Fig. 5: Regulation of Trx and GSTM1 promoter activity by TGF-β downregulation via a Smad complex.

a A375 and HPAC cell lines were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, Trx and GSTM1 promoter activities were analyzed by Chip assays using antibodies to AP-1 or Sp1. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (*p < 0.05; **p < 0.01). b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of AP-1, Sp1 and GAPDH were detected by western blot analysis using whole-cell lysates. c p-Smad2, Smad2, p-Smad3, Smad3, Smad4, and GAPDH were detected by western blot analysis. d A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, lysates were then subjected to immunoprecipitation with an anti‐AP1 or anti-Sp1 antibody to identify changes in Smad4 in complex formation. e A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, Smad4 localization was examined by confocal immunofluorescence staining. Representative confocal immunofluorescence staining of each infected A375 was performed as described in the Materials and methods section to confirm the localization of Smad4. f A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, Smad4 localization was examined in nuclear or cytosolic fractionation using nuclear (histone 1) or cytosol (actin) markers. g A375 and HPAC cell lines were transfected with negative control siRNA or Smad4 siRNA. After 48 h, transfected cells were detected with GSTM1, Trx, Smad4, and GAPDH. h Effects of NAC on Trx and GSTM1 expression in melanoma and pancreatic cancer cells expressing shTGF-β1 or -β2 adenovirus. A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 6 h, infected cells were treated with NAC (10 mM) for 42 h. Then, p-Smad2, p-Smad3, Trx, GSTM1, and GAPDH were detected by western blot analysis. i A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 6 h, infected cells were treated with NAC (10 mM) for 42 h. Then the expression levels of Trx and GSTM1 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (*p < 0.05; **p < 0.01)