT, 1 mM EGTA, 1 mM EDTA, 1 mM Na3 VO4 , 25 m MNaF, 1 mM PMSF, and protease inhibitor cocktail [Roche]). .. We precleared 500 μg clear cell lysate (1 μg protein/μl) with 40 μl ProteinG-Sepharose beads (Pierce, 1 hr at 4°C) and incubated with the relevant antibodies (rat anti-mouse-VEC 1:100, rabbit anti-mouse Raf-1, Cell Signaling; 1:100 overnight at 4°C).

Article Title: Sumoylation Regulates Nuclear Localization of Lipin-1? in Neuronal Cells
Article Snippet: .. Immunoprecipitation and immunoblotting Cells were harvested 48 h after transfection and lysed by brief sonication in lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 10% glycerol, complete Protease Inhibitor Cocktail (Roche Diagnostics)). .. Lysates were subjected to immunoprecipitation with anti-V5 agarose.

Article Title: Dendritic Cell-Derived Exosomes Promote Natural Killer Cell Activation and Proliferation: A Role for NKG2D Ligands and IL-15R?
Article Snippet: .. Immunoblotting Cells or exosomal proteins were extracted in lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% NP-40, 10 mM Na3VO4, 5 mM DTT, protease inhibitor cocktail tablet (Complete, Mini, EDTA-free, Roche)) for 30 min at 4°C. .. Nuclei and cell debris were removed by centrifugation.

Article Title: Ghrelin Ameliorates Angiotensin II-Induced Myocardial Fibrosis by Upregulating Peroxisome Proliferator-Activated Receptor Gamma in Young Male Rats
Article Snippet: .. Immunoblotting Total protein was extracted from ventricular tissue or harvested CFs by homogenizing in a lysis buffer containing 50 mmol/L HEPES, 5 mmol/L EDTA, 100 mmol/L NaCl, 1% Triton X-100, and 1x protease inhibitor cocktail (Roche, Mannheim, Germany) at pH 7.4. .. Protein concentrations were determined using a modified Bradford assay, and equal amounts of protein were separated by 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes.

Article Title: The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7
Article Snippet: .. IP For coimmunoprecipitations of tagged Treslin/TICRR and MTBP from 293T cells, a 10-cm plate of transiently transfected 293T cells were lysed in 5× pellet volume of lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, complete EDTA-free protease inhibitor cocktail [Roche, 05056489001], 0.1% Triton X-100, 2 mM 2-mercaptoethanol). .. For flag affinity purifications 50% of cell lysates was incubated with 1 μg anti-FLAG mouse monoclonal antibody or 1 μg mouse IgG coupled to 150 μg Protein G Dynabeads (Invitrogen, 100-04D).

Article Title: Matrix-Bound PAI-1 Supports Cell Blebbing via RhoA/ROCK1 Signaling
Article Snippet: .. Immunoblotting Cells were washed in phosphate-buffered saline (PBS) and lysed directly in 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40 supplemented with protease inhibitor cocktail ([Ref. 11836170001] Roche). .. Denatured proteins were separated by electrophoresis on 12% SDS-polyacrylamide or 4–12% Bis-Tris gels and transferred either to polyvinylidene fluoride or nitrocellulose membranes, then blocked with 5% non-fat dry milk-PBS at room temperature for 1 hour.

Infection:

Article Title: Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
Article Snippet: .. The integrin-recognizing ligands used in binding and infection competition analyses were vitronectin (R & D Systems), fibronectin (Roche), and laminin 511 (Biolamina). .. Human corneas were collected from two donors at autopsy and from three patients undergoing surgery (evisceration, n = 2; corneal transplantation, n = 1), with approval from the Regional Ethical Review Board in Umeå (Dnr 2010-373-31M).

other:

Article Title: Sensitization and synergistic anti-cancer effects of Furanodiene identified in zebrafish models
Article Snippet: Bevacizumab (lot #: H0126805) was bought from Roche, Switzerland.

