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

Figure 1.

Figure 1

Myosin-18B Knockout Cells Resemble the Cells with Mechanosensitive Defects

(A) Left panel shows the representative images of wild-type cells stained with phalloidin and myosin-18B antibody to visualize actin filaments and endogenous myosin-18B. Magnified images (corresponding to the white boxes) display myosin-18B distributions in wild-type cells. Scale bars, 10 and 2 μm in image and magnified images, respectively. Vsf, ventral stress fiber; Dsf, dorsal stress fiber. Right panel shows the line profiles along the yellow line in white box 1, demonstrating the colocalization of endogenous myosin-18B and F-actin at the ends of ventral stress fibers.

(B and C) Representative images of actin filaments visualized by phalloidin (B) and quantification of the total numbers of stress fibers and thick bundles and the length of dorsal stress fibers (C) in wild-type cells grown on glass cover slips (n = 31) or softer 5-kPa substrate (n = 19), and myosin-18B knockout (n = 22) and CaMKK2 inhibitor-treated cells (n = 27) grown on glass.

(D) Wild-type, myosin-18B knockout and CaMKK2 inhibitor-treated cells grown on crossbow-shaped micropatterns and stained with phalloidin. Scale bar, 10 μm in (B and D). In (B) and (D), orange arrows, dorsal stress fiber; blue brackets, transverse arcs; red arrows, ventral stress fiber.

(E) Based on the calculated lamella width and radius of curvature, the ventral stress fibers of myosin-18B-depleted (n = 20), CaMKK2 inhibitor-treated cells grown on glass cover slips (n = 18) and wild-type cells grown on soft substrate (n = 18) are significantly less contractile compared with the wild-type cells grown on glass cover slips (n = 36). The lamella width and radius of curvature are also measured in myosin-18B overexpression cells (n = 22) and further CaMKK2 inhibition-treated cells (n = 23). Values obtained from wild-type cells are normalized to 1.

(F and G) (F) Representative traction force maps and (G) quantification of cell-exerted strain energy, which indicates cell-exerted traction forces of wild-type (n = 28 for 26 kPa, n = 23 for 5 kPa), myosin-18B knockout (n = 21), and CaMKK2 inhibitor-treated cells (n = 25). Scale bar, 10 μm.

(H) The cells with myosin-18B overexpression are treated with CaMKK2 inhibitor. Scale bar, 10 μm. Quantification data are represented as mean ± SEM. ∗∗∗p < 0.001 (Mann-Whitney-Wilcoxon rank-sum test).