Figure 1.

A. Phase contrast photographs of PC-10 cells, two days after treatment with HDACIs then- hyperthermia for 6 h. The image showed limited number of recovered cells as well as the rounded and floating apoptotic cells in the colonies pre-treated with HDACIs then hyperthermia. B. Pre-treatment of PC-10 cells with HDACIs significantly increased hyperthermia-induced apoptosis. Comparative cytograms show Annexin V staining after hyperthermia in PC-10 cells pre-treated with nictotinamide (20 mM), Trichostatin A (300 nM) or both (upper panel). Summary of average annexin V results is concluded (lower panel). C. Bax expression levels, by Western analysis, in PC-10 cells after hyperthermia (6 h) pre-treated with different HDACIs. Lower band (18 KDa) indicates truncated, active, Bax only increased when cells treated with combination of HDACIs and hyperthermia. D. A representative western blot shows the amendment of Bax for the specific siRNA used. PC-10 cells were either transfected with Bax-si or cont-si twice. Actin immunoblot was used as a loading control (upper panel). Significant reduction of annexin V staining after hyperthermia and HDACIs in Bax KD cells compared with control(s) indicating that Bax is the key proapoptotic player in the double treatment-induced apoptosis (lower panel). Each data point represents the mean of three experiments; bars denote SD; ** indicates difference from control transfectant at P<0.01.