Fig. 4: Inhibition of myosin activity disrupts pancreatic ductal organoid rotation and actin stress fibers.

Ai Overlay of maximum projections of a rotating pancreatic ductal organoid (organoid #1) taken before the myosin inhibitor, Blebbistatin, treatment. The images were taken every 30 minutes for 5 hours. The transparent green arrow indicates the general direction of the rotation. Bar = 50 µm. ii Overlay of maximum projections of the same organoid taken 1 hour after the 200 µM Blebbistatin treatment, showing the significant slowdown of the rotation. The images were taken every 30 minutes for 7 hours. Bar = 50 µm. The nuclei were color-coded based on the time when they were taken. The red arrow points to the nuclei of non-viable cells within the lumen of the organoid, which are excluded from the subsequent nucleus tracking analyses. B The nuclei trajectory of the corresponding pancreatic ductal organoid before i and after ii Blebbistatin treatment. C Histogram plot showing the velocity distribution of the nuclei before and after the Blebbistatin treatment. The plot shows the decrease in cell velocity after the Blebbistatin treatment on organoid #1 (n = 29–59 nuclei per organoid from 5 to 7 hours of imaging period, i.e., 11–15 time points). D Average velocity of three independent pancreatic ductal organoids before and after the Blebbistatin treatment. The decrease in velocity was observed in all three organoids (mean ± SD, *p < 0.0001, N = 3 organoids from 3 independent experiments, n = 29–87 nuclei per organoid from 3–7 hours of imaging period, i.e., 7–15 time points). Ei Confocal maximum projection of the basal surface of pancreatic ductal organoid treated with 200 µM Blebbistatin for 12 hr and stained for F-actin, pMYL9, MYH9, and DNA. Bar = 50 µm. ii, iii Zoomed-in images and intensity profile showing the disrupted actin stress fibers and the accumulation of pMYL9 at the base of the stress fibers. The white arrow indicates the direction of the intensity profile, and the white dashed box indicates the area used to derive the intensity profile. Bar = 20 µm.