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

Figure 3.

Figure 3

Folate shifts toward mitochondrial metabolism for HSC activation.A, when LX-2 cells were fed [2,3,3-2H] serine, thymidine triphosphate (dTTP) with 2H was determined by mass spectrometry. dTTP with one deuterium (M + 1) is produced via the mitochondrial folate metabolism through SHMT2 and with two deuteriums (M + 2) via the cytosolic SHMT1. B, the percentage labeling of intracellular glycine when LX-2 were fed [2,3,3-2H]-serine in high (2 μM) and physiological (200 nM) FA levels. C, the ratio of dTTP M+1 and M+2 when LX-2 cells were fed [2,3,3-2H]]-serine in high and physiological FA levels. D, the subcellular distribution of Cy5-labeled folate in primary murine HSCs with TGF-β1(5 ng/ml) treatment for 36 h (scale bars represent 10 μm). E, flow cytometry analysis of FA-FITC in LX-2 cells after stimulation with control or TGF-β1(5 ng/ml) for 36 h, then treated with FA-Cy5 for 30 min. F, mRNA levels of SLC25A32 in LX-2 cells after treatment with TGF-β1(5 ng/ml) for 24 h. G, knockdown efficiency of three independent SLC25A32 siRNA in LX2 cells. H, immunoblotting for α-SMA and collagen α1(I) proteins expression in knockdown SLC25A32 LX-2 cells treatment with TGF-β1 (5 ng/ml) for 36 h. I, Western blot performed on cell lysates for phosphorylated SMAD2/3 in knockdown SLC25A32 LX-2 cells treated with or without TGF-β1 (5 ng/ml) for 30 min. The statistical significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001) was tested by student’s unpaired t test. FA, folic acid; HSC, hepatic stellate cell; SHMT, serine hydroxymethyltransferase.