Fig 2: RIPK3-induced apoptosis was dependent on RIPK1, FADD, and caspase-8.(A) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus the indicated agent for 24 hr. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (B–E) Cultured MCF7/TO-RIPK3 (wild type [WT], RIPK1-/-, Caspase8-/-, FADD-/-, and cFLIF-/-) cells were treated with DMSO or Dox induction for 36 hr. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *p<0.05, ***p<0.001. p-values were determined by two-sided unpaired Student’s t-tests. The cell lysates were analyzed by western blotting using antibodies against RIPK1, caspase-8, FADD, cFLIP, or β-actin (lower panel). Five independent knockout clones were test in each gene.

Fig 3: TY (5 μg/mL) induced the expression of the death factors/receptors Fas L/Fas and TRAIL/DR5. (A) The expression levels of FAS mRNA at 48 h after TY treatment estimated by qRT-PCR (left figure). Time-dependent protein expression of p-FADD and death factors/receptors Fas-L/Fas and TRAIL/DR5 were examined by Western blot analysis. (B) Time-dependent expression of MIR34a after treatment with TY estimated by qRT-PCR. Expression levels of MIR34a are shown as the relative ratios with respect to the RNU6B expression levels. (C) Ectopic expression of MIR34a inhibited cell growth dose-dependently and induced apoptosis according to Hoechst 33348 staining at 72 h after transfection. The apoptotic cells are indicated by white arrows. (D) Expression of MIR34a-targeting E2F3, SIRT1, and CyclinD1 was downregulated by the TY. (E) Anti-MIR34a (Anti; 10 and 20 nM) partly canceled the activation of caspase-8 and PARP cleavage elicited by co-transfection with MIR34a. (F) Silencing of E2F3 by siR-E2F3 or MIR32a increased in the expression levels of DR5, TRAIL, and Fas.

Fig 4: The SFF-CD95L induces the CD95-internalization in type I cellsa. Immunofluorescence analysis of SFF-CD95L induced CD95 internalization in HuT78 cells. The CD95L induced a time-dependent internalization of the CD95 receptor which is blocked by the dynamin-specific inhibitor Dynasore. b. Time course of intracellular CD95-receptosome trafficking (left panel) and corresponding non-magnetic fractions (right panel) in SKW6.4 cells. Magnetic and non-magnetic fractions derived after indicated times of CD95 internalization were analyzed for CD95, DISC proteins (FADD and Caspase-8), clathrin (Clathrin HC), early endosomes (Rab 4A) and lysosomes (Cathepsin D).

Fig 5: FADD immunoblot protein levels in primary T lymphocytes normalized to actin control as measured by densitometry