Acta2-CreER–labeled VSMCs contributed minimally to myofibroblasts and CAFs. All Acat2-creER;R26^tdTomato mice used in A–M were treated with tamoxifen for three consecutive days at 6 wk of age and analyzed at 8 wk of age (n = 3–5 mice per condition from three independent experiments). (A) Confocal images showing Tomato expression in pericentral- and periportal-venous cells, but not in periductal or perisinusoidal cells of Acta2-creER;R26^tdTomato livers. Blood vessels and bile ducts were labeled by anti-CD31 and anti-Epcam antibodies, respectively. Scale bar = 200 µm. (B) Confocal imaging revealed overlapping Tomato and aSma expression (detected by anti-aSma antibody) in the liver from Acta2-creER;R26^tdTomato mice. Scale bar = 50 µm. (C–G) Confocal images showing Tomato expression in Myh11⁺ VSMCs (C), but not in CD34⁺ (D) or Col1⁺ fibroblasts (E), Reln⁺ HSCs (F), or F4/80⁺ Kupffer cells (G) in Acta2-creER;R26^tdTomato livers. Arrows and arrowheads indicated VSMCs at portal veins and arteries, respectively (C). Please see Fig. S4 J for a magnified view of E. Scale bar = 50 µm. (H–M) Confocal images of liver sections from CCl₄-treated (H and I), DDC-treated (J and K), and ICC-burdened (L and M) Acta2-creER;R26^tdTomato mice, stained with DAPI and anti-Col1 antibody. Scale bar = 200 µm. Data represent the mean ± SD (I, K, and M).