Fig. 8.
A RAB35/PI3K axis is necessary for triggering CDR formation. a PI3K inhibition impairs RAB35-induced CDR formation. pSLIK-HA-RAB35-human-MEFs were treated or not with doxycycline to induce the transgene expression and incubated with vehicle or LY294002. Still, phase contrast images from time-lapse sequence (Supplementary Movie 14) are shown. Red arrows indicate CDRs. Scale bar, 50 µm. The number of CDR/cell is the mean ± SEM (n > 30 cells/condition, three independent experiments). ****p < 0.0001. RAB35 expression was verified by IF. b Genetic ablation of the regulatory subunits of PI3K abrogates the RAB35-dependent CDR formation. Control or p85α−/− pβ−/− double KO MEFs (p85−/−)47 were infected with pSLIK-HA-RAB35 to express RAB35 in a doxycycline-inducible fashion imaged by time-lapse phase contrast microscopy. Left: Still phase contrast images from time-lapse sequence (Supplementary Movie 15). Red arrows indicate CDRs. Scale bar, 50 µm. Middle graph: The number of CDR/cell is the mean ± SEM (n = 50 cells/condition, two independent experiments). ****p < 0.0001; *p < 0.05. Right graph: RAB35 expression was verified by qRTPCR. Data are the fold increase of RAB35 mRNA levels of doxycycline-treated with respect to untreated ones. c, d RAB35 regulates AKT activity. c Serum-starved control (shCtrl) and Rab35-silenced (shRab35) cells were stimulated or not for 10′ with PDGF. Cell lysates were analysed by immunoblotting to detect the indicated total or phosphorylated proteins. Vinculin is a loading control. d Lysates from pSLIK-HA-RAB35-human-MEFs treated or not with doxycycline were immunoblotted as in c. e RAB35 co-immunoprecipitates with endogenous p85α. Serum-starved p85α−/− pβ−/− double KO cells (p85−/−) and control MEFs were stimulated or not for 10’ with PDGF. Cell lysates (1 mg) were immunoprecipitated with anti-p85α monoclonal antibody or irrelevant immunoglobulin G (IgG). Cell lysates (50 μg) and immunoprecipitates (IPs) were immunoblotted with the indicated antibodies. f Active RAB35 interacts with p85α. Serum-starved HeLa cells transfected with RAB35 WT, or RAB35 S22N or RAB35Q67L fused to EGFP were mock treated (−) or stimulated with HGF (+) for 10′. Cell lysates (1 mg) were immunoprecipitated with anti-p85α antibody or irrelevant immunoglobulin G (IgG). Lysates (50 μg) and immunoprecipitates (IPs) were immunoblotted with the indicated antibodies. p-values are from paired Student’s t-test