Fig 2: FAIM regulated the expression of c-FLIP in MSCs under OGD conditions. (a–b) Immunoblots and densitometric quantitation of cleaved caspase-8 and cleaved caspase-9 protein levels in MSCsVec and MSCsFAIM under OGD conditions. (c–d) Immunoblots and densitometric quantitation of c-FLIP protein levels in MSCsVec and MSCsFAIM under OGD conditions. (e–f) c-FLIP protein levels after siRNA-mediated knockdown of FAIM or scrambled siRNA administration followed by OGD stimulation for 12 h. The results of densitometric quantification are shown on the right. The data are shown as the mean ± SD. Ns indicates not significant; * denotes P < 0.05, **P < 0.01.

Fig 3: PP6 negatively modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL.A HOIP-/- or HOIP reconstituted MEFs were treated with TNFa for indicated periods time. Cells were lysed with NP-40 buffer and immunoprecipitated with anti-TNFR1 antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. B PP6+/+ and PP6-/- L929 cells with Flag-RIPK1 stable expression were treated with TNFa for indicated periods of time. Cells were lysed with NP-40 buffer and immunoprecipitated with anti-Flag beads. The isolated complex I was eluted with urea lysis buffer. The eluted complex I was further immunoprecipitated with chain specific M1 or K63 ubiquitin antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. C Flag-RIPK1 was co-expressed with LUBAC (HA-HOIP/Myc-HOIL/Flag-Sharpin) or HA-PP6 in 293T cells for 20 h. Cells were lysed with urea lysis buffer and immunoprecipitated with chain specific M1 ubiquitin antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. D Flag-tagged c-FLIPL was co-expressed with LUBAC (HA-HOIP/Myc-HOIL/Flag-Sharpin) or HA-PP6 in 293T cells for 20 h. Cells were treated with PS341 8 h after transfection. Cells were lysed with urea lysis buffer and immunoprecipitated with chain specific M1 ubiquitin antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. Concentrations of reagents used: TNFa (T), 50 ng/ml; PS341, 100 nM.

Fig 4: Melanoma-associated PP6 inactivating mutations reduce the sensitivity to TNFa.A PP6 was removed by specific sgRNA in B16-F10 (murine melanoma cell), CT-26 (murine colorectal carcinoma cells), E0771 (murine breast adenocarcinoma cells) and HT-29 (human colorectal adenocarcinoma cell) tumor cells. Cells were then treated with TNFa/5Z-7 for 6 h. B PP6-/- MEFs were reconstituted with control vector, WT-PP6, D84N-PP6, or melanoma-associated G112E-PP6, H114Y-PP6, and G189R-PP6. Cells were then treated with TNFa/zVAD/5Z-7 and TNFa/5Z-7 for 4 h or TNFa/CHX for 10 h. The panel on the right showed the efficiency of PP6 reconstitution. C, D Control vector, WT-PP6, D84N-PP6, or melanoma-associated G112E-PP6, H114Y-PP6, and G189R-PP6 reconstituted PP6-/- MEFs were treated with TNFa/5Z-7 or TNFa/CHX for indicated periods of time. Cells were lysed with RIPA buffer and analyzed by western blotting with indicated antibodies. E A schematic model shows that PP6 negatively modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL to promote TNFa-mediated cell death. Concentrations of reagents used: TNFa (T), 50 ng/ml; CHX (C), 2 µg/ml; zVAD (Z), 50 µM; 5Z-7, 300 nM. The cell death in (A, B) was measured by CellTiter-Glo assay. Data represent mean ± SD of three independent experiments (Student’s t test *P < 0.05; **P < 0.01; ***P < 0.001).

Fig 5: CFLAR is a genetic vulnerability and restrains apoptosis in CUX1-deficient AML.a Cflar transcript expression by qRT-PCR in Cux1+/-;Flt3ITD and Flt3ITD Mac1+ cells. Expression was normalized to Gapdh. Biological and technical triplicates were analyzed. b Immunoblot showing increased CFLAR expression in whole bone marrow (top) and Mac1+ myeloid cells (bottom) from Flt3ITD and Cux1+/-;Flt3ITD mice. Vinculin was used as a loading control. The dashed line indicates splicing of non-adjacent lanes from the same blot. The experiment was performed twice. c Growth assays using 2 × 104 c-Kit+ cells from two mice of the indicated genotypes plated in triplicate. Doxycycline (0.1 µg/ml, Dox) and/or Nec-1 (20 µM) was added and the number of live cells per well was determined by Annexin-V/DAPI staining 48 h later. d Annexin-V/DAPI staining to determine numbers of early- and late-apoptotic cells in doxycycline-treated cells from c. e Quantification of colonies derived from plating 500 c-Kit+ cells transduced with doxycycline-inducible Cflar-targeting shRNA lentiviruses from indicated mice. Cells from two mice were plated in duplicate on cytokine-supplemented methylcellulose in the presence or absence of doxycycline. Colonies containing more than 50 cells were scored seven days after plating. f Immunoblot of lysates from Cux1+/-;Flt3ITD c-Kit+ cells transduced with doxycycline-inducible Cflar-targeting shRNA lentiviruses with and without 48 h of doxycycline treatment showing differences in caspase-8 and -3 activation. The immunoblot was performed once. g Immunoblot of lysates from Flt3ITD c-Kit+ cells showing effect of stable CFLAR expression on caspase-8 and -3 activation. The immunoblot was performed once. h Experimental outline of bone marrow transplant assay to assess selective impact of Cflar depletion on Cux1+/-;Flt3ITD cells compared with control (Con), Cux1+/- and Flt3ITD cells. Hematopoietic progenitor cKit+ cells from CD45.2+ mice of each genotype were infected with doxycycline-inducible Cflar-targeting shRNA lentiviruses and 105 puromycin-selected cells were transplanted with 105 wild-type CD45.1+CD45.2+ bone marrow support cells into lethally irradiated CD45.1+CD45.2+ recipient mice. After four weeks, mice were fed a doxycycline-containing diet to induce Cflar depletion or maintained on a normal diet. Donor-derived CD45.2+ bone marrow cells were quantified two weeks later by flow cytometry. i Quantification of donor-derived bone marrow CD45.2+ cells in recipient mice (n = 6, per group) transplanted with control (Con), Cux1+/-, Flt3ITD or Cux1+/-;Flt3ITD c-Kit+ cells with and without doxycycline-induced Cflar depletion. j Spleen weights normalized to body weight of mice at the termination of the transplant. All plots show mean + /± s.e.m.; ns, not significant; two-way (c) or one-way (d, j) ANOVA with Tukey’s test for multiple comparisons, Two-tailed, unpaired t-test (a, e, i).