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Cell Signaling Technology Inc cleaved caspase 12
Activation of apoptosis in Tmem30a KO mice. a – c Western blots a and quantitation b , c manifested the elevated expression of cleaved <t>caspase-12</t> and cleaved caspase-3 in cerebellum of WT and KO mice at P16 and P20, respectively, and GAPDH antibody as loading control. ( n = 6,***, p
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1) Product Images from "Disruption of Tmem30a results in cerebellar ataxia and degeneration of Purkinje cells"

Article Title: Disruption of Tmem30a results in cerebellar ataxia and degeneration of Purkinje cells

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0938-6

Activation of apoptosis in Tmem30a KO mice. a – c Western blots a and quantitation b , c manifested the elevated expression of cleaved caspase-12 and cleaved caspase-3 in cerebellum of WT and KO mice at P16 and P20, respectively, and GAPDH antibody as loading control. ( n = 6,***, p
Figure Legend Snippet: Activation of apoptosis in Tmem30a KO mice. a – c Western blots a and quantitation b , c manifested the elevated expression of cleaved caspase-12 and cleaved caspase-3 in cerebellum of WT and KO mice at P16 and P20, respectively, and GAPDH antibody as loading control. ( n = 6,***, p

Techniques Used: Activation Assay, Mouse Assay, Western Blot, Quantitation Assay, Expressing

2) Product Images from "Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol"

Article Title: Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol

Journal: Toxins

doi: 10.3390/toxins11010042

Cytotoxic properties on mammalian cells. The viability of B16-F10, GL261, SK-N-BE, CT26, and C 2 C 12 cells treated with increasing concentrations of AN1, AN2, and climacostol for 24 h was assessed by MTT assay. Data are expressed by setting the absorbance of the reduced MTT in the absence of compounds as 100%. The data points represent the mean ± SEM of results obtained from 4–6 independent experiments.
Figure Legend Snippet: Cytotoxic properties on mammalian cells. The viability of B16-F10, GL261, SK-N-BE, CT26, and C 2 C 12 cells treated with increasing concentrations of AN1, AN2, and climacostol for 24 h was assessed by MTT assay. Data are expressed by setting the absorbance of the reduced MTT in the absence of compounds as 100%. The data points represent the mean ± SEM of results obtained from 4–6 independent experiments.

Techniques Used: MTT Assay

Apoptosis in melanoma cells. Immunofluorescence imaging of cleaved-caspase 3 (punctate green pattern) in B16-F10 cells cultured in the presence of 30 μg/mL AN1, AN2, and climacostol or vehicle (CTRL) for 9 h. DAPI (blue) and phalloidin (red) were used for nuclei and cytoskeleton detection, respectively. The images are representative of six independent experiments. Inserts represent enlarged image details. Scale bar = 20 µm.
Figure Legend Snippet: Apoptosis in melanoma cells. Immunofluorescence imaging of cleaved-caspase 3 (punctate green pattern) in B16-F10 cells cultured in the presence of 30 μg/mL AN1, AN2, and climacostol or vehicle (CTRL) for 9 h. DAPI (blue) and phalloidin (red) were used for nuclei and cytoskeleton detection, respectively. The images are representative of six independent experiments. Inserts represent enlarged image details. Scale bar = 20 µm.

Techniques Used: Immunofluorescence, Imaging, Cell Culture

3) Product Images from "Restoring PUMA induction overcomes KRAS-mediated resistance to anti-EGFR antibodies in colorectal cancer"

Article Title: Restoring PUMA induction overcomes KRAS-mediated resistance to anti-EGFR antibodies in colorectal cancer

Journal: Oncogene

doi: 10.1038/s41388-018-0289-x

PUMA induction by anti-EGFR antibodies is mediated by p73 (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); phospho-AKT (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.
Figure Legend Snippet: PUMA induction by anti-EGFR antibodies is mediated by p73 (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); phospho-AKT (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.

Techniques Used: Transfection, Expressing, Western Blot, Plasmid Preparation, Construct, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

KRAS -mediated resistance to anti-EGFR antibodies abrogates PUMA induction (A) MTS analysis of parental (red) and cetuximab-resistant (Cmab-R, black) DiFi cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. (B) Western blotting of cleaved (C) caspase-3 in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab or Pmab for 24 hr. (C) Western blotting of indicated proteins in the parental and Cmab-R DiFi cells. Lysates of Cmab-R DiFi cells were prepared from cells cultured in medium with 10 nM cetuximab (Cmab+), or without cetuximab (Cmab-) for 6 days. p-EGFR (Y1068); p-AKT (S473); p-ERK1/2 (T202/Y204). (D) Western blotting of indicated Bcl-2 family proteins in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab for 24 hr. (E) Western blotting of phosphorylated (p-p73, Y99) and total p73 in the parental and Cmab-R DiFi cells treated with 10 nM Cmab for 8 hr. (F) Parental and Cmab-R DiFi cells transfected with control empty or HA-p73α-expressing vector were treated with 10 nM cetuximab for 8 hr. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (G) Parental and Cmab-R DiFi cells were infected with EGFP-PUMA-expressing adenovirus (Ad-PUMA) at the indicated MOI for 24 hr. Upper, analysis of apoptosis by nuclear fragmentation; lower , western blotting of PUMA. Results were expressed as means ± s.e.m. of triplicates in two independent experiments. *** P
Figure Legend Snippet: KRAS -mediated resistance to anti-EGFR antibodies abrogates PUMA induction (A) MTS analysis of parental (red) and cetuximab-resistant (Cmab-R, black) DiFi cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. (B) Western blotting of cleaved (C) caspase-3 in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab or Pmab for 24 hr. (C) Western blotting of indicated proteins in the parental and Cmab-R DiFi cells. Lysates of Cmab-R DiFi cells were prepared from cells cultured in medium with 10 nM cetuximab (Cmab+), or without cetuximab (Cmab-) for 6 days. p-EGFR (Y1068); p-AKT (S473); p-ERK1/2 (T202/Y204). (D) Western blotting of indicated Bcl-2 family proteins in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab for 24 hr. (E) Western blotting of phosphorylated (p-p73, Y99) and total p73 in the parental and Cmab-R DiFi cells treated with 10 nM Cmab for 8 hr. (F) Parental and Cmab-R DiFi cells transfected with control empty or HA-p73α-expressing vector were treated with 10 nM cetuximab for 8 hr. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (G) Parental and Cmab-R DiFi cells were infected with EGFP-PUMA-expressing adenovirus (Ad-PUMA) at the indicated MOI for 24 hr. Upper, analysis of apoptosis by nuclear fragmentation; lower , western blotting of PUMA. Results were expressed as means ± s.e.m. of triplicates in two independent experiments. *** P

Techniques Used: Western Blot, Cell Culture, Transfection, Expressing, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Infection

4) Product Images from "PIM kinases mediate resistance of glioblastoma cells to TRAIL by a p62/SQSTM1-dependent mechanism"

Article Title: PIM kinases mediate resistance of glioblastoma cells to TRAIL by a p62/SQSTM1-dependent mechanism

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1293-3

PIM kinases knockdown abolishes TRAIL-induced TRAIL-R2/DR5 internalization, favoring caspase-8 activation. A Expression of death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) were determined by western blot in U87MG cells after PIM2/3 knockdown. B Plasma membrane levels of TRAIL-R2/DR5 following PIM knockdown were analyzed in U87MG by flow cytometry as described in the materials and methods section. C U87MG cells were transfected with a scrambled oligonucleotide or siRNAs targeting PIM2 and PIM3 and treated with TRAIL (500 ng/mL) for the indicated times. Caspase-8 activation was determined by western blot. D U87MG transfected either with a scrambled oligonucleotide or small interfering RNAs (siRNAs) targeting PIM2/3 were treated with TRAIL (500 ng/mL) for the indicated times. Cell surface TRAIL-R2/DR5 levels were assessed by flow cytometry as described under materials and methods
Figure Legend Snippet: PIM kinases knockdown abolishes TRAIL-induced TRAIL-R2/DR5 internalization, favoring caspase-8 activation. A Expression of death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) were determined by western blot in U87MG cells after PIM2/3 knockdown. B Plasma membrane levels of TRAIL-R2/DR5 following PIM knockdown were analyzed in U87MG by flow cytometry as described in the materials and methods section. C U87MG cells were transfected with a scrambled oligonucleotide or siRNAs targeting PIM2 and PIM3 and treated with TRAIL (500 ng/mL) for the indicated times. Caspase-8 activation was determined by western blot. D U87MG transfected either with a scrambled oligonucleotide or small interfering RNAs (siRNAs) targeting PIM2/3 were treated with TRAIL (500 ng/mL) for the indicated times. Cell surface TRAIL-R2/DR5 levels were assessed by flow cytometry as described under materials and methods

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

PIM kinases-dependent phosphorylation of p62/SQSTM1 in Ser332 controls TRAIL-R2/DR5 levels in GBM cells. A U87MG cells were transfected with a scrambled oligonucleotide or p62/SQSTM1 small interfering RNAs (siRNAs) for 48 h and then TRAIL-R2/DR5 levels were analyzed by western blot. B Western blot analysis of TRAIL-R2/DR5 levels in U87MG cells treated with chloroquine for the indicated times. C U87MG cells were transfected with the indicated siRNAs for 48 h prior to treatment with biotin-TRAIL (1000 ng/mL) for 90 min. TRAIL DISC was pulled down with streptavidin-agarose beads as indicated in the materials and methods section and proteins associated to DISC were determined by western blot. D U87MG cells were transiently transfected with the indicated plasmids using jetPRIME reagent. After transfection, cells were treated overnight with or without SGI-1776 (5 μM) and protein expression was analyzed by western blot. E U87MG cells were transfected with plasmids p62/SQSTM1 wild-type and p62/SQSTM1 S233E mutant as described. After transfection, cells were treated for 48 h with SGI-1776 (5 μM), TRAIL (500 ng/mL), or both. Apoptosis was determined by flow cytometry
Figure Legend Snippet: PIM kinases-dependent phosphorylation of p62/SQSTM1 in Ser332 controls TRAIL-R2/DR5 levels in GBM cells. A U87MG cells were transfected with a scrambled oligonucleotide or p62/SQSTM1 small interfering RNAs (siRNAs) for 48 h and then TRAIL-R2/DR5 levels were analyzed by western blot. B Western blot analysis of TRAIL-R2/DR5 levels in U87MG cells treated with chloroquine for the indicated times. C U87MG cells were transfected with the indicated siRNAs for 48 h prior to treatment with biotin-TRAIL (1000 ng/mL) for 90 min. TRAIL DISC was pulled down with streptavidin-agarose beads as indicated in the materials and methods section and proteins associated to DISC were determined by western blot. D U87MG cells were transiently transfected with the indicated plasmids using jetPRIME reagent. After transfection, cells were treated overnight with or without SGI-1776 (5 μM) and protein expression was analyzed by western blot. E U87MG cells were transfected with plasmids p62/SQSTM1 wild-type and p62/SQSTM1 S233E mutant as described. After transfection, cells were treated for 48 h with SGI-1776 (5 μM), TRAIL (500 ng/mL), or both. Apoptosis was determined by flow cytometry

Techniques Used: Transfection, Western Blot, Expressing, Mutagenesis, Flow Cytometry, Cytometry

Involvement of TRAIL-R2/DR5 in TRAIL-induced caspase-8 activation and apoptosis upon inhibition of PIM kinases function. Caspase-8 activation, cFLIP L cleavage, and PEA-15 levels were assessed by western blot in U87MG cells extracts after overnight treatment with 5 μM SGI-1776 ( A ) or PIM silencing with siRNAs ( B ) followed by 6-h treatment with TRAIL (500 ng/mL). C , D U87MG cells were transfected for 48 h either with a scrambled oligonucleotide or with the indicated small interfering RNAs (siRNAs) targeting TRAIL-R1/DR4 or TRAIL-R2/DR5. In C cells were then treated with SGI-1776 (5 μM) overnight prior to incubation for 6 h in medium with TRAIL (500 ng/mL). Caspase-8 activation was determined by western blot. In D cells were treated with SGI-1776 (5 μM) and TRAIL (500 ng/mL) for 48 h. Hypodiploid apoptotic cells were determined by flow cytometry
Figure Legend Snippet: Involvement of TRAIL-R2/DR5 in TRAIL-induced caspase-8 activation and apoptosis upon inhibition of PIM kinases function. Caspase-8 activation, cFLIP L cleavage, and PEA-15 levels were assessed by western blot in U87MG cells extracts after overnight treatment with 5 μM SGI-1776 ( A ) or PIM silencing with siRNAs ( B ) followed by 6-h treatment with TRAIL (500 ng/mL). C , D U87MG cells were transfected for 48 h either with a scrambled oligonucleotide or with the indicated small interfering RNAs (siRNAs) targeting TRAIL-R1/DR4 or TRAIL-R2/DR5. In C cells were then treated with SGI-1776 (5 μM) overnight prior to incubation for 6 h in medium with TRAIL (500 ng/mL). Caspase-8 activation was determined by western blot. In D cells were treated with SGI-1776 (5 μM) and TRAIL (500 ng/mL) for 48 h. Hypodiploid apoptotic cells were determined by flow cytometry

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

5) Product Images from "Curcumin-Mediated Degradation of S-Phase Kinase Protein 2 Induces Cytotoxic Effects in Human Papillomavirus-Positive and Negative Squamous Carcinoma Cells"

Article Title: Curcumin-Mediated Degradation of S-Phase Kinase Protein 2 Induces Cytotoxic Effects in Human Papillomavirus-Positive and Negative Squamous Carcinoma Cells

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2018.00399

Curcumin mediated accumulation of ubiquitinated proteins via suppression of F-box protein Skp2 in HNSCC cell lines (A) Curcumin-mediated ubiquitination of various proteins. SCC25, FaDu, and SCC090 cells were treated with indicated doses of curcumin for 24 h. Equal amounts of protein lysates were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies of anti-ubiquitin and GAPDH as indicated (B) Curcumin treatment down-regulated the expression of Skp2 and enhanced the level of p27 and P21. SCC25, FaDu, and SCC090 cells were treated with various doses of curcumin for 24 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immuno-blotted with antibodies against Skp2, p27, p21, and GAPDH as indicated (C) Skp2 siRNA transfection downregulates Skp2 and accumulated p27 and p21. FaDu and SCC090 cells were transfected with Scrambled siRNA (100 pmol) and Skp2 siRNA (50 and 100 pmol) using Lipofectamine 2000 as described in Materials and Methods. After 48 h of transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against Skp2, p27, p21, and GAPDH as indicated.
Figure Legend Snippet: Curcumin mediated accumulation of ubiquitinated proteins via suppression of F-box protein Skp2 in HNSCC cell lines (A) Curcumin-mediated ubiquitination of various proteins. SCC25, FaDu, and SCC090 cells were treated with indicated doses of curcumin for 24 h. Equal amounts of protein lysates were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies of anti-ubiquitin and GAPDH as indicated (B) Curcumin treatment down-regulated the expression of Skp2 and enhanced the level of p27 and P21. SCC25, FaDu, and SCC090 cells were treated with various doses of curcumin for 24 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immuno-blotted with antibodies against Skp2, p27, p21, and GAPDH as indicated (C) Skp2 siRNA transfection downregulates Skp2 and accumulated p27 and p21. FaDu and SCC090 cells were transfected with Scrambled siRNA (100 pmol) and Skp2 siRNA (50 and 100 pmol) using Lipofectamine 2000 as described in Materials and Methods. After 48 h of transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against Skp2, p27, p21, and GAPDH as indicated.

Techniques Used: SDS Page, Expressing, Lysis, Transfection

6) Product Images from "Renal Damaging Effect Elicited by Bicalutamide Therapy Uncovered Multiple Action Mechanisms As Evidenced by the Cell Model"

Article Title: Renal Damaging Effect Elicited by Bicalutamide Therapy Uncovered Multiple Action Mechanisms As Evidenced by the Cell Model

Journal: Scientific Reports

doi: 10.1038/s41598-019-39533-3

Dose dependent effect of co-treatment with testosterone and bicalutamide on the survival and apoptotic pathways in the RMC cells. ( a ) TNF-α and TNFR. ( b ) Total and phosphorated PI3K, PDGF-R, and Akt. ( c ) Caspase-3 and cleaved caspase-3. Experiment was performed in triplicate and statistically treated (n = 3). The symbol ‘*’ compares within the same group; and ‘#’ compares among groups. * p
Figure Legend Snippet: Dose dependent effect of co-treatment with testosterone and bicalutamide on the survival and apoptotic pathways in the RMC cells. ( a ) TNF-α and TNFR. ( b ) Total and phosphorated PI3K, PDGF-R, and Akt. ( c ) Caspase-3 and cleaved caspase-3. Experiment was performed in triplicate and statistically treated (n = 3). The symbol ‘*’ compares within the same group; and ‘#’ compares among groups. * p

Techniques Used:

Expression of signaling proteins and Annexin V% affected by testosterone and bicalutamide. ( a ) PI3K and Akt proteins. ( b ) The expression of p-Akt and total Akt when treated as indicated. ( c ) Apoptotic effect of SC79 and a combination of SC79 plus MK2206. The dose(s) used were: testosterone (T) 100 nM; bicalutamide (7.5, 15, 30 and 60 μM); SC79 10 μM, and MK2206 1 μM. Triplicate experiments were statistically treated. Data expressed in mean ± S.D. (n = 3).
Figure Legend Snippet: Expression of signaling proteins and Annexin V% affected by testosterone and bicalutamide. ( a ) PI3K and Akt proteins. ( b ) The expression of p-Akt and total Akt when treated as indicated. ( c ) Apoptotic effect of SC79 and a combination of SC79 plus MK2206. The dose(s) used were: testosterone (T) 100 nM; bicalutamide (7.5, 15, 30 and 60 μM); SC79 10 μM, and MK2206 1 μM. Triplicate experiments were statistically treated. Data expressed in mean ± S.D. (n = 3).

Techniques Used: Expressing

7) Product Images from "Sodium nitrite-derived nitric oxide protects rat testes against ischemia/reperfusion injury"

Article Title: Sodium nitrite-derived nitric oxide protects rat testes against ischemia/reperfusion injury

Journal: Asian Journal of Andrology

doi: 10.4103/aja.aja_76_18

Immunohistochemistry (×400) and western blot analysis of caspase-3 in ipsilateral testis. ( a ) The sham group has few caspase-3-positive cells. The number of stained cells is significantly higher in groups ( b ) B and ( d ) G than that in Group A. ( c ) The number of stained cells is significantly lower in Group C. Scale bars = 50 μm. ( e ) The number of caspase-3-positive cells per seminiferous tubule is shown. ( f ) The western blot image for caspase-3 in left testis is shown. ( g ) The relative caspase-3/β-actin expression in Groups A and C is significantly lower than that in Group B. a P
Figure Legend Snippet: Immunohistochemistry (×400) and western blot analysis of caspase-3 in ipsilateral testis. ( a ) The sham group has few caspase-3-positive cells. The number of stained cells is significantly higher in groups ( b ) B and ( d ) G than that in Group A. ( c ) The number of stained cells is significantly lower in Group C. Scale bars = 50 μm. ( e ) The number of caspase-3-positive cells per seminiferous tubule is shown. ( f ) The western blot image for caspase-3 in left testis is shown. ( g ) The relative caspase-3/β-actin expression in Groups A and C is significantly lower than that in Group B. a P

Techniques Used: Immunohistochemistry, Western Blot, Staining, Expressing

8) Product Images from "Repression of Noxa by Bmi1 contributes to deguelin‐induced apoptosis in non‐small cell lung cancer cells, et al. Repression of Noxa by Bmi1 contributes to deguelin‐induced apoptosis in non‐small cell lung cancer cells"

Article Title: Repression of Noxa by Bmi1 contributes to deguelin‐induced apoptosis in non‐small cell lung cancer cells, et al. Repression of Noxa by Bmi1 contributes to deguelin‐induced apoptosis in non‐small cell lung cancer cells

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13908

Deguelin inhibits the binding of Bmi1 to the Noxa gene locus in NSCLC cells. A, Schematic representation of the Noxa locus and the location of primers (#1 to #4) used in Ch IP experiments and exons are indicated. B‐D, NCI ‐H23 (B), NCI ‐H1299 (C), and NCI ‐H460 (D) cells were treated with DMSO or the indicated concentrations of deguelin for 48 hours and subjected to Ch IP assays with an antibody against Bmi1 or normal rabbit IgG. The precipitated DNA fragments were subjected to PCR analysis to test for the presence of sequences corresponding to the Noxa gene locus. Input material (10%) was shown for comparison
Figure Legend Snippet: Deguelin inhibits the binding of Bmi1 to the Noxa gene locus in NSCLC cells. A, Schematic representation of the Noxa locus and the location of primers (#1 to #4) used in Ch IP experiments and exons are indicated. B‐D, NCI ‐H23 (B), NCI ‐H1299 (C), and NCI ‐H460 (D) cells were treated with DMSO or the indicated concentrations of deguelin for 48 hours and subjected to Ch IP assays with an antibody against Bmi1 or normal rabbit IgG. The precipitated DNA fragments were subjected to PCR analysis to test for the presence of sequences corresponding to the Noxa gene locus. Input material (10%) was shown for comparison

Techniques Used: Binding Assay, Polymerase Chain Reaction

Involvement of Bmi1 in deguelin‐induced Noxa expression in NSCLC cells. A, Deguelin increases the Noxa promoter activity in NSCLC cells. Dual luciferase reporter assays of plasmid DNA encoding a fragment of human Noxa promoter in NSCLC cells were performed as described in Materials and Methods. NCI ‐H23, NCI ‐H1299, and NCI ‐H460 cells were transfected with the Noxa promoter reporter plasmid ( pGL 3‐Noxa‐N1 ) or pGL 3‐Basi c vector and then exposed to deguelin for 48 hours. Firefly luciferase readings were normalized to Renilla luciferase to correct for transfection efficiency. The Noxa promoter‐driven luciferase activities were expressed as fold induction over the activity of pGL 3‐Basic vector. Data represent mean ± SD from two independent experiments performed in triplicate. * P
Figure Legend Snippet: Involvement of Bmi1 in deguelin‐induced Noxa expression in NSCLC cells. A, Deguelin increases the Noxa promoter activity in NSCLC cells. Dual luciferase reporter assays of plasmid DNA encoding a fragment of human Noxa promoter in NSCLC cells were performed as described in Materials and Methods. NCI ‐H23, NCI ‐H1299, and NCI ‐H460 cells were transfected with the Noxa promoter reporter plasmid ( pGL 3‐Noxa‐N1 ) or pGL 3‐Basi c vector and then exposed to deguelin for 48 hours. Firefly luciferase readings were normalized to Renilla luciferase to correct for transfection efficiency. The Noxa promoter‐driven luciferase activities were expressed as fold induction over the activity of pGL 3‐Basic vector. Data represent mean ± SD from two independent experiments performed in triplicate. * P

Techniques Used: Expressing, Activity Assay, Luciferase, Plasmid Preparation, Transfection

Effect of deguelin on the expression of apoptotic‐related proteins in NSCLC cells. A, NCI ‐H23, NCI ‐H1299, and NCI ‐H460 NSCLC cells were treated with DMSO or the indicated concentrations of deguelin in medium containing 10% of FBS for 48 hours. After treatment, attached and floating cells were harvested. Expression of the indicated proteins was analysed by Western blotting with specific antibodies. β‐actin was used as a loading control. B, H1299‐sh GFP , and H1299‐shNoxa stable cells were treated with deguelin as indicated, whole cell extract was analysed by Western blotting with specific antibodies. C, H1299‐sh GFP , and H1299‐shBmi1 stable cells were treated with deguelin as indicated, whole cell extract was analysed by Western blotting with specific antibodies. D and E, H1299 cells were treated with a panel of inhibitors as indicated, Western blotting was performed to detect apoptosis. F, H1299, and A549 cells were transfected with the Bmi1 plasmid, whole cell extract, cytosolic fractions, and mitochondrial fractions were subjected to Western blotting analysis with specific primary antibodies
Figure Legend Snippet: Effect of deguelin on the expression of apoptotic‐related proteins in NSCLC cells. A, NCI ‐H23, NCI ‐H1299, and NCI ‐H460 NSCLC cells were treated with DMSO or the indicated concentrations of deguelin in medium containing 10% of FBS for 48 hours. After treatment, attached and floating cells were harvested. Expression of the indicated proteins was analysed by Western blotting with specific antibodies. β‐actin was used as a loading control. B, H1299‐sh GFP , and H1299‐shNoxa stable cells were treated with deguelin as indicated, whole cell extract was analysed by Western blotting with specific antibodies. C, H1299‐sh GFP , and H1299‐shBmi1 stable cells were treated with deguelin as indicated, whole cell extract was analysed by Western blotting with specific antibodies. D and E, H1299 cells were treated with a panel of inhibitors as indicated, Western blotting was performed to detect apoptosis. F, H1299, and A549 cells were transfected with the Bmi1 plasmid, whole cell extract, cytosolic fractions, and mitochondrial fractions were subjected to Western blotting analysis with specific primary antibodies

