Figure 9

BA increases AIF nuclear localization and expression of AIFsh in PC cells. A. Western blot analysis showing increase in nuclear AIF (57 kd) in LNCaP (48 h) and DU145 (72 h) cells treated with MB compared to M, B, and control (C) cells. B. Western blot analysis of total protein lysates showing increase in AIFsh (35 kd) in MB, DB, and B treated LNCaP (48 h), DU145, and PC3 (72 h) compared to M, D, or C. Coomassie blue stain of total protein is loading control. C. Immunofluorescence showing increase in nuclear localization of AIF in DU145 cells treated with DB compared to C cells (72 h). In control DU145 cells, AIF (green) is cytoplasmic and the merge with nuclear PI stain (red) shows little nuclear localization. In DB treated DU145 cells, the merge of AIF and PI shows some nuclear localization (arrows; yellow). (×200). D. RT-qPCR analysis showing increase in AIFsh mRNA in LNCaP cells treated 48 h with MB, DB, or B alone compared to M or D alone. AIFsh mRNA levels were relative to C = 1. n = 5, three independent experiments; *, P < 0.05. E. Knockdown of AIF reduces cell death in LNCaP cells treated with MB and DB. Trypan blue exclusion assay showing that LNCaP/shAIF-2 (AIF) cells undergo less cell death compared to control LNCaP/shGFP (C) after treatment for 24 h with MB or DB. n = 6, three independent experiments; *, P < 0.02. Western blot analysis showing knockdown of AIF and AIFsh proteins in LNCaP/shAIF-2 results in lower cleaved PARP after MB or DB treatment compared to control LNCaP/shGFP cells. Coomassie blue stain of total protein is loading control.