TGF-β1 suppresses mRNA expression of Nrf2 in HSCs.

<p>(A) hTERT-HSCs were exposed to 1–5 ng/mL TGF-β1 for 48 hours. mRNA was extracted using TRIzol conventional procedure and fold changes were calculated as 2^(-ΔΔCT) for each sample and control and expressed as mean fold change ± SD (N = 3). Beta-2-microglobulin (B2M) was used as reference gene for each sample. The results show a significant downregulation of Nrf2, Keap1 and Nqo1 after exposure to TGF-β1. (B) Primary HSCs were exposed to 1 ng/mL TGF-β1, 1 μM SB-525334 (SB52) and/or 5 ng/mL PDGF-AB for 48 hours. mRNA was extracted using TRIzol conventional procedure and fold changes were calculated as 2^(-ΔΔCT) for each sample and control and expressed as mean fold change ± SD (N = 3 different batches). Beta-2-microglobulin (B2M) was used as reference gene for each sample. The results show a significant downregulation of Nrf2, and Nqo1 following exposure to TGF-β1. SB52 induces upregulation of Nrf2 and Nqo1, efficiently inhibiting the TGF-β1 effect. (C-D) The protein levels of Nrf2, Nqo1 and actin beta were analysed by Western blot analysis after exposure to 0–10 ng/mL TGF-β1 in hTERT-HSC (C) and primary HSCs (D) for 24 and 48 hours. (E-F) The protein levels of Keap1 and Tubulin were analysed by Western blot analysis after exposure to 0–10 ng/mL TGF-β1 in hTERT-HSC (E) and primary HSCs (F) for 24 and 48 hours.