Fig 2: ABT-263 fails to disrupt the interaction between BCL-XL and BAK in the absence of activator BH3s.(a) H2171 and SW1271 cells were treated with vehicle, 1 μM ABT-263 or 1 μM ABT-199 for 48 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). (b) H2171 cells, transfected with scrambled siRNA (siSCR) or siRNA against BIM, were untreated or treated with ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). The expression of BIM was assessed by an anti-BIM immunoblot. (c) SV40-transformed wild-type or Bid−/−Bim−/−Puma−/−Noxa−/− QKO MEFs, infected with retrovirus expressing shRNA against luciferase or Mcl-1, were untreated or treated with ABT-263 or ABT-199 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). (d) SV40-transformed wild-type or Bid−/−Bim−/−Puma−/−Noxa−/− QKO MEFs stably expressing GFP, HA-BCL-XL or HA-BCL-2, untreated or treated with ABT-263, were subjected to anti-HA immunoprecipitation. The input (5%) and immunoprecipitates were assessed by immunoblot analysis. (e) H2171 and SW1271 cells, untreated or treated with ABT-263, were subjected to anti-HA immunoprecipitation. The input (5%) and immunoprecipitates were assessed by immunoblot analysis. Arrow indicates a cross-reactive band. (f) H2171 cells stably expressing GFP, HA-BCL-2, HA-BCL-XL, or HA-BCL-XL BH1 mutant were untreated or treated with 1 μM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). EC50 of ABT-263 in H2171 cells was assessed by CellTiter-Glo assays at 48 h. The expression of HA-tagged BCL-2 and BCL-XL protein was assessed by immunoblot analysis. (g) DMS53 cells stably expressing GFP, HA-BCL-2, HA-BCL-XL or HA-BCL-XL BH1 mutant were untreated or treated with 1 μM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). EC50 of ABT-263 in H2171 cells was assessed by CellTiter-Glo assays at 48 h. The expression of HA-tagged BCL-2 and BCL-XL protein was assessed by immunoblot analysis. ***P<0.001 (Student’s t-test). Unprocessed original scans of blots are shown in Supplementary Fig. 11.

Fig 3: AKT-mediated phosphorylation of BAX and PUMA facilitates proteasomal degradation by FBXL20.A, immunoblot monitoring expression levels of PUMA, BAX, pAKT(S473), AKT, and FBXL20 in MCF7 cells treated with indicated concentrations of AKT inhibitor for 12 h. Experiment was repeated three times. B, immunoblot monitoring the expression levels of PUMA, BAX, FBXL20, and AKT in MCF7 cells expressing either NS or two unrelated AKT shRNAs (shAKT-1, shAKT-2). Experiment was repeated two times. C, immunoblot monitoring PUMA and BAX in MCF7 cells transfected with either vector or DDK-FBXl20. At 36 h post transfection, cells were treated with AKT inhibitor (5 μM) for additional 12 h. Experiment was repeated two times. D, coimmunoprecipitation monitoring endogenous interaction of FBXL20 with PUMA and BAX in the presence or absence of AKT inhibitor. E and F, K48-linked ubiquitination of PUMA in MCF7 cells expressing either vector or DDK-FBXL20 treated with AKT inhibitor(5 μM) for 12 h. Whole cell lysates were immunoprecipitated with either anti-PUMA (panel E) or anti-BAX antibody (panel F). Immunoprecipitates and input protein extracts were immunoblotted with indicated antibodies. G, MCF7 cells were coexpressing DDK-FBBXL20 and FLAG-PUMA or FLAG-PUMA(S10A) as indicated for 48 h. Whole cell protein extracts were immunoblotted for the indicated proteins. Experiment was repeated two times. H, MCF7 cells were coexpressing DDK-FBBXL20 and GFP-BAX or GFP-BAX (S184A) as indicated for 48 h. Whole cell protein extracts were immunoblotted for the indicated proteins. Experiment was repeated two times. I, K48-linked ubiquitination of PUMA in MCF7 cells coexpressing DKK-FBXL20 and FLAG-PUMA or FLAG-PUMA(S10). Whole cell lysates were immunoprecipitated with anti-FLAG and immunoprecipitates were immunoblotted with K48-Ub antibody. Experiment was repeated two times. For all immunoblot analysis tubulin was monitored as a loading control. IP, immunoprecipitation.

Fig 4: Activation of mitochondrial control of apoptosis by DCN-expressing Ad(A) Western blot; U343 and A549 cells were infected with dE1 or dE1/DCN at an MOI of 20. Cell lysates were probed with antibodies against PUMA, BAX, Bcl-2, and cytochrome C. β-actin was used as an internal control to show equal protein loading. The expression levels of proteins were semi-quantitatively analyzed with the ImageJ software. (B) Western blot; The U343 and A549 cells were infected with dE1, or dE1/DCN at an MOI of 20. Cell lysates were probed with antibodies against cleaved caspase and PARP. β-actin was used as an internal control to show equal protein loading. The expression levels of proteins were semi-quantitatively analyzed with the ImageJ software. (C) Immunocytochemistry; U343 cells were infected with dE1, or dE1/DCN at an MOI of 20. At 2 days post infection, fixed cells were probed with antibodies against cytochrome C using Alexa Fluor 488 anti-cleaved caspase-3 (green), and the nuclei were counterstained with DAPI (blue). (D) Western blot; U343 and A549 cells were infected with dE1 or dE1/DCN at an MOI of 20. Cell lysates were probed with antibodies against cleaved caspase and PARP. β-actin was used as an internal control to show equal protein loading. The expression levels of proteins were semi-quantitatively analyzed with the ImageJ software.

Fig 5: LC3B-II was consistently increased but not p53 activation after tenovin-6 treatment in DLBCL cell lines(A) The levels of LC3B, SQSTM1/p62 (p62), acetylated p53 [Ac-p53 (K382)], and p53 in the indicated cell lines after tenovin-6 treatment. (B) The levels of phospho-p53 (Ser15) [P-p53 (S15)], p53, Bax, Puma, p21 in OCI-Ly1 cells following tenovin-6 treatment. (C) The kinetics of LC3B, SQSTM1/p62, P-p53 (S15), p53 and p27 levels in OCI-Ly1 cells following treatment with 5 μM tenovin-6. (D) Immunofluorescence staining of LC3B punta in OCI-Ly1 cells following treatment with 2.5 μM or 5 μM tenovin-6 for 16 h.