Journal: Cell Death & Disease

Article Title: cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

doi: 10.1038/s41419-022-04574-6

Figure Lengend Snippet: A HCT116 cells were transfected with scrambled oligonucleotide (Sc) or FLIP L , FLIP S , or FLIP DUAL siRNAs for 48 h prior to TG (100 nM) treatment for the indicated times and apoptosis was determined by subG1 analysis. cFLIP knockdown was assessed in whole-cell extracts by western blotting. GAPDH was used as protein loading control. B HCT116 pBABE-ø or pBABE-cFLIP L cells were transfected for 48 h with scrambled oligonucleotide (Sc) or FLIP L siRNAs and apoptosis was determined after a further 24 h of thapsigargin treatment (ns = not statistically significant; **** p ≤ 0.0001; two-way ANOVA. Tukey’s multiple comparisons test). cFLIP L levels were assessed by western blotting. C HCT116 cells were either non-transfected (−) or transfected with scrambled oligonucleotide (Sc) or cFLIP L siRNA for 48 h prior to TG treatment for the indicated times. cFLIP protein levels and caspase-8 processing were determined in whole-cell extracts by western blotting. GAPDH levels were used as protein loading control. Blots are representative of three independent experiments.

Article Snippet: Primers and probes used were: cFLIP L (AIN1EV0), cFLIP S (Ss03391532_m1) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT1, Hs01003267_m1).

Techniques: Transfection, Knockdown, Western Blot, Control

Journal: Cell Death & Disease

Article Title: cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

doi: 10.1038/s41419-022-04574-6

Figure Lengend Snippet: A Cultures of HCT116 cells growing in 2D or as spheroids (10-days) were treated with TG (100 nM) for the indicated times. cFLIP L levels were determined in whole-cell extracts by western blotting. Quantification of FLIP loss after ER stress was performed using Image Lab TM 6.0 software by taking its respective untreated cultures as control at each time point. B To compare cFLIP L stability, 2D and 3D cultures were treated with CHX (5 μg/mL) for the indicated times (upper panel). cFLIP levels were determined in whole-cell extracts by western blotting. Levels of cFLIP L were quantified using GAPDH as protein-loading control through Image Lab TM 6.0 software and referred to time 0 h levels. As control of efficiency of CHX treatment in inhibiting protein synthesis in 10-day-old HCT116 spheroids (3D), puromycin was added for 10 min and its incorporation was determined in whole-cell extracts by western blotting as indicated in the scheme (lower panel). Ponceau staining confirms that the same amount of protein was present in each lane.

Article Snippet: Primers and probes used were: cFLIP L (AIN1EV0), cFLIP S (Ss03391532_m1) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT1, Hs01003267_m1).

Techniques: Western Blot, Software, Control, Staining

Journal: Cell death and differentiation

Article Title: Caspase-10: a molecular switch from cell-autonomous apoptosis to communal cell death in response to chemotherapeutic drug treatment.

doi: 10.1038/cdd.2017.164

Figure Lengend Snippet: Figure 5 RIP1 and TRAF2 are dispensable for 5FU-induced apoptosis, but required for FADDosome formation. (a) Western blot of protein lysates of HCT.shC10 cells, treated with 5FU, probed with cIAP1 antibodies. (b) Apoptosis measurements after 5FU treatment of the following stable knockdown clones: HCT.shctrl, HCT.shRIP1, HCT.shC10, HCT. sC10.shctrl and HCT.shC10.shRIP1. Data are plotted as mean ± S.E.M. (n ≥3). (c) (Left) HCT.shctrl, HCT.shC10 and HCT.shRIP1 cells were treated with 5FU and the resulting protein lysates immuno-blotted and probed with IκB-α and cIAP1 antibodies. (Right) TNF-α was measured in HCT.shRIP1 cells treated with 5FU. Data are plotted as mean ± S.E. M. (n ≥3). (d) Caspase-8 IP in 5FU-treated HCT116 cells. Resulting precipitates were analysed by western blot for RIP1, TRAF2 and caspase-8. Input controls are shown on the left. (e) (Left) Apoptosis measurements in HCT.shTRAF2 cells following 5FU treatment. (Right) TNF-α levels were measured in HCT.shTRAF2 cells treated with 5FU. Data are plotted as mean ± S.E.M. (n ≥3). (f) EGFP, ΔRING-TRAF2 and TRAF2 were overexpressed in HCT116 cells and 24 h later the resulting protein extracts tested by Western blot for cFLIP. (g) Degradation of cFLIP was analysed in cells overexpressing EGFP, ΔRING-TRAF2 or TRAF2 following 5FU treatment. ΔRING-TRAF2 and TRAF2 expression is also shown.

Article Snippet: TRAF2, pCMV6.FADD.MYC and cFLIP were bought from Origene (Rockville, MD, USA).

Techniques: Western Blot, Knockdown, Clone Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Cellular FLICE-like Inhibitory Protein (c-FLIP) and PS1-associated Protein (PSAP) Mediate Presenilin 1-induced γ-Secretase-dependent and -independent Apoptosis, Respectively *

doi: 10.1074/jbc.M115.640177

Figure Lengend Snippet: PS1 stimulates γ-secretase-dependent turnover of c-FLIP, and PS1-induced apoptosis was partially blocked by γ-secretase Inhibitor. A, HeLa, H4, and DU145 cells were transfected with either PS1 or LacZ in the presence or absence of the general caspase inhibitor Z-VAD (100 μm) for 24 h. The top panel shows expression of LacZ. Panel 2 shows PARP cleavage. Panels 3–5 show activation of caspase-9, caspase-3, and caspase-8, respectively. Panel 6 was probed for PS1. Panel 7 was probed for c-FLIPL and c-FLIPS. The bottom panel was probed for actin as a loading control. B, PS1-induced apoptosis was inhibited by transition state γ-secretase inhibitor but not affected by non-transition state inhibitor. Cells were transiently transfected with either PS1 or LacZ in the presence or absence of γ-secretase inhibitors for 24 h. L685,458 (lanes 3, 8, and 11), compound E (lane 4), and DAPT (lane 5) were used at 0.5 μm, 5 nm, and 0.5 μm, respectively. C, intact HeLa cells were treated with L-685,458 at different concentrations. D, cells were transfected with different PS1 variants for 16 h, and cell lysates were subjected to immunoprecipitation (IP) using anti-PS1N antibody and probed with anti-c-FLIP. The third panel is a longer exposure of the top panel for visualizing c-FLIPL in the cell lysate.

Article Snippet: The general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), caspase-8 inhibitor Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone (Z-IETD-fmk), and antibody against c-FLIP (NF6) were purchased from Enzo Life Sciences.

Techniques: Transfection, Expressing, Activation Assay, Immunoprecipitation