Journal: Journal of Biological Chemistry

Article Title: Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m115.655548

Figure Lengend Snippet: FIGURE 5. NOTCH3 protects smooth muscle cells from cell death. HAoSMCs with lentivirally overexpressed NICD2, NICD3, and GFP, or siRNA knockdown of NOTCH2 (siN2), NOTCH3 (siN3), and control (siCTL) were exposed to 50 J/cm2 UV radiation and collected for protein 9 h later. A and B, Western blots to detect NOTCH2 and NOTCH3 expression, and level of total and cleaved caspase3 with tubulin used as loading control, n 3. C and D, caspase3 enzymatic activity assay using fluorescent caspase substrate AFC-DEVD, n 4. Significance determined by one-way ANOVA; *, p 0.05.

Article Snippet: Lysates were then used in a caspase3 enzymatic activity assay using AFCDEVD substrate (MP Biomedicals) as described previously (43).

Techniques: Knockdown, Control, Western Blot, Expressing, Enzyme Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m115.655548

Figure Lengend Snippet: FIGURE 7. Genetic deletion of Notch3 has unique effects on the survival of mouse aortic smooth muscle cells. A, mRNA expression of pro-survival genes in aortas of wild-type (WT), Notch2-deficient (Notch2fl/fl; MCC/), and Notch3-mutant (Notch3/) adult mice. qPCR with relative expression compared with GAPDH, n 4. B, SMCs were isolated and cultured from aortas of wild-type, Notch2, and Notch3-deficient mice and used in enzymatic caspase3 activity assays, n 4. C, total and phosphorylated (phospho)-ERK was measured in cultured smooth muscle cells following serum challenge, with tubulin used as loading control, n 4. Significance determined by one-way ANOVA, *, p 0.05.

Article Snippet: Lysates were then used in a caspase3 enzymatic activity assay using AFCDEVD substrate (MP Biomedicals) as described previously (43).

Techniques: Expressing, Mutagenesis, Isolation, Cell Culture, Activity Assay, Control

Journal: Oncotarget

Article Title: Sensitization of glycoengineered interferon-β1a-resistant cancer cells by cFLIP inhibition for enhanced anti-cancer therapy

doi: 10.18632/oncotarget.14573

Figure Lengend Snippet: The cells were treated with 100 ng/mL R27T and collected at the indicated times post-treatment. A, B . Caspase-8 activity was assessed by protease activity assays in OVCAR-3 (A) or HeLa (B) cells (*** P < 0.001 compared to the untreated group). C, D . For the detection of caspase-3/8/9, cFLIPL, and cFLIPS, western blot analysis was performed in OVCAR-3 (C) or HeLa cells (D). β-actin was used as the loading control. E . OVCAR-3 cells were treated with R27T (100 ng/mL) alone or in combination with 50 μM zVAD for 72 h, and cell viability was measured by WTS assays. Data are presented as the means ± SD of three independent experiments (*** P < 0.001 compared to the untreated group). F . OVCAR-3 cells transfected with control or cFLIPS vector were treated with 100 ng/mL R27T for 72 h. Cell viability was analyzed by WTS assays (*** P < 0.001 compared to the untreated group).

Article Snippet: After centrifugation, the supernatant was collected and incubated with 1 μg of anti-caspase-8 antibody (Acris Antibodies, Herford, Germany) in the presence of 20 μL Dynabead protein G (Thermo Scientific).

Techniques: Activity Assay, Western Blot, Transfection, Plasmid Preparation

Journal: Acta Pharmacologica Sinica

Article Title: Pretreatment with low-dose gadolinium chloride attenuates myocardial ischemia/reperfusion injury in rats

doi: 10.1038/aps.2015.156

Figure Lengend Snippet: GdCl3 5 μmol/L and nifedipine 1 μmol/L inhibited A/R-induced cardiomyocytes apoptosis via the inhibition of the death receptor-related signaling pathway. (A) Representative images of Western blots and quantitative analyses of cleaved caspase-8; (B) Representative images of Western blots and quantitative analyses of DR5, Fas, and FADD. Mean±SD. n=3. bP<0.05, cP<0.01 vs control. eP<0.05, fP<0.01 vs A/R.

Article Snippet: Supernatants were assayed immediately with a caspase-3 activity ELISA kit (Applygen, Beijing, China), caspase-8 activity ELISA kit (Applygen, Beijing, China), and Fas ELISA kit (CUSABIO, Shanghai, China).

Techniques: Inhibition, Western Blot

Journal: Acta Pharmacologica Sinica

Article Title: Pretreatment with low-dose gadolinium chloride attenuates myocardial ischemia/reperfusion injury in rats

doi: 10.1038/aps.2015.156

Figure Lengend Snippet: GdCl3 attenuated I/R-induced myocardial apoptosis in vivo. (A) Representative photomicrographs of TUNEL staining in myocardial tissues from sham-operated rats or rats with different pretreatments before I/R as indicated. Scale bar=30 μm; arrows in each panel indicate cells positive for apoptosis; (B) Bar graph shows the percentages of TUNEL-positive nuclei in the sham, I/R and drug-treated groups; (C) Cardiomyocyte apoptosis was assessed on the basis of caspase-3 activity; (D) Cardiomyocyte apoptosis was assessed on the basis of caspase-8 activity; (E) Cardiomyocyte apoptosis was assessed on the basis of Fas levels; (F–H) Representative images of Western blots and quantitative analyses of apoptosis-related signaling molecules (DR5, FADD, and cytochrome c). All values are presented as the means±SD. n=6–8. bP<0.05, cP<0.01 vs sham. eP<0.05, fP<0.01 vs I/R.

Article Snippet: Supernatants were assayed immediately with a caspase-3 activity ELISA kit (Applygen, Beijing, China), caspase-8 activity ELISA kit (Applygen, Beijing, China), and Fas ELISA kit (CUSABIO, Shanghai, China).

Techniques: In Vivo, TUNEL Assay, Staining, Activity Assay, Western Blot