Fig. 1: The radiosensitizing effect of the pan-HDAC inhibitor panobinostat in bladder cancer cell lines.

a Clonogenic survival of human BC cells (BFTC909, UMUC-3, T24) treated with panobinostat (PAN) followed by irradiation (Gy). Colonies were stained with 0.25% crystal violet at day 7, and the survival rate was calculated. b Immunofluorescence staining with anti-γH2AX antibody was performed in cells with (PAN) or without (Ctrl) 10 nM panobinostat treatment followed by 5 Gy irradiation (RT). The average number of γH2AX foci per cell was analysed at 0, 0.5, 2, 4 and 24 h, and representative images at 2 h are shown below. c The cell cycle distribution of T24 cells treated with RT (5 Gy), panobinostat (10 nM), and a combination of RT and panobinostat (RT + PAN) was analysed at 16 h by flow cytometry. The percentages of cells within G0/G1, S, and G2/M phases are shown. Data are presented as the means ± SD from three independent biological replicates. Student’s t-test was used to determine significant differences. *P < 0.05; **P < 0.01, compared to control. #P < 0.05 compared to RT. d Western blotting analysis for class I (HDAC1, HDAC2, HDAC3) and class II (HDAC4, HDAC5/7, HDAC6) HDACs in BFTC909, UMUC-3 and T24 cells. GAPDH was used as the internal control. HDAC6 protein expression was quantified by ImageJ. e Cell migration and invasion abilities were examined under a microscope based on the numbers of stained migrated (16 h) and invaded (20 h) cells on the lower Transwell membrane. Representative images of BFTC909, UMUC-3 and T24 cells are shown below. f Western blotting for class I and class II HDACs and acetyl-histone H3 (Lys9) (H3K9ac) in T24 cells with or without 50 nM panobinostat treatment at 16 h. g Western blotting for HDAC6, H3K9ac, acetyl-α-tubulin, phospho-PTEN, PTEN, phospho-AKT, AKT, Rad51 and p21 expression in T24 cells treated with RT (5 Gy), panobinostat (50 nM), and the combination of RT and panobinostat at 16 h.