Figure 4: The reversion effects of Neferine on TGF-β1-induced EMT model in HCC cells.

(a,b) HCC cells were firstly treated with TGF-β1 for 48 hrs to induce EMT before the administration of Neferine (48 hrs) and/or OXA (48 hrs). mRNA and protein expression of epithelial marker (E-cadherin), mesenchymal markers (N-cadherin & Vimentin), and EMT promoting transcription factor (Snail, Slug, Twist, Zeb1), which was determined by qRT-PCR and by Western blot in TGF-β1-treated HCC cells treated with Neferine and/or OXA. Original blots of high-contrast blots are presented in Supplementary Fig. S5. (c) Representative double immunofluorescence staining for expression and co-localization of E-cadherin and Vimentin in TGF-β1-treated HCC cells treated with Neferine and/or OXA. (Original magnification: ×400). (d) HCC cells treated with 10 ng/ml TGF-β1 (TGF-β1 groups) or co-treated with 10 ng/ml TGF-β1 and Neferine (TGF-β1 + Neferine groups: applied Neferine to TGF-β1-treated HCC cells for 48 hrs). In Neferine + TGF-β1 groups, HCC cells were pre-treated with Neferine for 48 hrs before the administration of TGF-β1). Migration abilities of HepG2 and Bel-7402 cells were inhibited by Neferine in vitro and were canceled by TGF-β1 in wound healing assays. #: TGF-β1 vs. control, p < 0.01, respectively; + : TGF-β1 vs. TGF-β1 + Neferine, p < 0.001, respectively; Δ: TGF-β1 + Neferine vs. Neferine + TGF-β1, p < 0.001, respectively. (e) Invasion abilities of HepG2 and Bel-7402 cells examined by transwell assays were inhibited by Neferine in vitro and were canceled by TGF-β1. TGF-β1 groups: treated HCC cells with 10 ng/ml TGF-β1 for 48 hrs; TGF-β1 + Neferine groups: applied Neferine to TGF-β1-treated HCC cells for 48 hrs; Neferine + TGF-β1 groups: pre-treated HCC cells with Neferine for 48 hrs before the administration of TGF-β1. #: TGF-β1 vs. control, p < 0.01, respectively; + : TGF-β1 vs. TGF-β1 + Neferine, p < 0.001, respectively; Δ: TGF-β1 + Neferine vs. Neferine + TGF-β1, p < 0.01, respectively. ^**p < 0.01, ^***p < 0.001.