Figure 3. Senescence-induced reduction of DDIT4 is restored by HDAC4 overexpression. (A) Human dermal fibroblasts were transfected with negative control scrambled siRNA (NC) or HDAC4 siRNA. (B) For overexpression of HDAC4, cells were treated with pcDNA3 or HDAC4 plasmid DNA and the medium was replaced after 6 h. (A–D) RT-PCR and western blotting were performed to determine the expression of DDIT4 and HDAC4. Data are presented as mean ± SE (n = 5, * P <0.05, ** P <0.01, *** P <0.001 vs. NC, pcDNA3, or Control). (E, F) After senescence was induced, cells were transfected with pcDNA3 or HDAC4 overexpression vector. DDIT4, p21, and HDAC4 protein expression were analyzed by western blotting. Beta-actin and alpha-tubulin served as a loading control. Data are presented as mean ± SE using Student’s t-test (n = 5, * P <0.05, ** P <0.01, *** P <0.001 control vs H2O2; # P <0.05, ## P <0.01, pcDNA3 vs HDAC4). C: control.