Journal: The Journal of Cell Biology

Article Title: Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

doi: 10.1083/jcb.201111063

Figure Lengend Snippet: Bnip3 cooperates with AIF to induce apoptosis and cavitation. (A) 4-d AIF y/+ and AIF y/− EBs were analyzed by immunoblotting for AIF. Actin was used as a loading control. (B) EBs were cultured for 1–4 d and analyzed by immunoblotting for cleaved caspase-3 (cas-3) and actin. Ablation of AIF inhibited caspase-3 activation. (C) Live-phase micrographs show cavitation delay in AIF y/− EBs cultured for 4, 5, and 7 d. After 10 d, most of the AIF y/− EBs were cavitated similar to AIF y/+ EBs. Bars, 100 µm. (D) 4-d EBs were immunostained for cleaved caspase-3. F-actin was stained with rhodamine-phalloidin to show the apical actin belt. Ablation of AIF inhibited apoptosis of the core cells. 5-d EBs were immunostained for the apical marker MUPP1. Apical polarization of the AIF y/− epiblast was not affected despite delayed lumen clearance. (E) AIF y/− ES cells were stably transfected with Bnip3 shRNA (Bnip3 knockdown [KD]) or GFP. 5-d EBs were analyzed by immunoblotting for Bnip3 and cleaved caspase-3. Bnip3 silencing in AIF y/− EBs further inhibited caspase-3 activation. (F) AIF y/+ EBs expressing GFP and AIF y/− EBs stably transfected with Bnip3 shRNA or GFP were cultured for 4, 5, and 7 d. EB cavitation was quantitated by phase microscopy. EB cavitation was significantly delayed in the absence of AIF. Knockdown of Bnip3 in AIF y/− EBs nearly blocked cavitation. n = 6 independent experiments with a total of 529–808 EBs counted for each group. Error bars represent the mean ± SD. *, P < 0.01 versus AIF y/+ GFP; # , P < 0.01 versus AIF y/− GFP.

Article Snippet: Four pGFP-V-RS–based shRNA vectors targeting to mouse Bnip3 and four pRFP-V-RS–based vectors targeting to Bim together with scrambled controls were purchased from OriGene.

Techniques: Western Blot, Cell Culture, Activation Assay, Staining, Marker, Stable Transfection, Transfection, shRNA, Expressing, Microscopy

Journal: Frontiers in Immunology

Article Title: Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor

doi: 10.3389/fimmu.2019.01164

Figure Lengend Snippet: Expression of RANKL in osteoblasts and osteoclastogenesis in co-cultures of bone marrow macrophages and calvarial osteoblasts induced by OSM are dependent on Shc1 and STAT3. Osteoblasts in which the Shc1 gene (A) or the Stat3 gene (D) was knocked-down by siRNA were treated with OSM at 100 ng/mL for 24 h before analysis of Tnfsf11 gene expression. Osteoblasts transfected with scrambled RNA (siSCR) were similarly treated and analyzed. Osteoblasts in which Shc1 (B,C) or Stat3 (E,F) was knocked down (or siSCR as control) were exposed to OSM (100 ng/mL) or vehicle and co-cultured with BMMs for 3 days before TRAP staining and counting of TRAP + MuOCL (A,C,D,F) . Values represent means for four wells and SEM is shown as vertical bars. Significant differences are indicated by horizontal lines where ** P < 0.01; *** P < 0.001; analyzed by two-way ANOVA followed by Tukey post hoc-test . The difference in OSM-induced response with and without silencing analyzed by two-way ANOVA was statistically significant [interaction P -value in A ( P < 0.0001), C ( P < 0.005), D ( P < 0.0001) and F ( P < 0.0001)]. Scale bar in (B,E) is 50 μm.

Article Snippet: Recombinant mouse LIF, mouse OSM, bone morphogenetic protein-2 (BMP-2), macrophage colony-stimulating factor (M-CSF), RANKL (amino acids 158–316; cat. no. 462-TEC) and the ELISA kits for mouse RANKL and mouse OPG were purchased from R&D Systems, Abingdon, UK; bacterial collagenase type I from Worthington Biochemical Corp., Lakewood, NJ, USA; α-MEM, FBS, L-glutamine, and oligonucleotide primers from Invitrogen, Stockholm, Sweden; RNAqueous®-4PCR RNA isolation kit from Ambion, Inc., Austin TX, USA; 1st strand cDNA synthesis Kit and PCR Core Kit from Roche, Mannheim, Germany; DYEnamic ET terminator cycle sequencing kit from GE Healthcare, Uppsala, Sweden; QIAquick PCR Purification kit was from Qiagen Ltd., Crawley, West Sussex, England; TaqMan Universal PCR Master Mix and TaqMan probes from Applied Biosystems, Foster City, CA, USA; all primary and secondary antibodies used are specified in ; anti-IgG-HRP secondary antibodies used for Western blot were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; culture dishes and multi-well plates from Costar, Cambridge, MA, USA, or Nunc International Corp., Naperville, IL, USA.

