Figure 5. NOX4 downstream autocrine TGF-β is necessary to maintain the activated phenotype of cultured Mdr2^−/−/p19^ARF−/− .

MFB. MFB isolated from fibrotic Mdr2^−/−/p19^ARF−/− livers were used. (A) Real time PCR of Mdr2^−/−/p19^ARF−/− MFB treated for 72 hours with 10 μM LY36494 (TRβI inhibitor): NOX4, α-SMA (Acta2), vimentin (vim), fibronectin (Fn1), collagen I (Col1a1) and TGF-β₁ (Tgfb1). In B–D, Mdr2^−/−/p19^ARF−/− MFB were transfected with either an unsilencing siRNA (uns siRNA) or a specific siRNA for NOX4 (NOX4 siRNA) for 72 hours, when it was performed: (B) Real time PCR of the indicated genes. (C) Immunofluorescence staining of F-actin, α-SMA and vimentin. (D) Western blot of total lysates. β-actin was used as loading control. Data represent the mean ± SEM of three to six independent experiments, and were calculated relative to untreated MFB (A) or unsilencing-transfected cells (B) (*p<0.05; **p<0.01).