Sonication:

Article Title: Sumoylation Regulates Nuclear Localization of Lipin-1? in Neuronal Cells
Article Snippet: .. Immunoprecipitation and immunoblotting Cells were harvested 48 h after transfection and lysed by brief sonication in lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 10% glycerol, complete Protease Inhibitor Cocktail (Roche Diagnostics)). .. Lysates were subjected to immunoprecipitation with anti-V5 agarose.

Binding Assay:

Article Title: Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
Article Snippet: .. The integrin-recognizing ligands used in binding and infection competition analyses were vitronectin (R & D Systems), fibronectin (Roche), and laminin 511 (Biolamina). .. Human corneas were collected from two donors at autopsy and from three patients undergoing surgery (evisceration, n = 2; corneal transplantation, n = 1), with approval from the Regional Ethical Review Board in Umeå (Dnr 2010-373-31M).

MTT Assay:

Article Title: Conjugate of PAMAM Dendrimer, Doxorubicin and Monoclonal Antibody—Trastuzumab: The New Approach of a Well-Known Strategy
Article Snippet: Amine terminated PAMAM G4 dendrimer, doxorubicin hydrochloride, PBS (phosphate buffered saline), FBS (fetal bovine serum) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were purchased from Sigma-Aldrich. .. Herceptin (trastuzumab) was a gift from Roche Poland.

Isolation:

Article Title: Geometry sensing by dendritic cells dictates spatial organization and PGE2-induced dissolution of podosomes
Article Snippet: The following chemicals were used: fibronectin (Roche, Mannheim, Germany), gelatin, laminin and poly-l -lysine (PLL) (Sigma), polytetrafluoroethylene (Teflon), polystyrene (PS), polyethylene naphthalate (PEN), and impact modified poly(methyl methacrylate) (PMMA) (Goodfellow, Bad Nauheim, Germany). .. N. meningitides H44/76 was isolated from a patient with invasive meningococcal disease (kindly provided by Dr. P van der Ley, Laboratory of Vaccine Research, Netherlands Vaccine Institute, Bilthoven, The Netherlands).

Transfection:

Article Title: A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli
Article Snippet: Paragraph title: Cell Culture, DNA Transfection, and Cell Fractionation ... The cells were then washed with ice-cold isotonic saline and lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM KCl, 0.5% Triton X-100 with Roche mini-complete protease inhibitor) by repeated vortexing at maximal setting and keeping the cells on ice for 10 min. Lysing cells with Triton X-100 at isotonic salt concentrations has been shown to be a rapid and effective way to isolate purified nuclei .

Article Title: Sumoylation Regulates Nuclear Localization of Lipin-1? in Neuronal Cells
Article Snippet: .. Immunoprecipitation and immunoblotting Cells were harvested 48 h after transfection and lysed by brief sonication in lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 10% glycerol, complete Protease Inhibitor Cocktail (Roche Diagnostics)). .. Lysates were subjected to immunoprecipitation with anti-V5 agarose.

Article Title: The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7
Article Snippet: .. IP For coimmunoprecipitations of tagged Treslin/TICRR and MTBP from 293T cells, a 10-cm plate of transiently transfected 293T cells were lysed in 5× pellet volume of lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, complete EDTA-free protease inhibitor cocktail [Roche, 05056489001], 0.1% Triton X-100, 2 mM 2-mercaptoethanol). .. For flag affinity purifications 50% of cell lysates was incubated with 1 μg anti-FLAG mouse monoclonal antibody or 1 μg mouse IgG coupled to 150 μg Protein G Dynabeads (Invitrogen, 100-04D).

Labeling:

Article Title: Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
Article Snippet: Species C HAdV-5 (strain Ad75) and species D HAdV-37 (strain 1477) virions were propagated with or without 35 S labeling in A549 cells, as described elsewhere ( ). .. The integrin-recognizing ligands used in binding and infection competition analyses were vitronectin (R & D Systems), fibronectin (Roche), and laminin 511 (Biolamina).

Purification:

Article Title: A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli
Article Snippet: .. The cells were then washed with ice-cold isotonic saline and lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM KCl, 0.5% Triton X-100 with Roche mini-complete protease inhibitor) by repeated vortexing at maximal setting and keeping the cells on ice for 10 min. Lysing cells with Triton X-100 at isotonic salt concentrations has been shown to be a rapid and effective way to isolate purified nuclei . .. The lysates were centrifuged for 10 min at 5,000 g, the extra-nuclear faction was transferred to a new tube and the nuclear pellets were washed with lysis buffer.