Techniques: Expressing, Gene Expression, Transfection, Control, Cell Culture, Staining

Journal: Frontiers in Immunology

Article Title: Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor

doi: 10.3389/fimmu.2019.01164

Figure Lengend Snippet: OSM and, to a lesser extent, LIF stimulate bone resorption and RANKL production in calvarial bones, calvarial osteoblasts and stromal cells. (A) Mouse calvarial bones were cultured in the absence or the presence of LIF or OSM (both 0.1–100 ng/mL) and bone resorption was assessed by 45 Ca release after a 5-day culture period. (B) RT-qPCR was performed using mRNA extracted from calvarial bones treated with either LIF or OSM (both at 100 ng/mL) for 24 h to assess the expression of Tnfsf11 . (C) Protein expression of RANKL after 48 h was also analyzed in calvarial bone treated with LIF or OSM (both at 100 ng/L). (D) Mouse calvarial osteoblasts were incubated in the absence (Co) or the presence of LIF (100 ng/mL) or OSM (100 ng/mL) for 48 h and expression of Tnfsf11 was analyzed by semi-quantitative RT-PCR. (E) The expression of Tnfsf11 mRNA in calvarial osteoblasts stimulated by LIF and OSM a different concentrations (0.1-100 ng/mL) was performed using quantitative RT-PCR. (F) The mRNA expression of Osmr and Lifr in osteoblasts was compared at three different time points. (G) The receptor components Il6st, Lifr and Osmr are expressed in ST-2 stromal cells as assessed by RT-PCR. (H) The mRNA expression of Tnfsf11 in ST-2 cells cultured without (Co) or with LIF or OSM (both at 100 ng/mL) for 48 h was analyzed. Values represent means for six bones (calvarial bones) or four wells (cell culture experiments) and SEM is shown as vertical bars. * , ** , and *** , indicate significant difference compared to untreated (Co) cells, * P < 0.05, ** P < 0.01, and *** P < 0.001, respectively. Statistical significance was determined by ANOVA using Levene's homogeneity test followed by Dunnett's T3 post-hoc tests vs. Co. In (F) , Tukey post-hoc test was used to compare all groups and no statistical difference was observed.

Article Snippet: Recombinant mouse LIF, mouse OSM, bone morphogenetic protein-2 (BMP-2), macrophage colony-stimulating factor (M-CSF), RANKL (amino acids 158–316; cat. no. 462-TEC) and the ELISA kits for mouse RANKL and mouse OPG were purchased from R&D Systems, Abingdon, UK; bacterial collagenase type I from Worthington Biochemical Corp., Lakewood, NJ, USA; α-MEM, FBS, L-glutamine, and oligonucleotide primers from Invitrogen, Stockholm, Sweden; RNAqueous®-4PCR RNA isolation kit from Ambion, Inc., Austin TX, USA; 1st strand cDNA synthesis Kit and PCR Core Kit from Roche, Mannheim, Germany; DYEnamic ET terminator cycle sequencing kit from GE Healthcare, Uppsala, Sweden; QIAquick PCR Purification kit was from Qiagen Ltd., Crawley, West Sussex, England; TaqMan Universal PCR Master Mix and TaqMan probes from Applied Biosystems, Foster City, CA, USA; all primary and secondary antibodies used are specified in ; anti-IgG-HRP secondary antibodies used for Western blot were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; culture dishes and multi-well plates from Costar, Cambridge, MA, USA, or Nunc International Corp., Naperville, IL, USA.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor

doi: 10.3389/fimmu.2019.01164

Figure Lengend Snippet: OSM, but not LIF, stimulates osteoclastogenesis in bone marrow cell cultures. Mouse bone marrow cells (BMC) were cultured in the absence (Co) or the presence of LIF, or OSM (both at 100 ng/mL), PTH or 1,25(OH) 2 -vitamin D3 (D3; both at 10 −8 M) for 7 days before staining (A) and counting (B) of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts (TRAP + MuOCL). Quantitative real-time PCR analysis of mRNA expression of cathepsin K ( Ctsk , C ), TRAP ( Acp5 , D ), calcitonin receptor ( Calcr , E ) RANKL ( Tnfsf11 , F ) and OPG ( Tnfsf11b , G ) in BMC cultured without (Co) or with LIF or OSM (both at 100 ng/mL) for 7 days. Values represent means for four wells and SEM is shown as vertical bars. *** , indicates significant difference compared to untreated (Co) cells, *** P < 0.001. Statistical significance was determined by ANOVA using Levene's homogeneity test followed by Dunnett's 2-sided (B–F) or Dunnett's T3 (G) post-hoc test.