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

Expressions of Bmi1 and Noxa in human non‐small cell lung cancer. A, Western blot analysis was performed to examine Bmi1 and Noxa expressions in several NSCLC cell lines and normal MRC 5 lung cells. β‐actin was used as a loading control. B, Bmi1 and Noxa protein levels in six representative NSCLC cases was assessed by Western blot analysis. β‐actin was used as a loading control. N, normal adjacent tissue; T, tumour ( left panel ). Western blotting determined Bmi1 and Noxa protein levels in the malignant and the corresponding normal adjacent tissues of 22 NSCLC patients ( right panel ). The intensity was evaluated using Image J ( NIH ) computer software. *** P
Figure Legend Snippet: Expressions of Bmi1 and Noxa in human non‐small cell lung cancer. A, Western blot analysis was performed to examine Bmi1 and Noxa expressions in several NSCLC cell lines and normal MRC 5 lung cells. β‐actin was used as a loading control. B, Bmi1 and Noxa protein levels in six representative NSCLC cases was assessed by Western blot analysis. β‐actin was used as a loading control. N, normal adjacent tissue; T, tumour ( left panel ). Western blotting determined Bmi1 and Noxa protein levels in the malignant and the corresponding normal adjacent tissues of 22 NSCLC patients ( right panel ). The intensity was evaluated using Image J ( NIH ) computer software. *** P

Techniques Used: Western Blot, Software

Deguelin suppresses tumour growth in vivo. A and B, Deguelin significantly inhibited tumour growth in H1299 (A) and A549 (B) xenograft mouse models. C and D, The tumour weight from vehicle‐ and deguelin‐treated group was measured in H1299 (C) and A549 (D) xenograft mouse models. E, Immunohistochemical staining examination of Ki67, Bmi1, Noxa, and cleaved caspase‐3 in H1299 tumour sections from the mice of the vehicle‐ or deguelin‐treated group. All panels are of the same magnification (** P
Figure Legend Snippet: Deguelin suppresses tumour growth in vivo. A and B, Deguelin significantly inhibited tumour growth in H1299 (A) and A549 (B) xenograft mouse models. C and D, The tumour weight from vehicle‐ and deguelin‐treated group was measured in H1299 (C) and A549 (D) xenograft mouse models. E, Immunohistochemical staining examination of Ki67, Bmi1, Noxa, and cleaved caspase‐3 in H1299 tumour sections from the mice of the vehicle‐ or deguelin‐treated group. All panels are of the same magnification (** P

Techniques Used: In Vivo, Immunohistochemistry, Staining, Mouse Assay

9) Product Images from "The DEK Oncoprotein Functions in Ovarian Cancer Growth and Survival"

Article Title: The DEK Oncoprotein Functions in Ovarian Cancer Growth and Survival

Journal: Neoplasia (New York, N.Y.)

doi: 10.1016/j.neo.2018.10.005

Quantification of immunoblot band intensity from Figure 3 . CAOV3, OVCAR8, or OVCAR3 cells were infected with lentivirus expressing control or DEK-targeting shRNAs. Twenty-four hours after infection, cells were treated with cisplatin, doxorubicin, or panobinostat for 48 hours. After treatment, cells were lysed, and samples containing equal amounts of protein were analyzed by immunoblotting with indicated antibodies. The histograms show quantification of cleaved caspase 9, cleaved caspase 3, or Mcl-1 expression normalized to shControl mock. For at least two independent experiments, densitometry was quantified using ImageJ. Mean and standard deviation are shown. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc test, * P
Figure Legend Snippet: Quantification of immunoblot band intensity from Figure 3 . CAOV3, OVCAR8, or OVCAR3 cells were infected with lentivirus expressing control or DEK-targeting shRNAs. Twenty-four hours after infection, cells were treated with cisplatin, doxorubicin, or panobinostat for 48 hours. After treatment, cells were lysed, and samples containing equal amounts of protein were analyzed by immunoblotting with indicated antibodies. The histograms show quantification of cleaved caspase 9, cleaved caspase 3, or Mcl-1 expression normalized to shControl mock. For at least two independent experiments, densitometry was quantified using ImageJ. Mean and standard deviation are shown. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc test, * P

Techniques Used: Infection, Expressing, Standard Deviation

10) Product Images from "SLC25A22 Promotes Proliferation and Metastasis of Osteosarcoma Cells via the PTEN Signaling Pathway"

Article Title: SLC25A22 Promotes Proliferation and Metastasis of Osteosarcoma Cells via the PTEN Signaling Pathway

Journal: Technology in Cancer Research & Treatment

doi: 10.1177/1533033818811143

A, SLC25A22 promotes the invasion and metastasis of osteosarcoma cells by altering the PTEN signaling pathway. Wound healing assay: stable SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells were plated in 6-well plates for wound healing experiments, and healing was observed and photographed at 24 hours. B, Cell invasion assay: stable SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells were plated in Transwells and the invaded cells were stained with crystal violet and photographed. C, E-cadherin, vimentin, and MMP-9 protein levels were detected by Western blot in SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells. D, PTEN, phosphorylated Akt, and phosphorylated FAK were detected by Western blot in SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells. E, Control and SLC25A22 knockdown Saos-2 cells were used to construct a lung metastasis model, and HE staining showed 2 groups of lung metastases. F, Comparison of the number of lung metastatic cells in the control and SLC25A22 knockdown groups. G, The number of mice with lung metastases in the two groups, control and SLC25A22 knockdown lung metastasis model mice.
Figure Legend Snippet: A, SLC25A22 promotes the invasion and metastasis of osteosarcoma cells by altering the PTEN signaling pathway. Wound healing assay: stable SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells were plated in 6-well plates for wound healing experiments, and healing was observed and photographed at 24 hours. B, Cell invasion assay: stable SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells were plated in Transwells and the invaded cells were stained with crystal violet and photographed. C, E-cadherin, vimentin, and MMP-9 protein levels were detected by Western blot in SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells. D, PTEN, phosphorylated Akt, and phosphorylated FAK were detected by Western blot in SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells. E, Control and SLC25A22 knockdown Saos-2 cells were used to construct a lung metastasis model, and HE staining showed 2 groups of lung metastases. F, Comparison of the number of lung metastatic cells in the control and SLC25A22 knockdown groups. G, The number of mice with lung metastases in the two groups, control and SLC25A22 knockdown lung metastasis model mice.

Techniques Used: Wound Healing Assay, Invasion Assay, Staining, Western Blot, Construct, Mouse Assay

11) Product Images from "Adjudin synergizes with paclitaxel and inhibits cell growth and metastasis by regulating the sirtuin 3–Forkhead box O3a axis in human small‐cell lung cancer"

Article Title: Adjudin synergizes with paclitaxel and inhibits cell growth and metastasis by regulating the sirtuin 3–Forkhead box O3a axis in human small‐cell lung cancer

Journal: Thoracic Cancer

doi: 10.1111/1759-7714.12976

Overexpression of sirtuin 3 (SIRT3) enhanced the efficacy of Adjudin in small‐cell lung cancer cells. ( a ) Western blot analysis of SIRT3 and Forkhead box O3a (FOXO3a) in vector and SIRT3‐overexpressed transfected cells. After transfection for 24 hours, cells were treated with Adjudin (60 μM) for another 24 hours. ( b ) Western blot analysis of SIRT3, FOXO3A, cleaved caspase‐3 and EMT‐related marker (E‐cadherin, N‐cadherin and vimentin) levels vehicle ( ) ctrl, ( ) vector, and ( ) SIRT3, and Adjudin ( ) ctrl, ( ) Vector, and ( ) SIRT3. ( c ) Apoptotic cells were assessed by nuclear fragmentation and condensation (arrows) using DAPI staining. Cells were treated as described in ( b ) NCI‐H446 ( ) vehicle, and ( ) Adjudin and DMS114 ( ) vehicle, and ( ) Adjudin. ( d ) Cell proliferation was detected using Cell Counting Kit‐8 assays. Cells were treated as described in B NCI‐H446 ( ) vector, and ( ) SIRT3 and DMS114 ( ) vector, and ( ) SIRT3. ( e ) Scratch NCI‐H446 ( ) vehicle, and ( ) Adjudin and ( f ) Transwell assays were used to evaluate cell migration NCI‐H446 ( ) vehicle, and ( ) Adjudin and DMS114 ( ) vehicle, and ( ) Adjudin. Cells were treated similarly as in Fig 4 b, with Adjudin (40 μM). Values are presented as the mean ± SD, n = 3; one‐way anova test: * P
Figure Legend Snippet: Overexpression of sirtuin 3 (SIRT3) enhanced the efficacy of Adjudin in small‐cell lung cancer cells. ( a ) Western blot analysis of SIRT3 and Forkhead box O3a (FOXO3a) in vector and SIRT3‐overexpressed transfected cells. After transfection for 24 hours, cells were treated with Adjudin (60 μM) for another 24 hours. ( b ) Western blot analysis of SIRT3, FOXO3A, cleaved caspase‐3 and EMT‐related marker (E‐cadherin, N‐cadherin and vimentin) levels vehicle ( ) ctrl, ( ) vector, and ( ) SIRT3, and Adjudin ( ) ctrl, ( ) Vector, and ( ) SIRT3. ( c ) Apoptotic cells were assessed by nuclear fragmentation and condensation (arrows) using DAPI staining. Cells were treated as described in ( b ) NCI‐H446 ( ) vehicle, and ( ) Adjudin and DMS114 ( ) vehicle, and ( ) Adjudin. ( d ) Cell proliferation was detected using Cell Counting Kit‐8 assays. Cells were treated as described in B NCI‐H446 ( ) vector, and ( ) SIRT3 and DMS114 ( ) vector, and ( ) SIRT3. ( e ) Scratch NCI‐H446 ( ) vehicle, and ( ) Adjudin and ( f ) Transwell assays were used to evaluate cell migration NCI‐H446 ( ) vehicle, and ( ) Adjudin and DMS114 ( ) vehicle, and ( ) Adjudin. Cells were treated similarly as in Fig 4 b, with Adjudin (40 μM). Values are presented as the mean ± SD, n = 3; one‐way anova test: * P

Techniques Used: Over Expression, Western Blot, Plasmid Preparation, Transfection, Marker, Staining, Cell Counting, Migration

Adjudin upregulated Forkhead box O3a (FOXO3a) by activating sirtuin 3 (SIRT3). ( a ) Western blot analysis of SIRT3, FOXO3A, cleaved caspase‐3, and EMT‐related proteins. NCI‐H446 cells were transfected with FOXO3A siRNA or NC siRNA in SIRT3‐overexpressing NCI‐H446 cells. After 24 hours, cells were treated with Adjudin (60 μM) for an additional 24 hours. Then, cell lysates were harvested ( ) SIRT3+NC, ( ) SIRT3+NC, ( ) SIRT3+si‐FOXO3a, and ( ) SIRT3+si‐FOXO3a. ( b ) Apoptotic cells were assessed by nuclear fragmentation and condensation (arrows) using DAPI staining. Scale bar, 100 μm. Cells were treated as described in Fig 6 a ( ) Vehicle, and ( ) Adjudin. ( c ) Cell proliferation was detected using Cell Counting Kit‐8 assays. They were treated as described in ( a ) ( ) SIRT3+NC, and ( ) SIRT3+si‐FOXO3a. ( d ) Transwell assays were used to access cell migration. Cells were treated similarly as in Fig 6 a, with Adjudin (40 μM) ( ) vehicle, and ( ) Adjudin. Values are presented as the mean ± SD, n = 3; one‐way anova test: * P
Figure Legend Snippet: Adjudin upregulated Forkhead box O3a (FOXO3a) by activating sirtuin 3 (SIRT3). ( a ) Western blot analysis of SIRT3, FOXO3A, cleaved caspase‐3, and EMT‐related proteins. NCI‐H446 cells were transfected with FOXO3A siRNA or NC siRNA in SIRT3‐overexpressing NCI‐H446 cells. After 24 hours, cells were treated with Adjudin (60 μM) for an additional 24 hours. Then, cell lysates were harvested ( ) SIRT3+NC, ( ) SIRT3+NC, ( ) SIRT3+si‐FOXO3a, and ( ) SIRT3+si‐FOXO3a. ( b ) Apoptotic cells were assessed by nuclear fragmentation and condensation (arrows) using DAPI staining. Scale bar, 100 μm. Cells were treated as described in Fig 6 a ( ) Vehicle, and ( ) Adjudin. ( c ) Cell proliferation was detected using Cell Counting Kit‐8 assays. They were treated as described in ( a ) ( ) SIRT3+NC, and ( ) SIRT3+si‐FOXO3a. ( d ) Transwell assays were used to access cell migration. Cells were treated similarly as in Fig 6 a, with Adjudin (40 μM) ( ) vehicle, and ( ) Adjudin. Values are presented as the mean ± SD, n = 3; one‐way anova test: * P

Techniques Used: Western Blot, Transfection, Staining, Cell Counting, Migration

Knockdown of sirtuin 3 (SIRT3) partially restored the anticancer effect of Adjudin on small‐cell lung cancer. (a–c) Cells were treated with Adjudin as mentioned in Fig 1 a. Then, the expression of sirtuin 3 (SIRT3) and Forkhead box O3a (FOXO3a) was assessed by western blotting. ( a ) GAPDH was used as a loading control NCI‐H446 ( ) 0 μM, ( ) 20 μM, ( ) 40 μM, ( ) 60 μM, ( ) 80 μM, and ( ) 100 μM, and DMS114 ( ) 0 μM, ( ) 20 μM, ( ) 40 μM, ( ) 60 μM, ( ) 80 μM, and ( ) 100 μM. ( b ) The knockdown effect of SIRT3 silencing plasmid was determined by western blotting. NCI‐H446 and DMS114 cells were transfected with vehicle or SIRT3 silencing shRNA with Lipofectamine 3000 reagent for 24 hours, and then cells were exposed to Adjudin for another 24 hours, western blot analysis of cleaved‐caspase‐3 (c‐c‐3) (Adjudin: 60 μM) and EMT‐related protein (E‐cadherin, N‐cadherin, and vimentin) levels (Adjudin 40 μM) ( c ) Vehicle ( ) SCR, ( ) sh1, and ( ) sh2 and Adjudin ( ) SCR, ( ) sh1, and ( ) sh2. ( d ) Cells were treated as described in Fig 3 c (Adjudin: 60 μM). Afterwards, cell proliferation was detected using Cell Counting Kit‐8 assays NCI‐H466 ( ) SCR, ( ) sh1, and ( ) sh2 and DMS114 ( ) SCR, ( ) sh1, and ( ) sh2. ( e ) Apoptotic cells were assessed by nuclear fragmentation and condensation (arrows) using DAPI staining. Scale bar, 100 μm. Cells were treated similarly as in Fig 3 c ( ) vehicle, and ( ) Adjudin. (f) Transwell assays were used to access cell migration. Cells were treated similarly as in Fig 3 c, with Adjudin (40 μM) ( ) vehicle, and ( ) Adjudin. Data were representative of three independent experiments. Values are presented as the mean ± SD, n = 3; one‐way anova test: * P
Figure Legend Snippet: Knockdown of sirtuin 3 (SIRT3) partially restored the anticancer effect of Adjudin on small‐cell lung cancer. (a–c) Cells were treated with Adjudin as mentioned in Fig 1 a. Then, the expression of sirtuin 3 (SIRT3) and Forkhead box O3a (FOXO3a) was assessed by western blotting. ( a ) GAPDH was used as a loading control NCI‐H446 ( ) 0 μM, ( ) 20 μM, ( ) 40 μM, ( ) 60 μM, ( ) 80 μM, and ( ) 100 μM, and DMS114 ( ) 0 μM, ( ) 20 μM, ( ) 40 μM, ( ) 60 μM, ( ) 80 μM, and ( ) 100 μM. ( b ) The knockdown effect of SIRT3 silencing plasmid was determined by western blotting. NCI‐H446 and DMS114 cells were transfected with vehicle or SIRT3 silencing shRNA with Lipofectamine 3000 reagent for 24 hours, and then cells were exposed to Adjudin for another 24 hours, western blot analysis of cleaved‐caspase‐3 (c‐c‐3) (Adjudin: 60 μM) and EMT‐related protein (E‐cadherin, N‐cadherin, and vimentin) levels (Adjudin 40 μM) ( c ) Vehicle ( ) SCR, ( ) sh1, and ( ) sh2 and Adjudin ( ) SCR, ( ) sh1, and ( ) sh2. ( d ) Cells were treated as described in Fig 3 c (Adjudin: 60 μM). Afterwards, cell proliferation was detected using Cell Counting Kit‐8 assays NCI‐H466 ( ) SCR, ( ) sh1, and ( ) sh2 and DMS114 ( ) SCR, ( ) sh1, and ( ) sh2. ( e ) Apoptotic cells were assessed by nuclear fragmentation and condensation (arrows) using DAPI staining. Scale bar, 100 μm. Cells were treated similarly as in Fig 3 c ( ) vehicle, and ( ) Adjudin. (f) Transwell assays were used to access cell migration. Cells were treated similarly as in Fig 3 c, with Adjudin (40 μM) ( ) vehicle, and ( ) Adjudin. Data were representative of three independent experiments. Values are presented as the mean ± SD, n = 3; one‐way anova test: * P

Techniques Used: Expressing, Western Blot, Plasmid Preparation, Transfection, shRNA, Cell Counting, Staining, Migration

The prognostic and diagnostic values of sirtuin 3 (SIRT3) and Forkhead box O3a (FOXO3a) in lung cancer patients according to databases. ( a ) SIRT3 and FOXO3a levels in small cell lung cancer tumor and normal tissues from the GEO database (65 samples in total; 23 samples were small cell lung cancer tumors, and 42 samples were normal tissues; Student's t ‐test, * P
Figure Legend Snippet: The prognostic and diagnostic values of sirtuin 3 (SIRT3) and Forkhead box O3a (FOXO3a) in lung cancer patients according to databases. ( a ) SIRT3 and FOXO3a levels in small cell lung cancer tumor and normal tissues from the GEO database (65 samples in total; 23 samples were small cell lung cancer tumors, and 42 samples were normal tissues; Student's t ‐test, * P

Techniques Used: Diagnostic Assay

Adjudin synergized with paclitaxel in vitro . ( a ) The proliferation of NCI‐H446 and DMS114 cells was detected using Cell Counting Kit‐8 viability assays when Adjudin was combined with cisplatin, etoposide, irinotecan, or paclitaxel in a ratio of 1:1 after 48 hours as indicated (see Methods). The results reveal synergy with paclitaxel (combination indexes [CI] average 0.756) ( ) paclitaxel, ( ) Adjudin, and ( ) combination (1:1), antagonism with cisplatin (CI average 1.557) ( ) cisplatin, ( ) Adjudin, and ( ) combination (1:1), and addition with etoposide and irinotecan (CI 0.997and 1.09, respectively) ( ) etoposide, ( ) Adjudin, and ( ) combination (1:1) and ( ) irinotecan, ( ) Adjudin, and ( ) combination (1:1) in NCI‐H446. ( b ) Cell cycle analysis of NCI‐H446 cells treated with Adjudin (40 μM), paclitaxel (440 nM), and the combination after 24 hours. ( c ) The protein levels of sirtuin 3 (SIRT3) and Forkhead box O3a (FOXO3a) were analyzed by western blotting NCI‐H446 ( ) ctrl, ( ) Adjudin, ( ) paclitaxel, and ( ) combination (1:1) and DMS114 ( ) ctrl, ( ) Adjudin, ( ) paclitaxel, and ( ) combination (1:1). Cells were treated as described in Fig 7 b. Values are presented as the mean ± SD, n = 3; one‐way anova test: * P
Figure Legend Snippet: Adjudin synergized with paclitaxel in vitro . ( a ) The proliferation of NCI‐H446 and DMS114 cells was detected using Cell Counting Kit‐8 viability assays when Adjudin was combined with cisplatin, etoposide, irinotecan, or paclitaxel in a ratio of 1:1 after 48 hours as indicated (see Methods). The results reveal synergy with paclitaxel (combination indexes [CI] average 0.756) ( ) paclitaxel, ( ) Adjudin, and ( ) combination (1:1), antagonism with cisplatin (CI average 1.557) ( ) cisplatin, ( ) Adjudin, and ( ) combination (1:1), and addition with etoposide and irinotecan (CI 0.997and 1.09, respectively) ( ) etoposide, ( ) Adjudin, and ( ) combination (1:1) and ( ) irinotecan, ( ) Adjudin, and ( ) combination (1:1) in NCI‐H446. ( b ) Cell cycle analysis of NCI‐H446 cells treated with Adjudin (40 μM), paclitaxel (440 nM), and the combination after 24 hours. ( c ) The protein levels of sirtuin 3 (SIRT3) and Forkhead box O3a (FOXO3a) were analyzed by western blotting NCI‐H446 ( ) ctrl, ( ) Adjudin, ( ) paclitaxel, and ( ) combination (1:1) and DMS114 ( ) ctrl, ( ) Adjudin, ( ) paclitaxel, and ( ) combination (1:1). Cells were treated as described in Fig 7 b. Values are presented as the mean ± SD, n = 3; one‐way anova test: * P

Techniques Used: In Vitro, Cell Counting, Cell Cycle Assay, Western Blot

12) Product Images from "Roles of microRNA-21 and PDCD-4 in the pathogenesis of human uterine leiomyomas"

Article Title: Roles of microRNA-21 and PDCD-4 in the pathogenesis of human uterine leiomyomas

Journal: Fertility and sterility

doi: 10.1016/j.fertnstert.2012.05.040

Expression of phosphorylated EF-2 in UtM and UtLM cells following knockdown of miR-21. A) Basal ph-EF2 expression levels in UtM cells and UtLM cells. *Means ± SEM (n=3) ph-EF2 levels normalized to actin are different (P
Figure Legend Snippet: Expression of phosphorylated EF-2 in UtM and UtLM cells following knockdown of miR-21. A) Basal ph-EF2 expression levels in UtM cells and UtLM cells. *Means ± SEM (n=3) ph-EF2 levels normalized to actin are different (P

Techniques Used: Expressing

13) Product Images from "Caspase- and Serine Protease-dependent Apoptosis by the Death Domain of FADD in Normal Epithelial Cells V⃞"

Article Title: Caspase- and Serine Protease-dependent Apoptosis by the Death Domain of FADD in Normal Epithelial Cells V⃞

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-04-0207

FADD-DD inhibits Fas-induced apoptosis in prostate tumor cells. DU145 cells were infected with the YFP or YFP-FADD-DD adenoviruses, expression was induced by removing Dox, and then cells were treated with an anti-Fas agonistic antibody. (A) Cellular morphology indicating Fas-induced apoptosis that is inhibited by YFP-FADD-DD but not by YFP. (B) Total protein samples Western blotted with anticaspase 8. Fas stimulates caspase 8 as indicated by the appearance of the processed caspase 8 band that is inhibited by YFP-FADD-DD.
Figure Legend Snippet: FADD-DD inhibits Fas-induced apoptosis in prostate tumor cells. DU145 cells were infected with the YFP or YFP-FADD-DD adenoviruses, expression was induced by removing Dox, and then cells were treated with an anti-Fas agonistic antibody. (A) Cellular morphology indicating Fas-induced apoptosis that is inhibited by YFP-FADD-DD but not by YFP. (B) Total protein samples Western blotted with anticaspase 8. Fas stimulates caspase 8 as indicated by the appearance of the processed caspase 8 band that is inhibited by YFP-FADD-DD.

