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. 2021 Sep 28;220(11):e202006033. doi: 10.1083/jcb.202006033

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© 2021 Camillo et al.

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Figure 2. — Negative regulation of FA turnover, stress fiber formation, and ECM-elicited mechanosensing relies on FLRT2-binding OLF domain of LPHN2. (A–E) Confocal microscopy analysis (A) of vinculin (Vin; blue), and phalloidin-labeled F-actin (red; vinculin high-magnification images are on the bottom). Cells were first transduced with pCCL lentivirus (carrying GFP)-mediated overexpression (green) of silencing-resistant mouse WT or ΔOLF Lphn2 and then oligofected with either siCTL or siLPHN2 siRNAs. Scale bars, 10 µm; magnification scale bar, 5 µm. Confocal microscopy analysis reveals how lentiviral delivery of WT LPHN2, but not ΔOLF LPHN2 mutant, restores the phenotype of vinculin-containing FAs number, normalized on cell area (B) and size (expressed by maximum Feret diameter, C). The same rescue effect of WT LphnN2, but not ΔOLF Lphn2 mutant, occurs on stress fiber number, normalized on cell area (D) and amount (evaluated as mean gray intensity, E). Results are the mean ± SD of two independent experiments for a total of 15 ECs for each condition. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (F) XZ-STED confocal microscope cross-sectioning of stress fibers reveals how siLPHN2 ECs have thicker stress fibers in comparison to siCTL ECs, and the transduction of WT, but not ΔOLF Lphn2, rescues this phenotype. The cross-sectional area of stress fibers was measured with a mask, shown (image with white background) at the bottom each image, obtained with ImageJ starting from XZ STED confocal images after deconvolution (image with black background). Scale bar, 1 µm. Results are the mean ± SD of two independent experiments for a total of 11 ECs for each condition. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; **, P ≤ 0.01; ***, P ≤ 0.001. (G and H) Confocal microscopy analysis (G) of endogenous YAP (in red on the left) and TAZ (in red on the right) reveals how pCCL lentivirus (carrying GFP)-mediated delivery (green) of WT Lphn2, but not ΔOLF Lphn2 mutant, restores the nuclear (nucl)/cytoplasmic (cyto) ratio of both YAP (H, top panel) and TAZ (H, bottom panel). Scale bar, 20 µm. Representative images of ECs plated on FN (5 µg/ml)-coated 10 kPa stiff substrate are shown. Results are the mean ± SD of two independent experiments for a total of 22 ECs for each experimental condition (10 kPa and increasing 1, 3, and 5 µg/ml FN concentration). Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; NS, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (I) Real-time analysis of EC migration toward Coll I (xCELLigence RTCA DP system) reveals how only transduction with WT (green), but not ΔOLF LPHN2 (purple) rescues the higher migration rate of siLPHN2 ECs. Results are the mean ± SD of three independent experiments. Results were analyzed with two-way ANOVA and Bonferroni’s post hoc analysis; *, P ≤ 0.05; ^##, P ≤ 0.01; ^###, P ≤ 0.001. Max., maximum; n., number.