Article Snippet: Recombinant mouse LIF, mouse OSM, bone morphogenetic protein-2 (BMP-2), macrophage colony-stimulating factor (M-CSF), RANKL (amino acids 158–316; cat. no. 462-TEC) and the ELISA kits for mouse RANKL and mouse OPG were purchased from R&D Systems, Abingdon, UK; bacterial collagenase type I from Worthington Biochemical Corp., Lakewood, NJ, USA; α-MEM, FBS, L-glutamine, and oligonucleotide primers from Invitrogen, Stockholm, Sweden; RNAqueous®-4PCR RNA isolation kit from Ambion, Inc., Austin TX, USA; 1st strand cDNA synthesis Kit and PCR Core Kit from Roche, Mannheim, Germany; DYEnamic ET terminator cycle sequencing kit from GE Healthcare, Uppsala, Sweden; QIAquick PCR Purification kit was from Qiagen Ltd., Crawley, West Sussex, England; TaqMan Universal PCR Master Mix and TaqMan probes from Applied Biosystems, Foster City, CA, USA; all primary and secondary antibodies used are specified in ; anti-IgG-HRP secondary antibodies used for Western blot were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; culture dishes and multi-well plates from Costar, Cambridge, MA, USA, or Nunc International Corp., Naperville, IL, USA.

Techniques: Cell Culture, Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Immunology

Article Title: Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor

doi: 10.3389/fimmu.2019.01164

Figure Lengend Snippet: Lack of effect by OSM and LIF on osteoclast formation in bone marrow macrophage cultures. (A,B) BMMs were cultured in M-CSF (30 ng/mL) or differentiated to osteoclasts by addition of M-CSF + RANKL (30 and 4 ng/mL, respectively) in the presence or absence of LIF or OSM (both at 100 ng/mL) for 3 days before staining for tartarate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts (TRAP + MuOCL), which then were counted. No osteoclasts were formed in the absence of RANKL. (C) Quantitative PCR analyses of Il6st, Lifr , and Acp5 in BMM cultured for 72 h with M-CSF (30 ng/ml) or M-CSF+RANKL (30 and 4 ng/ml, respectively). Values represent means for four wells and SEM is shown as vertical bars. In (C) *** , indicates significant difference compared to cells treated with M-CSF (M), P < 0.001. In (B) Statistical significance was determined one-way ANOVA followed by Tukey's post-hoc test and in (C) , Student's t -test was used for comparison between M and M + R groups for each gene.

Article Snippet: Recombinant mouse LIF, mouse OSM, bone morphogenetic protein-2 (BMP-2), macrophage colony-stimulating factor (M-CSF), RANKL (amino acids 158–316; cat. no. 462-TEC) and the ELISA kits for mouse RANKL and mouse OPG were purchased from R&D Systems, Abingdon, UK; bacterial collagenase type I from Worthington Biochemical Corp., Lakewood, NJ, USA; α-MEM, FBS, L-glutamine, and oligonucleotide primers from Invitrogen, Stockholm, Sweden; RNAqueous®-4PCR RNA isolation kit from Ambion, Inc., Austin TX, USA; 1st strand cDNA synthesis Kit and PCR Core Kit from Roche, Mannheim, Germany; DYEnamic ET terminator cycle sequencing kit from GE Healthcare, Uppsala, Sweden; QIAquick PCR Purification kit was from Qiagen Ltd., Crawley, West Sussex, England; TaqMan Universal PCR Master Mix and TaqMan probes from Applied Biosystems, Foster City, CA, USA; all primary and secondary antibodies used are specified in ; anti-IgG-HRP secondary antibodies used for Western blot were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; culture dishes and multi-well plates from Costar, Cambridge, MA, USA, or Nunc International Corp., Naperville, IL, USA.

Techniques: Cell Culture, Staining, Real-time Polymerase Chain Reaction, Comparison

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Binding between AKAP79/AKAP150 and the N terminus of AC8. A , GST pull-downs using whole cell lysate from cells transiently expressing tagged AKAP proteins. Lane 1 , GST alone; lanes 2–4 , the following regions of AC8 fused to GST: 1–179 (N terminus), 582–703 (C1b domain), and 1106–1248 (C terminus), respectively. Lane 5 , input control (5%) to confirm expression of each construct. B , top , AKAP79-HA and AKAP150-HA transiently expressed in HEK293 cells were pulled down using GST-fused first half (residues 1–77) or second half (residues 73–179) of the AC8 N terminus. Bottom , immunoblotting of the GST-fused proteins used in the pull-downs in the top . The upper GST bands represent the full-length form of each protein. C , plot of densitometries from 3–6 repeats of blots presented in B to quantify binding of AKAP79/150 to GST-AC8 1–77 versus GST-AC8 73–179. Data are normalized to the intensity of the uppermost GST bands. Error bars , S.E. D , schematic diagram of GST-AC8 constructs used in A–C .

