Figure 4: ABT-263 fails to disrupt the interaction between BCL-XL and BAK in the absence of activator BH3s.
From: Targeting the differential addiction to anti-apoptotic BCL-2 family for cancer therapy
(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.
