Fig. 4: n-BANKs augmented eNOS activity in damaged endothelial cells to facilitate pericyte recruitment.

A Schematic illustration of the promoting effect of n-BANKs on eNOS activation in SVEC4–10 cells. B Total RNA isolated from SVEC4–10 cells after exposure to GTN or n-BANKs at 4 h after treatment was examined by real-time qPCR and the mRNA levels of eNOS were normalized to the control GAPDH mRNA levels (n = 3; Model versus GTN, **P = 0.0015; Model versus n-BANK, ****P < 0.0001; GTN versus n-BANK, ***P = 0.0006). SVEC4–10 cells were exposed to 500 μM CoCl₂ for 12 h prior to treatment. C Western blot of eNOS (phosphorylated and total) in SVEC4–10 cells (n = 3). D, E Flow cytometry analysis of NO levels in SVEC4–10 cells (n = 3; 0.17 h, **P = 0.0037; 1 h, ***P = 0.0003; 2 h, ****P < 0.0001; 4 h, ****P < 0.0001; 6 h, ****P < 0.0001). SVEC4–10 cells were exposed to 500 μM CoCl₂ for 12 h prior to treatment. F Release of NO in cell supernatant of SVEC4–10 cells was quantified using Griess reagent (n = 6; **P = 0.0022). G Schematic illustration of real-time cell migration assays. Damaged SVEC4–10 cells were placed in the lower chambers, and the primary pericytes were loaded in the upper chambers. H Migration kinetics of pericytes by real-time cell migration assays (n = 3; ****P < 0.0001). I Crystal violet assay and fluorescence imaging revealed pericyte recruitment ability by n-BANKs (n = 3). The non-migrating cells in the upper chambers were stained by crystal violet, and the migrating cells in the lower chambers were stained with Calcein-AM. Data are mean ± SD, * is P < 0.05, ** is P < 0.01, *** is P < 0.001, **** is P < 0.0001 by one-way or two-way ANOVA test.