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Binding Assay, Expressing, Control, Construct, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: AKAP disruption enhances CCE-induced AC8 activity. A , CCE-mediated increases in cAMP assessed using the FRET-based sensor Epac2-camps following knockdown of endogenous AKAP79 levels. Data are plotted as relative FRET ratio changes normalized to maximum signal response. Cells were pretreated with 200 n m Tg in Ca 2+ -free conditions, and CCE was induced upon the addition of 2 m m Ca 2+ to the bath solution. B , average Fura-2 traces from HEK-AC8 cells during CCE following transfection with AKAP150-HA, AKAP79-HA, or shRNA AKAP79. All data are normalized to maximal Fura-2 signal obtained upon the addition of 5 μ m ionomycin (Ca 2+ ionophore) and 5 m m Ca 2+ . C , effects of the AKAP/PKA disruptor peptide, St-Ht31 (10 μ m ), on CCE-stimulated AC8 activity assessed using Epac2-camps. Experiments were performed in the presence of 100 μ m IBMX, and CCE was induced upon the addition of 0.5 m m Ca 2+ . St-Ht31P (10 μ m ) was used as a negative control. D , analyses of the effects of AKAP79 knockdown or pharmacological AKAP disruption (St-Ht31) on AC8 activity. Plots of peak CCE-induced cAMP increase relative to control ( left chart ) and peak rate of cAMP production ( right chart ) are presented as mean ± S.E. ( error bars ). **, p < 0.001 using Students' t test.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Disruption, Activity Assay, Knockdown, Transfection, shRNA, Negative Control, Control

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Co-immunoprecipitation of endogenously expressed AC8 and AKAP150 in MIN6 cells. A , Western blot analysis to confirm the endogenous expression of AKAP150 ( left ) and AC8 ( right ) in MIN6 cells. B , immune complexes for endogenous AKAP150 co-purify with AC8 in MIN6 cell lysate. No AC8 band is seen in IgG controls. Antibodies used for immunoprecipitation ( IP ) and subsequent immunoblotting ( IB ) were specific to AKAP150 and AC8, respectively.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Immunoprecipitation, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Effects of AKAP150 on Ca 2+ -stimulated AC8 activity in MIN6 cells. A , Fura-2 data showing the standard protocol for inducing CCE in MIN6 cells. Cells were pretreated with 1 μ m Tg in Ca 2+ -free conditions for 3 min prior to the addition of 2 m m external Ca 2+ . The addition of 100 μ m 2-aminoethoxydiphenyl borate ( 2-APB ; a CCE inhibitor when used at high concentrations) from 1 min onward significantly reduced Ca 2+ entry (see bar chart inset ; ***, p < 0.001). Bi , Epac2-camps data showing the effects of AKAP150 overexpression on Ca 2+ -stimulated cAMP production compared with empty vector controls. 20 n m FSK and 100 μ m IBMX were present throughout. Data are plotted as a percentage of maximal FRET signal obtained using saturating cAMP concentrations. Bii , as above, except that the effects of lentiviral shRNA directed against AKAP150 were compared with scrambled shRNA controls. C , bar charts show mean ± S.E. values ( error bars ) for peak amplitude and rate of FRET ratio changes during CCE for data in B . *, p < 0.05 compared with shRNA controls.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Activity Assay, Over Expression, Plasmid Preparation, shRNA

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: A role for AKAP150 in the regulation of Ca 2+ -stimulated AC activity in hippocampal neurons. A , the basic design of the citrine-Epac2-camps-CFP (Ci/C-Epac2-camps) sensor used for hippocampal experiments and fluorescent images taken from three individual hippocampal neurons showing cytosolic expression of the cAMP sensor. Scale bar , 20 μm. B , in vitro calibrations of the Ci/C-Epac2-camps compared with the original Epac2-camps. Note the reduced pH sensitivity of the citrine-CFP version. C , comparison of Ca 2+ -stimulated AC activity in control, AKAP150-HA-expressing, and AKAP150 knockdown hippocampal neurons assessed using Ci/C-Epac2-camps. 1 μ m FSK was added in Ca 2+ -free conditions at 120 s with the readdition of 2 m m external Ca 2+ at 180 s to monitor Ca 2+ -dependent cAMP production. Maximum FRET ratio change was obtained by the subsequent addition of 10 μ m FSK, 10 μ m isoproterenol, and 100 μ m IBMX. D , overlay of control, AKAP150 overexpression, and shRNA AKAP150 data to compare Ca 2+ -stimulated AC activities. E , data analysis showing significant delay in Ca 2+ stimulation of cAMP production in neurons overexpressing AKAP150. Data represent mean ± S.E. ( error bars ) for each condition. n values are indicated on the graph . *, p < 0.01.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Activity Assay, Expressing, In Vitro, Comparison, Control, Knockdown, Over Expression, shRNA

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 1 mTORC2 affects rearrangement of the F-actin cytoskeleton in mouse fertilized eggs. (a) The development model of mouse fertilized eggs. G1 phase: 12–21 hours after injecting hCG; S phase: 21–27 hours after injecting hCG; G2 phase: 27–30 hours after injecting hCG; M phase: 30–33 hours after injecting hCG. (b) Staining for F-actin (red) and DNA (blue) revealed the organization of the F-actin cytoskeleton in mouse fertilized eggs transfected with RICTOR shRNA (F-actin: yellow arrow). Scale bar: 20 μm. (c) Western immunoblotting detection of mTORC2 in 200 mouse fertilized eggs treated with shRNA targeted against mTORC2. WB: Western blotting