Techniques Used: Infection, Expressing, Western Blot

14) Product Images from "Ginkgolides and bilobalide protect BV2 microglia cells against OGD/reoxygenation injury by inhibiting TLR2/4 signaling pathways"

Article Title: Ginkgolides and bilobalide protect BV2 microglia cells against OGD/reoxygenation injury by inhibiting TLR2/4 signaling pathways

Journal: Cell Stress & Chaperones

doi: 10.1007/s12192-016-0728-y

Effects of ginkgolides and bilobalide on the expression of Bcl-2,BAx,Bak, RIP3, Caspase 3, and PARP-1 levels in BV2 microglia cells after OGD/R. After exposure to OGD for 4 h, BV2 cells were reoxygenated for either 6 h or 6 h followed by treatment with 50 μg/ml GAPIs, GA, GB, GK, and BB respectively. Representative graph ( a ) and quantitative data on the ratio of Bax/Bcl-2 ( b ), C-Caspase-3/Caspase-3 ( d ), C-PARP-1/ PARP-1 ( e ) , and expression of Bak ( c ) and RIP3 ( f ) in BV2 microglia cells. GAPDH was used as the loading control. Western blot images are representative of three independent experiments. Each data point is a mean ± SD ( n = 3). △ P
Figure Legend Snippet: Effects of ginkgolides and bilobalide on the expression of Bcl-2,BAx,Bak, RIP3, Caspase 3, and PARP-1 levels in BV2 microglia cells after OGD/R. After exposure to OGD for 4 h, BV2 cells were reoxygenated for either 6 h or 6 h followed by treatment with 50 μg/ml GAPIs, GA, GB, GK, and BB respectively. Representative graph ( a ) and quantitative data on the ratio of Bax/Bcl-2 ( b ), C-Caspase-3/Caspase-3 ( d ), C-PARP-1/ PARP-1 ( e ) , and expression of Bak ( c ) and RIP3 ( f ) in BV2 microglia cells. GAPDH was used as the loading control. Western blot images are representative of three independent experiments. Each data point is a mean ± SD ( n = 3). △ P

Techniques Used: Expressing, Western Blot

15) Product Images from "Macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated CHOP induction in myeloma cells"

Article Title: Macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated CHOP induction in myeloma cells

Journal: International Journal of Oncology

doi: 10.3892/ijo.2013.1870

Induction of ER-stress-mediated apoptosis by inhibition of two major protein degradation systems in MM cells. Simultaneous inhibition of the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic results in over-loading ER-stress on MM cells. This leads to activation of ER-stress-mediated apoptotic signals, including CHOP and subsequent upregulation of proapoptotic genes.
Figure Legend Snippet: Induction of ER-stress-mediated apoptosis by inhibition of two major protein degradation systems in MM cells. Simultaneous inhibition of the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic results in over-loading ER-stress on MM cells. This leads to activation of ER-stress-mediated apoptotic signals, including CHOP and subsequent upregulation of proapoptotic genes.

Techniques Used: Inhibition, Activation Assay

Profiles of the ER-stress-related genes and the target genes transcriptionally regulated by CHOP in IM-9 and U266 cells after treatment with BZ and/or CAM. (A) The expression of ER-stress-related genes in IM-9 and U266 cells, including CHOP and proapoptotic genes transcriptionally regulated by CHOP, were assessed by quantitative real-time PCR during 48-h exposure to BZ (10 nM for IM-9 cells, 5 nM for U266 cells), CAM (50 μ g/ml) and BZ+CAM. The data of the real-time PCR products for each gene were standardized to GAPDH as an internal control. The expression levels were compared with those in untreated cells. (B) Immunoblotting with anti-CHOP mAb after combined treatment of U266 cells with BZ and CAM. U266 cells were treated with/without CAM (50 μ g/ml) in the presence or absence of BZ (5 nM) for 24 h. Cellular proteins were separated by 11.25%, then immunoblotted with anti-CHOP mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control.
Figure Legend Snippet: Profiles of the ER-stress-related genes and the target genes transcriptionally regulated by CHOP in IM-9 and U266 cells after treatment with BZ and/or CAM. (A) The expression of ER-stress-related genes in IM-9 and U266 cells, including CHOP and proapoptotic genes transcriptionally regulated by CHOP, were assessed by quantitative real-time PCR during 48-h exposure to BZ (10 nM for IM-9 cells, 5 nM for U266 cells), CAM (50 μ g/ml) and BZ+CAM. The data of the real-time PCR products for each gene were standardized to GAPDH as an internal control. The expression levels were compared with those in untreated cells. (B) Immunoblotting with anti-CHOP mAb after combined treatment of U266 cells with BZ and CAM. U266 cells were treated with/without CAM (50 μ g/ml) in the presence or absence of BZ (5 nM) for 24 h. Cellular proteins were separated by 11.25%, then immunoblotted with anti-CHOP mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control.

Techniques Used: Chick Chorioallantoic Membrane Assay, Expressing, Real-time Polymerase Chain Reaction

Cell growth inhibition of the CHOP −/− MEF cell line and the wild-type MEF cell line after treatment with BZ and EM or CAM. (A) After treatment with BZ (10 nM) for 24 h, cellular proteins were separated by 11.25% SDS-PAGE and immunoblotted with anti-CHOP mAb. (B) The CHOP −/− MEF cell line and the wild-type MEF cell line were cultured with BZ at various concentrations in the presence of absence of EM or CAM (at 25 μ g/ml) for 48 h. The number of viable cells was assessed by CellTiter Blue as described in Materials and methods. p
Figure Legend Snippet: Cell growth inhibition of the CHOP −/− MEF cell line and the wild-type MEF cell line after treatment with BZ and EM or CAM. (A) After treatment with BZ (10 nM) for 24 h, cellular proteins were separated by 11.25% SDS-PAGE and immunoblotted with anti-CHOP mAb. (B) The CHOP −/− MEF cell line and the wild-type MEF cell line were cultured with BZ at various concentrations in the presence of absence of EM or CAM (at 25 μ g/ml) for 48 h. The number of viable cells was assessed by CellTiter Blue as described in Materials and methods. p

Techniques Used: Inhibition, Chick Chorioallantoic Membrane Assay, SDS Page, Cell Culture

Expression profiles of ER-stress-related genes and the target genes transcriptionally regulated by CHOP after treatment with BZ and/or CAM in the wild-type MEF cell line and the CHOP −/− MEF cell line. ER-stress related genes in the wild-type MEF cell line and the CHOP −/− MEF cell line, including CHOP expressions, were assessed by quantitative real-time PCR during 48-h exposure to BZ (10 nM), CAM (50 μ g/ml) and BZ+CAM. The data of the real-time PCR products for each gene were standardized to GAPDH as an internal control. The expression levels were compared with those in untreated wild-type MEF cells.
Figure Legend Snippet: Expression profiles of ER-stress-related genes and the target genes transcriptionally regulated by CHOP after treatment with BZ and/or CAM in the wild-type MEF cell line and the CHOP −/− MEF cell line. ER-stress related genes in the wild-type MEF cell line and the CHOP −/− MEF cell line, including CHOP expressions, were assessed by quantitative real-time PCR during 48-h exposure to BZ (10 nM), CAM (50 μ g/ml) and BZ+CAM. The data of the real-time PCR products for each gene were standardized to GAPDH as an internal control. The expression levels were compared with those in untreated wild-type MEF cells.

Techniques Used: Expressing, Chick Chorioallantoic Membrane Assay, Real-time Polymerase Chain Reaction

16) Product Images from "The polygenetically inherited metabolic syndrome of male WOKW rats is associated with enhanced autophagy in adipose tissue"

Article Title: The polygenetically inherited metabolic syndrome of male WOKW rats is associated with enhanced autophagy in adipose tissue

Journal: Diabetology & Metabolic Syndrome

doi: 10.1186/1758-5996-5-23

Detection of Atg5 and Atg7 expression in visceral fat of WOKW and LEW.1 W rats by immunofluorescence. A significantly higher immunofluorescence intensity of Atg5 and Atg7 was found in visceral fat of WOKW rats compared with LEW.1 W control animals (Figure 2 A , B ). Both proteins could only be detected in stromal cells of the adipose tissue (arrow), rather in adipocytes directly (star; Figure 2 B and f). Scale bars: 15 μm (a - h); 30 μm (b’- h’).
Figure Legend Snippet: Detection of Atg5 and Atg7 expression in visceral fat of WOKW and LEW.1 W rats by immunofluorescence. A significantly higher immunofluorescence intensity of Atg5 and Atg7 was found in visceral fat of WOKW rats compared with LEW.1 W control animals (Figure 2 A , B ). Both proteins could only be detected in stromal cells of the adipose tissue (arrow), rather in adipocytes directly (star; Figure 2 B and f). Scale bars: 15 μm (a - h); 30 μm (b’- h’).

Techniques Used: Expressing, Immunofluorescence

Expression of autophagy markers Atg5 and Atg7 in visceral und subcutaneous fat of WOKW and LEW.1 W rats. Representative Western blots and corresponding densitometrical analyses (Figure 1 A , B ) of Atg5 and Atg7 in visceral and subcutaneous fat of WOKW rats compared to the LEW.1 W control animals. The significant increased expression of Atg5 and Atg7 in visceral fat WOKW rats vs. LEW.1 W control animals. Data from n = 6 are presented as mean ± SEM * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, according to the one-way analysis of variance together with the Newman-Keuls test. GAPDH was used as loading control. AU – arbitrary units.
Figure Legend Snippet: Expression of autophagy markers Atg5 and Atg7 in visceral und subcutaneous fat of WOKW and LEW.1 W rats. Representative Western blots and corresponding densitometrical analyses (Figure 1 A , B ) of Atg5 and Atg7 in visceral and subcutaneous fat of WOKW rats compared to the LEW.1 W control animals. The significant increased expression of Atg5 and Atg7 in visceral fat WOKW rats vs. LEW.1 W control animals. Data from n = 6 are presented as mean ± SEM * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, according to the one-way analysis of variance together with the Newman-Keuls test. GAPDH was used as loading control. AU – arbitrary units.

Techniques Used: Expressing, Western Blot

Expression of LC3-I and LC3-II proteins in visceral und subcutaneous fat WOKW and LEW.1 W rats. The over expression of the LC3-II (membrane bound) protein and a significantly higher LC3-II / LC3-I ratio in subcutaneous fat of WOKW rats vs. LEW.1 W control animals. Representative Western blots and corresponding densitometrical analyses (Figure 3 A ). The presence and location of the LC3 protein in subcutaneous fat of WOKW and LEW.1 W rats is additionally shown by immunostaining. As shown for Atg5 and Atg7, adipocytes were predominately negative for LC3 (star). Strong LC3 staining was found in stroma cells resembling macrophages (arrows; Figure 3 B ). Scale bars: 15 μm (a-d); 50 μm (b’-d’) (Figure 3 B ). Data from n = 6 are presented as mean ± SEM * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, according to the one-way analysis of variance together with the Newman-Keuls test. GAPDH was used as loading control.
Figure Legend Snippet: Expression of LC3-I and LC3-II proteins in visceral und subcutaneous fat WOKW and LEW.1 W rats. The over expression of the LC3-II (membrane bound) protein and a significantly higher LC3-II / LC3-I ratio in subcutaneous fat of WOKW rats vs. LEW.1 W control animals. Representative Western blots and corresponding densitometrical analyses (Figure 3 A ). The presence and location of the LC3 protein in subcutaneous fat of WOKW and LEW.1 W rats is additionally shown by immunostaining. As shown for Atg5 and Atg7, adipocytes were predominately negative for LC3 (star). Strong LC3 staining was found in stroma cells resembling macrophages (arrows; Figure 3 B ). Scale bars: 15 μm (a-d); 50 μm (b’-d’) (Figure 3 B ). Data from n = 6 are presented as mean ± SEM * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, according to the one-way analysis of variance together with the Newman-Keuls test. GAPDH was used as loading control.

Techniques Used: Expressing, Over Expression, Western Blot, Immunostaining, Staining

17) Product Images from "Tumor suppressor PDCD4 modulates miR-184-mediated direct suppression of C-MYC and BCL2 blocking cell growth and survival in nasopharyngeal carcinoma"

Article Title: Tumor suppressor PDCD4 modulates miR-184-mediated direct suppression of C-MYC and BCL2 blocking cell growth and survival in nasopharyngeal carcinoma

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.376

PDCD4 controls the expression of cell cycle and transcription factor C-JUN in NPC via PI3K/AKT/JNK pathway. ( a ) Overexpressed PDCD4 suppressed the expression of pPI3K, pAKT and JNK in clone 1 and 2 cells compared with scramble cells. ( b ) Reduced PDCD4 elevated the expression of pPI3K, pAKT and JNK in shPDCD4 SUNE1 and HONE1 cells compared with their respective controls. ( c ) Restored PDCD4 inhibited the expression of C-MYC, CCND1, pRB (Ser780), E2F1 and C-JUN in clone 1 and 2 cells compared with scramble cells. ( d ) Downregulated PDCD4 expression stimulated the C-MYC, CCND1, pRB (Ser780), E2F1 and C-JUN expression in shPDCD4 SUNE1 and HONE1 cells compared with their respective controls
Figure Legend Snippet: PDCD4 controls the expression of cell cycle and transcription factor C-JUN in NPC via PI3K/AKT/JNK pathway. ( a ) Overexpressed PDCD4 suppressed the expression of pPI3K, pAKT and JNK in clone 1 and 2 cells compared with scramble cells. ( b ) Reduced PDCD4 elevated the expression of pPI3K, pAKT and JNK in shPDCD4 SUNE1 and HONE1 cells compared with their respective controls. ( c ) Restored PDCD4 inhibited the expression of C-MYC, CCND1, pRB (Ser780), E2F1 and C-JUN in clone 1 and 2 cells compared with scramble cells. ( d ) Downregulated PDCD4 expression stimulated the C-MYC, CCND1, pRB (Ser780), E2F1 and C-JUN expression in shPDCD4 SUNE1 and HONE1 cells compared with their respective controls

Techniques Used: Expressing

MiR-184 directly targeted BCL2 and C-MYC suppressing cell cycle and stimulating cell apoptosis signals. ( a ) The introduction of miR-184 mimics suppressed the expression of pRB (ser 780), C-MYC, CCND1 and E2F1 in cell cycle. ( b ) Transfection of miR-184 mimics reduced BCL2 expression and induced the cleavage of CASP9, 3, 6, 7 and PARP in apoptosis pathway. ( c ) BCL2 and C-MYC expression was elevated with the transfection of miR-184 inhibitor at 100 nm in NPC SUNE1 and 5-8F cells. ( d ) Luciferase reporter assay was used to determine miR-184 directly targeting the 3′UTR of BCL2. ( e ) Luciferase reporter assay was used to determine miR-184 directly targeting the coding region of C-MYC. Data are presented as mean±S.D. of three independent experiments (* P
Figure Legend Snippet: MiR-184 directly targeted BCL2 and C-MYC suppressing cell cycle and stimulating cell apoptosis signals. ( a ) The introduction of miR-184 mimics suppressed the expression of pRB (ser 780), C-MYC, CCND1 and E2F1 in cell cycle. ( b ) Transfection of miR-184 mimics reduced BCL2 expression and induced the cleavage of CASP9, 3, 6, 7 and PARP in apoptosis pathway. ( c ) BCL2 and C-MYC expression was elevated with the transfection of miR-184 inhibitor at 100 nm in NPC SUNE1 and 5-8F cells. ( d ) Luciferase reporter assay was used to determine miR-184 directly targeting the 3′UTR of BCL2. ( e ) Luciferase reporter assay was used to determine miR-184 directly targeting the coding region of C-MYC. Data are presented as mean±S.D. of three independent experiments (* P

Techniques Used: Expressing, Transfection, Luciferase, Reporter Assay

18) Product Images from "Vaccinia Virus Induces Programmed Necrosis in Ovarian Cancer Cells"

Article Title: Vaccinia Virus Induces Programmed Necrosis in Ovarian Cancer Cells

Journal: Molecular Therapy

doi: 10.1038/mt.2013.195

Necrosis mediators following vaccinia infection. ( a ) Formation of a RIP1/RIP3/caspase 8 complex following infection with Lister-dTK (MOI 10) for 96 hours. Caspase-8 was immunoprecipitated and the presence of RIP1, RIP3, and caspase-8 detected by immunoblotting. Cells were treated with 100 µmol/l etoposide or 10 ng/ml TNF-α in the presence of zVAD-fmk (25 µmol/l) as positive controls for necrosis. ( b ) Expression of RIP1, RIP3, and caspase-8 was assessed following infection with Lister-dTK (MOI 10) for 72 hours. The proteasome inhibitor MG132 (10 µmol/l) was added 5 hours before cell lysates were collected, and p21 expression assessed as positive control for MG132 activity.
Figure Legend Snippet: Necrosis mediators following vaccinia infection. ( a ) Formation of a RIP1/RIP3/caspase 8 complex following infection with Lister-dTK (MOI 10) for 96 hours. Caspase-8 was immunoprecipitated and the presence of RIP1, RIP3, and caspase-8 detected by immunoblotting. Cells were treated with 100 µmol/l etoposide or 10 ng/ml TNF-α in the presence of zVAD-fmk (25 µmol/l) as positive controls for necrosis. ( b ) Expression of RIP1, RIP3, and caspase-8 was assessed following infection with Lister-dTK (MOI 10) for 72 hours. The proteasome inhibitor MG132 (10 µmol/l) was added 5 hours before cell lysates were collected, and p21 expression assessed as positive control for MG132 activity.

Techniques Used: Infection, Immunoprecipitation, Expressing, Positive Control, Activity Assay

TNF-α blockade, necrostatin, and RNAi knockdown of RIP1 and RIP3. ( a ) IGROV1 cells with stable shRNA-mediated TNF-α knockdown (IGROV1-shTNF) and control cells encoding scrambled shRNA (IGROV1-shControl) were infected with Lister-dTK in triplicate. Cell survival was assessed 72 hours postinfection (left). Experiments were repeated twice—representative data are presented. TNF-α expression was quantified by quantitative RT-PCR, normalized to 18S. Bars represent mean ± SD, n = 3. * P
Figure Legend Snippet: TNF-α blockade, necrostatin, and RNAi knockdown of RIP1 and RIP3. ( a ) IGROV1 cells with stable shRNA-mediated TNF-α knockdown (IGROV1-shTNF) and control cells encoding scrambled shRNA (IGROV1-shControl) were infected with Lister-dTK in triplicate. Cell survival was assessed 72 hours postinfection (left). Experiments were repeated twice—representative data are presented. TNF-α expression was quantified by quantitative RT-PCR, normalized to 18S. Bars represent mean ± SD, n = 3. * P

Techniques Used: shRNA, Infection, Expressing, Quantitative RT-PCR

19) Product Images from "Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway"

Article Title: Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway

Journal: PLoS ONE

doi: 10.1371/journal.pone.0059330

Increases in ROS induced by MMC triggers ER stress that can be blocked by antioxidants. (A) ROS levels were detected by using DCFH-DA and observed via fluorescence microscopy (200x magnification) 48 hours after MMC treatment. Nuclei are shown in blue and ROS staining in green. The fibroblasts were pretreated with 10 mM NAC, 10 mM GSH and 10 µM edaravone for 2 hours, respectively, then treated with MMC (0.4 mg/ml, 5 minutes) and incubated for 48 hours. (B) MDA generation was measured and is shown in the histogram. (C) The mean of DCF fluorescence intensity, which is indicative of ROS generation, was measured via flow cytometry and is depicted graphically as the relative fold increase of the control. (D) Cell viability and (E) apoptosis rates were examined 48 hours after MMC treatment using CCK-8 assays or Annexin V/propidium iodide double staining. (F) Western blot analysis of related proteins in fibroblasts 24 hours after MMC treatment. The expression of GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, PARP, cleaved PARP and β-actin (loading control) was analyzed. (G) The band intensities for GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, and cleaved PARP are shown as a histogram. The control group without MMC treatment was normalized to a value of 1.0-fold. The gel data results from the gels come from experiments performed in triplicate with similar results. The presented bar graphs shown in panels B, C, D and E are the average results from three different experiments. *P
Figure Legend Snippet: Increases in ROS induced by MMC triggers ER stress that can be blocked by antioxidants. (A) ROS levels were detected by using DCFH-DA and observed via fluorescence microscopy (200x magnification) 48 hours after MMC treatment. Nuclei are shown in blue and ROS staining in green. The fibroblasts were pretreated with 10 mM NAC, 10 mM GSH and 10 µM edaravone for 2 hours, respectively, then treated with MMC (0.4 mg/ml, 5 minutes) and incubated for 48 hours. (B) MDA generation was measured and is shown in the histogram. (C) The mean of DCF fluorescence intensity, which is indicative of ROS generation, was measured via flow cytometry and is depicted graphically as the relative fold increase of the control. (D) Cell viability and (E) apoptosis rates were examined 48 hours after MMC treatment using CCK-8 assays or Annexin V/propidium iodide double staining. (F) Western blot analysis of related proteins in fibroblasts 24 hours after MMC treatment. The expression of GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, PARP, cleaved PARP and β-actin (loading control) was analyzed. (G) The band intensities for GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, and cleaved PARP are shown as a histogram. The control group without MMC treatment was normalized to a value of 1.0-fold. The gel data results from the gels come from experiments performed in triplicate with similar results. The presented bar graphs shown in panels B, C, D and E are the average results from three different experiments. *P

Techniques Used: Fluorescence, Microscopy, Staining, Incubation, Multiple Displacement Amplification, Flow Cytometry, Cytometry, CCK-8 Assay, Double Staining, Western Blot, Expressing

The role of three UPR sensors in MMC-induced apoptosis in fibroblasts. Fibroblasts were transfected with the PERK, ATF6, IRE1 or non-targeting lentiviral-mediated shRNAs. (A) The expression levels of the targeted transcripts were determined by Western blotting with PERK, ATF6, IRE1 and β-actin antibodies. The presented data represents the results of two independent experiments. (B) Transfected cells were treated with MMC as described in the text and then cell viability was measured after 48 hours. (C) Apoptosis rates were determined via Annexin V/propidium iodide double staining and are shown in the bar graph. (D) After MMC treatment (0.4 mg/ml, 5 minutes) and incubation for 48 hours, equal amounts of the whole cell lysates were analyzed by Western blotting with antibodies specific for CHOP, BIM, cleaved caspase-3, and β-actin (loading control). This experiment was performed in triplicate. The data presented in panels B and C are the mean ± SD of three independent experiments, *P
Figure Legend Snippet: The role of three UPR sensors in MMC-induced apoptosis in fibroblasts. Fibroblasts were transfected with the PERK, ATF6, IRE1 or non-targeting lentiviral-mediated shRNAs. (A) The expression levels of the targeted transcripts were determined by Western blotting with PERK, ATF6, IRE1 and β-actin antibodies. The presented data represents the results of two independent experiments. (B) Transfected cells were treated with MMC as described in the text and then cell viability was measured after 48 hours. (C) Apoptosis rates were determined via Annexin V/propidium iodide double staining and are shown in the bar graph. (D) After MMC treatment (0.4 mg/ml, 5 minutes) and incubation for 48 hours, equal amounts of the whole cell lysates were analyzed by Western blotting with antibodies specific for CHOP, BIM, cleaved caspase-3, and β-actin (loading control). This experiment was performed in triplicate. The data presented in panels B and C are the mean ± SD of three independent experiments, *P

Techniques Used: Transfection, Expressing, Western Blot, Double Staining, Incubation

20) Product Images from "Glycyrrhizin Represses Total Parenteral Nutrition-Associated Acute Liver Injury in Rats by Suppressing Endoplasmic Reticulum Stress"

Article Title: Glycyrrhizin Represses Total Parenteral Nutrition-Associated Acute Liver Injury in Rats by Suppressing Endoplasmic Reticulum Stress

