(A) Representative confocal micrographs to show transferrin uptake (Tf, green), actin organisation (phalloidin, red) and DNA (blue) in control or Ect2-depleted cells. For each condition, a section though the middle or base of the mitotic cell with a neighbouring interphase cell is shown. Cells were transfected with control siRNA or one of two Ect2 siRNAs (Ect2_5 or Ect2_6). Scale bars, 10 µm. (B) Representative western blot to show the amount of Ect2 remaining after RNAi. Blots were probed for Ect2 and β-tubulin as a loading control. (C) Bar chart to show normalised transferrin uptake in interphase and mitotic (prometaphase) cells as quantified by confocal microscopy. Ncell = 5–8. (D) Representative confocal micrographs to show the distribution of F-actin (Phalloidin, red), G-actin (DNase I, green) and DNA (blue) in mitotic HeLa cells. The cells were transfected with control (GL2) or Ect2 (Ect2_5 or Ect2_6) siRNA. Scale bar, 10 µm. (E and F) Bar charts to summarise the quantification of F-actin ratio at the cell cortex vs the cytoplasm (E) or the amount of G-actin in the cytoplasm (F). Ncell = 40, Nexp = 3. (G) Summary of membrane tension measurements on mitotic HeLa cells transfected with control (GL2) or Ect2 (Ect2_5) siRNA. Relative tether force is shown for individual cells (dots) and the bar indicates the mean. (H) Bar chart to show normalised transferrin uptake in interphase and mitotic (prometaphase) cells. Note the inhibition by latrunculin B (LatB, 1 µM 30 min) of restored CME in mitotic cells depleted of Ect2. Ncell = 7–14. All bars show mean ± SEM, *p<0.05; **p<0.01, ***p<0.001. One-way ANOVA with Tukey’s post-hoc test, comparison to control RNAi, interphase.