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Staining, Transfection, shRNA, Western Blot

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 2 Akt1 regulates cell division and F-actin rearrangement in mouse fertilized eggs. (a) Western immunoblotting detection of Akt1 in 200 mouse fertilized eggs treated with siRNA targeted against Akt1. WB: Western blotting. (b) The division rate in cultured mouse embryos after 0.03 ng of mRNA-encoding Akt1-WT, myr-Akt1, Akt1-KD or Akt1 siRNA injection. The percentage of cells undergoing cell division and cell survival was calculated after manual counting under a dissecting microscope 35 hours after injection of human chorionic gonadotropin in female mice. The numbers of eggs undergoing cell division (hatched bars) or survival are given above each bar graph. (c) Cortical remodelling of F-actin is induced by Akt1 activation. Mouse fertilized eggs were stained with rhodamine-phalloidin (red fluorescence) to visualize F-actin cytoskeleton. Various scenarios were studied, which included cells injected with mRNA-encoding Akt1-WT, myr-Akt1, Akt1-KD or siRNA targeted against Akt1. Fertilized eggs were fixed and labelled with rhodamine-phalloidin (10 or 20 μmol/L; F-actin labelling, indicated by white arrows) and with Hoechst 33258 (1 mg/ml; DNA labelling) and imaged by laser-scanning confocal microscope. Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Western Blot, Cell Culture, Injection, Microscopy, Activation Assay, Staining, Fluorescence

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 4 Girdin is essential for rearrangement of the F-actin cytoskeleton and the development of mouse fertilized eggs. (a) Depletion of Girdin in mouse one-cell staged fertilized eggs by siRNA. Total cell extracts from control siRNA- and Girdin siRNA- injected one-cell staged fertilized eggs were subjected to Western blot analyses and immunodetection with anti-Girdin, anti-p-Girdin, anti-Akt1 and anti-actin antibodies. (b) The cleavage rate in cultured mouse embryos after Girdin siRNA injections shows that the total number of eggs undergoing cell division is given above each bar graph from three independent experiments. (c) F-actin cytoskeleton of embryos derived from fertilized eggs treated with Girdin siRNA. Mouse fertilized eggs were treated with control or Girdin siRNAs and fixed 48 hours later, followed by staining with rhodamine-phalloidin and anti-Girdin antibody. In situ validation of the interaction between Girdin and polymerized F-actin by confocal microscopy is shown. One-cell stage mouse fertilized eggs were treated with 10 or 20 μmol/L Girdin siRNA. Immunolocalization of Girdin is revealed by green staining (antibody), and immunolocalization of actin is revealed by red staining. Control fertilized eggs (21 hours after hCG): one-cell arrested embryos derived from fertilized eggs that were injected with Girdin siRNA 21 hours after hCG and cultured for 24 hours; control two-cell embryo: in control fertilized eggs, F-actin forms a regular ring in the cell cortex. In embryos derived from fertilized eggs treated with girdin siRNA, polymerized F-actin was observed in the cytoplasm and formed irregular patches that were scattered randomly in the cortex. Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Control, Injection, Western Blot, Immunodetection, Cell Culture, Derivative Assay, Staining, In Situ, Biomarker Discovery, Confocal Microscopy

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 3 The mTORC2/Akt1 pathway rearranges the F-actin cytoskeleton of one-cell stage fertilized eggs. Microinjection of Rictor shRNA then with myr-Akt1 mRNA into mouse one-cell stage embryos. Staining for F-actin (red) revealed the organization of the F-actin cytoskeleton in mouse fertilized eggs (F-actin is shown by the yellow arrow). Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Microinjection, shRNA, Staining

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 5 The Akt1/ Girdin pathway regulates rearrangement of the F-actin cytoskeleton of one-cell stage fertilized eggs. (a) One-cell stage mouse embryos were first microinjected with Akt1-WT mRNA, myr-Akt1 mRNA or siRNA, then stained with rhodamine-phalloidin (20 μmol/L; actin labelling as shown in red) and anti-P-Girdin (1:50; Girdin labelling; as shown in green). Merged colour detection between P-Girdin and polymerize F-actin appears as yellow stained images. The areas of co-localization are highlighted with yellow arrowheads. Scale bar: 20 μm. (b) 200 fertilized eggs were treated with mRNA coding for Akt1- WT, myr-Akt1, Akt1-KD or siRNA against Akt1. Western immunoblot analyses assessed following immunoreaction with anti-P-Girdin (upper panel) and anti-Girdin (lower panel) antibodies are shown. (c) One-cell stage mouse embryos were first microinjected with 0.03 ng of myr-Akt1 mRNA as described in the Materials and Methods, and then 1–2 hours later with Girdin siRNA, following which specimens were stained with rhodamine-phalloidin (20 μmol/L; actin labelling shown in red). Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Staining, Western Blot

