Figure S4.
FMNL2, FMNL3, DAAM1, and profilin differentially contribute to filopodia formation and stability. (A) Filopodia length (normalized to siCTR), coverage (relative filopodia number/perimeter), and lifetime (normalized to siCTR) was evaluated after knockdown of FMNL2, FMNL3, and DAAM1. One-way ANOVA with uncorrected Fisher posttest (n = 62, control cells; 62, siFMNL2 cells; 36, siFMNL3 cells; 39, siDAAM1 cells); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (B) Western blot (left) and filopodia FRAP experiments of siCTR cells (black) and siFMNL2 cells silenced with two different single siRNAs (turquoise). FRAP curves were compared by two-way ANOVA, t1/2 values using Student’s t test, and fascin mobility using two-way ANOVA; data were pooled from two independent experiments; n = 26 filopodia for siCTR and 27 and 28 for siFMNL2 1 and 2 (single), respectively; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (C) Evaluation of profilin-1 and -2 siRNA knockdown efficiency and specificity via Western blot (left). GAPDH and HSP70 served as loading control. Representative images of HeLa cells after knockdown of the indicated profilins are shown in right. Scale bar = 10 µm. (D) Fascin movement out of filopodia after profilin knockdown. mEOS2-fascin was photoconverted and monitored over time. Early and late phases were compared using two-way ANOVA with uncorrected Fisher posttest; shown are data from three independent experiments; *, P ≤ 0.05. Fascin t1/2 values and mobility versus immobility (in %) extracted from monoexponential single-curve fitting (n = 53, 58, or 30 filopodia). One- and two-way ANOVA; ****, P ≤ 0.0001; ns, not significant. (E) Filopodia length, coverage, and lifetime was evaluated after control siRNA or combined knockdown of profilin-1 and -2 (n = 63 and 35 cells, respectively). Student’s t test; data are representative of three or more independent experiments. (F) Filopodia flexibility depends on FMNL2. Filopodia of siCTR, siFMNL2, and siFMNL3 GFP-fascin HeLa cells were analyzed for the potential to bend by dividing the area of the traced filopodium with the convex area as outlined in the schematic illustration (left). Straight filopodia have a ratio close to 1; lower ratios indicate more filopodia bending. One-way ANOVA; *, P ≤ 0.05, n = 84, 75, and 54 single filopodia of ≥20 siCTR, siFMNL2, and siFMNL3 cells, respectively, from four independent experiments.

FMNL2, FMNL3, DAAM1, and profilin differentially contribute to filopodia formation and stability. (A) Filopodia length (normalized to siCTR), coverage (relative filopodia number/perimeter), and lifetime (normalized to siCTR) was evaluated after knockdown of FMNL2, FMNL3, and DAAM1. One-way ANOVA with uncorrected Fisher posttest (n = 62, control cells; 62, siFMNL2 cells; 36, siFMNL3 cells; 39, siDAAM1 cells); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (B) Western blot (left) and filopodia FRAP experiments of siCTR cells (black) and siFMNL2 cells silenced with two different single siRNAs (turquoise). FRAP curves were compared by two-way ANOVA, t1/2 values using Student’s t test, and fascin mobility using two-way ANOVA; data were pooled from two independent experiments; n = 26 filopodia for siCTR and 27 and 28 for siFMNL2 1 and 2 (single), respectively; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (C) Evaluation of profilin-1 and -2 siRNA knockdown efficiency and specificity via Western blot (left). GAPDH and HSP70 served as loading control. Representative images of HeLa cells after knockdown of the indicated profilins are shown in right. Scale bar = 10 µm. (D) Fascin movement out of filopodia after profilin knockdown. mEOS2-fascin was photoconverted and monitored over time. Early and late phases were compared using two-way ANOVA with uncorrected Fisher posttest; shown are data from three independent experiments; *, P ≤ 0.05. Fascin t1/2 values and mobility versus immobility (in %) extracted from monoexponential single-curve fitting (n = 53, 58, or 30 filopodia). One- and two-way ANOVA; ****, P ≤ 0.0001; ns, not significant. (E) Filopodia length, coverage, and lifetime was evaluated after control siRNA or combined knockdown of profilin-1 and -2 (n = 63 and 35 cells, respectively). Student’s t test; data are representative of three or more independent experiments. (F) Filopodia flexibility depends on FMNL2. Filopodia of siCTR, siFMNL2, and siFMNL3 GFP-fascin HeLa cells were analyzed for the potential to bend by dividing the area of the traced filopodium with the convex area as outlined in the schematic illustration (left). Straight filopodia have a ratio close to 1; lower ratios indicate more filopodia bending. One-way ANOVA; *, P ≤ 0.05, n = 84, 75, and 54 single filopodia of ≥20 siCTR, siFMNL2, and siFMNL3 cells, respectively, from four independent experiments.

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