Knockdown of Smad3 in NRK-52E cells inhibits TGF-β1-induced SET7/9 expression. NRK-52E cells were transfected with Smad3 siRNA (si-Smad3) or negative control (si-Neg) oligonucleotides. (A) SET7/9 mRNA levels determined by qRT-PCR in transfected NRK-52E cells with or without TGF-β1 (10 ng/mL, 24 hours). (B) Western blot analysis using SET7/9, (C) Smad3, and (D) phosphorylated Smad3 (p-Smad3) antibodies in transfected NRK-52E cells with or without TGF-β1 (10 ng/mL, 30 minutes or 24 hours). Total cell lysates were subjected to immunoblotting. Because p-Smad3 content peaked at 30 minutes after TGF-β1 stimulation, this time point was used only in p-Smad3 experiments. Quantification is shown in the lower panel (n=5 for each group). Data were analyzed by one-way ANOVA followed by the post hoc test using t test with Bonferroni correction. ##P<0.01. si-Neg, UUO mice with negative control oligonucleotides injection; si-Smad3, UUO mice with Smad3-siRNA injection.