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 6 The mTORC2/Akt1/Girdin pathway rearranges the F-actin cytoskeleton in one-cell stage fertilized eggs. (a) The effect of gene knockdown on the expression of Rictor and the phosphorylation of Girdin-1417. Extracts of mouse fertilized eggs were resolved by 6% SDS-PAGE, transferred to nitrocellulose and probed with phosphor- Girdin-Ser1417 antibody and Girdin antibody. (b) We examined Girdin phosphorylation in the fertilized eggs treated with Rictor shRNA, WT-Akt1 mRNA, Rictor shRNA and WT-Akt1 coinjection. In WT-Akt1 mRNA treated cells, we noted that Girdin was highly phosphorylated at 1417. But cells treated with Rictor siRNA and WT-Akt1 exhibited decreased phosphorylation of Girdin 1417

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Knockdown, Expressing, Phospho-proteomics, SDS Page, shRNA

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: BRD4 knockdown attenuates the proliferation and promotes the apoptosis of U251 cells. (A) A Cell counting kit-8 assay was performed to detect the proliferation of U251 cells transduced with BRD4-shRNA or Scr-shRNA. Each experiment was performed in triplicate. (B) An EdU assay was performed to determine cell proliferation rates following BRD4 knockdown. Nuclei were counterstained blue with DAPI. Red indicates the cells undergoing proliferation. Scale bar, 20 µ m. (C) Quantification of the EdU assay results demonstrated that U251 cell proliferation was reduced following BRD4 knockdown. (D) Representative plots and quantification of flow cytometry analysis of apoptosis following BRD4 knockdown in U251 cells. Data are presented as the mean ± standard deviation of three independent experiments. ** P<0.01. BRD4, bromodomain containing 4; sh, short hairpin; Scr, scrambled control; OD, optical density.

Article Snippet: Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as the control. siRNA targeting KRAS proto-oncogene GTPase (KRAS) and the negative control siRNA (cat. nos. sc-35731 and sc-37007, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, Cell Counting, Transduction, shRNA, EdU Assay, Flow Cytometry, Standard Deviation, Control

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: Ten key genes identified by global signal transduction network analysis.

Article Snippet: Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as the control. siRNA targeting KRAS proto-oncogene GTPase (KRAS) and the negative control siRNA (cat. nos. sc-35731 and sc-37007, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Transduction

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: Experimental validation of microarray results. (A) Reverse transcription-quantitative polymerase chain reaction results for the mRNA expression levels of the ten key genes identified by global signal transduction network analysis. (B) Western blotting validation of the protein expression changes of key genes in the BRD4-shRNA and the Scr-shRNA groups. GAPDH was used as an internal control. (C) Representative photographs and quantification of KRAS immunostaining in normal brain tissue and glioma tissues of grades II, III and IV. Scale bar, 20 µ m. (D) Western blot analysis of KRAS levels in HA and U251 cells. (E) KRAS silencing following siRNA transfection in U251 cells was confirmed by western blotting (at 72 h post-transfection). (F) A cell counting kit-8 assay was performed to detect the proliferation rates of siKRAS and or siCon-transfected U251 cells. Each experiment was performed in triplicate. (G) The apoptosis rates of siKRAS and siCon-transfected U251 cells were determined by TUNEL staining (red). Nuclei were counterstained with DAPI (blue). Scar bar, 50 µ m. Experimental data are presented as the mean ± standard deviation of at least three experiments. * P<0.05 and ** P<0.01. BRD4, bromodomain containing 4; sh, short hairpin; Scr, scrambled control; KRAS, KRAS proto-oncogene GTPase; HA, human astrocytes; si, small interfering; Con, control; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; OD, optical density.

Article Snippet: Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as the control. siRNA targeting KRAS proto-oncogene GTPase (KRAS) and the negative control siRNA (cat. nos. sc-35731 and sc-37007, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Biomarker Discovery, Microarray, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transduction, Western Blot, shRNA, Control, Immunostaining, Transfection, Cell Counting, TUNEL Assay, Staining, Standard Deviation

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Affinity purification of SirT7-binding factors identified the histone H2A variant mH2A1 by MS analysis (table S2). HA, hemagglutinin. ( B ) Schematic representation of the three mH2A isoforms, of which, only mH2A1.1 binds to ADP-ribose . ( C ) Endogenous SirT7 specifically immunoprecipitates mH2A1.1 in HEK293F cells. IgG, immunoglobulin G. ( D and E ) mH2A1.1 recognizes and binds ADP-ribosylated SirT7. Immunoprecipitation (IP) of bacterially expressed rSirT7 WT or N189A preincubated ± NAD + and added to nuclear extracts of HEK293F SirT7 KO cells (CRISPR-Cas9–mediated KO of SIRT7) expressing mH2A1.1 WT or G224E, a mutant deficient in ADP-ribose binding. Inputs (I) and elutions (E) are shown. A similar experiment with H187Y is shown in fig. S3A. ( F ) Top: Interaction between endogenous SirT7 and mH2A1.1 under high-stringency conditions upon different types of stress in HEK293 cells. C, untreated; IR, 7-gray ionizing irradiation; H 2 O 2 , oxidative stress. Western blot of the input and elution of immunoprecipitation experiments with anti-SirT7 antibody under these conditions. Bottom: Summary of the ADP-ribosylation events detected by MS in SirT7-FLAG expressed in the same cells and purified under the same stress conditions. Further details are shown in table in fig. S3B and in table S1. ( G ) Superose 6 gel filtration chromatography of nuclear endogenous proteins from nuclear extracts of HEK293F cells under normal cell growth (NT) or upon GS. Fraction numbers and approximate molecular weights (MW) are indicated. Western blot of SirT7 and mH2A1.1 are shown. ( H ) High-stringency immunoprecipitation of endogenous mH2A1.1 by WT or N189A SirT7 in HEK293F cells treated under normal conditions or under GS. Inputs (I) and elutions (E) are shown.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Affinity Purification, Binding Assay, Variant Assay, Immunoprecipitation, CRISPR, Expressing, Mutagenesis, Irradiation, Western Blot, Purification, Filtration, Chromatography

