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. 2021 May 4;8:633609. doi: 10.3389/fcvm.2021.633609

Figure 2.

Figure 2

PLXNA4 downregulation alters endothelial cellular phenotype. (A) mRNA expression of PLXNA4, PLXNA1, PLXNA2, and PLXNA3 in endothelial cells treated with a lentiviral non-targeting shRNA (mock) or a shRNA against PLXNA4 (PLXNA4 KD). Results are expressed as copies corrected for GAPDH. Mean ± S.E.M. of N = 27, *P < 0.05. (B) Representative overview pictures of mock-treated and PLXNA4 KD endothelial cells. (C) mRNA expression of ICAM-1, IL-6, VE-cadherin and integrinβ1 (ITGB1) in mock or PLXNA4 KD endothelial cells. Results are expressed as copies corrected for GAPDH. Mean ± S.E.M. of N > 4, *P < 0.05. (D) Immunoblots and quantification of ICAM-1 and GAPDH protein in mock control cells and PLXNA4 KD cells. Results are depicted as fold change in intensity. Mean ± S.E.M. of N = 7. (E–I) Immunofluorescent staining of F-actin (red), VE-cadherin (green) and nuclei (blue) in mock or PLXNA4 KD endothelial cells. (E) Representative fluorescent overview photographs. (F–I) Quantification of (F) F-actin fluorescent signal, (G) F-actin cellular distribution, (H) VE-cadherin fluorescent signal or (I) VE-cadherin cellular distribution. Fluorescent signal is quantified as (mean fluorescent intensity*area)/nuclei and expressed as fold change relative to control cells. The cellular distribution is quantified as mean intensity per pixel of the border or interior area and expressed as the ratio between border and interior. Mean ± S.E.M. of N = 3, *P < 0.05. (J) Immunoblots and quantification of VE-cadherin and GAPDH protein in control cells and PLXNA4 knockdown cells. Results are depicted as fold change in intensity. Mean ± S.E.M. of N = 4.