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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: J Physiol. 2020 Sep 11;598(20):4573–4590. doi: 10.1113/JP280111

Figure 1. S100A4 depletion inhibits NM myosin II heavy chain assembly and tension development in airway SM tissues in response to contractile stimulation.

Figure 1.

S100A4 was depleted or overexpressed in tracheal SM tissues using plasmids encoding shRNA or WT S100A4. A, typical immunoblots and mean expression levels of S100A4 protein in tracheal SM tissues (n = 12, p = 0.0001). B, contractile force in response to ACh before and after transfection. Depletion of S100A4 significantly inhibited tension development in response to 10−5 M ACh (n = 16, p = 0.0001). C, assembly of NM myosin II filaments was significantly inhibited by the depletion of S100A4 in ACh-stimulated tracheal SM tissues (NM myosin IIA, n = 6, p = 0.0001; NM myosin IIB (n = 5, p = 0.0264). NM myosin II assembly was determined by assessing the ratio of NM myosin II in the cytoskeletal (P) vs. soluble (S) fractions of tracheal tissue extracts. D, assembly of NM myosin II filaments was significantly inhibited by the depletion of S100A4 in ACh stimulated tracheal SM cells. In situ PLA fluorescence and PLA fluorescence merged with phase contrast is shown in freshly dissociated SM cells that were unstimulated (US) or stimulated with ACh. PLA fluorescence spots show interactions between NM myosin IIA and NM myosin IIB in cells dissociated from sham-treated tissues and in cells dissociated from tissues after S100A4 depletion. ACh stimulation resulted in a significant increase in the number of complexes between NM myosin IIA and NM myosin IIB at the cell cortex in sham-treated tissues but not in tissues treated with S100A4 shRNA [n = 16 (sham) or 17 (shRNA), p = 0.0001]. E, NM myosin II heavy chain Ser1943 phosphorylation increased significantly in response to 5 min stimulation with 10−5 M ACh in sham-treated tissues but not in S100A4 depleted tissues (n = 11, p = 0.0001). Data were analysed by a paired Student’s t test (A, B) or by one-way ANOVA with repeated measures (C–E). Values are the mean ± SD. *Significant difference between unstimulated (US) and ACh stimulated groups. **Significant difference between treatment groups. NS, no significant difference.