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. 2023 Jun 10;299(7):104909. doi: 10.1016/j.jbc.2023.104909

Figure 2.

Figure 2

FA supplementation sustains TGF-β1 signaling activation in HSCs.A, immunoblotting for SMAD2/3 phosphorylation levels of LX-2 cells with or without FA supplementation upon control or TGF-β1(5 ng/ml) treatment for indicated times. B, immunofluorescence detection of p-Smad2/3 levels in cell nuclei after FA supplementation (scale bars represent 40 μm). C and D, mRNA and protein expression analysis of α-SMA in LX-2 cells. LX-2 cells were treated with TGF-β1(5 ng/ml) with or without of FA supplementation. E, Western blots of phosphorylated SMAD2/3 in LX-2 cells with or without FA upon control or TGF-β1(5 ng/ml) treatment for 30 min. F, mouse primary HSCs were cultured with or without FA, then treated with TGF-β1(5 ng/ml) for 36 h. Flow cytometry analysis for α-SMA expression at 0 h, 12 h, 24 h, 36 h. G and H, mouse primary HSCs were stimulated multiple times by TGF-β1(5 ng/ml) at 0 h, 24 h, and 48 h, 72 h, and cells were depleted of FA at 24 h. Flow cytometry analysis for α-SMA and collagen α1(I) protein expression at 0 h, 24 h, 48 h, 72 h, 96 h. The statistical significance (∗p < 0.05, ∗∗p < 0.01) was tested by student’s unpaired t test. CM, complete medium; CM+FA, the addition of folic acid to complete medium; CM-FA, complete medium with folic acid removed; FA, folic acid; HSCs, hepatic stellate cells.