Fig 2: Iodine induces apoptosis through the death receptor pathway. (A) A representative Western blot of Fas, FasL, FADD, caspase-8, cleaved caspase-8, caspase-3, cleaved caspase-3, PARP and cleaved PARP in protein extracts of MG-63 and Saos-2 cells pretreated with iodine and released iodine solution for 12 h. (B) Densitometric analysis of Fas, FasL, FADD, caspase-8, cleaved caspase-8, caspase-3, cleaved caspase-3, PARP and cleaved PARP levels. The results are presented as the mean ± SD (n = 3) (*p < 0 0.05, **p < 0 0.01, ***p < 0 0.001) (SD: standard deviation).

Fig 3: 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 4: 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 5: NRF1 accumulation enhances FADD-dependent apoptosis and impairs efferocytosis in vivo. a–c The indicated xenograft tumor tissues were stained with anti-IBA1 (red) and TUNEL (green) (a) and quantified TUNEL + apoptotic cells (ACs) (b), and ratio of free ACs: macrophage-associated ACs (c). Scale bars, 25 µm. d Diagram showing the NRF1 binding site in FADD gene and oligonucleotides used in the ChIP assay. e ChIP assay was performed with IgG and antibody against NRF1 and indicated genes were analyzed by qRT-PCR. qRT-PCR results were normalized to the input. f The indicated cells were cultured under normoxia or hypoxia for 36 h and FADD mRNA levels were statistical analyzed. qRT-PCR results were normalized to the housekeeping gene B2M. g Wild-type or K230R stably expressed MDA-MB-231 cells were cultured under normoxia or hypoxia for 36 h. Cells were harvested and analyzed by western blotting with anti-FADD and anti-ACTIN antibodies. Right: densitometric quantification of FADD protein levels. h Three groups of fresh frozen tissues from indicated xenograft tumors were analyzed by western blotting with the anti-FADD and anti-ACTIN antibodies. i Wild-type or K230R stably expressed MDA-MB-231 cells were cultured under normoxia or hypoxia for 24 h and then were treated with 50 ng mL−1 TRAIL for additional 12 h. Cells were harvested and analyzed by western blotting with indicated antibodies. Bottom: densitometric quantification of cleaved Caspase-3 and cleaved PARP1 protein levels. For all panels, error bars indicate s.d. For panel (a–c), n = 5 mice, average of n = 5–10 pictures per mouse were statistically analyzed. For other panels, n = 3 biological replicates, average of n = 3 technical replicates for each biological replicate was used in (e–f). The two-tailed unpaired student t-test was used in (b–c) and (e). The two-tailed paired ratio t-test was used in (f–g) and (i)