Morphology and migration capacity of MKL1-deficient fibroblasts are not altered. (A) An absence of MKL1 in the index patient’s fibroblasts was observed by western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Fibroblasts were left unstimulated or were stimulated with transforming growth factor-β (TGF-β; 10 ng/mL) for 48 hours, and αSMA was detected by western blotting. GAPDH was used as loading control. (C) Confocal analysis of fibroblasts by staining for F-actin (green = F-actin [phalloidin], cyan = DNA [Hoechst]). Images were acquired with a Leica TCS SP8 confocal microscope. Scale bar, 75 µm. (D) Actin levels in MKL1-deficient neutrophils were determined by western blotting with anti-actin. GAPDH was used as loading control. (E) Migration capacity of fibroblasts from the index case was assessed using a scratch assay (mean + range, n = 3). (F) Presence of MKL2 in control and patient fibroblasts was confirmed by western blotting, whereas MKL2 was not observed in neutrophils. αTubulin was used as loading control. Significance of fibroblast migration was tested using the Student t test. C, control; ns, not significant; P, patient.