Relieving cells of high stiffness delays, but does not prevent, myofibroblast differentiation.
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A) Schematic illustration of protocol used for experiments in B and C. Cardiac fibroblasts were cultured on plastic or soft gels for 3, 9 or 15 days (d) and then analysed. B) Immunostaining for smooth muscle α-actin (SMA; green) in cardiac fibroblasts. Nuclei were stained with DAPI (blue). Scale bar 100μm. C) mRNA expression of myofibroblast markers and Tcf21 at 13 days after seeding. Y-axis value, 2-ΔCt, represents mRNA levels for each gene relative to 18S rRNA. ΔCt is the Ct value of the gene of interest minus the Ct value of 18S rRNA. D) Schematic illustration of protocol used for experiments in E and F. Cardiac fibroblasts were cultured on plastic for 9d where after they were either transferred to soft gels of kept on plastic for an additional 4d. E) Immunostaining for SMA (green) in cardiac fibroblasts. Nuclei were stained with DAPI (blue). Scale bar 100μm. F) mRNA expression of myofibroblast markers and Tcf21. Significant differences were determined by Student’s t-test corrected for multiple comparisons using Holm-Sidak test. N = 8 (C, plastic and 3d N = 4 (C, 9d), N = 3 (C, 15d) and N = 3 (F).