Induction of Smooth Muscle Differentiation in Fibroblasts by Modulation of Cytoplasmic Actin Ratio

Figures

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  Figure 1. β-Actin suppression alters the expression of cytoplasmic and smooth muscle actin isoforms in human fibroblasts. (a–d) Expression of actin isoforms in control culture and culture after suppression of β-actin (gingival fibroblasts, 9 days): (a) β-actin, (b) γ-actin, (c) α-SMA and (d) γ-SMA with the mean IF intensity for each comparison. Immunofluorescence microscopy. Scale bar: 15 µm. The graphs represent analysis of IF intensity, mean ± SEM, calculated from the results of three independent experiments; at least 20 fields of view were analyzed for each biological replicate; (e) expression of β-actin (left), γ-actin (middle), and α-SMA (right) in control cultures of gingival fibroblasts and subcutaneous fibroblasts. Immunofluorescence microscopy. Scale bar: 25 µm. The graphs represent percentage of cells with distinct α-SMA expression in gingival (upper graph) and subcutaneous (lower graph) fibroblasts, investigated in controls and in cells with downregulated β-actin, 9 days after infection, mean ± SEM, three independent experiments, at least 20 fields of view were analyzed for each biological replicate; (f) expression of α-SMA and γ-SMA in subcutaneous fibroblasts after suppression…

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  Figure 2. Markers of smooth muscle differentiation in human fibroblasts upon β-actin suppression. (a) IF staining for SM22, α-SMA, γ-SMA, SMM (smooth muscle myosin), and desmin (left to right) in human subcutaneous fibroblasts upon suppression of β-actin (16 days). Immunofluorescence microscopy. Scale bar: 15 µm; (b) end-point RT-PCR β-actin, γ-actin, α-SMA, γ-SMA, and smooth muscle myosin light chain (SMMLC) of human gingival (left) and human subcutaneous (right) fibroblasts (control and shRNA to β-actin, 16 days); (c) qRT-PCR analysis of cytoskeletal and contractile gene expression in human subcutaneous fibroblasts. Relative expression levels of ACTB (β-actin), ACTG1 (γ-actin), ACTA2 (α-SMA), ACTG2 (γ-SMA), MYL9 (smooth muscle myosin light chain), TAGLN (transgelin), and CALD1 (caldesmon) were measured in control cells and cells subjected to β-actin downregulation (shRNA) for 16 days, mean ± SEM, calculated from the results of three independent experiments; (d) Western blot analysis of β-actin, γ-actin, α-SMA, γ-SMA and SM22 in human gingival fibroblasts (control and shRNA to β-actin, 16 days). (e) Western blot analysis of β-actin, γ-actin, α-SMA, γ-SMA, SM22, and smooth muscle myosin…

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  Figure 3. Morphometric analysis of cells before and after β-actin suppression. (a) The morphology of human gingival fibroblasts in control and upon β-actin suppression (9 days, two magnifications). Immunofluorescence microscopy. γ-Actin—green, DAPI—blue. Scale bar: 25 µm; (b) alterations in cell area and circularity of human gingival fibroblasts in control and upon β-actin suppression (9 and 16 days, mean ± SEM, three independent experiments; at least 35 cells were analyzed for each biological replicate; (c,d) identification of α-actinin-1 and α-SMA following the prolonged β-actin suppression (16 days) in human subcutaneous fibroblasts. Immunofluorescence microscopy: (c) α-actinin-1; (d) α-SMA—red, DAPI—blue. Scale bar: 15 µm; (e–i) focal adhesions (FAs) and their characteristic proteins in human subcutaneous fibroblasts (control and shRNA to β-actin, 16 days). Immunofluorescence microscopy: (e) α-SMA—green, paxillin—red; (f) actin (phalloidin)—red, paxillin—green, DAPI—blue; (g) actin (phalloidin)—red, vinculin—green, DAPI—blue. Scale bar: 15 µm; (h) analysis of the number and area of FA structures in human subcutaneous fibroblasts (control and shRNA to β-actin, 16 days). Data are…

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  Figure 4. Immunofluorescence confocal microscopy of focal adhesions (FAs) in human subcutaneous fibroblasts before and after β-actin suppression. (a,b) IF staining for actin and paxillin/vinculin in FAs of human subcutaneous fibroblasts (shRNA to β-actin, 16 days). Actin (phalloidin)—red, paxillin/vinculin—green, DAPI—blue. LSM microscopy. Scale bars: 20 µm (a); 10 µm (upper panels) and 5 µm (upper panels) of (b).