Journal: Cell Death and Differentiation

Article Title: p53-mediated suppression of BiP triggers BIK-induced apoptosis during prolonged endoplasmic reticulum stress

doi: 10.1038/cdd.2017.96

Figure Lengend Snippet: p53 induces apoptosis during prolonged ER stress. ( a ) p53-null non-small cell lung carcinoma H1299 cell line expressing, or not, p53wt were treated with DMSO or 50 nM of thapsigargin (THAP) for 24 h and analyzed for apoptosis by flow cytometry after staining with Annexin V-FITC and propidium iodide (PI). Representative dot plots show the discrimination of viable cells (FITC− PI−, Q3), early apoptotic (FITC+ PI−, Q4) and late apoptotic or necrotic cells (FITC+ PI+, Q2). The percentage shown on each quadrant represents the number of cells in each group compared with the parent population considered for the analysis. Histogram shows the relative change in percentage of cells in early and late apoptosis/necrosis compared with empty vector (EV)-transfected and DMSO-treated cells, set to 1 (mean±S.D., n =5). Two-tailed paired t-test compared data as indicated, ** P <0.01. ( b ) H1299 cells were transfected as indicated. The p53wt colon carcinoma HCT116 cell line was treated, or not, with siRNA against p53. Western blots show the levels of cleaved PARP-1 (85-kDa fragment; CL-PARP) as an apoptotic marker. p53 isoforms were detected by ACMDD serum. , BiP was used as a positive control for UPR activation and β -actin as a loading control. Numbers below the blots correspond to relative quantification by densitometry compared with the reference point set to 1. ( c ) H1299 cells expressing, or not, p53wt were incubated with 50 nM thapsigargin (THAP) at indicated times. Levels of pro-apoptotic CHOP and apoptotic marker CL-PARP were detected by western blotting during indicated time points. Numbers below the blots correspond to relative quantification by densitometry compared with the reference point set to 1. ( d ) H1299 cells as in were transfected with siRNA targeting CHOP or control siRNA. Downregulation of CHOP was confirmed by western blotting and its effect on apoptosis induction was assessed by detection of CL-PARP. FL-PARP confirmed PARP-1 expression levels are not significantly affected. For all, western blots represent n ≥2

Article Snippet: Anti-cleaved PARP-1 rabbit pAb was from Cell Signaling Technology (Danvers, MA, USA), anti-BiP rabbit pAb and anti-CHOP mouse mAb were purchased from Abcam (Cambridge, UK), anti-BIK mouse mAb and anti-PARP-1 rabbit pAb were purchased from Santa Cruz (Dallas, TX, USA), anti- β -actin mouse mAbs were purchased from Sigma-Aldrich, anti-GFP mouse mAb was purchased from Roche.

Techniques: Expressing, Flow Cytometry, Staining, Plasmid Preparation, Transfection, Two Tailed Test, Western Blot, Marker, Positive Control, Activation Assay, Control, Quantitative Proteomics, Incubation

Journal: Cell Death and Differentiation

Article Title: p53-mediated suppression of BiP triggers BIK-induced apoptosis during prolonged endoplasmic reticulum stress

doi: 10.1038/cdd.2017.96

Figure Lengend Snippet: BiP prevents ER stress- and p53-induced apoptosis. ( a ) p53-null H1299 or p53-proficient HCT116 cells transfected with siRNA against BiP or control siRNA were treated as in and analyzed by FACS. Histograms show the relative change in percentage of cells in early and late apoptosis/necrosis compared with control siRNA-transfected and DMSO-treated cells, set to 1 (mean±S.D., n =4). Two-tailed paired t -test compared data as indicated, * P <0.05; ns, not significant. ( b ) H1299 and HCT116 cell lines were transfected or not with siRNA against BiP or control and treated as in . Levels of apoptotic marker CL-PARP and BiP were detected by western blotting. Numbers below the blots correspond to relative expression levels compared with the reference point set to 1. ( c ) Apoptosis induction was analyzed in H1299 transfected with BiP and/or p53wt, treated and stained with FITC and PI as described in . Histograms show the relative change in percentage of cells in early and late apoptosis/necrosis compared with EV-transfected and DMSO-treated cells, set to 1 (mean±S.D., n =5). Two-tailed paired t-test compared data as indicated, * P <0.05, ** P <0.01, ns non-significant. ( d ) Apoptosis induction was estimated by CL-PARP levels in H1299 treated as in . The pro-apoptotic CHOP shows no significant variation. Numbers below the blots correspond to relative quantification by densitometry compared with the reference point set to 1. For all, blots represent n ≥2

Article Snippet: Anti-cleaved PARP-1 rabbit pAb was from Cell Signaling Technology (Danvers, MA, USA), anti-BiP rabbit pAb and anti-CHOP mouse mAb were purchased from Abcam (Cambridge, UK), anti-BIK mouse mAb and anti-PARP-1 rabbit pAb were purchased from Santa Cruz (Dallas, TX, USA), anti- β -actin mouse mAbs were purchased from Sigma-Aldrich, anti-GFP mouse mAb was purchased from Roche.

Techniques: Transfection, Control, Two Tailed Test, Marker, Western Blot, Expressing, Staining, Quantitative Proteomics