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. 2020 Mar 11;23(4):100975. doi: 10.1016/j.isci.2020.100975

Figure 3.

Figure 3

Myosin-18B Depletion Compromises Mechanosensitive AMPK-VASP Phosphorylation

(A) Western blot analysis of endogenous total AMPK, VASP, phospho-AMPK, and phospho-VASP levels in the lysates of wild-type, myosin-18B knockout, and full-length rescue (FL) cells grown on glass cover slips and soft substrates (5 kPa). The quantifications are from three independent experiments. GAPDH is probed for equal sample loading.

(B) Representative images of actin filaments visualized by phalloidin, and quantification of total number of thick bundles in wild-type (n = 24) and myosin-18B knockout cells (n = 24) treated with AICAR on glass cover slips. Orange arrows, dorsal stress fiber; blue brackets, transverse arcs; red arrows, ventral stress fiber. Scale bar, 10 μm.

(C) The level of active RhoA detected by G-Lisa in wild-type, myosin-18B knockout, and full-length rescue cells grown on glass cover slips and soft substrates (5 kPa). Quantification is based on five independent experiments.

(D) Western blot analysis of endogenous total MLC and phospho-MLC levels in the lysates of wild-type, myosin-18B knockout, and full-length rescue cells grown on glass cover slips and soft substrates (5 kPa).

(E) Representative images of active MLC visualized by staining with phospho-MLC antibody in wild-type and myosin-18B knockout cells cultured on glass and 5-kPa matrix. Scale bar, 10 μm.

Quantification data are represented as mean ± SEM. Values obtained from wild-type cells are normalized to 1 in (A, C, D, and E). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (unpaired t test).