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms140612563

The effect of glycyrrhizin (GL) on liver histological SOCS3 and apoptotic pathway protein expression in rat livers. ( A ) Western blot analysis of proteins related to SOCS3 and the apoptotic pathway in the control, TPN and GL-pretreated groups; ( B ) SOCS3, CHOP and MAPK signaling protein expression were measured. The TPN group significantly increased SOCS3 protein expression, whereas SOC3 protein expression was decreased in the GL pretreatment groups. The phosphorylated of p38 MAPK and JNK proteins was increased in the TPN groups compared to the control and pretreatment groups. CHOP protein was also increased following TPN treatment. Equal amounts of protein from the total cell lysates of rat livers pretreated with GL were analyzed. β-actin served as an internal control. Protein levels were quantified using densitometric analysis and the control was set at 100%. The data depicted in bar graph are the mean ± SD of three independent measurements ( n = 6 in each group). * p
Figure Legend Snippet: The effect of glycyrrhizin (GL) on liver histological SOCS3 and apoptotic pathway protein expression in rat livers. ( A ) Western blot analysis of proteins related to SOCS3 and the apoptotic pathway in the control, TPN and GL-pretreated groups; ( B ) SOCS3, CHOP and MAPK signaling protein expression were measured. The TPN group significantly increased SOCS3 protein expression, whereas SOC3 protein expression was decreased in the GL pretreatment groups. The phosphorylated of p38 MAPK and JNK proteins was increased in the TPN groups compared to the control and pretreatment groups. CHOP protein was also increased following TPN treatment. Equal amounts of protein from the total cell lysates of rat livers pretreated with GL were analyzed. β-actin served as an internal control. Protein levels were quantified using densitometric analysis and the control was set at 100%. The data depicted in bar graph are the mean ± SD of three independent measurements ( n = 6 in each group). * p

Techniques Used: Expressing, Western Blot

The effect of glycyrrhizin (GL) on liver cytokine-inducible SOCS3 protein levels. ( A ) Immunohistochemical staining for SOCS3 in rat livers was analyzed in different liver zonal areas: control rats (I); rats with TPN infusion (II); GL-pretreated rats (10 mg/kg) (III) staining of the portal and terminal hepatic venues zones; ( B ) The quantitation of immunohistochemical staining of SOCS3 levels by average integrated optical density (AIOD). The positive stained area was evaluated from three randomly selected observation fields of each liver section. The data represent the mean ± SD ( n = 6/group). * p
Figure Legend Snippet: The effect of glycyrrhizin (GL) on liver cytokine-inducible SOCS3 protein levels. ( A ) Immunohistochemical staining for SOCS3 in rat livers was analyzed in different liver zonal areas: control rats (I); rats with TPN infusion (II); GL-pretreated rats (10 mg/kg) (III) staining of the portal and terminal hepatic venues zones; ( B ) The quantitation of immunohistochemical staining of SOCS3 levels by average integrated optical density (AIOD). The positive stained area was evaluated from three randomly selected observation fields of each liver section. The data represent the mean ± SD ( n = 6/group). * p

Techniques Used: Immunohistochemistry, Staining, Quantitation Assay

21) Product Images from "Effect of Serenoa repens (Permixon(R)) on the expression of inflammation-related genes: analysis in primary cell cultures of human prostate carcinoma"

Article Title: Effect of Serenoa repens (Permixon(R)) on the expression of inflammation-related genes: analysis in primary cell cultures of human prostate carcinoma

Journal: Journal of Inflammation (London, England)

doi: 10.1186/1476-9255-10-11

GADPH, IL-6, CCL-5 (RANTES), CCL-2, COX-1, COX-2 and iNOS RT-PCR products separated on a 2% agarose gel followed by ethidium bromide staining in PC3, LNCaP and primary cells lines. In PC3 (panel 1) and LNCaP (panel 2) analysis was performed at 8, 24 and 72 hours of incubation whereas in primary cell lines (panel 3) at 16 hours of interval. Here we report results obtained from one prostatectomy, representative of 40 independent experiments. The experiment was performed in untreated and LSESr treated (44 μg/ml and 88 μg/ml in PC3 and LNCaP and only 44 μg/ml in primary cultures) conditions. UN = untreated; LS = LSESr treated; T = primary cells culture derived from PC nodules; Ct = primary cells culture derived from normal prostate tissue surrounding the tumor.
Figure Legend Snippet: GADPH, IL-6, CCL-5 (RANTES), CCL-2, COX-1, COX-2 and iNOS RT-PCR products separated on a 2% agarose gel followed by ethidium bromide staining in PC3, LNCaP and primary cells lines. In PC3 (panel 1) and LNCaP (panel 2) analysis was performed at 8, 24 and 72 hours of incubation whereas in primary cell lines (panel 3) at 16 hours of interval. Here we report results obtained from one prostatectomy, representative of 40 independent experiments. The experiment was performed in untreated and LSESr treated (44 μg/ml and 88 μg/ml in PC3 and LNCaP and only 44 μg/ml in primary cultures) conditions. UN = untreated; LS = LSESr treated; T = primary cells culture derived from PC nodules; Ct = primary cells culture derived from normal prostate tissue surrounding the tumor.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Incubation, Derivative Assay

Cytitoxicity assay in LNCaP cell and PC3 lines. Results are presented at 48 and 72 hours of incubation, either in untreated or in Permixon treated (44 and 88 μg/ml) conditions. The graphic shows the reduction of cell growth according to the absorbance of treated and untreated cultures (XTT Cell Proliferation kit Sigma-Aldrich).
Figure Legend Snippet: Cytitoxicity assay in LNCaP cell and PC3 lines. Results are presented at 48 and 72 hours of incubation, either in untreated or in Permixon treated (44 and 88 μg/ml) conditions. The graphic shows the reduction of cell growth according to the absorbance of treated and untreated cultures (XTT Cell Proliferation kit Sigma-Aldrich).

Techniques Used: Incubation

Detection of caspase-3 in apoptotic cells. Immunocytochemistry (immunofluorescence) with anti-cleaved caspase3 by rhodamine indirect labelling. The upper panels are PC3 untreated ( a ) and treated ( b ) with LSESr for 16 hours. The lower panels are primary cells line untreated ( c ) and treated ( d ) with Permixon® for 16 hours. Untreated cells show no labelling with the anti-cleaved caspase-3 antibody and TRITC anti IgG. LSERSr treated cells show (arrows) labelling with anti-cleaved caspase-3.
Figure Legend Snippet: Detection of caspase-3 in apoptotic cells. Immunocytochemistry (immunofluorescence) with anti-cleaved caspase3 by rhodamine indirect labelling. The upper panels are PC3 untreated ( a ) and treated ( b ) with LSESr for 16 hours. The lower panels are primary cells line untreated ( c ) and treated ( d ) with Permixon® for 16 hours. Untreated cells show no labelling with the anti-cleaved caspase-3 antibody and TRITC anti IgG. LSERSr treated cells show (arrows) labelling with anti-cleaved caspase-3.

Techniques Used: Immunocytochemistry, Immunofluorescence

NF-κB detection. Immunofluorescence of p65 NF-κB in PC3 ( a, b, c ) and LNCaP ( d, e, f ) cell lines. In untreated condition ( a, d ) NF-κB is 100% detected in the cytoplasm of cells (arrows). After LSESr (44 μg/ml) treatment ( b, e ) = 24 hours and ( c, f ) = 48 hours of incubation more than 30% of NF-κB translocated at nuclear level (arrows).
Figure Legend Snippet: NF-κB detection. Immunofluorescence of p65 NF-κB in PC3 ( a, b, c ) and LNCaP ( d, e, f ) cell lines. In untreated condition ( a, d ) NF-κB is 100% detected in the cytoplasm of cells (arrows). After LSESr (44 μg/ml) treatment ( b, e ) = 24 hours and ( c, f ) = 48 hours of incubation more than 30% of NF-κB translocated at nuclear level (arrows).

Techniques Used: Immunofluorescence, Incubation

Cell counting by trypsan blue in LNCaP and PC3 cell lines. Results are presented at 24, 48 and 72 hours of incubation, either in untreated or in Permixon® treated (44 and 88 μg/ml) conditions. Cells were seeded at different concentration, lower for longer incubation time in order to avoid overgrowth. The same amount of cells was used to start each incubation time, control and treated cells. a) LNCaP. b) PC3. * = p
Figure Legend Snippet: Cell counting by trypsan blue in LNCaP and PC3 cell lines. Results are presented at 24, 48 and 72 hours of incubation, either in untreated or in Permixon® treated (44 and 88 μg/ml) conditions. Cells were seeded at different concentration, lower for longer incubation time in order to avoid overgrowth. The same amount of cells was used to start each incubation time, control and treated cells. a) LNCaP. b) PC3. * = p

Techniques Used: Cell Counting, Incubation, Concentration Assay

22) Product Images from "Specific deletion of glycogen synthase kinase-3? in the renal proximal tubule protects against acute nephrotoxic injury in mice"

Article Title: Specific deletion of glycogen synthase kinase-3? in the renal proximal tubule protects against acute nephrotoxic injury in mice

Journal: Kidney international

doi: 10.1038/ki.2012.239

HgCl 2 induced apoptosis is reduced in KO mice a) TUNEL nuclear staining in renal cortex, 24h after HgCl 2 treatment and b) quantitation which shows reduced TUNEL positive nuclei in KO group. c) Cleaved caspase 3 and activated Bax levels are lower by Western blot in KO kidney compared to WT (d e). **, P
Figure Legend Snippet: HgCl 2 induced apoptosis is reduced in KO mice a) TUNEL nuclear staining in renal cortex, 24h after HgCl 2 treatment and b) quantitation which shows reduced TUNEL positive nuclei in KO group. c) Cleaved caspase 3 and activated Bax levels are lower by Western blot in KO kidney compared to WT (d e). **, P

Techniques Used: Mouse Assay, TUNEL Assay, Staining, Quantitation Assay, Western Blot

23) Product Images from "Unexpected Role of the Steroid-Deficiency Protein Ecdysoneless in Pre-mRNA Splicing"

Article Title: Unexpected Role of the Steroid-Deficiency Protein Ecdysoneless in Pre-mRNA Splicing

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004287

Loss of Ecd interferes with splicing of spok pre-mRNA. (A) Schematic of Drosophila spok and phm gene loci. Open and colored boxes represent untranslated and translated exons, respectively; black arrows point in the direction of transcription. Colored arrowheads mark the positions of primer pairs used to discriminate between pre-mRNA and mRNA species by qRT-PCR: yellow, in exons separated by an intron; red, within an intron; green and blue, spanning exon-intron boundaries of the first (EI1) and second (EI2) spok exon, respectively. (B) Pre-mRNA:mRNA ratios, determined by qRT-PCR with primers depicted in (A), were significantly elevated for spok but not for phm in third-instar phm > ecd RNAi larvae and in ecd 1 mutants at 29°C. (C, D) spok and phm pre-mRNA (C) and mRNA (D) levels decreased in phm > ecd RNAi and ecd 1 larvae. Levels of α-tubulin 84B mRNA did not change upon ecd RNAi and expression of ecd itself was unaffected by the ecd 1 mutation (D). (E) RNAi knockdown of prp8 in the PG diminished spok mRNA, whereas unspliced transcripts accumulated, causing the pre-mRNA:mRNA ratio to rise dramatically. (F) Expression of hEcd in the Ecd-deficient PG restored normal spok transcription and pre-mRNA splicing as judged from restored levels of pre-mRNA, mRNA, and their ratio. In all experiments, qRT-PCR was performed with total RNA from whole larvae 6 days AEL, and levels of rp49 transcripts were used for normalization. The pre-mRNA:mRNA ratios in (B, E, F) were calculated from the normalized qRT-PCR data by dividing values obtained with intron primer sets (A, red triangles) or primers spanning exon-intron boundaries (green or blue triangles) with values obtained using mRNA-specific primers (yellow triangles). Data are mean ± S.E.M; n ≥4; * p
Figure Legend Snippet: Loss of Ecd interferes with splicing of spok pre-mRNA. (A) Schematic of Drosophila spok and phm gene loci. Open and colored boxes represent untranslated and translated exons, respectively; black arrows point in the direction of transcription. Colored arrowheads mark the positions of primer pairs used to discriminate between pre-mRNA and mRNA species by qRT-PCR: yellow, in exons separated by an intron; red, within an intron; green and blue, spanning exon-intron boundaries of the first (EI1) and second (EI2) spok exon, respectively. (B) Pre-mRNA:mRNA ratios, determined by qRT-PCR with primers depicted in (A), were significantly elevated for spok but not for phm in third-instar phm > ecd RNAi larvae and in ecd 1 mutants at 29°C. (C, D) spok and phm pre-mRNA (C) and mRNA (D) levels decreased in phm > ecd RNAi and ecd 1 larvae. Levels of α-tubulin 84B mRNA did not change upon ecd RNAi and expression of ecd itself was unaffected by the ecd 1 mutation (D). (E) RNAi knockdown of prp8 in the PG diminished spok mRNA, whereas unspliced transcripts accumulated, causing the pre-mRNA:mRNA ratio to rise dramatically. (F) Expression of hEcd in the Ecd-deficient PG restored normal spok transcription and pre-mRNA splicing as judged from restored levels of pre-mRNA, mRNA, and their ratio. In all experiments, qRT-PCR was performed with total RNA from whole larvae 6 days AEL, and levels of rp49 transcripts were used for normalization. The pre-mRNA:mRNA ratios in (B, E, F) were calculated from the normalized qRT-PCR data by dividing values obtained with intron primer sets (A, red triangles) or primers spanning exon-intron boundaries (green or blue triangles) with values obtained using mRNA-specific primers (yellow triangles). Data are mean ± S.E.M; n ≥4; * p

Techniques Used: Quantitative RT-PCR, Expressing, Mutagenesis

Ecd and Prp8 are required for expression of Spok in the PG. (A–D) Relative to control PG dissected 6 days AEL (A′, A″), expression of the Spok protein (B′) was undetected while the Phm signal (B″) was weakened in the PG of phm > ecd RNAi (B) and phm > prp8 RNAi (D′, D″) larvae. Note the moderate reduction in size of PG cells and nuclei in phm > ecd RNAi (B) compared to a more severe PG deterioration in phm > prp8 RNAi larvae (D). Expression of hEcd restored Spok and Phm expression (C′, C″) and improved the morphology of the Ecd-deficient PG (C). Cell membranes are decorated with CD8::GFP; DAPI stains the nuclei. Panels show single confocal sections. Scale bars, 20 µm. See Figure S1 for RNAi-mediated depletion of Ecd.
Figure Legend Snippet: Ecd and Prp8 are required for expression of Spok in the PG. (A–D) Relative to control PG dissected 6 days AEL (A′, A″), expression of the Spok protein (B′) was undetected while the Phm signal (B″) was weakened in the PG of phm > ecd RNAi (B) and phm > prp8 RNAi (D′, D″) larvae. Note the moderate reduction in size of PG cells and nuclei in phm > ecd RNAi (B) compared to a more severe PG deterioration in phm > prp8 RNAi larvae (D). Expression of hEcd restored Spok and Phm expression (C′, C″) and improved the morphology of the Ecd-deficient PG (C). Cell membranes are decorated with CD8::GFP; DAPI stains the nuclei. Panels show single confocal sections. Scale bars, 20 µm. See Figure S1 for RNAi-mediated depletion of Ecd.

Techniques Used: Expressing

24) Product Images from "Lasiodin Inhibits Proliferation of Human Nasopharyngeal Carcinoma Cells by Simultaneous Modulation of the Apaf-1/Caspase, AKT/MAPK and COX-2/NF-?B Signaling Pathways"

Article Title: Lasiodin Inhibits Proliferation of Human Nasopharyngeal Carcinoma Cells by Simultaneous Modulation of the Apaf-1/Caspase, AKT/MAPK and COX-2/NF-?B Signaling Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0097799

Inhibition of PI3K/AKT signaling by lasiodin. ( A ), CNE1 and CNE2 cells were treated with lasiodin at the indicated doses. After 24 hr treatment, the expressions of the phosphorylated or total protein of AKT and PI3K were detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. GAPDH was used as the control for sample loading. ( B ), CNE1 and CNE2 cells were treated with the AKT-selective inhibitor (LY294002, 5 µM) for 4 hr, and then treated with lasiodin at the indicated doses. After 48 hr treatment, cell viability was determined by the MTT assay. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P
Figure Legend Snippet: Inhibition of PI3K/AKT signaling by lasiodin. ( A ), CNE1 and CNE2 cells were treated with lasiodin at the indicated doses. After 24 hr treatment, the expressions of the phosphorylated or total protein of AKT and PI3K were detected by Western blotting. NPC cells treated with the vehicle control (DMSO) were used as the reference group. GAPDH was used as the control for sample loading. ( B ), CNE1 and CNE2 cells were treated with the AKT-selective inhibitor (LY294002, 5 µM) for 4 hr, and then treated with lasiodin at the indicated doses. After 48 hr treatment, cell viability was determined by the MTT assay. The figures are representative of three experiments. The data are presented as mean ± S.D. of three separate experiments. * P

Techniques Used: Inhibition, Western Blot, MTT Assay

25) Product Images from "Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication"

Article Title: Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication

Journal: Oncotarget

doi:

Silencing of Cx43 blocks GJIC and induces therapy resistance and clonogenicity (A) BxPc-3 cells were treated with non-specific siRNAs (siCO) or specific siRNAs directed against Cx43 (siCx43) for 3 days. The protein expression of Cx43 was analyzed by Western blot on days 1, 2 and 3 after incubation with siRNA or 3 d after incubation with siRNA at concentrations of 50, 100 or 200 pmol, as described in Fig. 3A. (B) BxPc-3 cells were treated with 50 pmol siRNA for 72 h followed by microinjection of Lucifer Yellow, and the monitoring of dye diffusion was performed as described in Fig. 1C. Pictures were extracted from the videos at distinct time points, and the gray values were calculated. The average gray value of the first neighbors (1) and second neighbors (2) and the gray value of the injected cell (0) are shown as the percentage over a period of 100 s. The data shown represent the means of 3 independent experiments. (C) The cells were treated with 50 pmol siRNA against Cx43 (siCx43) or non-specific control siRNA (siCO) for 72 h. Then, the cells were left untreated or were treated with gemcitabine for 24 h, followed by co-culture at a ratio of 1:1 for 24 h. The viability of untreated cells (CO, white bars) and of cells co-cultured with gemcitabine-treated cells (GEM-Byst, black bars) was measured using the MTT assay. (D) Three days after transfection with siCO or siCx43, 1×10 3 BxPc-3-GEM cells/well were seeded onto 6-well plates. The cells were grown without a change of the medium for 2 weeks, followed by the evaluation of fixed and Coomassie-stained colonies containing at least 50 cells. The percentage of plating efficiency was calculated with the formula 100× number of colonies/number of seeded cells. The data shown represent the means ± SD (**p
Figure Legend Snippet: Silencing of Cx43 blocks GJIC and induces therapy resistance and clonogenicity (A) BxPc-3 cells were treated with non-specific siRNAs (siCO) or specific siRNAs directed against Cx43 (siCx43) for 3 days. The protein expression of Cx43 was analyzed by Western blot on days 1, 2 and 3 after incubation with siRNA or 3 d after incubation with siRNA at concentrations of 50, 100 or 200 pmol, as described in Fig. 3A. (B) BxPc-3 cells were treated with 50 pmol siRNA for 72 h followed by microinjection of Lucifer Yellow, and the monitoring of dye diffusion was performed as described in Fig. 1C. Pictures were extracted from the videos at distinct time points, and the gray values were calculated. The average gray value of the first neighbors (1) and second neighbors (2) and the gray value of the injected cell (0) are shown as the percentage over a period of 100 s. The data shown represent the means of 3 independent experiments. (C) The cells were treated with 50 pmol siRNA against Cx43 (siCx43) or non-specific control siRNA (siCO) for 72 h. Then, the cells were left untreated or were treated with gemcitabine for 24 h, followed by co-culture at a ratio of 1:1 for 24 h. The viability of untreated cells (CO, white bars) and of cells co-cultured with gemcitabine-treated cells (GEM-Byst, black bars) was measured using the MTT assay. (D) Three days after transfection with siCO or siCx43, 1×10 3 BxPc-3-GEM cells/well were seeded onto 6-well plates. The cells were grown without a change of the medium for 2 weeks, followed by the evaluation of fixed and Coomassie-stained colonies containing at least 50 cells. The percentage of plating efficiency was calculated with the formula 100× number of colonies/number of seeded cells. The data shown represent the means ± SD (**p

Techniques Used: Expressing, Western Blot, Incubation, Diffusion-based Assay, Injection, Co-Culture Assay, Cell Culture, MTT Assay, Transfection, Staining

Sulforaphane induces Cx43 and inhibits CSC characteristics in primary CSCs (A) Staining of patient-derived frozen tissue from a ductal adenocarcinoma (Primary Tumor 22) and it´s derived xenograft from passage 3 (Xenograft P3) with Trichrome, c-Met or Cx43 phosphorylated at Ser 368 (Abcam), followed by microscopical evaluation under 400× magnification. The scale bar indicates 50 μm. (B) Representative picture of an anchorage-independent growing spheroidal culture established from a mouse xenograft derived from the primary patient tumor 22. The percentage of expression of the CSC markers c-Met and CD133 was determined as described in part C and is indicated. (C) One week after in vitro spheroidal culture, cells derived from pancreatic ductal adenocarcinoma 22 and 30 were left untreated or were treated with sulforaphane (10 μM). Twenty-four hours later, the cells were cytospinned to glass slides, and the expression of Cx43 phosphorylated at Ser 368 (Abcam), c-Met, CD133, E-cadherin and the cleaved, active fragment of Caspase-3 was examined by immunohistochemistry. The number of positive cells was quantified in 10 vision fields under 400× magnification and the means ± SD are shown in the diagrams. **p
Figure Legend Snippet: Sulforaphane induces Cx43 and inhibits CSC characteristics in primary CSCs (A) Staining of patient-derived frozen tissue from a ductal adenocarcinoma (Primary Tumor 22) and it´s derived xenograft from passage 3 (Xenograft P3) with Trichrome, c-Met or Cx43 phosphorylated at Ser 368 (Abcam), followed by microscopical evaluation under 400× magnification. The scale bar indicates 50 μm. (B) Representative picture of an anchorage-independent growing spheroidal culture established from a mouse xenograft derived from the primary patient tumor 22. The percentage of expression of the CSC markers c-Met and CD133 was determined as described in part C and is indicated. (C) One week after in vitro spheroidal culture, cells derived from pancreatic ductal adenocarcinoma 22 and 30 were left untreated or were treated with sulforaphane (10 μM). Twenty-four hours later, the cells were cytospinned to glass slides, and the expression of Cx43 phosphorylated at Ser 368 (Abcam), c-Met, CD133, E-cadherin and the cleaved, active fragment of Caspase-3 was examined by immunohistochemistry. The number of positive cells was quantified in 10 vision fields under 400× magnification and the means ± SD are shown in the diagrams. **p

Techniques Used: Staining, Derivative Assay, Expressing, In Vitro, Immunohistochemistry

Loss of Cx43 expression in cells with CSC features. (A) Proteins were harvested from PDA cells and immortalized pancreatic ductal cells (CRL-4023), and the expression of Cx26, Cx32, Cx36, Cx43 (anti-Cx43 Ab was from Invitrogen) and Cx45 was analyzed by Western blot. The expression of β-Actin (β-Act) served as a loading control. (B) The expression and cellular localization of Cx43 (Cell Signaling) was analyzed by double immunofluorescence staining using antibodies specific for Cx43. Co-staining with the cell surface protein EpCAM was performed to mark the cell surface. Cx43-positive cells were visualized with Alexa Fluor 488 (green), and EpCAM-positive cells were visualized with Alexa Fluor 594 (red). The arrows mark Cx43 expression on the cell surface. Cells were analyzed under 400× magnification using a Leica DMRB fluorescence microscope. Images of representative fields were captured using a Kappa CF 20/4 DX digital color camera (Kappa GmbH, Gleichen, Germany) and Kappa ImageBase 2.2 software. All photographs were taken at the same magnification, and the scale bar indicates 20 μm. (C) Electron microscopy of gap junction-like cell-cell contacts of BxPc-3 and AsPC-1 cells. The arrows indicate the extension of junctions with a narrow gap in BxPc-3 cells, and the inserts show details of the gaps at higher magnification. The scale bar indicates 1 μm.
Figure Legend Snippet: Loss of Cx43 expression in cells with CSC features. (A) Proteins were harvested from PDA cells and immortalized pancreatic ductal cells (CRL-4023), and the expression of Cx26, Cx32, Cx36, Cx43 (anti-Cx43 Ab was from Invitrogen) and Cx45 was analyzed by Western blot. The expression of β-Actin (β-Act) served as a loading control. (B) The expression and cellular localization of Cx43 (Cell Signaling) was analyzed by double immunofluorescence staining using antibodies specific for Cx43. Co-staining with the cell surface protein EpCAM was performed to mark the cell surface. Cx43-positive cells were visualized with Alexa Fluor 488 (green), and EpCAM-positive cells were visualized with Alexa Fluor 594 (red). The arrows mark Cx43 expression on the cell surface. Cells were analyzed under 400× magnification using a Leica DMRB fluorescence microscope. Images of representative fields were captured using a Kappa CF 20/4 DX digital color camera (Kappa GmbH, Gleichen, Germany) and Kappa ImageBase 2.2 software. All photographs were taken at the same magnification, and the scale bar indicates 20 μm. (C) Electron microscopy of gap junction-like cell-cell contacts of BxPc-3 and AsPC-1 cells. The arrows indicate the extension of junctions with a narrow gap in BxPc-3 cells, and the inserts show details of the gaps at higher magnification. The scale bar indicates 1 μm.

