Effects of dynein inhibition on maintenance and establishment of centrosome centration. (A–G) The activity of dynein molecular motors was inhibited inside Ptk2 and RPE1 cells through the microinjection of the 74.1 (1 mg/ml) inhibiting antibody. After microinjection, cells underwent a 50-min incubation period. In all the conditions, cells were fixed and stained for the microtubules, the centrosome, the Golgi apparatus, and actin. (H) Inhibition of dynein molecular motors activity inside control Ptk2 cells through the expression of a p150-DN construct. Cells were fixed 24 h after transfection and stained for the Golgi apparatus. (I) Three different centrosome recentering conditions. Two conditions inside PtK2 cytoplasts expressing GFP-tubulin: Nocodazole washout, Y27632 washout, Cytochalasin D washout (n = 70); Nocodazole present, Y27632 washout, Cytochalasin D washout (n = 81). One condition inside control PtK2 cytoplasts expressing the p150–DN–GFP construct: Nocodazole washout, Y27632 washout, Cytochalasin D washout (n = 67). In the graphs, horizontal bars represent the mean. P represents the P values, which were obtained from Kruskal-Wallis non-parametric tests (I) or Mann–Whitney non-parametric tests (C and F). (A) Effect of the injection of the 74.1 antibody on the morphology of the Golgi apparatus. Representative images of control Golgi apparatuses in fixed RPE1 cells (left). Representative images of Golgi apparatuses in fixed RPE1 cells 50 min after their microinjection with the 74.1 antibody (left). (B) Images of fixed PtK2 cells. Microtubule and actin networks in the control condition (top). Microtubule, and actin networks 50 min after the microinjection with the 74.1 antibody (bottom). (C) Effect of 74.1 microinjection inside PtK2 cells. Graph showing the distance between the centrosome and the centroid of the cell in the control (n = 44) and the 74.1 (n = 52) microinjected conditions. (D) Effect of the injection of 74.1 on the pericentrosomal microtubule network in PtK2 cells. Images of pericentrosomal networks in fixed control cells (top). Images of pericentrosomal networks 50 min after the microinjection with the 74.1 antibody (bottom). (E) Images of fixed RPE1 cells. Microtubule and actin networks in the control condition (top). Microtubule and actin networks 50 min after microinjection with the 74.1 antibody (bottom). (F) Effect of 74.1 microinjection inside RPE1 cells. Graph showing the distance between the centrosome and the centroid of the cell in the control (n = 66) and the 74.1 (n = 59) micro-injected conditions. (G) Effect of the injection of 74.1 on the pericentrosomal microtubule network in RPE1 cells. Images of pericentrosomal networks in control cells (top). Images of pericentrosomal networks 50 min after the microinjection with the 74.1 antibody (bottom). (H) Effect of p150-DN expression on the morphology of the Golgi apparatus. Images of Golgi apparatuses in fixed control PtK2 cells not expressing p150–DN–GFP. Images of Golgi apparatuses in fixed control Ptk2 cells 24 h after their transfection with p150-DN. (I) From left to right, the graph shows the total displacement of the centrosomes in the three different centrosome recentering conditions; graph showing the recentering ratio in the three different centrosome recentering conditions; graph showing the distance between the centrosome and the centroid at the end of the recentering experiments in the three different centrosome recentering conditions. (A, B, D, E, and G) All images are max projections, further processed using an unsharp mask and a gamma filter. (H) All images are max projections, further processed using an unsharp mask a gamma filter and a Gaussian blur filter.