Fig 2: Proposed bidirectional DED assembly mechanisms in forming the ternary complex of cFLIP, FADD, and procaspase-8 in determining cell fate.a Proposed DED assembly mechanism in the reverse hierarchical binding process. When cFLIP levels are elevated, some cFLIP molecules could initiate the formation of a transient cFLIP double-layer intermediate complex. This intermediate complex recruits endogenous Casp-8 and cFLIP to form a stable binary complex. The resultant cFLIP-Casp-8 binary complex subsequently utilizes the CBS to recruit endogenous FADD and loses excess cFLIP molecule, resulting in a ternary cFLIP-Casp-8-FADD complex. This complex, similar to the one formed through the hierarchical binding process, provides resistance to apoptosis and necroptosis. Together with the hierarchical binding process shown in (b), the reverse hierarchical binding process completes the bidirectional DED assembly mechanism. b Proposed DED assembly mechanism in the hierarchical binding process56. Upon high levels of FADD or a high local concentration of FADD, FADD initiates the formation of a transient binary intermediate complex with endogenous Casp-8. cFLIP targets the CBS of this intermediate complex to block apoptotic signaling, resulting in a ternary FADD-Casp-8-cFLIP complex. When cFLIP is depleted, the intermediate complex could recruit endogenous procaspase-8 to initiate apoptosis. Blue and green ribbons represent cFLIP and Casp-8, respectively. FADD molecules are colored as their counterparts in Fig. 1d.

Fig 3: Graphical abstract: Schematic diagram of the proposed mechanism; schematic diagram of the proposed mechanisms by which FAIM impairs the JNK-mediated, ubiquitination–proteasome-dependent degradation of c-FLIP, thereby effectively promoting MSC survival and ultimately ameliorating cardiac function and improving the prognosis of MI in vivo.

Fig 4: Regulation of c-FLIPL by PI(3)K/Akt signaling during ECM-detachment. (A) Measurement of c-FLIPL protein levels (in detached and attached conditions) in MCF-10A cells overexpressing oncogenes (HRASG12V, HER2, EGFR) as compared to empty vector (EV) control. (B) RT-qPCR measurement of CFLAR in MCF-10A cells overexpressing oncogenes versus EV control. (C) Measurement of c-FLIPL protein levels in MCF-10A cells overexpressing oncogenes (p110αE545K and myr-AKT) as compared to EV control. (D) Measurement of c-FLIPL levels after treatment of oncogene overexpressing MCF-10A cells with small molecule inhibitor of PI3Kα, BYL719 (10 μM). (E) Measurement of CFLAR levels in oncogene overexpressing MCF-10A cell treated with BYL719. Graphs show representative data from a minimum of three biological replicates and all western blotting experiments were independently repeated a minimum of three times with similar results. Statistical significance was determined using Student’s two-tailed t test. Error bars show standard deviation. All panels show cells grown in ECM-detached conditions.

Fig 5: miR-204-5p inhibitor protected AGS and MKN45 cells from TP/TNF-α–induced apoptosis. (A) Relative mRNA expression of miR-204-5p in cells transfected with miR-204-5p inhibitor and treated with TP (25 nM) and TNF-α (5 ng/ml) for 24 h. (B) Relative NF-κB activity of cells transfected with miR-204-5p inhibitor and treated with TP and TNF-α for 30 min. (C) Cell viability of AGS and MKN45 cells transfected with miR-204-5p inhibitor and treated with TP (25 nM) and TNF-α (5 ng/ml) for 24 h. (D–G) Representative western blots and relative intensity of protein bands of cleaved caspase-3, cleaved caspase-8, and FLIP in AGS and MKN45 cells transfected with miR-204-5p inhibitor and treated with TP (25 nM) and TNF-α (5 ng/ml), 24 h after TNF-α application, with β-actin as the loading control. Results were expressed as mean ± SEM, and statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparison test. *, # p < 0.05 (n ≥ 3). *p compared with the control group and # p compared with TP/TNF-α/miR-204-5p inhibitor-treated group.