Techniques Used: Expressing, Western Blot, Activated Clotting Time Assay, Double Immunofluorescence Staining, Staining, Fluorescence, Microscopy, Software, Electron Microscopy

Cx43 is downregulated in malignant pancreas tissue but not in normal pancreas tissue (A) The expression of Cx43 phosphorylated at Ser 368 (Ab from Abcam) was analyzed by immunohistochemistry in frozen 6-μm-thick tissue sections of non-malignant human pancreatic tissue derived from 6 organ donors. Labeled anti-rabbit polymer-HRP was used as the secondary antibody. (B) Likewise, the expression of Cx43 phosphorylated at Ser 279/282 was analyzed in the same tissues and by the same technique.
Figure Legend Snippet: Cx43 is downregulated in malignant pancreas tissue but not in normal pancreas tissue (A) The expression of Cx43 phosphorylated at Ser 368 (Ab from Abcam) was analyzed by immunohistochemistry in frozen 6-μm-thick tissue sections of non-malignant human pancreatic tissue derived from 6 organ donors. Labeled anti-rabbit polymer-HRP was used as the secondary antibody. (B) Likewise, the expression of Cx43 phosphorylated at Ser 279/282 was analyzed in the same tissues and by the same technique.

Techniques Used: Expressing, Immunohistochemistry, Derivative Assay, Labeling

Cx43 and E-Cadherin are downregulated in malignant but not in non-malignant parts of pancreas tissue from the same patients (A) The expression of total Cx43 (Invitrogen) was analyzed in the malignant and non-malignant parts of frozen 6-μm-thick tissue sections of pancreas tissue derived from 3 patients. (B) The expression of total Cx43 (Invitrogen) and E-cadherin (E-CAD) was analyzed by double immunofluorescence staining of malignant and non-malignant parts of a patient-derived PDA tissue section. Cx43-positive cells were visualized with Alexa Fluor 488 (green), and E-cadherin-positive cells were visualized with Alexa Fluor 594 (red). DAPI staining indicates the cell nuclei. Tissue sections were analyzed under 400× magnification. The scale bars indicate 50 μm.
Figure Legend Snippet: Cx43 and E-Cadherin are downregulated in malignant but not in non-malignant parts of pancreas tissue from the same patients (A) The expression of total Cx43 (Invitrogen) was analyzed in the malignant and non-malignant parts of frozen 6-μm-thick tissue sections of pancreas tissue derived from 3 patients. (B) The expression of total Cx43 (Invitrogen) and E-cadherin (E-CAD) was analyzed by double immunofluorescence staining of malignant and non-malignant parts of a patient-derived PDA tissue section. Cx43-positive cells were visualized with Alexa Fluor 488 (green), and E-cadherin-positive cells were visualized with Alexa Fluor 594 (red). DAPI staining indicates the cell nuclei. Tissue sections were analyzed under 400× magnification. The scale bars indicate 50 μm.

Techniques Used: Expressing, Derivative Assay, Double Immunofluorescence Staining, Staining

Sulforaphane enhances GJIC and Cx43 protein expression, which is prevented by inhibition of kinase activity (A) BxPc-3, BxPc-3-GEM and AsPC-1 cells were left untreated or were treated with sulforaphane (SF, 10 μM) and time points indicated. The proteins of all treatment groups were harvested at the same time point, and Western blot analysis was performed using antibodies to detect total Cx43 with an antibody, which does not detect Cx43 phosphorylated at Ser 368 (Invitrogen), Cx43 phosphorylated at Ser 368 (Abcam), Cx43 phosphorylated at Ser 279/282, E-cadherin and β-Actin. (B) BxPc-3 cells were treated with sulforaphane (SF, 10 μM) or were left untreated (CO). Twenty-four hours later, the cells were analyzed as described in Fig. 5B. The mean gray values of the 1 st and 2 nd neighbors of all time points are shown. (C) Electron microscopy of AsPC-1 cells, which were left either untreated or were treated with sulforaphane (SF, 10 μM) for 24 h, was performed as described in Fig. C. (D) AsPC-1 cells were left untreated or were treated with sulforaphane (SF, 10 μM) in the presence or absence of the GSK3-inhibitor BIO (10 μM), the PKC-inhibitor staurosporine (100 nM) or the JNK-inhibitor SP600125 (10 μM). Treatment with the inhibitors alone or with the solvent DMSO served as control. Proteins were harvested 24 h after treatment and the expression of total Cx43, Cx43 phosphorylated at Ser 368 and β-Actin was examined by Western blot analysis.
Figure Legend Snippet: Sulforaphane enhances GJIC and Cx43 protein expression, which is prevented by inhibition of kinase activity (A) BxPc-3, BxPc-3-GEM and AsPC-1 cells were left untreated or were treated with sulforaphane (SF, 10 μM) and time points indicated. The proteins of all treatment groups were harvested at the same time point, and Western blot analysis was performed using antibodies to detect total Cx43 with an antibody, which does not detect Cx43 phosphorylated at Ser 368 (Invitrogen), Cx43 phosphorylated at Ser 368 (Abcam), Cx43 phosphorylated at Ser 279/282, E-cadherin and β-Actin. (B) BxPc-3 cells were treated with sulforaphane (SF, 10 μM) or were left untreated (CO). Twenty-four hours later, the cells were analyzed as described in Fig. 5B. The mean gray values of the 1 st and 2 nd neighbors of all time points are shown. (C) Electron microscopy of AsPC-1 cells, which were left either untreated or were treated with sulforaphane (SF, 10 μM) for 24 h, was performed as described in Fig. C. (D) AsPC-1 cells were left untreated or were treated with sulforaphane (SF, 10 μM) in the presence or absence of the GSK3-inhibitor BIO (10 μM), the PKC-inhibitor staurosporine (100 nM) or the JNK-inhibitor SP600125 (10 μM). Treatment with the inhibitors alone or with the solvent DMSO served as control. Proteins were harvested 24 h after treatment and the expression of total Cx43, Cx43 phosphorylated at Ser 368 and β-Actin was examined by Western blot analysis.

Techniques Used: Expressing, Inhibition, Activity Assay, Western Blot, Electron Microscopy

26) Product Images from "Sulforaphane and TRAIL induce a synergistic elimination of advanced prostate cancer stem-like cells"

Article Title: Sulforaphane and TRAIL induce a synergistic elimination of advanced prostate cancer stem-like cells

Journal: International Journal of Oncology

doi: 10.3892/ijo.2014.2335

Sulforaphane, but not TRAIL, strongly inhibits stem cell-associated signaling and differentiation and their combination enhances these effects. (A) PC3 cells were treated as described in Fig. 1A . Twenty-four hours later, the proteins were isolated and a human pluripotent stem cell array was performed. The binding of proteins to antibodies spotted in duplicate on the membranes was detected using biotinylated secondary antibodies, streptavidin-HRP and chemiluminescence. After normalization to reference spots (positive control A1, A4 and F1; PBS-negative control E4), the pixel density was quantified using ImageJ software. (B) Proteins were also harvested, and a western blot analysis was performed to detect the expression of CXCR4, Jagged1, Notch 1, SOX2 and Nanog. The detection of β-actin served as a control for equal conditions. (C) For ALDH1 evaluation, PC3 cells were treated as described in Fig. 1A ; the activity of ALDH1 was determined by a substrate assay and the turnover was analyzed by flow cytometry. The data are presented as the percentage of ALDH1-positive cells ± SD ( * p
Figure Legend Snippet: Sulforaphane, but not TRAIL, strongly inhibits stem cell-associated signaling and differentiation and their combination enhances these effects. (A) PC3 cells were treated as described in Fig. 1A . Twenty-four hours later, the proteins were isolated and a human pluripotent stem cell array was performed. The binding of proteins to antibodies spotted in duplicate on the membranes was detected using biotinylated secondary antibodies, streptavidin-HRP and chemiluminescence. After normalization to reference spots (positive control A1, A4 and F1; PBS-negative control E4), the pixel density was quantified using ImageJ software. (B) Proteins were also harvested, and a western blot analysis was performed to detect the expression of CXCR4, Jagged1, Notch 1, SOX2 and Nanog. The detection of β-actin served as a control for equal conditions. (C) For ALDH1 evaluation, PC3 cells were treated as described in Fig. 1A ; the activity of ALDH1 was determined by a substrate assay and the turnover was analyzed by flow cytometry. The data are presented as the percentage of ALDH1-positive cells ± SD ( * p

Techniques Used: Isolation, Binding Assay, Positive Control, Negative Control, Software, Western Blot, Expressing, Activity Assay, Flow Cytometry, Cytometry

27) Product Images from "Dihydroartemisinin is a Hypoxia-Active Anti-Cancer Drug in Colorectal Carcinoma Cells"

Article Title: Dihydroartemisinin is a Hypoxia-Active Anti-Cancer Drug in Colorectal Carcinoma Cells

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2014.00116

BH3-only proteins Bim, Puma, Noxa, and BNIP3 are differentially regulated in response DHA in normoxia and hypoxia . HCT116 cells were treated with 0, 12.5, or 25 μM DHA under normoxic or under hypoxic conditions as indicated. Twenty-four hours later, whole cell lysates were made. Levels of anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family were analyzed by western blot. Induction of autophagy was accessed by western blot using an antibody against LC3B and a subsequent analysis of processed LC3B form (LC3B II, 14 kDa). Levels of anti-apoptotic Bcl-2 and Bcl-xL and pro-apoptotic Bax and Bak did not change in response to treatment with DHA under normoxic and hypoxic conditions. Levels of BH3-only protein Noxa were increased in response to DHA in normoxia as well as in hypoxia. Levels of Bim and Puma were only increased by DHA in normoxia, whereas levels of BNIP3 were elevated by DHA only in hypoxia. Induction of autophagy in response to DHA was observed only under normoxic conditions. Protein levels were analyzed by densitometry and normalized to β-actin levels. Relative protein levels are shown below the respective blots. Data show representative blots from at least two independent experiments.
Figure Legend Snippet: BH3-only proteins Bim, Puma, Noxa, and BNIP3 are differentially regulated in response DHA in normoxia and hypoxia . HCT116 cells were treated with 0, 12.5, or 25 μM DHA under normoxic or under hypoxic conditions as indicated. Twenty-four hours later, whole cell lysates were made. Levels of anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family were analyzed by western blot. Induction of autophagy was accessed by western blot using an antibody against LC3B and a subsequent analysis of processed LC3B form (LC3B II, 14 kDa). Levels of anti-apoptotic Bcl-2 and Bcl-xL and pro-apoptotic Bax and Bak did not change in response to treatment with DHA under normoxic and hypoxic conditions. Levels of BH3-only protein Noxa were increased in response to DHA in normoxia as well as in hypoxia. Levels of Bim and Puma were only increased by DHA in normoxia, whereas levels of BNIP3 were elevated by DHA only in hypoxia. Induction of autophagy in response to DHA was observed only under normoxic conditions. Protein levels were analyzed by densitometry and normalized to β-actin levels. Relative protein levels are shown below the respective blots. Data show representative blots from at least two independent experiments.

Techniques Used: Western Blot

28) Product Images from "Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome"

Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

Journal: Nature

doi: 10.1038/nature13585

p53 25,26,53,54/+  Embryos Exhibit Additional Features of CHARGE Syndrome and p53-Dependent Cellular Responses (a)  Double outlet right ventricle (DORV) in E13.5 p53 25,26,53,54/+  heart (50%, n=6). Top: Main pulmonary artery (MPA) connects via pulmonary valve (PV) to right ventricle (RV) in both control and p53 25,26,53,54/+  embryo. Bottom: Aorta (Ao) in control embryo connects to left ventricle (LV) via aortic valve (AV) Φ . Aorta in p53 25,26,53,54/+  embryo connects to RV via AV*. (b)  Abnormal atrioventricular cushions in E13.5 p53 25,26,53,54/+  heart (75%, n=4) fail to elongateinto mature mitral (mv, arrowhead) and tricuspid (tv, arrow) valves. RA: right atrium; LA: left atrium.  (c)  E13.5  p53 25,26,53,54/+  kidneys are smaller (79%), with fewer average glomeruli (13 vs. 3; n=5; arrows), than controls. (d) p53 25,26,53,54/+  embryonic phenotypes observed in CHARGE (+present, −absent).  (e)  Left: Cleaved-caspase 3 (CC3; Top) and p53 (Bottom) immunohistochemistry in E15.5 retinas. Arrows: CC3-positive cells. Right: CC3-positive cells per retinal area. ***p-value=0.007; one-tailed Welsh’s t-test (n=5).  (f)  BrdU immunofluorescence in E9.5 Pax3 +  NCCs (delineated by green-dotted line;  Extended-Data Fig. 6c ). Right: Percentage BrdU-positive cells per total Pax3 +  NCCs ***p-value=0.004 one-tailed Student’s t-test (n=4).
Figure Legend Snippet: p53 25,26,53,54/+ Embryos Exhibit Additional Features of CHARGE Syndrome and p53-Dependent Cellular Responses (a) Double outlet right ventricle (DORV) in E13.5 p53 25,26,53,54/+ heart (50%, n=6). Top: Main pulmonary artery (MPA) connects via pulmonary valve (PV) to right ventricle (RV) in both control and p53 25,26,53,54/+ embryo. Bottom: Aorta (Ao) in control embryo connects to left ventricle (LV) via aortic valve (AV) Φ . Aorta in p53 25,26,53,54/+ embryo connects to RV via AV*. (b) Abnormal atrioventricular cushions in E13.5 p53 25,26,53,54/+ heart (75%, n=4) fail to elongateinto mature mitral (mv, arrowhead) and tricuspid (tv, arrow) valves. RA: right atrium; LA: left atrium. (c) E13.5 p53 25,26,53,54/+ kidneys are smaller (79%), with fewer average glomeruli (13 vs. 3; n=5; arrows), than controls. (d) p53 25,26,53,54/+ embryonic phenotypes observed in CHARGE (+present, −absent). (e) Left: Cleaved-caspase 3 (CC3; Top) and p53 (Bottom) immunohistochemistry in E15.5 retinas. Arrows: CC3-positive cells. Right: CC3-positive cells per retinal area. ***p-value=0.007; one-tailed Welsh’s t-test (n=5). (f) BrdU immunofluorescence in E9.5 Pax3 + NCCs (delineated by green-dotted line; Extended-Data Fig. 6c ). Right: Percentage BrdU-positive cells per total Pax3 + NCCs ***p-value=0.004 one-tailed Student’s t-test (n=4).

Techniques Used: Immunohistochemistry, One-tailed Test, Immunofluorescence

p53 25,26,53,54/+  Embryo Tissues Display Increased Apoptosis and Decreased Proliferation (a)  Left: Immunofluorescence for Phospho-Histone H3 (red) in the retina of E13.5 control and  p53 25,26,53,54/+ embryos. Right: Quantification of Phospho-Histone H3 positive cells per retina area relative to littermate controls. **p-value=0.006 by one-tailed Welsh’s t-test (n=4). ( b ) Left: Immunohistochemistry for cleaved-caspase 3 (CC3) in thymi of control (left) and  p53 25,26,53,54/+  (right) embryos. Inset: close-up image of cleaved-caspase 3 positive region. Right: Quantification of CC3-positive cells per thymic area. *p-value=0.02 by one-tailed Student’s t-test (n=4).  (c)  Immunofluorescence for Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+  embryos was used to identify neural crest cells in   Figure 2f .  (d) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) and Pax3 (green) in neural crest cells of E9.5 control and  p53 25,26,53,54/+ embryos.  p53 25,26,53,54/+  embryos have more apoptotic (red) neural crest cells, as determined by Pax3-positive staining (green), compared to control littermates. Right: Quantification of CC3 positive cells per total neural crest cell number. p-value=0.14 by one-tailed Student’s t-test (n=4). (e)  Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) in otic vesicle of E9.5 control and  p53 25,26,53,54/+  embryos. Right: Quantification of CC3 positive cells per total cell number. *p-value=0.03 by one-tailed Student’s t-test (n=3). ( f ) Whole-mount cleaved-caspase 3 staining in E8.5 control and p53 25,26,53,54/+  embryos reveals enhanced apoptosis in the neuroepithelium of  p53 25,26,53,54/+  embryos (right) but not in controls (left). Close-up shows magnification of the caudal neuroepithelium (bottom). Arrows indicate cleaved-caspase 3 positive regions.
Figure Legend Snippet: p53 25,26,53,54/+ Embryo Tissues Display Increased Apoptosis and Decreased Proliferation (a) Left: Immunofluorescence for Phospho-Histone H3 (red) in the retina of E13.5 control and p53 25,26,53,54/+ embryos. Right: Quantification of Phospho-Histone H3 positive cells per retina area relative to littermate controls. **p-value=0.006 by one-tailed Welsh’s t-test (n=4). ( b ) Left: Immunohistochemistry for cleaved-caspase 3 (CC3) in thymi of control (left) and p53 25,26,53,54/+ (right) embryos. Inset: close-up image of cleaved-caspase 3 positive region. Right: Quantification of CC3-positive cells per thymic area. *p-value=0.02 by one-tailed Student’s t-test (n=4). (c) Immunofluorescence for Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+ embryos was used to identify neural crest cells in Figure 2f . (d) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) and Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+ embryos. p53 25,26,53,54/+ embryos have more apoptotic (red) neural crest cells, as determined by Pax3-positive staining (green), compared to control littermates. Right: Quantification of CC3 positive cells per total neural crest cell number. p-value=0.14 by one-tailed Student’s t-test (n=4). (e) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) in otic vesicle of E9.5 control and p53 25,26,53,54/+ embryos. Right: Quantification of CC3 positive cells per total cell number. *p-value=0.03 by one-tailed Student’s t-test (n=3). ( f ) Whole-mount cleaved-caspase 3 staining in E8.5 control and p53 25,26,53,54/+ embryos reveals enhanced apoptosis in the neuroepithelium of p53 25,26,53,54/+ embryos (right) but not in controls (left). Close-up shows magnification of the caudal neuroepithelium (bottom). Arrows indicate cleaved-caspase 3 positive regions.

Techniques Used: Immunofluorescence, One-tailed Test, Immunohistochemistry, Staining

29) Product Images from "The effects of phosphodiesterase-5 inhibitor sildenafil against post-resuscitation myocardial and intestinal microcirculatory dysfunction by attenuating apoptosis and regulating microRNAs expression: essential role of nitric oxide syntheses signaling"

Article Title: The effects of phosphodiesterase-5 inhibitor sildenafil against post-resuscitation myocardial and intestinal microcirculatory dysfunction by attenuating apoptosis and regulating microRNAs expression: essential role of nitric oxide syntheses signaling

Journal: Journal of Translational Medicine

doi: 10.1186/s12967-015-0550-9

Western blot of a expression of Bax, Bcl-2 and active caspase-3, cGMP, eNOS and iNOS proteins of myocardial tissue in the sham, SA and sildenafil groups at 24 h after ROSC. b Quantification of cGMP, eNOS and iNOS accumulation protein levels. The value represent mean ± SD. *p
Figure Legend Snippet: Western blot of a expression of Bax, Bcl-2 and active caspase-3, cGMP, eNOS and iNOS proteins of myocardial tissue in the sham, SA and sildenafil groups at 24 h after ROSC. b Quantification of cGMP, eNOS and iNOS accumulation protein levels. The value represent mean ± SD. *p

Techniques Used: Western Blot, Expressing

a Western blots of quantification of Bax, Bcl-2 and active caspase-3 protein levels of myocardial tissue in the sham, saline and sildenafil groups at 24 h after ROSC. b Expressions of Bcl-2/Bax proteins of myocardial tissue at 24 h after ROSC. c Quantification of mRNA expressions of caspase 3. The value represent mean ± SD. *p
Figure Legend Snippet: a Western blots of quantification of Bax, Bcl-2 and active caspase-3 protein levels of myocardial tissue in the sham, saline and sildenafil groups at 24 h after ROSC. b Expressions of Bcl-2/Bax proteins of myocardial tissue at 24 h after ROSC. c Quantification of mRNA expressions of caspase 3. The value represent mean ± SD. *p

Techniques Used: Western Blot

30) Product Images from "Forkhead Box F1 Is Essential for Migration of Mesenchymal Cells and Directly Induces Integrin-Beta3 Expression ▿"

Article Title: Forkhead Box F1 Is Essential for Migration of Mesenchymal Cells and Directly Induces Integrin-Beta3 Expression ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01736-06

Inhibition of Foxf1 by TAT-T7-Foxf1 DN protein causes decreased migration of MFLM-91U cells and reduces Itgβ3 expression. (A) Schematic drawing of TAT-T7-Foxf1 DN and control TAT-T7-Engrail constructs. Whd, winged helix domain. (B) The TAT-T7-Foxf1 DN fusion protein reduces Foxf1 transcriptional activity. U2OS cells were transiently transfected with a TATA-luciferase reporter construct and a CMV-Foxf1 expression plasmid and then treated with either TAT-T7-Foxf1 DN or control TAT-T7-Engrail protein. Cells were harvested 24 or 48 h later and processed for a dual luciferase assay to determine transcriptional activity. A P value of ≤0.05 is shown with double asterisks. (C) TAT-T7-Foxf1 DN protein localizes into cell nuclei. MFLM-91U cells were treated with either TAT-T7-Foxf1 DN or control TAT-T7-Engrail protein for 24 h and then fixed and stained with mouse monoclonal T7 antibodies followed by anti-mouse antibodies conjugated with TRITC (red). Slides were counterstained with DAPI (blue). (D) The TAT-T7-Foxf1 DN fusion protein does not alter the transcriptional activity of either Foxm1b or HNF-6 protein in cotransfection experiments. (E and F) TAT-T7-Foxf1 DN fusion protein inhibits the migration of MFLM-91U cells. (E) MFLM-91U cells were treated for 48 h with either TAT-T7-Foxf1 DN or control TAT-T7-Engrail protein. An agarose drop migration assay was performed as described in Materials and Methods. (F) Numbers of cells migrating from agarose were counted in six independent experiments and are presented as means ± SD. A P value of ≤0.05 is shown with double asterisks. (G) The TAT-T7-Foxf1 DN fusion protein reduces the expression of integrin-beta3. Total protein extracts were prepared from MFLM-91U cells treated for 48 h with either TAT-T7-Foxf1 DN or control TAT-T7-Engrail protein. Western blotting was used to determine expression levels of Itgβ3 and β-actin.
Figure Legend Snippet: Inhibition of Foxf1 by TAT-T7-Foxf1 DN protein causes decreased migration of MFLM-91U cells and reduces Itgβ3 expression. (A) Schematic drawing of TAT-T7-Foxf1 DN and control TAT-T7-Engrail constructs. Whd, winged helix domain. (B) The TAT-T7-Foxf1 DN fusion protein reduces Foxf1 transcriptional activity. U2OS cells were transiently transfected with a TATA-luciferase reporter construct and a CMV-Foxf1 expression plasmid and then treated with either TAT-T7-Foxf1 DN or control TAT-T7-Engrail protein. Cells were harvested 24 or 48 h later and processed for a dual luciferase assay to determine transcriptional activity. A P value of ≤0.05 is shown with double asterisks. (C) TAT-T7-Foxf1 DN protein localizes into cell nuclei. MFLM-91U cells were treated with either TAT-T7-Foxf1 DN or control TAT-T7-Engrail protein for 24 h and then fixed and stained with mouse monoclonal T7 antibodies followed by anti-mouse antibodies conjugated with TRITC (red). Slides were counterstained with DAPI (blue). (D) The TAT-T7-Foxf1 DN fusion protein does not alter the transcriptional activity of either Foxm1b or HNF-6 protein in cotransfection experiments. (E and F) TAT-T7-Foxf1 DN fusion protein inhibits the migration of MFLM-91U cells. (E) MFLM-91U cells were treated for 48 h with either TAT-T7-Foxf1 DN or control TAT-T7-Engrail protein. An agarose drop migration assay was performed as described in Materials and Methods. (F) Numbers of cells migrating from agarose were counted in six independent experiments and are presented as means ± SD. A P value of ≤0.05 is shown with double asterisks. (G) The TAT-T7-Foxf1 DN fusion protein reduces the expression of integrin-beta3. Total protein extracts were prepared from MFLM-91U cells treated for 48 h with either TAT-T7-Foxf1 DN or control TAT-T7-Engrail protein. Western blotting was used to determine expression levels of Itgβ3 and β-actin.

