Moesin depletion enhances the formation of TNTs and reduces MCA. (A–D) Effect of moesin depletion on TNT formation in mOCs (A and B) and hOCs (C and D). (A and C) Representative scanning electron microscopy images of mOCs (day 3) CTL versus moesin KO (A) and mononucleated hOCs (day 3) treated with nontargeting siRNA (siCTL) or targeting moesin (siMoesin) (C). White arrowheads show TNTs. (A) A giant mOC is colored in purple. Scale bar, 50 µm (A) and 20 µm (C). (B and D) Quantification of the percentage of cells forming thick and thin TNTs after immunofluorescence analysis in mOCs (B, n = 3 independent experiments) and hOCs (D, n = 4 donors) (see Fig. S1 B and Materials and methods), n > 250 cells per conditions, means ± SDs are shown. Statistical analysis is shown for thick TNTs. (E and F) Analysis of force by atomic force spectroscopy operated in dynamic tether pulling mode. (E) Force-velocity curve from dynamic tether pulling on CTL and moesin KO (MKO) mOCs. Data points are mean tether force ± SEM at 2, 5, 10 and 30 µm/s pulling velocity. At least 17 cells per condition were analyzed in 4 independent experiments. (F) Mean and SD of the MCA parameter Alpha obtained from fitting the Brochard-Wyart model (see Materials and methods for details).