Figure 3.
FLRT2 negatively regulates EC response to the ECM via LPHN2 and triggers cAMP/Rap1 signaling (A and B) Real-time analysis of EC migration toward Coll I, assessed with an xCELLigence RTCA DP system. (A) Endogenous FLRT2 silencing (siFLRT2) increases migration compared with siCTL ECs. Results are the mean ± SD of seven independent assays. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; *, P ≤ 0.05; ***, P ≤ 0.001. (B) Exogenous rhFLRT2 (800 ng/ml) inhibits siCTL but not siLPHN2 EC migration. For sake of simplicity, data from the same experiments are plotted in two separate graphs. Results are the mean ± SD of three independent assays. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; *, P ≤ 0.05 for siCTL ECs (left), whereas all differences in siLPHN2 ECs (right) were not significant; NS, P > 0.05. (C) Endogenous FLRT2 silencing in ECs decreases basal cAMP amount. Results are the mean ± SD of seven independent assays. Statistical analysis: two-tailed heteroscedastic Student’s t test; *, P ≤ 0.05. (D) Endogenous FLRT2 silencing (siFLRT2) in ECs decreases basal Rap1-GTP levels, and treatment of siFLRT2 ECs with exogenous rhFLRT2 (800 ng/ml) rescues the decrease in Rap1-GTP levels. Total Rap1 was employed to calculate the normalized OD (N.O.D.) levels of active Rap1-GTP. Results are the mean ± SD of four independent experiments (a representative one is shown). Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; **, P ≤ 0.01 for siCTL versus siFLRT2 and *, P ≤ 0.05 for siFLRT2 versus siFLRT2 + FLRT2. (E–I) Confocal microscopy analysis (E) of endogenous FLRT2 (green), vinculin (Vin; blue), and phalloidin-labeled F-actin (red; high-magnification images are on the right) reveals how, compared with siCTL ECs, FLRT2 silencing increases the number, normalized on cell area (F) and size (expressed by maximum Feret diameter, G) of vinculin-containing FAs and the number, normalized on cell area (H), and amount (evaluated as mean gray intensity, I) of F-actin stress fibers in siFLRT2 ECs. Scale bar, 10 µm (siCTL), 15 µm (siFLRT2). Results are the mean ± SD of four independent experiments for a total of 18 (siCTL) and 19 (siFLRT2) ECs. Statistical analysis: two-tailed heteroscedastic Student’s t test; **, P ≤ 0.01; ***, P < 0.001. Max., maximum; n., number.

FLRT2 negatively regulates EC response to the ECM via LPHN2 and triggers cAMP/Rap1 signaling (A and B) Real-time analysis of EC migration toward Coll I, assessed with an xCELLigence RTCA DP system. (A) Endogenous FLRT2 silencing (siFLRT2) increases migration compared with siCTL ECs. Results are the mean ± SD of seven independent assays. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; *, P ≤ 0.05; ***, P ≤ 0.001. (B) Exogenous rhFLRT2 (800 ng/ml) inhibits siCTL but not siLPHN2 EC migration. For sake of simplicity, data from the same experiments are plotted in two separate graphs. Results are the mean ± SD of three independent assays. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; *, P ≤ 0.05 for siCTL ECs (left), whereas all differences in siLPHN2 ECs (right) were not significant; NS, P > 0.05. (C) Endogenous FLRT2 silencing in ECs decreases basal cAMP amount. Results are the mean ± SD of seven independent assays. Statistical analysis: two-tailed heteroscedastic Student’s t test; *, P ≤ 0.05. (D) Endogenous FLRT2 silencing (siFLRT2) in ECs decreases basal Rap1-GTP levels, and treatment of siFLRT2 ECs with exogenous rhFLRT2 (800 ng/ml) rescues the decrease in Rap1-GTP levels. Total Rap1 was employed to calculate the normalized OD (N.O.D.) levels of active Rap1-GTP. Results are the mean ± SD of four independent experiments (a representative one is shown). Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; **, P ≤ 0.01 for siCTL versus siFLRT2 and *, P ≤ 0.05 for siFLRT2 versus siFLRT2 + FLRT2. (E–I) Confocal microscopy analysis (E) of endogenous FLRT2 (green), vinculin (Vin; blue), and phalloidin-labeled F-actin (red; high-magnification images are on the right) reveals how, compared with siCTL ECs, FLRT2 silencing increases the number, normalized on cell area (F) and size (expressed by maximum Feret diameter, G) of vinculin-containing FAs and the number, normalized on cell area (H), and amount (evaluated as mean gray intensity, I) of F-actin stress fibers in siFLRT2 ECs. Scale bar, 10 µm (siCTL), 15 µm (siFLRT2). Results are the mean ± SD of four independent experiments for a total of 18 (siCTL) and 19 (siFLRT2) ECs. Statistical analysis: two-tailed heteroscedastic Student’s t test; **, P ≤ 0.01; ***, P < 0.001. Max., maximum; n., number.

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