Establishment of mouse intestinal myofibroblast cell lines

Figure 1 Proliferation and contact inhibition of LmcMF and SmcMF. A, B: The cells were subcultured every 3 d, and the growth rate of LmcMF (A) and SmcMF (B) were assessed as described; C, D: Contact inhibition of LmcMF (C) and SmcMF (D).

Figure 2 Phase-contrast microscopic images of primary intestinal myofibroblasts, mouse embryonic fibroblasts, LmcMF, and SmcMF. A: Primary intestinal myofibroblasts; B: Mouse embryonic fibroblasts; C: LmcMF; D: SmcMF were cultured to confluence, and the phase-contrast microscopic images were taken. Representative images are shown. Scale bars indicate 200 μm.

Figure 3 Expression levels of characteristic markers in LmcMF and SmcMF. A, B: The expression levels of α-smooth muscle actin (α-SMA); C, D: Vimentin; E, F: Desmin; G: Type I collagen in primary intestinal myofibroblast (primary); LmcMF (A, C, and E), and SmcMF (B, D, and F) were investigated by Western blotting. Representative blots from 2 independent experiments are shown. Tubulin, actin, and valosin-containing protein (VCP) were used as loading controls.

Figure 4 Immunofluorescence staining for α-smooth muscle actin and vimentin in primary intestinal myofibroblasts, LmcMF, and SmcMF. Primary intestinal myofibroblasts (IMFs), LmcMF, and SmcMF were immunostained with α-smooth muscle actin (α-SMA) (A) and vimentin (B) (red). SYTOX Green was used for nuclear labeling (green). Representative images are shown. Scale bars indicate 40 μm.

Figure 5 Expression of lipopolysaccharide-related proteins and responses of primary intestinal myofibroblasts, LmcMF, and SmcMF to lipopolysaccharide. A: The expression levels of indicated proteins in primary intestinal myofibroblasts (IMFs) (primary), LmcMF, and SmcMF were investigated by Western blotting; B-D: Primary IMFs (B), LmcMF (C), and SmcMF (D) were stimulated with lipopolysaccharide (LPS) (20 ng/mL) for indicated periods. The IκBα degradation and p38 MAPK phosphorylation were determined by Western blotting. Representative blots from 3-4 independent experiments are shown. Actin was used as a loading control.