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. 2019 Oct 1;8(10):1184. doi: 10.3390/cells8101184

Figure 3.

Figure 3

Figure 3

(A) Validation of α–SMA, vimentin, peroxisome proliferator activated receptor-γ (PPARγ) and proliferating cell nuclear antigen (PCNA) expression in hepatic stellate cell (HSC-T6 cells applied with vimentin with RNA silencing (siVIM) or without (Mock) siVIM by Western blotting. GAPDH was used as an internal control. PVDF membrane stained with Coomassie blue R-250 was utilized to perform loading amount of proteins. The quantified results were indicated by the bar chart and represented the mean ± SD of three independent experiments (*** p < 0.001). Morphological changes of HSC-T6 cells with or without siVIM transfection by optical microscopy were demonstrated as the left figures. (B) Western blot analysis for phosphorylation and total protein levels of mitogen-activated protein kinases (MAPK) and protein kinase B (AKT) in HSC-T6 cells after administrating with or without siVIM. The quantification of phosphorylation in each lane was normalized by total protein levels (** p < 0.01, *** p < 0.001). (C) Knockdown of vimentin retarded wound closure. Representative phase-contrast micrographs of closure of scratch-wounded confluent cultures of mock- or siVIM-transfected HSC-T6 cells at a time point immediately after wounding and 24 h post-wounding. The migration rate was calculated by the percentage (%) and indicated with the bar chart (*** p < 0.001). (D) Western blot analysis for phosphorylation and total protein levels of Cdc42, Rac1/2/3, and Rho A/B/C in HSC-T6 cells after administrating with or without siVIM. The quantification of phosphorylation in each lane was normalized by total protein levels. GAPDH was used as an internal control. The quantified results were indicated by the bar chart and represented the mean ± SD of three independent experiments (* p < 0.05, *** p < 0.001).