Article Title: Impact of HIV on Cell Survival and Antiviral Activity of Plasmacytoid Dendritic Cells
Article Snippet: HIV p24 assays 2–4×108 total PBMCs from HIV-infected individuals were stimulated overnight with anti-CD3 antibody and rIL-2 (Roche). .. CD4+ and CD8+ T cells were then purified by negative selection using a column-based separation technique (StemCell Technologies) as previously described .

Lysis:

Article Title: A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli
Article Snippet: .. The cells were then washed with ice-cold isotonic saline and lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM KCl, 0.5% Triton X-100 with Roche mini-complete protease inhibitor) by repeated vortexing at maximal setting and keeping the cells on ice for 10 min. Lysing cells with Triton X-100 at isotonic salt concentrations has been shown to be a rapid and effective way to isolate purified nuclei . .. The lysates were centrifuged for 10 min at 5,000 g, the extra-nuclear faction was transferred to a new tube and the nuclear pellets were washed with lysis buffer.

Article Title: Immunodeficiency, auto-inflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency
Article Snippet: .. Cell lysis, immunoprecipitation and immunoblotting Cells were lysed in a buffer containing 30 mM Tris-HCl pH 7.5, 120 mM NaCl, 2 mM KCl, 1% Triton X-100 and 2 mM EDTA supplemented with protease and phosphatase inhibitors (Complete and PhoStop, Roche). .. For immunoprecipitations, antibodies (5 μg) were added to 1 mg of total protein extract and incubated overnight at 4°C.

Article Title: Sumoylation Regulates Nuclear Localization of Lipin-1? in Neuronal Cells
Article Snippet: .. Immunoprecipitation and immunoblotting Cells were harvested 48 h after transfection and lysed by brief sonication in lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 10% glycerol, complete Protease Inhibitor Cocktail (Roche Diagnostics)). .. Lysates were subjected to immunoprecipitation with anti-V5 agarose.

Article Title: Gremlin-1 associates with fibrillin microfibrils in vivo and regulates mesothelioma cell survival through transcription factor slug
Article Snippet: .. SDS–PAGE and immunoblotting Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.2% sodium deoxycholate) containing protease inhibitors (Roche, Mannheim, Germany) for 15 min on ice. .. Protein concentrations were measured using a BCA protein assay Kit (Pierce, Rockfors, IL, USA).

Article Title: Dendritic Cell-Derived Exosomes Promote Natural Killer Cell Activation and Proliferation: A Role for NKG2D Ligands and IL-15R?
Article Snippet: .. Immunoblotting Cells or exosomal proteins were extracted in lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% NP-40, 10 mM Na3VO4, 5 mM DTT, protease inhibitor cocktail tablet (Complete, Mini, EDTA-free, Roche)) for 30 min at 4°C. .. Nuclei and cell debris were removed by centrifugation.

Article Title: Ghrelin Ameliorates Angiotensin II-Induced Myocardial Fibrosis by Upregulating Peroxisome Proliferator-Activated Receptor Gamma in Young Male Rats
Article Snippet: .. Immunoblotting Total protein was extracted from ventricular tissue or harvested CFs by homogenizing in a lysis buffer containing 50 mmol/L HEPES, 5 mmol/L EDTA, 100 mmol/L NaCl, 1% Triton X-100, and 1x protease inhibitor cocktail (Roche, Mannheim, Germany) at pH 7.4. .. Protein concentrations were determined using a modified Bradford assay, and equal amounts of protein were separated by 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes.

Article Title: The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7
Article Snippet: .. IP For coimmunoprecipitations of tagged Treslin/TICRR and MTBP from 293T cells, a 10-cm plate of transiently transfected 293T cells were lysed in 5× pellet volume of lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, complete EDTA-free protease inhibitor cocktail [Roche, 05056489001], 0.1% Triton X-100, 2 mM 2-mercaptoethanol). .. For flag affinity purifications 50% of cell lysates was incubated with 1 μg anti-FLAG mouse monoclonal antibody or 1 μg mouse IgG coupled to 150 μg Protein G Dynabeads (Invitrogen, 100-04D).

