Figure 1.
Rab5C depletion significantly inhibits cell migration.
A) DIC images of stable Rab5 isoform knock-down (KD) HeLa cells taken with light microscope at 40X magnification (left panel). Arrows indicate membrane ruffles. KD of Rab5 isoforms (right panel) in these stable cell lines is shown in the immunoblots following SDS-PAGE as described in Experimental Procedures. B) 0.5–1 mm width wounds were made on a monolayer of HeLa stable control or Rab5 isoform KD cells. 5–7 wounded spots in each dish were imaged with time-lapse microscope every 5 minutes for 20 hours. C) The percentage of wound closure (left panel) was calculated from images acquired at time 0 and 16 hours with ImageJ. For each sample, at least 5 images were used to calculate the percentage of wound closure in each experiment. The graph represents the Mean± S.E. from four independent experiments. U937 cells (right panel) transiently transfected with siRNA against Rab5 isoforms were seeded in the upper chamber of the Transwell plates and allowed to migrate towards 10% FBS in the bottom chamber for 24 hours. Migrated cells were measured as indicated in Material and Methods. Data are normalized to initial seeding cell numbers. The graph represents the Mean± S.E. from four independent experiments. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test.(*P<0.05, ***P<0.001)
Figure 2.
Loss of Rab5C reduced translocation of Rac1 to cell periphery and Rac1 activation in response to EGF stimulation.
A) HeLa cells stably expressing GFP-Rac1 were transfected with scrambled or Rab5 isoform siRNAs. 48 hours post-transfection, cells were seeded onto coverslips imprinted with crossbow micro-patterns. 3-D GFP-Rac1 images of at least 40 micro-patterned cells were acquired for each sample. Each GFP-Rac1 3-D image stack was subjected to Maximum intensity projection and then grey scale normalization. Next, the max projections of 88 GFP-Rac1 images (from two independent experiments) were aligned and averaged (upper panel, in pseudo-color). Image subtraction was carried out between averaged image of scramble control and that of individual Rab5 isoform siRNA-treated sample. The resulting image after subtraction (siScr minus siRab5) is shown in pseudo-color (bottom panel). B) HeLa cells stably expressing GFP-Rac1 were transfected with scrambled or Rab5 isoform siRNAs. 48 hours post-transfection, cells were separated into membrane (Mem) and cytosolic (Cyt) fractions as described in Material and Method. Relative amounts of GFP-Rac1 in each fraction were analyzed by SDS-PAGE and Western blot. Densitometry of the bands was quantified using AlphaEaseFC 4.0 software. The numbers represent the ratio of GFP-Rac1 in cytosol or membrane/total. C) HeLa cells were transfected with scrambled or Rab5 isoform-specific siRNA. 48 hours post-transfection, cells were starved and then stimulated with EGF for two minutes. Cell lysates were prepared and subjected to Rac1 pull-down assays. Proteins were eluted, separated by SDS-PAGE and blotted for Rac. Total lysates were also probed for Rac1 to determine the total Rac1 level is equal in all samples. The intensity of the bands from western blots was quantified with AlphaEaseFc 4.0 software. The relative amount of Rac-GTP from pull-downs was normalized to that of total Rac1 from total cell lysates. The adjacent graph represents the mean value ± S.E. from four independent experiments. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test. (*P<0.05, **P<0.01)
Figure 3.
PI3K signaling in response to Rab5 isoform depletion.
A) HeLa cells were transfected with GFP (as negative control) or Rab5 isoform-specific siRNA. 48 hours post-transfection, cells were starved and then stimulated with EGF for indicated times. Cell lysates were subjected to SDS-PAGE and probed with antibodies as indicated. Band intensity was quantified with AlphaEaseFc 4.0 software. Bars represent the mean value ± S.E. from four independent experiments. Analysis was carried out with a two-way ANOVA, Bonferroni’s post-test. P<0.05. B) HeLa cells were transfected with indicated siRNA. 48 hours post-transfection, cells were seeded onto micropatterned coverslips coated with fibronectin, and then allowed to spread out for 2 hours in starvation medium. Starved cells were stimulated with 10 % FCS for 3 minutes and then fixed for PIP3-FITC antibody immuno-staining. Images shown here are average projections of PIP3 staining from 30–35 cells.
Figure 4.
Depletion of Rab5C reduces cell adhesion.
A) HeLa cells were seeded on coverslips O/N and then transfected with GFP or Rab5 isoform siRNAs. The focal adhesion complex was visualized by immunostaining with vinculin antibody. The numbers of focal adhesion complexes were determined with ImageJ. The graph represents Mean± S.E. from 30 cells. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test. P<0.0001. B) HeLa cells transfected with GFP or Rab5 isoform siRNAs were re-suspended and re-plated on fibronectin-coated plates for indicated times. At the end of each time point, cell lysates were extracted and prepared for SDS-PAGE and Western bloting. The activation of focal adhesion kinase was determined with phospho-FAK antibody. Total levels of FAK were not determined. The data represents Mean± S.E. from three independent experiments. Analysis was carried out with a two-way ANOVA, Bonferroni’s post-test. P<0.05.