Figure S1.
Endogenous LPHN2 expression, silencing, and rescue impact on endothelial cell adhesion, migration, and signaling. (A) Upon surface biotinylation, LPHN2 was immunoprecipitated (IP) with a rabbit anti-LPHN2 pAb from EC lysates and revealed in Western blot (WB) by means of HRP streptavidin. Pre-immune serum (PIS) was employed for control purposes. The cleaved extracellular portion of LPHN2 appears as an ∼130 kD protein band. (B) HEK 293T cells were transfected with an empty vector or increasing amounts (2, 4, and 6 µg) of HA-Lphn2-EGFP construct. Cell lysates were then analyzed by Western blot with either anti-HA (left) or anti-GFP (right) Ab. The 230-kD, 130-kD, and 97-kD protein bands correspond to uncleaved full-length, cleaved N-terminal extracellular-only portion, and cleaved C-terminal portion of the transfected HA-Lphn2-EGFP protein, respectively. In the anti-GFP Western blot, the 27-kD band corresponds to the GFP whose cDNA was present in the control empty vector. (C) Real-time quantitative PCR analysis of LPHN2 mRNA in siCTL or siLPHN2 human ECs relative to the housekeeping genes GAPDH and TBP and normalized on siCTL levels. Results are themean ± SD of nine independent assays. Statistical analysis: two-tailed heteroscedastic Student’s t test; ***, P ≤ 0.001. (D) Real-time analysis of control (siCTL) or LPHN2 (siLPHN2) silenced EC migration toward FN was assessed with an xCELLigence RTCA DP system. Results are the mean ± SD of four independent assays. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; NS, P > 0.05; *, P ≤ 0.05. (E) Real-time quantitative PCR analysis of LPHN2 mRNA in siCTL or siLPHN2 human ECs rescued or not (CTL) with mouse WT or ΔOLF Lphn2 relative to the housekeeping genes GAPDH and TBP and normalized on siCTL levels. Results are the mean ± SD of four independent assays. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; ***, P ≤ 0.001. Right: Transduced and silenced ECs were also lysed and WT HA-Lphn2-pCCL and ΔOLF HA-Lphn2-pCCL protein expression levels analyzed by Western blot with anti-HA Ab. (F–H) Confocal microscopy analysis (F) of endogenous paxillin (Pax; blue), and phalloidin-labeled F-actin (red) reveals how, compared with siCTL ECs, LPHN2 silencing increases the number, normalized on cell area (G) and size (expressed by maximum Feret diameter, H) of paxillin-containing FAs. Scale bars, 10 µm. Results are the mean ± SD of two independent experiments for a total of 18 (siCTL) and 18 (siLPHN2) ECs. Statistical analysis: two-tailed heteroscedastic Student’s t test; *, P ≤ 0.05; ***, P < 0.001. (I) LPHN2 silencing in human ECs does not affect basal GTP loading of RhoA small GTPase. Total Rho was used to calculate the normalized OD (N.O.D.) levels of active Rho-GTP. Results are the mean ± SD of three independent assays. Statistical analysis: two-tailed heteroscedastic Student’s t test; NS, P > 0.05. (L) Mean fluorescence intensity of VE-cadherin (VE-cad+) intercellular staining (in green in Fig. 5 A) in siCTL ECs seeded on 10 kPa substrates coated with increasing amounts of FN (1, 3, and 5 µg/ml). The VE-cadherin intercellular recruitment was not affected by FN density. Results are the mean ± SD of two independent experiments. Statistical analysis: two-way ANOVA with Bonferroni’s post hoc analysis; NS, P > 0.05. (M) Mean fluorescence intensity of VE-cad+ intercellular staining (in green in Fig. 5 B) in lentivirally delivered WT or ΔOLF Lphn2 in ECs seeded on 10 kPa substrates coated with FN (5 µg/ml). The VE-cadherin intercellular recruitment was not affected by LPHN2 silencing. Results are the mean ± SD of two independent experiments. Statistical analysis: two-way ANOVA with Bonferroni’s post hoc analysis; NS, P > 0.05. Max., maximum; n., number.

