Figure 6.

Effect of HDAC inhibitors on the expression of specificity protein (Sp) 1/Sp3 and histone acetylation. (A) Western blot of histone modification, NOX4, and Sp1/Sp3 protein levels in HPAECs exposed to the indicated HDAC inhibitors. The same amount of protein was loaded in each well. Digital adjustment of brightness and contrast was applied to the whole blot images using Adobe. (B and C) Analysis of Sp1 and Sp3 expression in response to HDAC inhibitors. HPAECs were incubated with HDAC-42 (1 μM), scriptaid (8 μM), or TSA (1.5 μM) for 24 hours. Levels of mRNA were analyzed using quantitative RT-PCR and normalized to GAPDH expression. Data are presented as mean ± SD. (D and E) Identification of scriptaid responsive promoter elements within the EC-SOD gene. 5′-Regions of the human (A) and mouse (B) EC-SOD gene were cloned in front of luciferase (Luc) reporter gene in pGL3-Basic vector. The promoter–reporter constructs were transiently transfected into HPAECs, and firefly luciferase activity was measured 18 hours later. Reporter activity was normalized to Renilla luciferase activity produced by the cotransfection of control plasmid pRL-CMV. Results shown as mean ± SD from at least two independent transfection experiments, each performed in quadruplicate. The hatched oval represents the Sp1/Sp3 consensus binding site. *P < 0.001; ^#P < 0.01 (t test).