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Levels of three mH2A isoforms in WCE of NIH3T3 cells transfected with scramble shRNA or mH2A1 shRNA and cultured under normal or GS conditions. ( B ) Levels of endogenous SirT7 and histone H3 in chromatin and nucleoplasm fractions purified from Wt and Sirt7 −/− MEFs cultured under normal or GS conditions during the indicated times. ( C ) Venn diagrams showing the intersection of SIRT7-associated genes with mH2A1-enriched genes in Wt cells under NT (top) or GS (bottom). SirT7-associated and mH2A1-enriched genes were derived from GREAT analysis (fig. S3B and Materials and Methods). ( D ) Average enrichment of mH2A1 at all genes in Wt (left) or SirT7 −/− MEF (right) cells under NT (black) or GS (red) conditions. Data are expressed as the log 2 ratio of reads per kilobase of transcript per million mapped reads–normalized ChIP/input signals. ( E ) Distribution of sites occupied by SirT7 upon GS around the TSS by GREAT analysis. The values for each bin from the TSS are shown above each bar (TSS: −5 kb, 5 to 50 kb, 50 to 500 kb, and >500 kb). ( F ) KEGG cell signaling pathways for SirT7-associated genes mapped by GREAT analysis under GS in MEF cells. The signaling pathways were ranked by their combined score provided by Enrichr analysis. cGMP-PKG, guanosine 3′,5′-monophosphate–protein kinase G; cAMP, cyclic adenosine 3′,5′-monophosphate; TCA, tricarboxylic acid. ( G ) SirT7 ChIP-qPCR (quantitative polymerase chain reaction) analysis of SirT7 binding sites associated with mH2A1 at distal regions upon shRNA-mediated down-regulation of mH2A1 under normal and GS conditions in NIH3T3 cells. The amplified regions (red) and their distance to each gene are indicated in the upper part of each graph. Each SirT7 ChIP was normalized with respect to its own input. SEM from n = 4. Two-tailed t test (* P < 0.05 and *** P < 0.005).

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Transfection, shRNA, Cell Culture, Purification, Derivative Assay, Protein-Protein interactions, ChIP-qPCR, Real-time Polymerase Chain Reaction, Binding Assay, Amplification, Two Tailed Test

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Heat map showing RNA expression changes relative to NT (as log 2 magnitude of difference between GS and NT) conditions in Wt and SirT7-deficient MEFs. ( B ) Pipeline applied to RNA-seq data to filter genes associated with SirT7/mH2A1 in Wt and Sirt7 −/− MEF cells treated under GS or NT. The analysis was restricted to genes that (i) were associated with SirT7 via GREAT and were mH2A1-enriched, (ii) showed a log 2 fold change (FC) of expression between WT-GS and WT-NT >0.6, and (iii) showed a difference between WT and KO log 2 FC (GS versus NT) of >0.3 (table S3). ( C ) mH2A1 ChIP-seq signals across ctgf , a gene differentially enriched in mH2A1 upon GS in Wt MEFs compared with NT. ( D ) Top: Real-time qPCR (RT-qPCR) analysis of genes regulated by SirT7 upon GS and NT. The expression of SirT7 in SirT7 −/− MEFs was rescued by retroviral-mediated gene transfer of SirT7 WT, H187Y(HY), N189A(NA), and empty vector (−). SEM from n = 4. Two-tailed t tests (* P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001). Bottom: Relative mH2A1 enrichment (GS versus NT) by ChIP-qPCR analysis at specific regions around the TSS of the indicated genes [ gbp6 , −6 kb; necab1 , −3 kb; lair1 , −27.5 kb; ctgf , +600 base pairs (bp); adra2a , −5 kb; and nrip3 , +7 kb]. SEM from n = 3. One-way analysis of variance (ANOVA) (* P <0.05, ** P < 0.01, and *** P < 0.005). a.u., arbitrary units. ( E ) Chromatin state transitions induced by GS in Wt and SirT7-deficient cells. The colors of the arrows indicate the frequency (%) of the transition as stated in the color scale (right). Bottom right: State map illustrating the specific combination of mH2A1 and/or H3K27me3 in the four chromatin states defined in the analysis. U1, without H3K27me3 or mH2A1; U2, mH2A1; U3, H3K27me3; U4, enriched by H3K27me3 and mH2A1.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: RNA Expression, RNA Sequencing, Expressing, ChIP-sequencing, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Two Tailed Test, ChIP-qPCR

