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. Author manuscript; available in PMC: 2015 Sep 15.
Published in final edited form as: Clin Cancer Res. 2014 Jul 28;20(18):4849–4860. doi: 10.1158/1078-0432.CCR-14-0034

Fig. 3. BEZ235/panobinostat lethality involves Mcl-1 down-regulation, and perturbations in Bim, Bax, and Bak.

Fig. 3

(A) Western blot analysis on cells treated for 8 hr with BEZ235 (200 nM) ± panobinostat (10 nM for HBL-1 and OCI-LY18; 15 nM for SU-DHL4). (B) Bax and Bak conformational changes in SU-DHL4 cells following 16 hr treatment as in A. C) cell growth and viability in SU-DHL4 and SU-DHL16 transiently transfected with siRNA against Mcl-1 (si-Mcl-1), or non-silencing siRNA (si-NS), and exposed to panobinostat for 16 hr. Error Bars: S.D of 3 independent experiments; * P < 0.05. D) SU-DHL4 and SU-DHL16 cells in which Bim was knocked down using lentiviral particles carrying shRNA constructs against Bim or non-silencing shRNA (sh-NS) were exposed to BEZ235 (200 nM for SUDHL4 and 25 nM for SU-DHL16) ± panobinostat (15 nM for SU-DHL4 and 7.5 nM for SU-DHL16) for 24 hr after which the extent of cell death was determined using Annexin V/PI assay. Error Bars: S.D of 3 separate experiments; * P < 0.01 for SU-DHL16 and P < 0.02 for SU-DHL4. E) Annexin V/PI assay in SU-DHL4 cells stably transfected with shRNA constructs against Bax (shBax), Bak (shBak), or non-silencing shRNA (sh-NS) following 24 hr treatment with 200 nM BEZ235 ± 15 nM panobinostat. Inset: Western blot on non-treated cells. Each sample was run twice. Error Bars: S.D of 3 separate experiments; * P < 0.05.