SDS Page:

Article Title: Angiogenic Sprouting Requires the Fine Tuning of Endothelial Cell Cohesion by the Raf-1/Rok-? Complex
Article Snippet: Immunoprecipitation and Immunoblotting Cells were washed once with ice-cold PBS and lysed in IP-buffer (20 mM Tris/acetate [pH7.0], 0.27 M sucrose, 1% Triton X-100, 1 mM DTT, 1 mM EGTA, 1 mM EDTA, 1 mM Na3 VO4 , 25 m MNaF, 1 mM PMSF, and protease inhibitor cocktail [Roche]). .. For immunoblotting, cell lysates and immunoprecipitates were subjected to SDS-PAGE and blotted to nitrocellulose membranes subsequently probed with α-actin, α-14-3-3 α-pMYPT1, α-Rok-α, and α-pCofilin (Santa Cruz); α-Raf-1, α-α-catenin, α-β-catenin, and α-p120catenin (BD Transduction Laboratories); α-VEC (BD PharMingen); and α-pMLC2 and α-pERK (Cell Signaling).

Article Title: Gremlin-1 associates with fibrillin microfibrils in vivo and regulates mesothelioma cell survival through transcription factor slug
Article Snippet: .. SDS–PAGE and immunoblotting Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.2% sodium deoxycholate) containing protease inhibitors (Roche, Mannheim, Germany) for 15 min on ice. .. Protein concentrations were measured using a BCA protein assay Kit (Pierce, Rockfors, IL, USA).

Article Title: Dendritic Cell-Derived Exosomes Promote Natural Killer Cell Activation and Proliferation: A Role for NKG2D Ligands and IL-15R?
Article Snippet: Immunoblotting Cells or exosomal proteins were extracted in lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% NP-40, 10 mM Na3VO4, 5 mM DTT, protease inhibitor cocktail tablet (Complete, Mini, EDTA-free, Roche)) for 30 min at 4°C. .. Aliquots of protein extracts were solubilised in Laemmli loading buffer and resolved by 10% SDS-PAGE and transferred onto nitrocellulose or PVDF membranes (Bio-Rad Laboratories).

Article Title: Ghrelin Ameliorates Angiotensin II-Induced Myocardial Fibrosis by Upregulating Peroxisome Proliferator-Activated Receptor Gamma in Young Male Rats
Article Snippet: Immunoblotting Total protein was extracted from ventricular tissue or harvested CFs by homogenizing in a lysis buffer containing 50 mmol/L HEPES, 5 mmol/L EDTA, 100 mmol/L NaCl, 1% Triton X-100, and 1x protease inhibitor cocktail (Roche, Mannheim, Germany) at pH 7.4. .. Protein concentrations were determined using a modified Bradford assay, and equal amounts of protein were separated by 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes.

Plasmid Preparation:

Article Title: A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli
Article Snippet: The cells at 60–80% confluency were transfected with indicated plasmid(s) using Lipofectamine 2000 (Invitrogen). .. The cells were then washed with ice-cold isotonic saline and lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM KCl, 0.5% Triton X-100 with Roche mini-complete protease inhibitor) by repeated vortexing at maximal setting and keeping the cells on ice for 10 min. Lysing cells with Triton X-100 at isotonic salt concentrations has been shown to be a rapid and effective way to isolate purified nuclei .

In Vitro:

Article Title: Conjugate of PAMAM Dendrimer, Doxorubicin and Monoclonal Antibody—Trastuzumab: The New Approach of a Well-Known Strategy
Article Snippet: Flasks and multiwell plates for in vitro studies were obtained from Nunc (Life Technologies Polska Sp. z o. o., Warsaw, Poland). .. Herceptin (trastuzumab) was a gift from Roche Poland.

Selection:

Article Title: Impact of HIV on Cell Survival and Antiviral Activity of Plasmacytoid Dendritic Cells
Article Snippet: HIV p24 assays 2–4×108 total PBMCs from HIV-infected individuals were stimulated overnight with anti-CD3 antibody and rIL-2 (Roche). .. CD4+ and CD8+ T cells were then purified by negative selection using a column-based separation technique (StemCell Technologies) as previously described .