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Model validation studies in WT and Sirt7 −/− mice fed AL or calorie restricted (CR, 30%) for 8 weeks. ( B ) RT-qPCR analysis of the indicated genes in liver samples from Wt and Sirt7 −/− mice fed AL or CR. Three animals were analyzed for each condition. Each quantification was generated from three replicates. Probabilities are those associated with one-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.005). ( C ) Levels of SirT7 in liver samples from Wt and Sirt7 −/− mice AL and CR after subcellular fractionation. WCE and chromatin fractions are shown. ( D ) SirT7 immunoprecipitation of mH2A1 in the same liver samples. Inputs (I) and elutions (E) are shown. ( E ) Autophagy activity in the Wt and Sirt7 −/− livers under AL or CR monitored by levels of formation of LCIII-2. Left: A representative Western blot of n = 5 replicates used in the quantification shown. Right: Quantification of the relative accumulation of LCIII-2 compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) analyzed with a two-tailed t test (* P < 0.05). GAPDH was used as a loading control, as we did not detect a significant alteration of the levels of the protein in our conditions (data not shown). ( F ) Similar analysis ( n = 5) of Beclin-1 as in (E). ( G ) Model proposed for the dual SirT7/mH2A regulatory axis in GS. On the basis of our data, we speculate that this axis is also involved in CR and aging. Nutrient stress induces SirT7 auto-mADPRT, which leads to mH2A-dependent recruitment of SirT7 to distal regulatory regions and subsequent mH2A enrichment around the associated genes. This axis plays a key role in CR in vivo and possibly in aging by modulating key signaling pathways.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Generated, Fractionation, Immunoprecipitation, Activity Assay, Western Blot, Two Tailed Test, Control, In Vivo, Protein-Protein interactions

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 2. Effects of integrin β4 knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co-culture system for 24 h. Then, the levels of integrin β4 in the VECs were examined using the immunofluorescence assay. The bar graph shows the relative intensity of integrin β4 in the single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of integrin β4 in VECs. The levels of integrin β4 were determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L integrin β4-specific siRNA or with scramble siRNA for 48 h. After the addition of SPC, the effects of the integrin β4 knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed by the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as the ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 3. Effects of Fyn knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co- culture system for 24 h. Then, the levels of Fyn in the VECs were examined using the immunofluorescence assay. The bar shows the relative intensity of Fyn in single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of Fyn in VECs. The value of Fyn was determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), the cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L Fyn-specific siRNA or with scramble siRNA for 48 h. After the SPC was added, the effects of the Fyn knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed using the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as a ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 4. Effects of integrin β4 knockdown on the changes in Fyn levels in VECs. (A) VECs were treated with 10–40 nmol/L integrin β4 siRNA or scramble siRNA for 48 h. The levels of integrin β4 in the VECs were determined by the immunofluorescence assay, and the bar graph shows the relative fluorescent intensity of integrin β4 per cell as determined by laser scanning confocal microscopy (bP<0.05 vs scramble ctrl, n=3). (B) After 10–40 nmol/L integrin β4 siRNA or scramble siRNA were treated for 48 h, the Fyn levels in the VECs were assessed using western blotting (cP<0.01 vs scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Immunofluorescence, Confocal Microscopy, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 5. Effects of integrin β4 knockdown on NO production in VECs. (A) VECs were cultured under normal conditions or with siRNA for 48 h and stimulated with SPC for 0, 3, 15, or 30 min, and the supernatant was used for the NO level assay using the NO Detection Kit. In the control group (ctrl), the cells were cultured in M199 medium with 0.3% (v/v) ethanol instead of SPC. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA in the presence of SPC. The bar graph shows the changes in NO levels in VECs that had been treated with SPC (bP<0.05 vs ctrl at 3 min, eP<0.05 vs ctrl at 15 min, n=5) or with siRNA in the presence of SPC (hP<0.05 vs scramble ctrl at 3 min, kP<0.05 vs scramble ctrl at 15 min, n=5). (B) The viabilities of VECs were measured by MTT, and no significant changes were observed (n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Transfection

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 6. Effects of integrin β4 knockdown in VECs on changes in ROCK levels in co-cultured VSMCs. VSMCs that had been co-cultured with VECs that were treated with or without integrin β4-specific siRNA (β4) or scramble control siRNA in the presence of SPC were immunostained for ROCK, α-SMA, or both. The bar graph shows the relative fluorescence intensity of ROCK per VSMC as determined by laser scanning confocal microscopy (bP<0.05 vs SPC-non-stimulated VSMCs; eP<0.05 vs SPC- stimulated scramble ctrl in co-cultured system, n=4).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Fluorescence, Confocal Microscopy