Fig. 5

Endothelial quiescence depth varies with laminar flow exposure time and positively correlates with p27 expression levels. A, Representative images of human umbilical vein endothelial cell (HUVEC) with indicated conditions and stained for p27 (inhibitor), p21 (inhibitor), Ki67 (proliferation marker), and DAPI (4′,6-diamidino-2-phenylindole; nucleus) in white. Scale bar, 50 µm. White arrow, flow direction. B through D, Quantification of percent p27+ ( B), p21+ cells ( C), or Ki67+ cells ( D). n=3 replicates, 5 images per condition per replicate. E, Representative images of HUVEC under indicted conditions and after 5-ethynyl-2′-deoxyuridine (EdU) incorporation (red, S phase) and stained for phospho-retinoblastoma protein (pRB; blue, interphase) and with DAPI (4′,6-diamidino-2-phenylindole; white, nuclear mask), flow establishment (Flow-E; 15 d/cm2, 16 hours); flow maintenance (Flow-M; 15 d/cm2, 72 hours). Scale bar, 50 µm. White arrow, flow direction. F, Quantification of EdU+ cells under indicated conditions. n=3 replicates, 5 images per condition per replicate. G, Quantification of pRB+ cells under indicated conditions. n=3 replicates, 5 images per condition per replicate. H, Representative images of HUVEC with indicated siRNA treatments and conditions after EdU incorporation (red, S-phase) and staining with DAPI (white, nuclear mask). Scale bar, 50 µm. White arrow, flow direction. I, Quantification of EdU+ cells with indicated conditions. n=3 replicates, 5 images per condition per replicate. Statistics, 1-way ANOVA with Tukey multiple comparisons test ( B through D, F, and G) and 2-way ANOVA with Tukey multiple comparisons test ( I). NT indicates non-targeting; and siRNA, small interfering RNA.