Figure 5. Functional properties of CHIR- and DMSO-treated endothelial cells (ECs).

(A) Flow cytometry analysis of CD31 expression in Passage 1 ECs following the dextran internalization assay. CD31⁺ cells were gated for further analysis. (B) Flow cytometry analysis of 10 kDa dextran-Alexa Fluor 488 (AF488) abundance in CD31⁺ cells. Cells were treated with DMSO or CHIR for 6 days prior to the assay. Representative plots from cells incubated with dextran for 2 hr at 37°C are shown. (C) Quantification of 10 kDa dextran-AF488 geometric mean fluorescence intensity in CD31⁺ cells. Treatment and assay conditions were as described in (B). Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA. (D) Quantification of the coefficient of variation (CV) of 10 kDa dextran-AF488 fluorescence intensity in CD31⁺ cells. Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA. (E) Transendothelial electrical resistance (TEER) of Passage 3 ECs. The x-axis indicates the number of days after seeding cells on Transwell inserts. Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different shape. p-value: two-way ANOVA. (F) Permeability of Passage 3 ECs to sodium fluorescein. Points represent replicate wells from two independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA.

Figure 5—figure supplement 1. Endocytosis dependence of dextran uptake.

(A, B) Flow cytometry analysis of 10 kDa dextran-Alexa Fluor 488 (AF488) abundance in DMSO-treated CD31⁺ cells. Representative plots from cells incubated with dextran for 2 hr at 37°C (same plot shown in Figure 5B) or 4°C (A) or cells incubated with dextran and indicated inhibitors (B) are shown. (C) Quantification of 10 kDa dextran-AF488 geometric mean fluorescence intensity in CD31⁺ cells. Treatment and assay conditions were as described in (B). Points represent replicate wells from two independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-values: two-way ANOVA followed by Dunnett’s test. CPZ: chlorpromazine. (D) Confocal immunocytochemistry analysis of caveolin-1 expression and 10 kDa dextran internalization in Passage 1 DMSO-treated endothelial cells (ECs). Hoechst nuclear counterstain is overlaid. Single confocal Z-slices from two representative fields are shown. XZ and YZ projections derived from serial Z-slices with 0.25 μm spacing are shown at right and below. Arrowheads indicate dextran⁺ caveolin-1⁺ puncta. Scale bars: 10 μm.