Fig 2: CD44 and Spectrin Restrict Caveolar Mobility which, in Turn, Regulates Diffusion of Selectin(A) TNF-α-activated endothelial cells immunostained for P/E-selectin (green) and caveolin-1 (red). Area denoted in left panel is magnified in center panels (scale bars, 1 μm). Pearson’s correlation coefficient of individual cells is indicated in the far-right panel.(B) Distribution of caveolin-1 (red) and βI+βII-spectrin (green) imaged using STED. Scale bar, 1 μm.(C) Ratio of apical/basolateral fluorescence of immunostained caveolin-1 and KRas(tail)-GFP in cells treated with non-targeted, CD44, or βI+βII-spectrin siRNA. n = 3; ≥25 cells per experiment.(D) Representative trajectories of apical caveolin-1-RFP under the indicated conditions. Color coding is as in Figure 1. Scale bars, 50 nm.(E) DC-MSS classification of caveolin-1-RFP trajectories as in (D) for cells transfected with non-targeted, CD44, or βI+βII-spectrin siRNAs. n = 3; ≥25 cells per experiment.(F) Diffusion coefficient determined for apical caveolin-1-RFP of non-targeted siRNA, CD44-transfected, or βI+βII-spectrin siRNA-transfected cells; n = 3, ≥25 cells.(G) Diffusion coefficients for E-selectin-GFP tagged with anti-GFP-Cy3B nanobodies in cells treated with vehicle or 10 mM methyl-β-cyclodextrin (MβCD) for 30 min. n = 3; ≥20 cells per experiment.(H) Confinement areas for E-selectin-GFP in cells treated with non-targeted, CD44, or βI+βII-spectrin siRNA ± 10 mM MβCD; n = 3; ≥20 cells per experiment.

Fig 3: Induction of MIR181B expression by methotrexate (MTX) or adenosine is adenosine receptor A3 (ADORA3) dependent.(A) Knockdown for adenosine receptors A1, A2A, A2B, and A3 in HUVECs was performed to analyze MIR181B expression. three biological replicates. One-way ANOVA. (B) MIR181B expression in HUVECs transfected with Ctl-siRNA or ADORA3 siRNA after treatment with MTX (10 µM) or Ad (50 µM) or (C) treatment with TNF-a (10 ng/ml) alone or in combination MTX (10 µM) or Ad (50 µM). Three biological replicates. One-way ANOVA and Unpaired two-tailed Student t test. (D) Western blot analyses of VCAM-1, ICAM-1, and E-Selectin expression in HUVECs transfected with Ctl-siRNA or ADORA3 siRNA in the presence of TNF-a (10 ng/ml) in combination with either MTX (10 µM) or Ad (50 µM). Three biological replicates. Unpaired two-tailed Student t test. (E) in the presence of miRNA negative control (NS-m) or MIR181B mimics (181b-m) stimulated with TNF-a (10 ng/ml) or in combination with MTX (10 µM) or Ad (50 µM). Please see Figure 2—source data 1. Three biological replicates. Unpaired two-tailed Student t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. n.s. indicated non significance. All values represent mean ± SEM.Figure 2—source data 1.

Fig 4: VCAM-1 and E-selectin in rs2071559 A>G. Expression of (A) VCAM-1 and (B) E-selectin in rs2071559 A>G. Human umbilical vein endothelial cells were exposed to flow for 17 h followed by tumor necrosis factor-a stimulation for 2 h. Cells were fixed and stained for VCAM-1 or E-selectin. Data are expressed as the median with 25th and 75th percentiles. Whiskers indicate 5th and 95th percentiles (Tukey-style). VCAM-1, vascular cell adhesion molecule-1; CTCF, corrected total cell fluorescence; lam, laminar; n-u, non-uniform.

Fig 5: VCAM-1 and E-selectin in rs1870377 T>A. Expression of (A) VCAM-1 and (B) E-selectin in rs1870377 T>A. Human umbilical vein endothelial cells were exposed to flow for 17 h followed by tumor necrosis factor-α stimulation for 2 h. Cells were fixed and stained for VCAM-1 or E-selectin. Significant differences in the expression of VCAM-1 were observed between A/A and T/T genotypes under non-uniform shear stress, measured by Kruskal-Wallis one-way ANOVA on Ranks (0.023) with a post-hoc test (Bonferroni). Data are expressed as the median with 25th and 75th percentiles. Whiskers indicate 5th and 95th percentiles (Tukey-style). *P<0.05. VCAM-1, vascular cell adhesion molecule-1; CTCF, corrected total cell fluorescence; lam, laminar; n-u, non-uniform.