Figure 2. Suppression of ATF4 expression by siRNA transfection inhibited TBHP-induced apoptosis of HTMCs. HTMCs were transfected with siRNA specific to ATF4 (si-ATF4) or to scramble sequences (si-sc) for 48 h, after which they were exposed to 50 μM of TBHP for additional 12 h. (A) The suppression efficiency of ATF4 using si-RNA transfection and the expression of its downstream target CHOP were determined by Western blot analysis (mean ± SEM, n = 3). (B) Levels of cleaved caspase-3 were examined by Western blot. Intensities of protein expression were quantified, normalized against the level of total caspase-3 and expressed as relative changes to protein abundance in cells transfected with scramble control (si-sc) (mean ± SEM, n = 3). (C) Apoptotic cells were examined by TUNEL staining (red, TUNEL; blue, DAPI; scale bar, 100 μm). (D) Numbers of apoptotic cells were quantified and expressed as the percentage of TUNEL-positive to DAPI-positive cells (mean ± SEM, n = 3). *P<0.05 and **P<0.01 vs. TBHP+si-sc. TBHP+si-sc, exposure of HTMCs transfected with si-sc to TBHP. TBHP+si-ATF4, exposure of HTMCs transfected with si-ATF4 to TBHP.