Research Paper Volume 14, Issue 12 pp 5097—5115

Figure 4. HDAC inhibitors trigger CRT translocation to the nucleus in liver cancer cells overexpressing CRT. (E–G) HCC cells treated with vehicle, transfected with CRT-plasmid, or HDAC inhibitors alone or in combination exhibit DNA damage, as determined by TUNEL assays. Scale bar = 100 μm. Upper panel: Green fluorescence indicates CRT-transfected HCC cells. HCC cells are stained with fluorescein-labeled dUTP according to the protocols reported in the Methods section. Red fluorescence indicates TUNEL-positive cells. Lower panel: HCC cells in the upper panel are stained with 4',6-diamidino-2-phenylindole (DAPI) to identify cell nuclei. (H) Rate of apoptosis is calculated as the percentage of TUNEL-positive cells among the total number of counted parental and resistant cells (mean ± SD, n = 3). (I–K) Cells are seeded in an 8-well chamber and treated with vehicle, CRT-plasmid, or HDAC inhibitors alone or in combination. HDAC inhibitors trigger CRT translocation to the nucleus and stimulate calcium accumulation, analyzed by confocal microscopy. Scale bar = 10 μm. Mean values are significantly different than that in control group. *p< 0.05, **p< 0.01, ***p< 0.001.