Fig 2: Respiratory syncytial virus (RSV) infection promotes phospho-myosin light chain 2 (pMLC2) expression. 16HBE14o (16HBE) cells were infected for 48 h with medium control (C), live RSV (R; 0.5 multiplicity of infection), or UV-inactivated RSV (UV-RSV; U) followed by cell detachment and seeding on collagen type I-coated plates. After 2 h, nonattached cells were washed off, and attached cells were fixed and immunolabeled with pMLC2 antibody (A). Scale bar = 5 µm. Total cell fluorescence was measured using ImageJ software (B). Western blot analysis of 16HBE control and RSV-infected cells was performed using total cell lysates. Expression of pMLC2 and total MLC2 were analyzed by Western blot, followed by densitometric analysis, normalized to total MLC2 and GAPDH, respectively (C-E). Data are presented as means ± SD; n = 3 independent experiments. Student’s two-tailed t test was used for the results shown in B (n = 4); a paired Student’s one-tailed t test was used for the results shown in D and E. *P < 0.05 and ***P < 0.001 versus the respective control as determined.

Fig 3: Effect of acrolein on transcriptional levels of Rocks, Mypt1, and Mlc2 in TR-MUL5 cells. The mRNA expression levels of (A) Rock1, (B) Rock2, (C) Mypt1, and (D) Mlc2 when TR-MUL5 cells were incubated with acrolein (0–50 µM, n = 6 each). Values represent mean ± SEM; *P < 0.05; **P < 0.01; N.S., not significant.

Fig 4: Thrombin activates acto-myosin interaction by phosphorylation of myosin light chain-2 (MLC2).(A) Immunocytochemistry of PAR1 (red) and phosphorylated MLC2 (Ser19, green) in human myometrial cells. (B, C) Immunoblots of phosphorylated MLC2 (p-MLC2), total MLC2, and ß-actin of myometrial cells. Myometrial cells were treated with thrombin (2 U/mL) as a function of time (B), or pretreated with 100 nM PAR1 inhibitor (SCH79797) for 1 h, and then treated with 2 U/mL of thrombin for 30 min (C). The experiments were repeated three times.

Fig 5: Localization of YAP and non-muscle myosin associated with cell deformation. (a) Immunostaining for YAP, Megalin, and DAPI in WT and Camsap3dc/dc kidneys at P22. Fluorescence signals of DAPI and YAP were scanned along the line drawn in the enlarged images, and the spatial changes in fluorescence intensity are shown in blue for DAPI and green for YAP. Dashed lines indicate the approximate area of the nucleus. Arrowheads indicate the position of nucleus in representative cells. (b) Ratio of nuclear YAP intensity to cytoplasmic YAP intensity [+/+ , n = 81 cells; dc/dc (dilated), n = 43 cells; dc/dc (non-dilated), n = 53 cells]. Box plots indicate first quartile, median, and third quartile values. ***p < 0.001, t-test. (b) Immunostaining for MLC2, actin, and DAPI in WT and Camsap3dc/dc kidneys at P22. Arrowheads indicate apical accumulation of MLC2 in cystic tubules.