Fig. 2

Intracellular nitric oxide detection by LP; Effects of eNOS phosphorylation and angiogenesis. A Cell proliferation was measured via BrdU assay. LP increased HUVEC proliferation. A statistically significant increase in the proliferation was observed with treatment time. **P < 0.01; NS = not significant. Intracellular NO analyzed by (B) flow cytometry, and (C) fluorescence images of control and LP-treated cells captured using DAF-FM probes Bar graph presents mean ± standard deviation of three independent experiments. ***P < 0.001. D Effects of simulated LP on tube formation in HUVEC. Representative images captured after incubation for 6 h. After cultured cells that had been incubated under control conditions or treated with LP were seeded onto Matrigel, the cells were stained with Calcein-AM and inspected under fluorescence light; scale bar = 1000 μm. Quantification of tube formation. The ImageJ plugin software was used to determine the total length of the tube-like structures in images. The bar graph shows the pixelated tube formation and the mean value. N = 5, ***P < 0.001. E, F The expression levels of VEGFA were measured by real-time PCR (n = 5, values are presented as mean ± SD). G, H eNOS signaling is involved in the LP-induced angiogenic pathway. Immunoblotting was performed to examine phosphorylated eNOS (Ser1177). LP induced the phosphorylation of eNOS (Ser1177) in a dose and time-dependent manner. Bar graph (I) showing the results of immunoblot analysis performed with anti-p-AKT, AKT, p-ERK, and ERK antibodies. We immediately cropped the target blots according to reference markers, and an α-tubulin antibody was used for normalization