Fig 2: Yoda-1 modifies myosin light chain phosphorylation (P-MYL9) in PSCs. (A) Immunocytochemistry images of PSCs treated with vehicle (0.2% DMSO; Ctrl) or 5 µM Yoda1 for 1 h, indicating P-MYL9 (red) and NMIIA (green) of different cells. Scatter plot indicates the total cellular fluorescence intensity of P-MYL9 normalized to NMIIA fluorescence (n = 90, N = 3). (B) Representative Western blots of P-MYL2 and MYL-2, compared to GAPDH, respectively, without or with 5 µM Yoda1 treatment. Scatter plot shows the results for individual PSC lysates (Ctrl: n = 5, N = 5; Yoda1 n = 4, N = 4). *p < 0.05.

Fig 3: MLC20 regulates the recruitment of c-Ab, cortactin, Pfn-1, and Abi1 to the cell edge. (A) Ctrl and MLC20 KD cells were plated onto collagen-coated coverslips for 30 min followed by immunofluorescence and fluorescence analysis. MLC20 KD attenuates the localization of c-Abl, cortactin (cort), Pfn-1, Abi1, and F-actin at the cell edge. The arrows point to the leading edge. Scale bar: 10 µm. (B) Illustration of quantitative analysis. An average of at least 5 line scans across each cell is used for analysis. Relative Intensity (RI) = Edge Intensity (EdI)/Cortex Intensity (CI). (C) Data are mean values of experiments from at least 20 cells for each group. Error bars indicate SD. One-way ANOVA was used for statistical analysis. ** p < 0.01; * p < 0.05.

Fig 4: Smooth muscle myosin localizes at the tip of lamellipodia. (A) Human airway smooth muscle (HASM) cells were plated onto collagen-coated coverslips for 30 min and stained for 20-kDa myosin light chain (MLC20) and F-actin. MLC20 is found at the leading cell edge. In addition, F-actin is also localized at the cell edge. (B) Myosin-11 (MYH11) colocalizes with MLC20 at the leading edge. (C) Phosphorylated MLC20 (pMLC20) is found to position at the leading edge. The arrows point to the leading edge. Scale bar: 10 µm.

Fig 5: Knockdown (KD) of MLC20 or MYH11 attenuates cell migration. (A) Human airway smooth muscle (HASM) cells treated with control (Ctrl) siRNA or MLC20 siRNA were evaluated by immunoblot analysis. MLC siRNA reduces the expression of MLC20 in HASM cells. Data are mean values of experiments from five cultures from three donors. Error bars indicate SD. (B) The migration of HASM cells was examined by using the wound healing assay. n = six experiments from three donors. Error bars indicate SD. (C) Cells were treated with Ctrl or Crispr/Cas9 constructs followed by immunoblot analysis. Data are the mean values of experiments from four cultures from three donors. Error bars indicate SD. (D) MYH11 KD reduces the migration of HASM cells. n = six experiments from three donors. Error bars indicate SD. ** p < 0.01. Student’s t-test was used for statistical analysis.