Figure 4.

Comparison of RhoB and CaM roles in endosomes. (A) Twenty-four hours after transfection with myc-RhoB wt, COS1 cells were incubated with EGF (100 ng/ml; 15 min) at 37°C, fixed, and triple-stained with anti-myc (Alexa Fluor 488), phalloidin-TRITC and anti-EGFR (Cy5). (B) COS1 cells were transfected with pSUPER RhoB, and, after 72 h of knockdown, cells were incubated with W13 (5 μg/ml; 60 min) at 37°C. EEA1 was detected with specific antibody followed by Alexa Fluor 594. The extent of RhoB depletion is shown by Western blotting. (C) NRK cells were preincubated with W13 (5 μg/ml; 30 min), treated with the ROCK inhibitor Y27632 (20 μM) for the next 45 min and with transferrin-TRITC (50 μg/ml) for the last 15 min at 37°C. After fixation, cells were labeled with phalloidin-Alexa Fluor 350 and anti-EEA1 (Alexa Fluor 488). Bar, 10 μm. (D) COS1 cells were transfected and treated as described in A, adding 5 μM rottlerin for 30 min. Bar, 10 μm. On the right, quantification of the number of RhoB-overexpressing cells presenting 5 or more enlarged endosomes (diameter >350 nm) from two independent experiments (performed as described above; n = 900 cells) is represented. Each data point represents value averaged (±SD). ***p < 0.001 for Student's t test, indicating statistical significance of differences between EGF- and EGF- + rottlerin-treated cells.