In Situ:

Article Title: Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
Article Snippet: The integrin-specific antibodies used for flow cytometry, in situ immunohistochemistry, colocalization, binding, and infection competition analyses were the following: MAbs anti-α2 (clone P1E6 [mouse]), anti-α3 (clones P1B5 [mouse] and ASC-1 [mouse]), anti-α4 (clones P4C2 [mouse] and PS/2 [rat]), and anti-α5 (clone P1D6 [mouse]), all from Merck Millipore; anti-α6 MAbs (clones GoH3 [rat] and MP 4F10 [mouse]) from Abcam; MAb anti-αV (clone 272-17E6 [mouse]) from Thermo Fisher Scientific; MAb anti-β1 (clone P5D2 [mouse]) and PAb anti-β1 (AF1778 [goat]), both from R & D Systems; MAb anti-β3 (clone MHF4 [mouse]) from Abnova; MAb anti-β4 (clone 422325 [mouse]) from R & D Systems; and PAb anti-β5 (H00003693D01P [rabbit]) from Abnova. .. The integrin-recognizing ligands used in binding and infection competition analyses were vitronectin (R & D Systems), fibronectin (Roche), and laminin 511 (Biolamina).

Immunoprecipitation:

Article Title: Angiogenic Sprouting Requires the Fine Tuning of Endothelial Cell Cohesion by the Raf-1/Rok-? Complex
Article Snippet: .. Immunoprecipitation and Immunoblotting Cells were washed once with ice-cold PBS and lysed in IP-buffer (20 mM Tris/acetate [pH7.0], 0.27 M sucrose, 1% Triton X-100, 1 mM DTT, 1 mM EGTA, 1 mM EDTA, 1 mM Na3 VO4 , 25 m MNaF, 1 mM PMSF, and protease inhibitor cocktail [Roche]). .. We precleared 500 μg clear cell lysate (1 μg protein/μl) with 40 μl ProteinG-Sepharose beads (Pierce, 1 hr at 4°C) and incubated with the relevant antibodies (rat anti-mouse-VEC 1:100, rabbit anti-mouse Raf-1, Cell Signaling; 1:100 overnight at 4°C).

Article Title: Immunodeficiency, auto-inflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency
Article Snippet: .. Cell lysis, immunoprecipitation and immunoblotting Cells were lysed in a buffer containing 30 mM Tris-HCl pH 7.5, 120 mM NaCl, 2 mM KCl, 1% Triton X-100 and 2 mM EDTA supplemented with protease and phosphatase inhibitors (Complete and PhoStop, Roche). .. For immunoprecipitations, antibodies (5 μg) were added to 1 mg of total protein extract and incubated overnight at 4°C.

Article Title: Sumoylation Regulates Nuclear Localization of Lipin-1? in Neuronal Cells
Article Snippet: .. Immunoprecipitation and immunoblotting Cells were harvested 48 h after transfection and lysed by brief sonication in lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 10% glycerol, complete Protease Inhibitor Cocktail (Roche Diagnostics)). .. Lysates were subjected to immunoprecipitation with anti-V5 agarose.

Staining:

Article Title: Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells
Article Snippet: We also used MAb anti-adenovirus hexon (clone B025/AD51 [mouse] from Abcam; for staining of HAdV-infected, artificial cornea) and MAb anti-GD1a (clone EM9 [mouse]; a kind gift from Hugh Willison; for competition binding and infection experiments). .. The integrin-recognizing ligands used in binding and infection competition analyses were vitronectin (R & D Systems), fibronectin (Roche), and laminin 511 (Biolamina).

BIA-KA:

Article Title: Gremlin-1 associates with fibrillin microfibrils in vivo and regulates mesothelioma cell survival through transcription factor slug
Article Snippet: SDS–PAGE and immunoblotting Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.2% sodium deoxycholate) containing protease inhibitors (Roche, Mannheim, Germany) for 15 min on ice. .. Protein concentrations were measured using a BCA protein assay Kit (Pierce, Rockfors, IL, USA).

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