Endogenous LPHN2 expression, silencing, and rescue impact on endothelial cell adhesion, migration, and signaling. (A) Upon surface biotinylation, LPHN2 was immunoprecipitated (IP) with a rabbit anti-LPHN2 pAb from EC lysates and revealed in Western blot (WB) by means of HRP streptavidin. Pre-immune serum (PIS) was employed for control purposes. The cleaved extracellular portion of LPHN2 appears as an ∼130 kD protein band. (B) HEK 293T cells were transfected with an empty vector or increasing amounts (2, 4, and 6 µg) of HA-Lphn2-EGFP construct. Cell lysates were then analyzed by Western blot with either anti-HA (left) or anti-GFP (right) Ab. The 230-kD, 130-kD, and 97-kD protein bands correspond to uncleaved full-length, cleaved N-terminal extracellular-only portion, and cleaved C-terminal portion of the transfected HA-Lphn2-EGFP protein, respectively. In the anti-GFP Western blot, the 27-kD band corresponds to the GFP whose cDNA was present in the control empty vector. (C) Real-time quantitative PCR analysis of LPHN2 mRNA in siCTL or siLPHN2 human ECs relative to the housekeeping genes GAPDH and TBP and normalized on siCTL levels. Results are themean ± SD of nine independent assays. Statistical analysis: two-tailed heteroscedastic Student’s t test; ***, P ≤ 0.001. (D) Real-time analysis of control (siCTL) or LPHN2 (siLPHN2) silenced EC migration toward FN was assessed with an xCELLigence RTCA DP system. Results are the mean ± SD of four independent assays. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; NS, P > 0.05; *, P ≤ 0.05. (E) Real-time quantitative PCR analysis of LPHN2 mRNA in siCTL or siLPHN2 human ECs rescued or not (CTL) with mouse WT or ΔOLF Lphn2 relative to the housekeeping genes GAPDH and TBP and normalized on siCTL levels. Results are the mean ± SD of four independent assays. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; ***, P ≤ 0.001. Right: Transduced and silenced ECs were also lysed and WT HA-Lphn2-pCCL and ΔOLF HA-Lphn2-pCCL protein expression levels analyzed by Western blot with anti-HA Ab. (F–H) Confocal microscopy analysis (F) of endogenous paxillin (Pax; blue), and phalloidin-labeled F-actin (red) reveals how, compared with siCTL ECs, LPHN2 silencing increases the number, normalized on cell area (G) and size (expressed by maximum Feret diameter, H) of paxillin-containing FAs. Scale bars, 10 µm. Results are the mean ± SD of two independent experiments for a total of 18 (siCTL) and 18 (siLPHN2) ECs. Statistical analysis: two-tailed heteroscedastic Student’s t test; *, P ≤ 0.05; ***, P < 0.001. (I) LPHN2 silencing in human ECs does not affect basal GTP loading of RhoA small GTPase. Total Rho was used to calculate the normalized OD (N.O.D.) levels of active Rho-GTP. Results are the mean ± SD of three independent assays. Statistical analysis: two-tailed heteroscedastic Student’s t test; NS, P > 0.05. (L) Mean fluorescence intensity of VE-cadherin (VE-cad+) intercellular staining (in green in Fig. 5 A) in siCTL ECs seeded on 10 kPa substrates coated with increasing amounts of FN (1, 3, and 5 µg/ml). The VE-cadherin intercellular recruitment was not affected by FN density. Results are the mean ± SD of two independent experiments. Statistical analysis: two-way ANOVA with Bonferroni’s post hoc analysis; NS, P > 0.05. (M) Mean fluorescence intensity of VE-cad+ intercellular staining (in green in Fig. 5 B) in lentivirally delivered WT or ΔOLF Lphn2 in ECs seeded on 10 kPa substrates coated with FN (5 µg/ml). The VE-cadherin intercellular recruitment was not affected by LPHN2 silencing. Results are the mean ± SD of two independent experiments. Statistical analysis: two-way ANOVA with Bonferroni’s post hoc analysis; NS, P > 0.05. Max., maximum; n., number.

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