Fig. 6
Acto-myosin force organization regulates PLK4 recruitment at the centrosome and centriole duplication. a Images of cells expressing C1-GFP immunostained for PLK4 (scale bar = 5 μm). Dashed boxes indicate zoomed section presented in panels on the right of the pictures. Zoomed panels (scale bar = 2 μm): from top to bottom: C1-GFP, total PLK4, Merge. b, c PLK4 mean fluorescent intensity for all cells analyzed. Between 20 to 30 cells were analyzed for all the conditions. d Representative pictures of C1-GFP HeLa cells arrested in G1 phase with a double thymidine block and then analyzed for presence of duplicated centrosomes (4C and more) at different time points from the release of the thymidine block. Cells were either untreated (Control) or treated with 10 μM Blebbistatin (Bleb). Red asterisks indicate misduplicated centrosomes and green asterisks indicate correct duplication. Pictures were analyzed as maximal projection of the z-stack spanning through all the volume of the cell. e, f Quantification of duplicated centrioles (4C and more than 4C) at the time point at 10 h from Thymidine release for H, Tripod and T, and 18 h for Square (Square n = 20 cells; H n = 25 cells; Tripod n = 24 cells; T n = 25 cells) and for the corresponding blebbistatin (Bleb) treated cells (Square + Bleb n = 20 cells; H + Bleb n = 20 cells; Tripod + Bleb n = 20 cells; T + Bleb n = 20 cells). Quantification for Square and H shapes (e) and Tripod and T (f). Error bars represent s.e. g Representative pictures of C1-GFP arrested in G1 phase with a double thymidine block and then analyzed after 16 h of CentrinoneB (CenB) treatment versus untreated (scale bar = 2 μm). h Quantification of more than four centrioles and more than four centrioles in untreated and 16 h CentrinoneB (CenB) treated Square and Tripod cells (square n = 20 cells; Square + CenB n = 20 cells; Tripod n = 20 cells; Tripod + CenB n = 23 cells). Plain bars represent percentages of cells with four centrioles, patterned bars represent percentage of cells with more than four centriole. * is the statistics (p < 0.05) for treated vs untreated % of cells with more than 4C in Square and in Tripod. Error bars represent s.e. p-values were obtained with unpaired two-tailed t-test; *p < 0.05, ***p < 0.001
