LARG and mDia1 Link G_12/13 to Cell Polarity and Microtubule Dynamics

Supplemental Materials

This article contains the following supporting material:

• Supplementary Figure 1 - Gα_12/13-deficient MEFs fail to polarize in response to LPA. Concentration-response analysis in WT (A) and Gα_12/13-deficient (B) MEFs after addition of 1 nM LPA (?), 10 nM LPA (?), 100 nM LPA (?) and 1000 nM LPA (x) in serum free medium. Results represent the mean of 3 independent experiments. (C) WT MEFs were starved, scratched and treated with 100 nM LPA for 1 hour. Fixed cells were probed with GFP alone (upper panel) or GFP-RBD in the absence or presence of 100 nM LPA as indicated. Scale bar 10 µm. (D) Quantifications of cells with GFP-RBD at the front before and after LPA treatment are shown as means ± S.E.M. of 3 independent experiments. (E) GFP-RBD fluorescent intensities are shown as arbitrary units over cell distance corresponding to the lines drawn through cells (top panel: treated, bottom panel: untreated cells). (F) Starved WT MEFs were either untreated or treated with 100 nM LPA. MTOC orientation in at least 200 cells was scored (6 hours post wounding). Data represent means ± S.E.M. of 3 independent experiments. Representative staining for MTOC (green), DAPI (blue) α-tubulin (red) are shown for untreated (G) and treated (H) cells. Merged images with α-tubulin are shown. A dotted, white line indicates the wound. Scale bar 10 µm.
• Supplementary Figure 2 - NIH 3T3 fibroblast polarization. (A) Time course experiment in NIH 3T3 fibroblasts showing the percentage of polarized MTOCs over 8 hours. MTOC reorientation in these cells takes longer then WT MEFs and it does not attain a level of 85 % positive cells, as with WT MEFs. (B) NIH 3T3 fibroblasts were transfected with control siRNA or siRNA against LARG, RhoA or mDia1 and were either left untreated or treated with 0.5 µM TAT-C3 for 18 hours or pretreated with 10 µM Y27632 for 30 minutes and percentage of positive MTOCs was counted 8 hours after wounding. MTOC polarization was determined by scoring at least 200 cells. Data represent means ± S.E.M. of 3 independently performed experiments. (C) Cell extracts were immunoblotted for the indicated proteins. Representative images are shown of (D) untreated NIH 3T3 cells or treated with (E) TAT-C3 or (F) Y27632 stained for MTOC (green) and DAPI (blue). Representative images of cells after treatment with control siRNA (G), siRNA against LARG (H), RhoA (I) or mDia1 (J) and staining for MTOC (green) and DAPI (blue) are shown. Merged images with α-tubulin (red) show the leading edge. Scale bar 10 µm.
• Supplementary Figure 3 - LARG is localized along microtubule tracks. (A) Leading edge WT MEFs were microinjected with GFP. Representative TIRF image of GFP (green) together with α-tubulin staining and merged are shown. (B) Leading edge WT cells were microinjected with GFP-LARG (green) and representative TIRF images are shown with α-tubulin staining (red) and merged. (C) WT MEFs were microinjected with GFP-PDZ-RGS at the leading edge. Representative image shows GFP-PDZ-RGS (green), α-tubulin (red) and merged. (D) WT MEFs were microinjected with GFP-RGS in the leading edge and representative image shows GFP-RGS (green) with α-tubulin staining (red) and merged. Scale bar 2µm.
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