Fig 2: FLNA effect on cell proliferation, adhesion and cell migration and Rap1 involvement(A) FLNA siRNA-treated cells showed a strong decrease in Cyclin D1 expression and cell proliferation in H727 cells not via Rap1. H727 cells were transfected with control siRNA, FLNA siRNA alone or together with Rap1 siRNA. The graph shows the quantification of Cyclin D1 normalized to GAPDH. Data represent mean ± SD of three independent experiments. One-way ANOVA; Bonferroni post test * =p<0.05 vs C- siRNA. (B) The graph shows BrdU incorporation during DNA synthesis in H727 cells transfected with C-siRNA, FLNA siRNA alone or together with Rap1 siRNA. Data represent mean ± SD of three independent experiments. One-way ANOVA; Bonferroni post test **=p<0.01 vs C- siRNA. (C) H727 cells transfected with C-siRNA or FLNA siRNA alone or together with Rap1 siRNA were incubated in complete medium for 90 min at 37°C. Experiments carried out in H727 cells were repeated at least 4 times. The graph shows the quantification of adherent cells. Values represent mean (±SD). One-way ANOVA; Bonferroni post test **=p<0.01 vs C-siRNA. (D) H727 cells transfected with C-siRNA, FLNA siRNA alone or together with Rap1 were incubated for 24 h with serum-free medium. Migratory cells were quantified by fluorometric plate reader. Experiments were performed in triplicate 3 times. Values represent mean (±SD); One-way ANOVA; Bonferroni post test **=p<0.01 vs C-siRNA.

Fig 3: The interaction with Filamin protects Refilins from degradation. (A) Sub-confluent FLNA-deficient M2 cells (lanes 1-3) and A7 cells (lanes 4-6) were not transfected (lanes 1,4) or transfected with Myc-tagged RefilinA (lanes 2,5) or RefilinB (lanes 3,6). Total cell extracts were analysed by western blot with anti-Myc and anti-FLNA antibodies. (B) M2 cells (left panel) and A7 cells (right panel) transfected with Myc-tagged RefilinA were fixed and stained with anti-Myc antibodies. (C) Sub-confluent FLNA-deficient M2 cells co-transfected with 19-22 FLNA-HA and either Myc-tagged control protein (Miret) (lane 1), RefilinA-?BD1 to -?BD4 mutants (lanes 2-5) or wild-type RefilinA (lane 6) were immunoprecipitated with rat anti-HA antibody. Total cell extracts (Total) and immunoprecipitates (IP-HA) were analysed by western blot using mouse anti-HA and mouse anti-Myc. In A and C, asterisks indicate to a non-specific band.

Fig 4: RefilinB co-localizes with FLNA at the plasma membrane and membrane protrusions in NG2 oligodendrocyte precursor cells. (A,B) High-density OPC cultures were fixed in methanol and double immunostained with guinea pig anti-RefilinB (red) and mouse anti-FLNA (green) antibodies. In B, nuclei were stained with Hoescht. Scale bar: 10 µm (A) and 20 µm (B). In B, arrows point to cell with no RefilinB immunostaining.

Fig 5: Effect of FLNA on VEGF expression and in vitro release in H727 cells(A&B) Representative immunoblot of VEGF demonstrates that FLNA silencing reduces VEGF expression in primary PNTs cells and H727 cells, respectively. The graph shows the ratio of VEGF/GAPDH normalized to C- siRNA. Experiments were repeated at least 2-3 times. *p<0.05, **p<0.01 vs C- siRNA. (C) ELISA analysis of VEGF release in supernatant by c-siRNA and FLNA siRNA H727 cells. All data are expressed as mean ± SD of three independent experiments. **=p <0.01, **=p<0.01 vs C- siRNA.