Techniques Used: Inhibition, Migration, Expressing, Construct, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Staining, Cotransfection, Western Blot

Foxf1 depletion does not influence DNA replication and cell survival in vitro. (A and B) siFoxf1 transfection does not alter DNA replication and apoptosis in MFLM-91U cells. (A) MFLM-91U cells were transfected for 72 h with either siFoxf1 or control siRNA and then fixed and used for immunostaining with antibodies against cleaved caspase-3 (top panels) followed by TRITC-conjugated secondary antibodies (red). siRNA-transfected cells were treated with BrdU for 15 min and then fixed and immunostained with mouse monoclonal antibodies specific for BrdU (bottom panels) followed by anti-mouse antibody conjugated with fluorescein isothiocyanate (green). Cell nuclei were counterstained with 4′-6′-diamidino-2-phenylindole (DAPI) (blue). The numbers of caspase-3- and BrdU-positive cells were counted in five random microscope fields (magnification, ×400). (B) Three distinct siRNA transfections were used to determine the mean percentages of cells ± SD. (C) Transfection of Foxf1 siRNA into MEFs effectively diminishes expression of endogenous Foxf1. Total RNA was prepared 72 h after transfection with either Foxf1-specific siRNA (siFoxf1) or control siRNA and then analyzed for Foxf1 expression by RT-PCR. (D) Foxf1 depletion does not influence numbers of caspase-3- and BrdU-positive MEFs.
Figure Legend Snippet: Foxf1 depletion does not influence DNA replication and cell survival in vitro. (A and B) siFoxf1 transfection does not alter DNA replication and apoptosis in MFLM-91U cells. (A) MFLM-91U cells were transfected for 72 h with either siFoxf1 or control siRNA and then fixed and used for immunostaining with antibodies against cleaved caspase-3 (top panels) followed by TRITC-conjugated secondary antibodies (red). siRNA-transfected cells were treated with BrdU for 15 min and then fixed and immunostained with mouse monoclonal antibodies specific for BrdU (bottom panels) followed by anti-mouse antibody conjugated with fluorescein isothiocyanate (green). Cell nuclei were counterstained with 4′-6′-diamidino-2-phenylindole (DAPI) (blue). The numbers of caspase-3- and BrdU-positive cells were counted in five random microscope fields (magnification, ×400). (B) Three distinct siRNA transfections were used to determine the mean percentages of cells ± SD. (C) Transfection of Foxf1 siRNA into MEFs effectively diminishes expression of endogenous Foxf1. Total RNA was prepared 72 h after transfection with either Foxf1-specific siRNA (siFoxf1) or control siRNA and then analyzed for Foxf1 expression by RT-PCR. (D) Foxf1 depletion does not influence numbers of caspase-3- and BrdU-positive MEFs.

Techniques Used: In Vitro, Transfection, Immunostaining, Microscopy, Expressing, Reverse Transcription Polymerase Chain Reaction

Foxf1 directly regulates the mouse Itgβ3 promoter through the bp −871 to −815 Itgβ3 promoter region. (A) Schematic drawing of Itgβ3 promoter constructs. Schematically shown are LUC reporter constructs that use the bp −900 Itgβ3 promoter region (includes a Foxf1 binding site) and the bp −793 Itgβ3 promoter region (without the Foxf1 binding site) to drive expression of the LUC reporter. (B) Foxf1 induces Itgβ3 promoter activity in cotransfection assays. We transiently transfected HEK-293 cells (which do not express endogenous Foxf1) with CMV-Foxf1 expression vector and the bp −900 Itgβ3-LUC or bp −793 Itgβ3-LUC reporter plasmid. Cells were harvested at 48 h after transfection and processed for dual luciferase assays to determine luciferase activity. Transcriptional induction is shown as the increase ( n -fold) relative to that for CMV-empty vector (± SD). A P value of ≤0.05 is shown with double asterisks. (C) ChIP assays show that Foxf1 protein binds to the Itgβ3 promoter region in the context of endogenous DNA. Cross-linked chromatin was prepared from untreated MFLM-91U cells or MFLM-91U cells transfected with siFoxf1 for 72 h. The cross-linked and sonicated chromatin was then immunoprecipitated with antibodies (Ab) specific to Foxf1, Foxf2, Foxm1b, or P-selectin (control). The IP genomic DNA was analyzed for the amount of mouse Itgβ3 promoter DNA by use of PCR analysis with primers specific to the mouse Itgβ3 promoter region (bp −988 to −867).
Figure Legend Snippet: Foxf1 directly regulates the mouse Itgβ3 promoter through the bp −871 to −815 Itgβ3 promoter region. (A) Schematic drawing of Itgβ3 promoter constructs. Schematically shown are LUC reporter constructs that use the bp −900 Itgβ3 promoter region (includes a Foxf1 binding site) and the bp −793 Itgβ3 promoter region (without the Foxf1 binding site) to drive expression of the LUC reporter. (B) Foxf1 induces Itgβ3 promoter activity in cotransfection assays. We transiently transfected HEK-293 cells (which do not express endogenous Foxf1) with CMV-Foxf1 expression vector and the bp −900 Itgβ3-LUC or bp −793 Itgβ3-LUC reporter plasmid. Cells were harvested at 48 h after transfection and processed for dual luciferase assays to determine luciferase activity. Transcriptional induction is shown as the increase ( n -fold) relative to that for CMV-empty vector (± SD). A P value of ≤0.05 is shown with double asterisks. (C) ChIP assays show that Foxf1 protein binds to the Itgβ3 promoter region in the context of endogenous DNA. Cross-linked chromatin was prepared from untreated MFLM-91U cells or MFLM-91U cells transfected with siFoxf1 for 72 h. The cross-linked and sonicated chromatin was then immunoprecipitated with antibodies (Ab) specific to Foxf1, Foxf2, Foxm1b, or P-selectin (control). The IP genomic DNA was analyzed for the amount of mouse Itgβ3 promoter DNA by use of PCR analysis with primers specific to the mouse Itgβ3 promoter region (bp −988 to −867).

Techniques Used: Construct, Binding Assay, Expressing, Activity Assay, Cotransfection, Transfection, Plasmid Preparation, Luciferase, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Polymerase Chain Reaction

Depletion of Foxf1 levels by siRNA causes diminished mesenchyme migration. (A) Foxf1 is expressed in mesenchymal cell lines. Total RNA was prepared from mouse fetal lung mesenchymal cells (MFLM-4 and MFLM-91U), human osteosarcoma U2OS cells, or MEFs and then examined for Foxf1 and cyclophilin (cycloph.) levels by RT-PCR. (B) Transfection of Foxf1 siRNA into MFLM-91U cells effectively diminishes expression of endogenous Foxf1. Total RNA was prepared 72 h after transfection with either Foxf1-specific siRNA (siFoxf1) or control (cont.) siRNA and then analyzed for expression levels of Foxf1, Foxf2, Foxm1b, and cyclophilin by RT-PCR. (C and D) Transfection of siFoxf1 reduces Foxf1 protein levels in MFLM-91U cells. Nuclear protein extracts were prepared 48 or 72 h after transfection with either siFoxf1 or control siRNA and then analyzed for Foxf1 or Foxf2 by Western blot analysis. Levels of β-actin were determined using total protein extract. Foxf1 expression was normalized to its corresponding β-actin level and expressed relative to untreated cells. Values are means ± SD. (E and F) Foxf1 depletion reduces cell migration. (E) MFLM-91U cells were transfected for 72 h with either siFoxf1 or control siRNA and then placed into an agarose drop. Cell migration was assayed in the next 12 or 24 h using a phase-contrast microscope. (F) Numbers of cells migrating from the agarose drop were counted in six independent experiments and expressed as means ± SD. P values of
Figure Legend Snippet: Depletion of Foxf1 levels by siRNA causes diminished mesenchyme migration. (A) Foxf1 is expressed in mesenchymal cell lines. Total RNA was prepared from mouse fetal lung mesenchymal cells (MFLM-4 and MFLM-91U), human osteosarcoma U2OS cells, or MEFs and then examined for Foxf1 and cyclophilin (cycloph.) levels by RT-PCR. (B) Transfection of Foxf1 siRNA into MFLM-91U cells effectively diminishes expression of endogenous Foxf1. Total RNA was prepared 72 h after transfection with either Foxf1-specific siRNA (siFoxf1) or control (cont.) siRNA and then analyzed for expression levels of Foxf1, Foxf2, Foxm1b, and cyclophilin by RT-PCR. (C and D) Transfection of siFoxf1 reduces Foxf1 protein levels in MFLM-91U cells. Nuclear protein extracts were prepared 48 or 72 h after transfection with either siFoxf1 or control siRNA and then analyzed for Foxf1 or Foxf2 by Western blot analysis. Levels of β-actin were determined using total protein extract. Foxf1 expression was normalized to its corresponding β-actin level and expressed relative to untreated cells. Values are means ± SD. (E and F) Foxf1 depletion reduces cell migration. (E) MFLM-91U cells were transfected for 72 h with either siFoxf1 or control siRNA and then placed into an agarose drop. Cell migration was assayed in the next 12 or 24 h using a phase-contrast microscope. (F) Numbers of cells migrating from the agarose drop were counted in six independent experiments and expressed as means ± SD. P values of

Techniques Used: Migration, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Western Blot, Microscopy

Foxf1 deficiency is associated with reduced integrin-beta3 expression and decreased cell adhesion. (A to C) siFoxf1 transfection causes reduced expression of the Itgβ3 subunit. MFLM-91U cells were transfected for 72 h with either siFoxf1 or control siRNA duplexes and then either fixed or used for preparation of total protein and RNA. (A) Total RNA was analyzed for expression levels of Itgβ3 and cyclophilin by RT-PCR analysis. (B and C) Western blotting showed significantly decreased levels of Itgβ3 protein in siFoxf1-transfected cells. Each individual sample was normalized to its corresponding β-actin level. Three independent experiments were used to determine means ± SD. A P value of
Figure Legend Snippet: Foxf1 deficiency is associated with reduced integrin-beta3 expression and decreased cell adhesion. (A to C) siFoxf1 transfection causes reduced expression of the Itgβ3 subunit. MFLM-91U cells were transfected for 72 h with either siFoxf1 or control siRNA duplexes and then either fixed or used for preparation of total protein and RNA. (A) Total RNA was analyzed for expression levels of Itgβ3 and cyclophilin by RT-PCR analysis. (B and C) Western blotting showed significantly decreased levels of Itgβ3 protein in siFoxf1-transfected cells. Each individual sample was normalized to its corresponding β-actin level. Three independent experiments were used to determine means ± SD. A P value of

Techniques Used: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

Conditional expression of Foxf1 DN protein in transgenic MEFs decreases cell migration and reduces expression of integrin-beta3. (A) Nuclear localization of Foxf1 DN protein in transgenic MEFs infected with Ad-TA. Transgenic TetO-Foxf1 DN or WT MEFs were infected with Ad-TA (Tet-off system) or control Ad-LacZ. Cells were then fixed and used for immunofluorescent staining with T7 antibody (red) followed by DAPI counterstaining (blue) to visualize cell nuclei. (B) Schematic drawing of the TetO-Foxf1 DN transgene, containing the CMV-TetO promoter and the Foxf1 DNA binding winged helix domain (Whd) fused with Engrail transcriptional repressor domain and T7 tag sequences. (C) Foxf1 transcriptional activity is reduced in transgenic TetO-Foxf1 DN MEFs after infection with Ad-TA. Transgenic or WT MEFs were transiently transfected with 6× Foxf1 TATA-luciferase reporter plasmid and CMV-Foxf1 expression vector and then infected with either Ad-TA or control Ad-LacZ. Cells were harvested 48 h later and processed for a dual luciferase assay to determine luciferase activity. Transcriptional activity levels are presented as means ± SD. A P value of ≤0.05 is shown with an asterisk. (D) Western blotting shows increased expression of Foxf1 DN protein in transgenic TetO-Foxf1 DN MEFs infected with Ad-TA. Total protein extracts were prepared from transgenic or WT MEFs infected with either Ad-TA or control Ad-LacZ. The positive control was MFLM-91U cells that were transiently transfected with TetO-Foxf1 DN plasmid and then infected with Ad-TA. (E) Western blotting shows decreased expression of Itgβ3 in transgenic TetO-Foxf1 DN MEFs infected with Ad-TA. Protein levels of β-actin were used as loading controls. (F and G) Activation of the Foxf1 DN transgene reduces cell migration in transgenic TetO-Foxf1 DN MEFs. Transgenic or WT MEFs were infected for 48 h with either Ad-TA or Ad-LacZ and then used for an agarose drop migration assay. Twelve hours later, numbers of migrating cells were counted in six independent agarose drops. A P value of ≤0.05 is shown with an asterisk.
Figure Legend Snippet: Conditional expression of Foxf1 DN protein in transgenic MEFs decreases cell migration and reduces expression of integrin-beta3. (A) Nuclear localization of Foxf1 DN protein in transgenic MEFs infected with Ad-TA. Transgenic TetO-Foxf1 DN or WT MEFs were infected with Ad-TA (Tet-off system) or control Ad-LacZ. Cells were then fixed and used for immunofluorescent staining with T7 antibody (red) followed by DAPI counterstaining (blue) to visualize cell nuclei. (B) Schematic drawing of the TetO-Foxf1 DN transgene, containing the CMV-TetO promoter and the Foxf1 DNA binding winged helix domain (Whd) fused with Engrail transcriptional repressor domain and T7 tag sequences. (C) Foxf1 transcriptional activity is reduced in transgenic TetO-Foxf1 DN MEFs after infection with Ad-TA. Transgenic or WT MEFs were transiently transfected with 6× Foxf1 TATA-luciferase reporter plasmid and CMV-Foxf1 expression vector and then infected with either Ad-TA or control Ad-LacZ. Cells were harvested 48 h later and processed for a dual luciferase assay to determine luciferase activity. Transcriptional activity levels are presented as means ± SD. A P value of ≤0.05 is shown with an asterisk. (D) Western blotting shows increased expression of Foxf1 DN protein in transgenic TetO-Foxf1 DN MEFs infected with Ad-TA. Total protein extracts were prepared from transgenic or WT MEFs infected with either Ad-TA or control Ad-LacZ. The positive control was MFLM-91U cells that were transiently transfected with TetO-Foxf1 DN plasmid and then infected with Ad-TA. (E) Western blotting shows decreased expression of Itgβ3 in transgenic TetO-Foxf1 DN MEFs infected with Ad-TA. Protein levels of β-actin were used as loading controls. (F and G) Activation of the Foxf1 DN transgene reduces cell migration in transgenic TetO-Foxf1 DN MEFs. Transgenic or WT MEFs were infected for 48 h with either Ad-TA or Ad-LacZ and then used for an agarose drop migration assay. Twelve hours later, numbers of migrating cells were counted in six independent agarose drops. A P value of ≤0.05 is shown with an asterisk.

Techniques Used: Expressing, Transgenic Assay, Migration, Infection, Staining, Binding Assay, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Western Blot, Positive Control, Activation Assay

31) Product Images from "Eupafolin enhances TRAIL-mediated apoptosis through cathepsin S-induced down-regulation of Mcl-1 expression and AMPK-mediated Bim up-regulation in renal carcinoma Caki cells"

Article Title: Eupafolin enhances TRAIL-mediated apoptosis through cathepsin S-induced down-regulation of Mcl-1 expression and AMPK-mediated Bim up-regulation in renal carcinoma Caki cells

Journal: Oncotarget

doi: 10.18632/oncotarget.11604

Effects of eupafolin on expression levels of apoptosis-related proteins Caki cells were treated with the indicated concentrations of eupafolin for 24 h. The protein levels of Mcl-1, Bcl-xL, Bcl-2, Bim, Bax, DR5, FADD, c-FLIP, cIAP1, cIAP2, XIAP, and actin were determined by Western blotting. The level of actin was used as a loading control.
Figure Legend Snippet: Effects of eupafolin on expression levels of apoptosis-related proteins Caki cells were treated with the indicated concentrations of eupafolin for 24 h. The protein levels of Mcl-1, Bcl-xL, Bcl-2, Bim, Bax, DR5, FADD, c-FLIP, cIAP1, cIAP2, XIAP, and actin were determined by Western blotting. The level of actin was used as a loading control.

Techniques Used: Expressing, Western Blot

32) Product Images from "Effects of tristetraprolin on doxorubicin (adriamycin)-induced experimental kidney injury through inhibiting IL-13/STAT6 signal pathway"

Article Title: Effects of tristetraprolin on doxorubicin (adriamycin)-induced experimental kidney injury through inhibiting IL-13/STAT6 signal pathway

Journal: American Journal of Translational Research

doi:

Effect of Tristetraprolin (TTP) on cell apoptosis markers (Bax, Bcl-2 and Caspase-3) in HK-2 and NRK-52E cells after treatment of DOX. Western blot for the detection of Bax, Bcl-2, Cleaved-caspase3, Caspase3 and GAPDH in (A) HK-2 and (B) NRK-52E cells. The bar graphs represented the relative expressions compared to GAPDH. All values were expressed as means ± SD, n=5, **P
Figure Legend Snippet: Effect of Tristetraprolin (TTP) on cell apoptosis markers (Bax, Bcl-2 and Caspase-3) in HK-2 and NRK-52E cells after treatment of DOX. Western blot for the detection of Bax, Bcl-2, Cleaved-caspase3, Caspase3 and GAPDH in (A) HK-2 and (B) NRK-52E cells. The bar graphs represented the relative expressions compared to GAPDH. All values were expressed as means ± SD, n=5, **P

Techniques Used: Western Blot

33) Product Images from "Cabozantinib induces PUMA-dependent apoptosis in colon cancer cells via AKT/GSK-3β/NF-κB signaling pathway"

Article Title: Cabozantinib induces PUMA-dependent apoptosis in colon cancer cells via AKT/GSK-3β/NF-κB signaling pathway

Journal: Cancer Gene Therapy

doi: 10.1038/s41417-019-0098-6

p65 mediates Cabozantinib induced PUMA induction. a HCT116 cells were treated with 5 μmol/L Cabozantinib at indicated time points. p73, p-STAT1, STAT1, p-FoxO3a, and FoxO3a expression was analyzed by western blotting. b HCT116 cells were treated with 5 μmol/L Cabozantinib at indicated time points. p-p65 (S536) and p65 expression was analyzed by western blotting. c HCT116 cells were transfected with either a control scrambled siRNA or a p65 siRNA for 24 h, and then treated with 5 μmol/L Cabozantinib for 24 h. p65 and PUMA expression was probed by western blotting. d HCT116 cells were pretreated with 10 μmol/L BAY11-7082 for 1 h, and then with 5 μmol/L Cabozantinib for 24 h. Nuclear fractions were isolated from cells and analyzed for p65 expression by western blotting. Lamin A/C and β-actin were used as controls for loading and fractionation. e HCT116 cells were pretreated with 10 μmol/L BAY11-7082 for 1 h, and then with 5 μmol/L Cabozantinib for 24 h. p-p65 (S536) and PUMA expression were analyzed by western blotting. f Chromatin immunoprecipitation (ChIP) was performed using anti-p65 antibody on HCT116 cells following Cabozantinib treatment for 12 h. ChIP with the control IgG was used as a control. PCR was carried out using primers surrounding the p65 binding sites in the PUMA promoter
Figure Legend Snippet: p65 mediates Cabozantinib induced PUMA induction. a HCT116 cells were treated with 5 μmol/L Cabozantinib at indicated time points. p73, p-STAT1, STAT1, p-FoxO3a, and FoxO3a expression was analyzed by western blotting. b HCT116 cells were treated with 5 μmol/L Cabozantinib at indicated time points. p-p65 (S536) and p65 expression was analyzed by western blotting. c HCT116 cells were transfected with either a control scrambled siRNA or a p65 siRNA for 24 h, and then treated with 5 μmol/L Cabozantinib for 24 h. p65 and PUMA expression was probed by western blotting. d HCT116 cells were pretreated with 10 μmol/L BAY11-7082 for 1 h, and then with 5 μmol/L Cabozantinib for 24 h. Nuclear fractions were isolated from cells and analyzed for p65 expression by western blotting. Lamin A/C and β-actin were used as controls for loading and fractionation. e HCT116 cells were pretreated with 10 μmol/L BAY11-7082 for 1 h, and then with 5 μmol/L Cabozantinib for 24 h. p-p65 (S536) and PUMA expression were analyzed by western blotting. f Chromatin immunoprecipitation (ChIP) was performed using anti-p65 antibody on HCT116 cells following Cabozantinib treatment for 12 h. ChIP with the control IgG was used as a control. PCR was carried out using primers surrounding the p65 binding sites in the PUMA promoter

Techniques Used: Expressing, Western Blot, Transfection, Isolation, Fractionation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay

34) Product Images from "The Myc 3′ Wnt-Responsive Element Suppresses Colonic Tumorigenesis"

Article Title: The Myc 3′ Wnt-Responsive Element Suppresses Colonic Tumorigenesis

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00969-13

The Myc 3′ WRE controls proliferation but not apoptosis in the colons of Apc Min/ + mice. (A) Ki67- and cleaved caspase 3 (CASP3)-stained sections of preneoplastic colons and colonic tumors from Apc Min/ + (top) and Apc Min/ + Myc 3′ WRE −/− (bottom) mice. Representative images are shown ( n = 4 mice analyzed per genotype). Arrowheads indicate CASP3 + cells. (B) Quantification of Ki67 + (left) and CASP3 + (right) cells in preneoplastic colons of Apc Min/ + and Apc Min/ + Myc 3′ WRE −/− mice ( n = 4 mice analyzed, with cells counted in a total of 100 crypts per genotype). (C) Same as panel B except that Ki67 + and CASP3 + cells were analyzed in tumor sections prepared from mice with the indicated genotypes ( n = 4 mice, with 40 fields of view examined per genotype). In panels B and C, errors are standard errors of the means (**, P
Figure Legend Snippet: The Myc 3′ WRE controls proliferation but not apoptosis in the colons of Apc Min/ + mice. (A) Ki67- and cleaved caspase 3 (CASP3)-stained sections of preneoplastic colons and colonic tumors from Apc Min/ + (top) and Apc Min/ + Myc 3′ WRE −/− (bottom) mice. Representative images are shown ( n = 4 mice analyzed per genotype). Arrowheads indicate CASP3 + cells. (B) Quantification of Ki67 + (left) and CASP3 + (right) cells in preneoplastic colons of Apc Min/ + and Apc Min/ + Myc 3′ WRE −/− mice ( n = 4 mice analyzed, with cells counted in a total of 100 crypts per genotype). (C) Same as panel B except that Ki67 + and CASP3 + cells were analyzed in tumor sections prepared from mice with the indicated genotypes ( n = 4 mice, with 40 fields of view examined per genotype). In panels B and C, errors are standard errors of the means (**, P

Techniques Used: Mouse Assay, Staining

35) Product Images from "SHP2 Inhibition Abrogates Adaptive Resistance to KRASG12C-Inhibition and Remodels the Tumor Microenvironment of KRAS-Mutant Tumors"

Article Title: SHP2 Inhibition Abrogates Adaptive Resistance to KRASG12C-Inhibition and Remodels the Tumor Microenvironment of KRAS-Mutant Tumors

