Fig. 3.

Inhibition of ROS reduces HDAC4 nuclear export. A: NHLFs were transfected with GFP-HDAC4-expressing plasmids using electroporation and then treated with TGF-β₁ ± DPI for 24 h. Green fluorescent protein (GFP)-positive cells were counted for each treatment. The results represent the percentage of cells lacking nuclear GFP (*P < 0.05 vs. control, **P < 0.05 vs. TGF-β₁ + DMSO). B: immunostaining of endogenous HDAC4 in NHLFs. NHLFs were treated with DPI for 2 h before TGF-β₁ treatment for 12 h. C: immunoblot analysis of HDAC4 disulfide bond formation. The upper blot, “HDAC4 S=S,” represents BIAM HDAC4 (BIAM-labeled HDAC4 disulfide bonds). The lower blot, “Input,” indicates total HDAC4. D: immunoblot analysis showing HDAC4 distribution within cytoplasm and nuclear compartments. E: immunostaining of HDAC4 localization. NHLFs were pretreated with nontargeting control (NC) siRNA or NOX4 siRNA for 48 h and then treated with or without TGF-β₁ for 24 h. Results represent at least 2 independent assays. Arrows indicate nuclei. DM, DMSO.