Fig. 4. Entinostat downregulates FLIP and overcomes resistance to apoptosis induced by IAP antagonists.

a Western Blot of FLIP, cIAP1, cIAP2, XIAP expression in PC3 and DU145 cells following 0 (DMSO), 1, 6, 12 or 24 h treatment with 1 or 2.5 μM Entinostat. b Western blot analysis of FLIP expression in VCaP cells following 24 h treatment with 0, 0.1, 0.5, 1.0, 2.5 or 5 μM Entinostat (Ent). c Western blot analysis of FLIP, FADD, Procaspase-8, HSP90 and HDAC1 in cytoplasmic (cC) and nuclear (nN) fractions following 24 h treatment with 2.5 μM Entinostat(Ent). d Annexin-V/PI flow cytometry analysis of PC3, DU145 and VCaP cells upon 24 h pre-treatment with 1 or 2.5 μM Entinostat (EEnt) followed by 24 h with 1 µM TL32711(TL) +/−10 ng/mL TNFα. e Cell viability assay following 24 h pretreatment with 0, 0.1, 0.5, 1.0 and 2.5 μM Entinostat followed by 48 h with TL32711 + TNFα. f Left: Annexin-V/PI flow cytometry analysis in PC3 cells retrovirally infected to stably overexpress empty vector(EV), wild-type FLIP(sS)(FS WT), wild-type FLIP(lL) (FL WT) and FADD-binding-deficient F114A mutant FLIP(sS) (FS F114) after 24 h 2.5 μM Entinostat(Ent) in combination with 1 µM TL32711(TL) and 10 ng/mL TNFα, Right: Western blot analysis of FLIP expression in in PC3 cells retrovirally infected to stably overexpress empty vector(EV), wild-type FLIP(S)(FS WT), wild-type FLIP(L) (FL WT) and FADD-binding-deficient F114A mutant FLIP(S) (FS F114) following 24 h 2.5 μM Entinostat *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001