Journal: bioRxiv

doi: 10.1101/2020.05.30.125138

Combined ARS1620/SHP2 inhibition is highly efficacious in PDAC models in vivo . A , Pancreas tumors were established in syngeneic mice by orthotopic injections of KCP cells, and 14 days later, mice were treated with vehicle, SHP099, ARS1620 or both drugs (Combo), as depicted. Tumor weight was quantified in a cohort at Day 0 (baseline) and in treated mice at Day 10. B , Immunoblots of KCP-derived tumor lysates showing effects of the indicated treatments on KRAS G12C -GTP, pERK, and DUSP6 levels. C , ERK-dependent gene expression, assessed by RNAseq, in KCP tumors treated for 3 days, as indicated in A (colors indicate log2FC). D-E , Time-dependent increase in RTK (D) and RTK ligands (E) gene expression in KCP-derived orthotopic tumors after vehicle, SHP099, ARS1620 and Combo treatment at Day 3, determined by RNAseq (colors represent log2FC). F , H E, Masson Trichome, CD31, pERK, Ki67 and cleaved Caspase 3 staining and quantification in KCP tumor sections from mice after 10 days of treatment, as indicated. G , KCP tumors were established in syngeneic mice and allowed to grow to much larger size before treatments were initiated, as depicted in the scheme. Tumor weight was quantified in one cohort before treatment, in another cohort after 12 days of treatment, and after drug withdrawal, at Day 27, as indicated. H , Kaplan-Meier curve of KCP tumor-bearing mice after withdrawal of the indicated drugs (top). Tumor growth curve after withdrawal of indicated treatment at day 12 (bottom). H , Response of sub-cutaneous NY53 patient-derived xenograft to treatment with vehicle, SHP099, ARS1620 or both drugs. For all experiments, drug doses were: SHP099 (75 mg/kg body weight, daily), ARS1620 (200 mg/kg body weight, daily) or both drugs (daily). Data represent mean ± SD; *P
Figure Legend Snippet: Combined ARS1620/SHP2 inhibition is highly efficacious in PDAC models in vivo . A , Pancreas tumors were established in syngeneic mice by orthotopic injections of KCP cells, and 14 days later, mice were treated with vehicle, SHP099, ARS1620 or both drugs (Combo), as depicted. Tumor weight was quantified in a cohort at Day 0 (baseline) and in treated mice at Day 10. B , Immunoblots of KCP-derived tumor lysates showing effects of the indicated treatments on KRAS G12C -GTP, pERK, and DUSP6 levels. C , ERK-dependent gene expression, assessed by RNAseq, in KCP tumors treated for 3 days, as indicated in A (colors indicate log2FC). D-E , Time-dependent increase in RTK (D) and RTK ligands (E) gene expression in KCP-derived orthotopic tumors after vehicle, SHP099, ARS1620 and Combo treatment at Day 3, determined by RNAseq (colors represent log2FC). F , H E, Masson Trichome, CD31, pERK, Ki67 and cleaved Caspase 3 staining and quantification in KCP tumor sections from mice after 10 days of treatment, as indicated. G , KCP tumors were established in syngeneic mice and allowed to grow to much larger size before treatments were initiated, as depicted in the scheme. Tumor weight was quantified in one cohort before treatment, in another cohort after 12 days of treatment, and after drug withdrawal, at Day 27, as indicated. H , Kaplan-Meier curve of KCP tumor-bearing mice after withdrawal of the indicated drugs (top). Tumor growth curve after withdrawal of indicated treatment at day 12 (bottom). H , Response of sub-cutaneous NY53 patient-derived xenograft to treatment with vehicle, SHP099, ARS1620 or both drugs. For all experiments, drug doses were: SHP099 (75 mg/kg body weight, daily), ARS1620 (200 mg/kg body weight, daily) or both drugs (daily). Data represent mean ± SD; *P

Techniques Used: Inhibition, In Vivo, Mouse Assay, Western Blot, Derivative Assay, Expressing, Staining

36) Product Images from "Spiny mice (Acomys) exhibit attenuated hallmarks of aging and rapid cell turnover after UV exposure in the skin epidermis"

Article Title: Spiny mice (Acomys) exhibit attenuated hallmarks of aging and rapid cell turnover after UV exposure in the skin epidermis

Journal: bioRxiv

doi: 10.1101/2020.05.07.083287

Efficient removal of damaged and dying skin epidermal cells through rapid turnover and apoptosis in acute UVB-exposed A. cahirinus . A , representative immunofluorescence images of epidermal thymine dimer ( T-T dimer ) labeling of skin from control ( sham ) and UV-irradiated M. musculus and A. cahirinus , collected 24 and 48 hours after exposure. B and C , quantification of thymine dimer labeling in ( B ) basal epidermis and ( C ) suprabasal epidermis. D , representative immunofluorescence images of epidermal γH2AX labeling . E and F , quantification γH2AX labeling in ( D ) basal epidermis and ( E ) suprabasal epidermis. G , representative immunofluorescence images of epidermal cleaved caspase-3 ( CC3) labeling. Positive cells are indicated by the pink arrows. H and I , quantification of cleaved caspase-3 labeling in ( H ) basal and ( I) suprabasal epidermis. Data points are biological replicates. Lines indicate group means. *, significantly different from the indicated group ( p
Figure Legend Snippet: Efficient removal of damaged and dying skin epidermal cells through rapid turnover and apoptosis in acute UVB-exposed A. cahirinus . A , representative immunofluorescence images of epidermal thymine dimer ( T-T dimer ) labeling of skin from control ( sham ) and UV-irradiated M. musculus and A. cahirinus , collected 24 and 48 hours after exposure. B and C , quantification of thymine dimer labeling in ( B ) basal epidermis and ( C ) suprabasal epidermis. D , representative immunofluorescence images of epidermal γH2AX labeling . E and F , quantification γH2AX labeling in ( D ) basal epidermis and ( E ) suprabasal epidermis. G , representative immunofluorescence images of epidermal cleaved caspase-3 ( CC3) labeling. Positive cells are indicated by the pink arrows. H and I , quantification of cleaved caspase-3 labeling in ( H ) basal and ( I) suprabasal epidermis. Data points are biological replicates. Lines indicate group means. *, significantly different from the indicated group ( p

Techniques Used: Immunofluorescence, Labeling, Irradiation

37) Product Images from "AI boosted molecular MRI for apoptosis detection in oncolytic virotherapy"

Article Title: AI boosted molecular MRI for apoptosis detection in oncolytic virotherapy

Journal: bioRxiv

doi: 10.1101/2020.03.05.977793

Histology validation. a. T 2 -weighted image of an OV-treated mouse, 72 hours post virus inoculation. b. Semisolid macro-molecules proton volume fraction (f ss ) map, overlaid atop the T 2 -weighted image at the ipsilateral side. c. Similarly overlaid amide proton exchange-rate (k sw ) and d. amide proton volume fraction (f s ) maps. e. Immunohistochemistry image stained for Herpes Simplex Virus (HSV) presence (brown). f. HE stained image, demonstrating the tumor location (pink). g. Caspase-3 immunohistochemistry image, demonstrating the apoptotic tumor region (brown). h. Coomassie Blue stained image, demonstrating reduced protein concentration in the apoptotic tumor core. The dashed lines in images b-d, and f-h, generally depict the tumor (b, f) and apoptotic (c, d, g, h) regions borders, respectively.
Figure Legend Snippet: Histology validation. a. T 2 -weighted image of an OV-treated mouse, 72 hours post virus inoculation. b. Semisolid macro-molecules proton volume fraction (f ss ) map, overlaid atop the T 2 -weighted image at the ipsilateral side. c. Similarly overlaid amide proton exchange-rate (k sw ) and d. amide proton volume fraction (f s ) maps. e. Immunohistochemistry image stained for Herpes Simplex Virus (HSV) presence (brown). f. HE stained image, demonstrating the tumor location (pink). g. Caspase-3 immunohistochemistry image, demonstrating the apoptotic tumor region (brown). h. Coomassie Blue stained image, demonstrating reduced protein concentration in the apoptotic tumor core. The dashed lines in images b-d, and f-h, generally depict the tumor (b, f) and apoptotic (c, d, g, h) regions borders, respectively.

Techniques Used: Immunohistochemistry, Staining, Protein Concentration

38) Product Images from "Mitochondria regulate Drosophila intestinal stem cell differentiation through FOXO"

Article Title: Mitochondria regulate Drosophila intestinal stem cell differentiation through FOXO

Journal: bioRxiv

doi: 10.1101/2020.02.12.946194

Strategy for studying the role of mitochondrial ETC system in intestinal stem cells. (A) Tracking ISCs with the esg ts Flp-Out ( esg ts F/O ) labelling system. (Upper) “ esg ts F/O “flies (i.e., w ; esg-GAL4, tub-GAL80 ts UAS-nlsGFP; UAS-flp, act > CD2 > GAL4 ) were grown at 18 ºC, exposed to a 2-hr heat shock at 37 ºC two days after eclosion into adults, and maintained at 29 ºC until assessment. Representative images show no GFP expression in the midgut of esg ts F/O adults before heat shock and a large percentage of GFP + cells 20 days after heat shock. (Lower) Schematics of ISC labelling: heat shock and maintenance at 29 ºC reverse the inhibition of the GAL4/UAS expression system by GAL80 ts , leading to continuous expression of GFP in esg + ISCs and their lineage via Flp activation of Act-GAL4, without affecting other already differentiated midgut epithelial cells. Differentiation of the GFP + ISCs into EBs, ECs and EEs can be tracked based on cell shape and the expression of specific cell markers. Green: GFP; Red: Delta antibody staining for ISC, Su(H)GBE-lacZ for EB and Pdm1 antibody staining for EC; Magenta: Prospero antibody staining for EE; Blue: DAPI. Scale bars: 200 µm for whole-gut views, 10 µm for close-up views. (B) Mitochondrial DNA clonal analysis. Wild type (wt) mtDNA carries a unique restriction XhoI site in the cytochrome c oxidase locus, mt:CoI . The mt:CoI T300I mutation is resistant to XhoI cleavage but disrupts COXI function at high temperature (temperature-sensitive lethal). The mt:CoI syn mutant mtDNA is also resistant to XhoI cleavage due to a synonymous mutation that does not affect COXI function. Using the esg ts F/O system, we can drive the expression of a mitochondria-targeted form of XhoI ( mitoXhoI ) in the ISCs of flies carrying either a heteroplasmic mixture of wt and mt:CoI T300I mtDNAs (mutant group), or a homoplasmic population of mt:CoI syn mtDNA (control group). The heteroplasmic mixture is more than 80% WT and behaves as wild type even at 29 ºC. After heat shock and maintenance at 29 ºC, WT mtDNAs are eliminated by mitoXhoI cleavage in ISCs only—the ECs and EEs that formed before the heat shock do not express esg and remain heteroplasmic. The control group carrying the homoplasmic mt:CoI syn mutation, which is resistant to mitoXhoI cleavage, retains wild-type COXI function at both 18 ºC and 29 ºC. This method allows the disruption of ETC system in the GFP + ISC cells without affecting the GFP - midgut epithelial tissues. (C) Representative esg ts F/O tracking pattern in the midgut of control flies, showing patches of GFP + EBs, EEs and ECs over a background of non GFP tissues, reflecting past ISC proliferation/differentiation events. Green: GFP; Red: Su(H)GBE-lacZ; Magenta: Prospero. Scale bar: 10 µm.
Figure Legend Snippet: Strategy for studying the role of mitochondrial ETC system in intestinal stem cells. (A) Tracking ISCs with the esg ts Flp-Out ( esg ts F/O ) labelling system. (Upper) “ esg ts F/O “flies (i.e., w ; esg-GAL4, tub-GAL80 ts UAS-nlsGFP; UAS-flp, act > CD2 > GAL4 ) were grown at 18 ºC, exposed to a 2-hr heat shock at 37 ºC two days after eclosion into adults, and maintained at 29 ºC until assessment. Representative images show no GFP expression in the midgut of esg ts F/O adults before heat shock and a large percentage of GFP + cells 20 days after heat shock. (Lower) Schematics of ISC labelling: heat shock and maintenance at 29 ºC reverse the inhibition of the GAL4/UAS expression system by GAL80 ts , leading to continuous expression of GFP in esg + ISCs and their lineage via Flp activation of Act-GAL4, without affecting other already differentiated midgut epithelial cells. Differentiation of the GFP + ISCs into EBs, ECs and EEs can be tracked based on cell shape and the expression of specific cell markers. Green: GFP; Red: Delta antibody staining for ISC, Su(H)GBE-lacZ for EB and Pdm1 antibody staining for EC; Magenta: Prospero antibody staining for EE; Blue: DAPI. Scale bars: 200 µm for whole-gut views, 10 µm for close-up views. (B) Mitochondrial DNA clonal analysis. Wild type (wt) mtDNA carries a unique restriction XhoI site in the cytochrome c oxidase locus, mt:CoI . The mt:CoI T300I mutation is resistant to XhoI cleavage but disrupts COXI function at high temperature (temperature-sensitive lethal). The mt:CoI syn mutant mtDNA is also resistant to XhoI cleavage due to a synonymous mutation that does not affect COXI function. Using the esg ts F/O system, we can drive the expression of a mitochondria-targeted form of XhoI ( mitoXhoI ) in the ISCs of flies carrying either a heteroplasmic mixture of wt and mt:CoI T300I mtDNAs (mutant group), or a homoplasmic population of mt:CoI syn mtDNA (control group). The heteroplasmic mixture is more than 80% WT and behaves as wild type even at 29 ºC. After heat shock and maintenance at 29 ºC, WT mtDNAs are eliminated by mitoXhoI cleavage in ISCs only—the ECs and EEs that formed before the heat shock do not express esg and remain heteroplasmic. The control group carrying the homoplasmic mt:CoI syn mutation, which is resistant to mitoXhoI cleavage, retains wild-type COXI function at both 18 ºC and 29 ºC. This method allows the disruption of ETC system in the GFP + ISC cells without affecting the GFP - midgut epithelial tissues. (C) Representative esg ts F/O tracking pattern in the midgut of control flies, showing patches of GFP + EBs, EEs and ECs over a background of non GFP tissues, reflecting past ISC proliferation/differentiation events. Green: GFP; Red: Su(H)GBE-lacZ; Magenta: Prospero. Scale bar: 10 µm.

Techniques Used: Expressing, Inhibition, Activation Assay, Staining, Mutagenesis

39) Product Images from "SMARCA4 supports the oncogenic landscape of KRAS-driven lung tumors"

Article Title: SMARCA4 supports the oncogenic landscape of KRAS-driven lung tumors

Journal: bioRxiv

doi: 10.1101/2020.04.18.043927

SMARCA4 loss impedes malignancy of KRASG12D-driven mouse lung tumors tumors (A) IHC staining of TTF1, FOXA2 and HMGA2 in lung tumors of indicated genotypes at indicated time points post Adeno-Cre infection. (B) p-AKT and p-ERK1/2 staining in sections of mouse lung tumors of indicated genotypes 6 months post Adeno Cre. (C-D) Representative IHC of Cleaved Caspase 3 and Ki67 of indicated genotypes at indicated time points. (E) Quantification of Ki67 positive cells in lung tumors of indicated genotypes (n=12 random fields). Significance calculated by paired two-tailed Student’s t -test between the respective genotypes is indicated. #-not significant, **
Figure Legend Snippet: SMARCA4 loss impedes malignancy of KRASG12D-driven mouse lung tumors tumors (A) IHC staining of TTF1, FOXA2 and HMGA2 in lung tumors of indicated genotypes at indicated time points post Adeno-Cre infection. (B) p-AKT and p-ERK1/2 staining in sections of mouse lung tumors of indicated genotypes 6 months post Adeno Cre. (C-D) Representative IHC of Cleaved Caspase 3 and Ki67 of indicated genotypes at indicated time points. (E) Quantification of Ki67 positive cells in lung tumors of indicated genotypes (n=12 random fields). Significance calculated by paired two-tailed Student’s t -test between the respective genotypes is indicated. #-not significant, **

Techniques Used: Immunohistochemistry, Staining, Infection, Two Tailed Test

SMARCA4 promotes transformative properties of human lung cells. (A) Immuno-precipitation against the core SWI/SNF subunit, BAF57 was performed followed by western blot (B) Density map showing spectral count of SWI/SNF peptides detected in mass spectrometric analysis of control and SMARCA4 reconstituted A549 extracts. Mass spectrometry was performed following immunoprecipitation of SWI/SNF complex using BAF57 antibody. (C) Western blots of fractions 1-20 of the glycerol gradient sedimentation analysis assessing the migration of BRM-/BRG1-containing BAF complexes at 2MDa and PBAF complexes at 4MDa in A549 control or SMARCA4 reconstituted A549 cells. (D) qPCR (E) Western blot assessing expression of EMT genes in A549 control or SMARCA4 reconstituted A549 cells. (F) Ectopic expression of SMARCA4 in BRG1 deficient human lung tumor line, A549 augments colony formation in soft agar. (G) Experimental scheme of doxycycline induced SMARCA4 knockdown in AALE-KRAS G12D cells (H) Western blot showing decreased p-AKT upon depletion of SMARCA4 (dox +) in AALE-Kras G12D cells. (I) Doxycycline inducible depletion of SMARCA4 in AALE-Kras G12D cells reduced colony formation in soft agar. *
Figure Legend Snippet: SMARCA4 promotes transformative properties of human lung cells. (A) Immuno-precipitation against the core SWI/SNF subunit, BAF57 was performed followed by western blot (B) Density map showing spectral count of SWI/SNF peptides detected in mass spectrometric analysis of control and SMARCA4 reconstituted A549 extracts. Mass spectrometry was performed following immunoprecipitation of SWI/SNF complex using BAF57 antibody. (C) Western blots of fractions 1-20 of the glycerol gradient sedimentation analysis assessing the migration of BRM-/BRG1-containing BAF complexes at 2MDa and PBAF complexes at 4MDa in A549 control or SMARCA4 reconstituted A549 cells. (D) qPCR (E) Western blot assessing expression of EMT genes in A549 control or SMARCA4 reconstituted A549 cells. (F) Ectopic expression of SMARCA4 in BRG1 deficient human lung tumor line, A549 augments colony formation in soft agar. (G) Experimental scheme of doxycycline induced SMARCA4 knockdown in AALE-KRAS G12D cells (H) Western blot showing decreased p-AKT upon depletion of SMARCA4 (dox +) in AALE-Kras G12D cells. (I) Doxycycline inducible depletion of SMARCA4 in AALE-Kras G12D cells reduced colony formation in soft agar. *

Techniques Used: Immunoprecipitation, Western Blot, Mass Spectrometry, Sedimentation, Migration, Real-time Polymerase Chain Reaction, Expressing

40) Product Images from "Opa1 overexpression protects from early onset Mpv17-/--related mouse kidney disease"

Article Title: Opa1 overexpression protects from early onset Mpv17-/--related mouse kidney disease

Journal: bioRxiv

doi: 10.1101/2020.03.18.996561

Caspase 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p
Figure Legend Snippet: Caspase 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p

Techniques Used: Staining, Isolation, Immunohistochemistry, Derivative Assay

Related Articles

Digital Holographic Microscopy:

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Article Title: SIRT3 Activation by Dihydromyricetin Suppresses Chondrocytes Degeneration via Maintaining Mitochondrial Homeostasis
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Immunohistochemistry:

Article Title: LGR5, a novel functional glioma stem cell marker, promotes EMT by activating the Wnt/β-catenin pathway and predicts poor survival of glioma patients
Article Snippet: .. In addition, quantitative analysis of IHC was used to determine the expression of LGR5, Ki67, Active-β-catenin, SOX2 and CD44. .. The expression level of LGR5 was statistically significant between the shCtrl and shLGR5 groups (P < 0.001, Fig. ) and between the shCtrl and Wnt-C59 groups (P < 0.001, Fig. ).

Negative Control:

Article Title: SOX2, a stemness gene, induces progression of NSCLC A549 cells toward anchorage-independent growth and chemoresistance to vinblastine
Article Snippet: .. In consistent with this finding, recent studies showed that SOX2 overexpression increased cell proliferation and tumorigenicity., Importantly, knockdown of SOX2 decreases the phosphorylation level of AKT kinase compared to negative control siRNA in both 2D and 3D cultures ( ). .. As aberrant activation of Akt in NSCLC is a frequent and early event that correlates with tumor progression, drug resistance, and poor prognosis, – we propose that upregulation of SOX2 expression contributes to this abnormal activation of Akt in A549 cell spheroid, leading to cell survival.

Cell Culture:

Article Title: Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol
Article Snippet: .. Immunofluorescence Detection of Caspase 3 Activity As previously detailed [ ], B16-F10 cells cultured in 120 mm coverslips were fixed in 4% paraformaldehyde in 0.1 M PB, pH 7.4, for 10 min and overnight stained with anti-cleaved-caspase 3 antibody (Cell Signaling Technology, Danvers, MA, USA), in PB containing 0.5% Triton X-100. .. Cells were then stained with the appropriate Alexa Fluor secondary antibodies (Life Technologies, Monza, Italy), for 1 h and cover-slipped in a ProLong Gold Antifade Mountant (Life Technologies), stained with fluorescein phalloidin (cytoskeleton detection) (Life Technologies) and DAPI (nuclei detection) (Sigma-Aldrich, St. Louis, MO, USA).

Pyrolysis Gas Chromatography:

Article Title: SS-31 Provides Neuroprotection by Reversing Mitochondrial Dysfunction after Traumatic Brain Injury
Article Snippet: .. The dilution ratio of used antibodies was listed as follows: eIF2α (1 : 1000, CST), p-eIF2α (1 : 1000, CST), Bcl-2 (1 : 1000, CST), Bax (1 : 1000, CST), cytochrome c (1 : 1000, CST), P53 (1 : 500, Proteintech), PGC-1α (1 : 2000, Proteintech), SIRT1 (1 : 1000, CST), H3 (1 : 1000, CST), and β -actin (1 : 5000, Bioworld). .. Mitochondrial MDA and SOD Content Determination of mitochondrial malondialdehyde (MDA) and superoxide dismutase (SOD) contents was performed by following the manufacturer's instructions (Nanjing Jiancheng Biochemistry Co., Nanjing, China).

other:

Article Title: SOX2, a stemness gene, induces progression of NSCLC A549 cells toward anchorage-independent growth and chemoresistance to vinblastine
Article Snippet: Although the LUAD A549 cells used in our study are distinct in their histological, molecular, and clinical presentation from LUSC and SCLC, our study strongly suggests that SOX2 universally mediates a major tumorigenic effect on NSCLC regardless of histological heterogeneity.

Activity Assay:

Article Title: Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol
Article Snippet: .. Immunofluorescence Detection of Caspase 3 Activity As previously detailed [ ], B16-F10 cells cultured in 120 mm coverslips were fixed in 4% paraformaldehyde in 0.1 M PB, pH 7.4, for 10 min and overnight stained with anti-cleaved-caspase 3 antibody (Cell Signaling Technology, Danvers, MA, USA), in PB containing 0.5% Triton X-100. .. Cells were then stained with the appropriate Alexa Fluor secondary antibodies (Life Technologies, Monza, Italy), for 1 h and cover-slipped in a ProLong Gold Antifade Mountant (Life Technologies), stained with fluorescein phalloidin (cytoskeleton detection) (Life Technologies) and DAPI (nuclei detection) (Sigma-Aldrich, St. Louis, MO, USA).

Expressing:

Article Title: LGR5, a novel functional glioma stem cell marker, promotes EMT by activating the Wnt/β-catenin pathway and predicts poor survival of glioma patients
Article Snippet: .. In addition, quantitative analysis of IHC was used to determine the expression of LGR5, Ki67, Active-β-catenin, SOX2 and CD44. .. The expression level of LGR5 was statistically significant between the shCtrl and shLGR5 groups (P < 0.001, Fig. ) and between the shCtrl and Wnt-C59 groups (P < 0.001, Fig. ).

Marker:

Article Title: Disruption of Tmem30a results in cerebellar ataxia and degeneration of Purkinje cells
Article Snippet: .. Cleaved caspase-12, which is a typical ER stress-induced apoptosis marker, was upregulated at P16 in the KO cerebellum (Fig. ), suggested that the prolonged ER stress triggered the caspase cascade before cell apoptosis. .. Moreover, both immunofluorescence and immunoblotting results demonstrated that the expression level of cleaved caspase-3, which is an important marker of cell apoptosis, was elevated from P16 to P20 (Fig. ).

Staining:

Article Title: Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol
Article Snippet: .. Immunofluorescence Detection of Caspase 3 Activity As previously detailed [ ], B16-F10 cells cultured in 120 mm coverslips were fixed in 4% paraformaldehyde in 0.1 M PB, pH 7.4, for 10 min and overnight stained with anti-cleaved-caspase 3 antibody (Cell Signaling Technology, Danvers, MA, USA), in PB containing 0.5% Triton X-100. .. Cells were then stained with the appropriate Alexa Fluor secondary antibodies (Life Technologies, Monza, Italy), for 1 h and cover-slipped in a ProLong Gold Antifade Mountant (Life Technologies), stained with fluorescein phalloidin (cytoskeleton detection) (Life Technologies) and DAPI (nuclei detection) (Sigma-Aldrich, St. Louis, MO, USA).

Over Expression:

Article Title: SOX2, a stemness gene, induces progression of NSCLC A549 cells toward anchorage-independent growth and chemoresistance to vinblastine
Article Snippet: .. In consistent with this finding, recent studies showed that SOX2 overexpression increased cell proliferation and tumorigenicity., Importantly, knockdown of SOX2 decreases the phosphorylation level of AKT kinase compared to negative control siRNA in both 2D and 3D cultures ( ). .. As aberrant activation of Akt in NSCLC is a frequent and early event that correlates with tumor progression, drug resistance, and poor prognosis, – we propose that upregulation of SOX2 expression contributes to this abnormal activation of Akt in A549 cell spheroid, leading to cell survival.

Immunofluorescence:

Article Title: Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol
Article Snippet: .. Immunofluorescence Detection of Caspase 3 Activity As previously detailed [ ], B16-F10 cells cultured in 120 mm coverslips were fixed in 4% paraformaldehyde in 0.1 M PB, pH 7.4, for 10 min and overnight stained with anti-cleaved-caspase 3 antibody (Cell Signaling Technology, Danvers, MA, USA), in PB containing 0.5% Triton X-100. .. Cells were then stained with the appropriate Alexa Fluor secondary antibodies (Life Technologies, Monza, Italy), for 1 h and cover-slipped in a ProLong Gold Antifade Mountant (Life Technologies), stained with fluorescein phalloidin (cytoskeleton detection) (Life Technologies) and DAPI (nuclei detection) (Sigma-Aldrich, St. Louis, MO, USA).

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    p53 25,26,53,54/+  Embryos Exhibit Additional Features of CHARGE Syndrome and p53-Dependent Cellular Responses (a)  Double outlet right ventricle (DORV) in E13.5 p53 25,26,53,54/+  heart (50%, n=6). Top: Main pulmonary artery (MPA) connects via pulmonary valve (PV) to right ventricle (RV) in both control and p53 25,26,53,54/+  embryo. Bottom: Aorta (Ao) in control embryo connects to left ventricle (LV) via aortic valve (AV) Φ . Aorta in p53 25,26,53,54/+  embryo connects to RV via AV*. (b)  Abnormal atrioventricular cushions in E13.5 p53 25,26,53,54/+  heart (75%, n=4) fail to elongateinto mature mitral (mv, arrowhead) and tricuspid (tv, arrow) valves. RA: right atrium; LA: left atrium.  (c)  E13.5  p53 25,26,53,54/+  kidneys are smaller (79%), with fewer average glomeruli (13 vs. 3; n=5; arrows), than controls. (d) p53 25,26,53,54/+  embryonic phenotypes observed in CHARGE (+present, −absent).  (e)  Left: Cleaved-caspase 3 (CC3; Top) and p53 (Bottom) immunohistochemistry in E15.5 retinas. Arrows: CC3-positive cells. Right: CC3-positive cells per retinal area. ***p-value=0.007; one-tailed Welsh’s t-test (n=5).  (f)  BrdU immunofluorescence in E9.5 Pax3 +  NCCs (delineated by green-dotted line;  Extended-Data Fig. 6c ). Right: Percentage BrdU-positive cells per total Pax3 +  NCCs ***p-value=0.004 one-tailed Student’s t-test (n=4).
    Whole Mount Cleaved Caspase 3 Staining, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Combined ARS1620/SHP2 inhibition is highly efficacious in PDAC models in vivo . A , Pancreas tumors were established in syngeneic mice by orthotopic injections of KCP cells, and 14 days later, mice were treated with vehicle, SHP099, ARS1620 or both drugs (Combo), as depicted. Tumor weight was quantified in a cohort at Day 0 (baseline) and in treated mice at Day 10. B , Immunoblots of KCP-derived tumor lysates showing effects of the indicated treatments on KRAS G12C -GTP, pERK, and DUSP6 levels. C , ERK-dependent gene expression, assessed by RNAseq, in KCP tumors treated for 3 days, as indicated in A (colors indicate log2FC). D-E , Time-dependent increase in RTK (D) and RTK ligands (E) gene expression in KCP-derived orthotopic tumors after vehicle, SHP099, ARS1620 and Combo treatment at Day 3, determined by RNAseq (colors represent log2FC). F , H E, Masson Trichome, CD31, pERK, Ki67 and cleaved <t>Caspase</t> 3 staining and quantification in KCP tumor sections from mice after 10 days of treatment, as indicated. G , KCP tumors were established in syngeneic mice and allowed to grow to much larger size before treatments were initiated, as depicted in the scheme. Tumor weight was quantified in one cohort before treatment, in another cohort after 12 days of treatment, and after drug withdrawal, at Day 27, as indicated. H , Kaplan-Meier curve of KCP tumor-bearing mice after withdrawal of the indicated drugs (top). Tumor growth curve after withdrawal of indicated treatment at day 12 (bottom). H , Response of sub-cutaneous NY53 patient-derived xenograft to treatment with vehicle, SHP099, ARS1620 or both drugs. For all experiments, drug doses were: SHP099 (75 mg/kg body weight, daily), ARS1620 (200 mg/kg body weight, daily) or both drugs (daily). Data represent mean ± SD; *P
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    p53 25,26,53,54/+  Embryos Exhibit Additional Features of CHARGE Syndrome and p53-Dependent Cellular Responses (a)  Double outlet right ventricle (DORV) in E13.5 p53 25,26,53,54/+  heart (50%, n=6). Top: Main pulmonary artery (MPA) connects via pulmonary valve (PV) to right ventricle (RV) in both control and p53 25,26,53,54/+  embryo. Bottom: Aorta (Ao) in control embryo connects to left ventricle (LV) via aortic valve (AV) Φ . Aorta in p53 25,26,53,54/+  embryo connects to RV via AV*. (b)  Abnormal atrioventricular cushions in E13.5 p53 25,26,53,54/+  heart (75%, n=4) fail to elongateinto mature mitral (mv, arrowhead) and tricuspid (tv, arrow) valves. RA: right atrium; LA: left atrium.  (c)  E13.5  p53 25,26,53,54/+  kidneys are smaller (79%), with fewer average glomeruli (13 vs. 3; n=5; arrows), than controls. (d) p53 25,26,53,54/+  embryonic phenotypes observed in CHARGE (+present, −absent).  (e)  Left: Cleaved-caspase 3 (CC3; Top) and p53 (Bottom) immunohistochemistry in E15.5 retinas. Arrows: CC3-positive cells. Right: CC3-positive cells per retinal area. ***p-value=0.007; one-tailed Welsh’s t-test (n=5).  (f)  BrdU immunofluorescence in E9.5 Pax3 +  NCCs (delineated by green-dotted line;  Extended-Data Fig. 6c ). Right: Percentage BrdU-positive cells per total Pax3 +  NCCs ***p-value=0.004 one-tailed Student’s t-test (n=4).

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 25,26,53,54/+ Embryos Exhibit Additional Features of CHARGE Syndrome and p53-Dependent Cellular Responses (a) Double outlet right ventricle (DORV) in E13.5 p53 25,26,53,54/+ heart (50%, n=6). Top: Main pulmonary artery (MPA) connects via pulmonary valve (PV) to right ventricle (RV) in both control and p53 25,26,53,54/+ embryo. Bottom: Aorta (Ao) in control embryo connects to left ventricle (LV) via aortic valve (AV) Φ . Aorta in p53 25,26,53,54/+ embryo connects to RV via AV*. (b) Abnormal atrioventricular cushions in E13.5 p53 25,26,53,54/+ heart (75%, n=4) fail to elongateinto mature mitral (mv, arrowhead) and tricuspid (tv, arrow) valves. RA: right atrium; LA: left atrium. (c) E13.5 p53 25,26,53,54/+ kidneys are smaller (79%), with fewer average glomeruli (13 vs. 3; n=5; arrows), than controls. (d) p53 25,26,53,54/+ embryonic phenotypes observed in CHARGE (+present, −absent). (e) Left: Cleaved-caspase 3 (CC3; Top) and p53 (Bottom) immunohistochemistry in E15.5 retinas. Arrows: CC3-positive cells. Right: CC3-positive cells per retinal area. ***p-value=0.007; one-tailed Welsh’s t-test (n=5). (f) BrdU immunofluorescence in E9.5 Pax3 + NCCs (delineated by green-dotted line; Extended-Data Fig. 6c ). Right: Percentage BrdU-positive cells per total Pax3 + NCCs ***p-value=0.004 one-tailed Student’s t-test (n=4).

    Article Snippet: Whole-mount cleaved-caspase 3 staining was performed as described with anti-cleaved-caspase 3 antibody (Cell Signaling #9664) and developed with DAB (Vector Labs).

    Techniques: Immunohistochemistry, One-tailed Test, Immunofluorescence

    p53 25,26,53,54/+  Embryo Tissues Display Increased Apoptosis and Decreased Proliferation (a)  Left: Immunofluorescence for Phospho-Histone H3 (red) in the retina of E13.5 control and  p53 25,26,53,54/+ embryos. Right: Quantification of Phospho-Histone H3 positive cells per retina area relative to littermate controls. **p-value=0.006 by one-tailed Welsh’s t-test (n=4). ( b ) Left: Immunohistochemistry for cleaved-caspase 3 (CC3) in thymi of control (left) and  p53 25,26,53,54/+  (right) embryos. Inset: close-up image of cleaved-caspase 3 positive region. Right: Quantification of CC3-positive cells per thymic area. *p-value=0.02 by one-tailed Student’s t-test (n=4).  (c)  Immunofluorescence for Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+  embryos was used to identify neural crest cells in   Figure 2f .  (d) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) and Pax3 (green) in neural crest cells of E9.5 control and  p53 25,26,53,54/+ embryos.  p53 25,26,53,54/+  embryos have more apoptotic (red) neural crest cells, as determined by Pax3-positive staining (green), compared to control littermates. Right: Quantification of CC3 positive cells per total neural crest cell number. p-value=0.14 by one-tailed Student’s t-test (n=4). (e)  Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) in otic vesicle of E9.5 control and  p53 25,26,53,54/+  embryos. Right: Quantification of CC3 positive cells per total cell number. *p-value=0.03 by one-tailed Student’s t-test (n=3). ( f ) Whole-mount cleaved-caspase 3 staining in E8.5 control and p53 25,26,53,54/+  embryos reveals enhanced apoptosis in the neuroepithelium of  p53 25,26,53,54/+  embryos (right) but not in controls (left). Close-up shows magnification of the caudal neuroepithelium (bottom). Arrows indicate cleaved-caspase 3 positive regions.

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 25,26,53,54/+ Embryo Tissues Display Increased Apoptosis and Decreased Proliferation (a) Left: Immunofluorescence for Phospho-Histone H3 (red) in the retina of E13.5 control and p53 25,26,53,54/+ embryos. Right: Quantification of Phospho-Histone H3 positive cells per retina area relative to littermate controls. **p-value=0.006 by one-tailed Welsh’s t-test (n=4). ( b ) Left: Immunohistochemistry for cleaved-caspase 3 (CC3) in thymi of control (left) and p53 25,26,53,54/+ (right) embryos. Inset: close-up image of cleaved-caspase 3 positive region. Right: Quantification of CC3-positive cells per thymic area. *p-value=0.02 by one-tailed Student’s t-test (n=4). (c) Immunofluorescence for Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+ embryos was used to identify neural crest cells in Figure 2f . (d) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) and Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+ embryos. p53 25,26,53,54/+ embryos have more apoptotic (red) neural crest cells, as determined by Pax3-positive staining (green), compared to control littermates. Right: Quantification of CC3 positive cells per total neural crest cell number. p-value=0.14 by one-tailed Student’s t-test (n=4). (e) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) in otic vesicle of E9.5 control and p53 25,26,53,54/+ embryos. Right: Quantification of CC3 positive cells per total cell number. *p-value=0.03 by one-tailed Student’s t-test (n=3). ( f ) Whole-mount cleaved-caspase 3 staining in E8.5 control and p53 25,26,53,54/+ embryos reveals enhanced apoptosis in the neuroepithelium of p53 25,26,53,54/+ embryos (right) but not in controls (left). Close-up shows magnification of the caudal neuroepithelium (bottom). Arrows indicate cleaved-caspase 3 positive regions.

    Article Snippet: Whole-mount cleaved-caspase 3 staining was performed as described with anti-cleaved-caspase 3 antibody (Cell Signaling #9664) and developed with DAB (Vector Labs).

    Techniques: Immunofluorescence, One-tailed Test, Immunohistochemistry, Staining

    Combined ARS1620/SHP2 inhibition is highly efficacious in PDAC models in vivo . A , Pancreas tumors were established in syngeneic mice by orthotopic injections of KCP cells, and 14 days later, mice were treated with vehicle, SHP099, ARS1620 or both drugs (Combo), as depicted. Tumor weight was quantified in a cohort at Day 0 (baseline) and in treated mice at Day 10. B , Immunoblots of KCP-derived tumor lysates showing effects of the indicated treatments on KRAS G12C -GTP, pERK, and DUSP6 levels. C , ERK-dependent gene expression, assessed by RNAseq, in KCP tumors treated for 3 days, as indicated in A (colors indicate log2FC). D-E , Time-dependent increase in RTK (D) and RTK ligands (E) gene expression in KCP-derived orthotopic tumors after vehicle, SHP099, ARS1620 and Combo treatment at Day 3, determined by RNAseq (colors represent log2FC). F , H E, Masson Trichome, CD31, pERK, Ki67 and cleaved Caspase 3 staining and quantification in KCP tumor sections from mice after 10 days of treatment, as indicated. G , KCP tumors were established in syngeneic mice and allowed to grow to much larger size before treatments were initiated, as depicted in the scheme. Tumor weight was quantified in one cohort before treatment, in another cohort after 12 days of treatment, and after drug withdrawal, at Day 27, as indicated. H , Kaplan-Meier curve of KCP tumor-bearing mice after withdrawal of the indicated drugs (top). Tumor growth curve after withdrawal of indicated treatment at day 12 (bottom). H , Response of sub-cutaneous NY53 patient-derived xenograft to treatment with vehicle, SHP099, ARS1620 or both drugs. For all experiments, drug doses were: SHP099 (75 mg/kg body weight, daily), ARS1620 (200 mg/kg body weight, daily) or both drugs (daily). Data represent mean ± SD; *P

    Journal: bioRxiv

    Article Title: SHP2 Inhibition Abrogates Adaptive Resistance to KRASG12C-Inhibition and Remodels the Tumor Microenvironment of KRAS-Mutant Tumors

    doi: 10.1101/2020.05.30.125138

    Figure Lengend Snippet: Combined ARS1620/SHP2 inhibition is highly efficacious in PDAC models in vivo . A , Pancreas tumors were established in syngeneic mice by orthotopic injections of KCP cells, and 14 days later, mice were treated with vehicle, SHP099, ARS1620 or both drugs (Combo), as depicted. Tumor weight was quantified in a cohort at Day 0 (baseline) and in treated mice at Day 10. B , Immunoblots of KCP-derived tumor lysates showing effects of the indicated treatments on KRAS G12C -GTP, pERK, and DUSP6 levels. C , ERK-dependent gene expression, assessed by RNAseq, in KCP tumors treated for 3 days, as indicated in A (colors indicate log2FC). D-E , Time-dependent increase in RTK (D) and RTK ligands (E) gene expression in KCP-derived orthotopic tumors after vehicle, SHP099, ARS1620 and Combo treatment at Day 3, determined by RNAseq (colors represent log2FC). F , H E, Masson Trichome, CD31, pERK, Ki67 and cleaved Caspase 3 staining and quantification in KCP tumor sections from mice after 10 days of treatment, as indicated. G , KCP tumors were established in syngeneic mice and allowed to grow to much larger size before treatments were initiated, as depicted in the scheme. Tumor weight was quantified in one cohort before treatment, in another cohort after 12 days of treatment, and after drug withdrawal, at Day 27, as indicated. H , Kaplan-Meier curve of KCP tumor-bearing mice after withdrawal of the indicated drugs (top). Tumor growth curve after withdrawal of indicated treatment at day 12 (bottom). H , Response of sub-cutaneous NY53 patient-derived xenograft to treatment with vehicle, SHP099, ARS1620 or both drugs. For all experiments, drug doses were: SHP099 (75 mg/kg body weight, daily), ARS1620 (200 mg/kg body weight, daily) or both drugs (daily). Data represent mean ± SD; *P

    Article Snippet: IHC for pERK (Cell Signaling, 4370), CD31 (Cell Signaling, D8V9E), Cleaved Caspase 3 (Cell Signaling, D3E9), Ki67 (Spring Biosciences, SP6), αSMA (Abcam, ab5694) was performed on sections from paraformaldehyde-fixed tumors.

    Techniques: Inhibition, In Vivo, Mouse Assay, Western Blot, Derivative Assay, Expressing, Staining

    Histology validation. a. T 2 -weighted image of an OV-treated mouse, 72 hours post virus inoculation. b. Semisolid macro-molecules proton volume fraction (f ss ) map, overlaid atop the T 2 -weighted image at the ipsilateral side. c. Similarly overlaid amide proton exchange-rate (k sw ) and d. amide proton volume fraction (f s ) maps. e. Immunohistochemistry image stained for Herpes Simplex Virus (HSV) presence (brown). f. HE stained image, demonstrating the tumor location (pink). g. Caspase-3 immunohistochemistry image, demonstrating the apoptotic tumor region (brown). h. Coomassie Blue stained image, demonstrating reduced protein concentration in the apoptotic tumor core. The dashed lines in images b-d, and f-h, generally depict the tumor (b, f) and apoptotic (c, d, g, h) regions borders, respectively.

    Journal: bioRxiv

    Article Title: AI boosted molecular MRI for apoptosis detection in oncolytic virotherapy

    doi: 10.1101/2020.03.05.977793

    Figure Lengend Snippet: Histology validation. a. T 2 -weighted image of an OV-treated mouse, 72 hours post virus inoculation. b. Semisolid macro-molecules proton volume fraction (f ss ) map, overlaid atop the T 2 -weighted image at the ipsilateral side. c. Similarly overlaid amide proton exchange-rate (k sw ) and d. amide proton volume fraction (f s ) maps. e. Immunohistochemistry image stained for Herpes Simplex Virus (HSV) presence (brown). f. HE stained image, demonstrating the tumor location (pink). g. Caspase-3 immunohistochemistry image, demonstrating the apoptotic tumor region (brown). h. Coomassie Blue stained image, demonstrating reduced protein concentration in the apoptotic tumor core. The dashed lines in images b-d, and f-h, generally depict the tumor (b, f) and apoptotic (c, d, g, h) regions borders, respectively.

    Article Snippet: Slides were incubated with primary antibodies against HSV 1 (B0114, Dako; 1:100 dilution) or cleaved caspase 3 (9579, Cell Signaling Technology; 1:150) diluted as recommended by the manufacturer with TBST.

    Techniques: Immunohistochemistry, Staining, Protein Concentration

    Doxycycline Reduces Cleaved Caspase 3 Levels (A) Immunofluorescence of iPSC line AS7192-9 two days after induction with staining for Hoechst 33342, Ki-67, Cleaved Caspase 3 (CC3), and combined (first column) shows greatly decreased CC3 in the presence of doxycycline (n=3). At this time point, there is a slight decrease in Ki-67. First two rows are 10x with scale bar at 400μm and bottom two rows 20x with 200μm scale bar. Data were analyzed by Student’s t test and presented as mean ± SEM. * p

    Journal: bioRxiv

    Article Title: Doxycycline Significantly Enhances Induction of iPSCs to Endoderm by Enhancing survival via AKT Phosphorylation

    doi: 10.1101/2020.04.13.034595

    Figure Lengend Snippet: Doxycycline Reduces Cleaved Caspase 3 Levels (A) Immunofluorescence of iPSC line AS7192-9 two days after induction with staining for Hoechst 33342, Ki-67, Cleaved Caspase 3 (CC3), and combined (first column) shows greatly decreased CC3 in the presence of doxycycline (n=3). At this time point, there is a slight decrease in Ki-67. First two rows are 10x with scale bar at 400μm and bottom two rows 20x with 200μm scale bar. Data were analyzed by Student’s t test and presented as mean ± SEM. * p

    Article Snippet: Immunohistochemistry and Cell staining IPSCs and iHeps were fixed using 4% paraformaldehyde for 15 min, permeabilized using 0.1% triton X-100 for 10 minutes followed by 3x PBS (phosphate buffered saline, Corning Life Sciences) rinses and blocked with 1.5% bovine serum albumin for 1 h. Samples were incubated with primary antibody overnight (List of all antibodies with dilutions 1:100 - 1:., OCT3/4 (Santa Cruz, #sc-9081, 1:500, SOX2 (Proteintech, #11064-1-AP, 1:1000), NANOG (Proteintech, #14295-1-AP, 1:1000), SSEA (Proteintech, #19497-1-AP, 1:500), TRA 1-60 (Proteintech, #18150-1-AP, 1:500), HNFα4 (Proteintech, #26245-1-AP, 1:500, Albumin (Bethyl, #A80-129A, 1:500, AFP (Thermo Scientific, #RB-365-A1, 1:500), ki-67 (8D5) (Cell Signaling, #9449, 1:800), Cleaved Caspase-3 (Cell Signaling, #9661, 1:400)) in blocking solution.

    Techniques: Immunofluorescence, Staining

    Doxycycline Reduces Apoptosis and Increases Mitochondrial Mass (A) Immunofluorescence of iPSC line 1023-5 on day 2 of induction for Hoechst 33342, Ki-67, Cleaved Caspase 3 (CC3), and combined shows greatly decreased CC3 staining with doxycycline treatment. At this early time point, there is a small but significant decrease in Ki-67. First two rows are 10x with scale bar at 400μm and bottom two rows 20x with 200μm scale bar. (B) Mitotracker Orange staining of iPSCs with no induction and with induction shows that addition of Doxycycline increases the quantity of polarized mitochondria compared to vehicle alone in three separate iPSC lines (AS7192-7, 1023-5, and AS7017-2) (n=3). Scale bar represents 100μm. Data were analyzed by Student’s t test and presented as mean ± SEM. * p

    Journal: bioRxiv

    Article Title: Doxycycline Significantly Enhances Induction of iPSCs to Endoderm by Enhancing survival via AKT Phosphorylation

    doi: 10.1101/2020.04.13.034595

    Figure Lengend Snippet: Doxycycline Reduces Apoptosis and Increases Mitochondrial Mass (A) Immunofluorescence of iPSC line 1023-5 on day 2 of induction for Hoechst 33342, Ki-67, Cleaved Caspase 3 (CC3), and combined shows greatly decreased CC3 staining with doxycycline treatment. At this early time point, there is a small but significant decrease in Ki-67. First two rows are 10x with scale bar at 400μm and bottom two rows 20x with 200μm scale bar. (B) Mitotracker Orange staining of iPSCs with no induction and with induction shows that addition of Doxycycline increases the quantity of polarized mitochondria compared to vehicle alone in three separate iPSC lines (AS7192-7, 1023-5, and AS7017-2) (n=3). Scale bar represents 100μm. Data were analyzed by Student’s t test and presented as mean ± SEM. * p

    Article Snippet: Immunohistochemistry and Cell staining IPSCs and iHeps were fixed using 4% paraformaldehyde for 15 min, permeabilized using 0.1% triton X-100 for 10 minutes followed by 3x PBS (phosphate buffered saline, Corning Life Sciences) rinses and blocked with 1.5% bovine serum albumin for 1 h. Samples were incubated with primary antibody overnight (List of all antibodies with dilutions 1:100 - 1:., OCT3/4 (Santa Cruz, #sc-9081, 1:500, SOX2 (Proteintech, #11064-1-AP, 1:1000), NANOG (Proteintech, #14295-1-AP, 1:1000), SSEA (Proteintech, #19497-1-AP, 1:500), TRA 1-60 (Proteintech, #18150-1-AP, 1:500), HNFα4 (Proteintech, #26245-1-AP, 1:500, Albumin (Bethyl, #A80-129A, 1:500, AFP (Thermo Scientific, #RB-365-A1, 1:500), ki-67 (8D5) (Cell Signaling, #9449, 1:800), Cleaved Caspase-3 (Cell Signaling, #9661, 1:400)) in blocking solution.

    Techniques: Immunofluorescence, Staining