Fig 2: Effects of tunicamycin and LPS on lung endothelial permeability. (A) BPAECs were grown on gold-plated ECIS arrays to form a confluent monolayer. Those cells were exposed to either vehicle (VEH) (0.1% DMSO) or tunicamycin (TM) (0.5 μM). A gradual decrease in BPAEC permeability (increased TEER) was observed in the TM-treated cells. n = 3 per group; means ± SEM. (B) BPAECs were treated with either vehicle (0.1% DMSO) or TM (0.5 μM) for 24 h prior to treatment with either vehicle (PBS) or LPS (10 μg/mL). LPS exposure decreased TEER values (increased permeability), while TM pretreatment prevented LPS-triggered barrier dysfunction as reflected in the higher TEER values. * p < 0.05 vs. vehicle (VEH) and $ p < 0.05 vs. LPS. n = 3 per group; means ± SEM. (C) BPAECs were seeded onto transwell inserts of a 24-well culture plate. After 24 h, the cells were treated with either vehicle (VEH) (0.1% DMSO) or TM (1 µM) for 48 h. LPS (1 µg/mL) was added in the media for 1 h; followed by the addition of 70 kDa FITC-dextran (1 mg/mL). 20 min after FITC-dextran addition, 100 mL of basal media was removed and the fluorescence intensity was measured. * p < 0.05 vs. vehicle (VEH) and $ p < 0.05 vs. LPS. Means ± SEM, n = 3. (D) Western blot analysis of phosphorylated VE-cadherin (pVE-cad) and VE-cadherin (VE-cad) in BPAEC treated with either vehicle (0.1% DMSO) or TM (1 μΜ) prior to a 1 h exposure to either vehicle (PBS) or LPS (1 μg/mL). The blots shown are representative of three independent experiments. The signal intensity of the protein bands was analyzed by densitometry. Protein levels of pVE-cad were normalized to total VE-cad. * p < 0.05 vs. vehicle (VEH) and $ p < 0.05 vs. LPS. Means ± SEM.

Fig 3: NOTCH1 is polarized downstream of flow. a Endothelial cells (HAECs) subjected to high laminar flow (20 dynes cm-2) display significant cellular elongation, revealed by VECAD (white) and distribution of NOTCH1 intracellular domain (ICD, red) and extracellular domain (ECD, green) at the downstream pole downstream pole, as marked by solid white arrows. Dashed line marks a cell nucleus confirming the nuclear presence of NOTCH1 ICD without ECD. Scale bar = 10 µm. b Downstream pole of an endothelial cell subjected to high laminar flow showing NOTCH1 ECD (green) and ICD (red). Co-localization of ECD and ICD is indicated by the solid arrowhead and intracellular activated NOTCH1 is marked by the open arrowhead; scale bar = 3 µm. c Upon flow-direction reversal, NOTCH1 ICD and ECD protein redistributes to the new downstream pole; scale bar = 10 µm. Kinetics of the protein redistribution was measured and the typical time for repolarization (dashed lines) was ~30 min (n = 116 individual cells for each time point measured). d Arterial denudation injury of wildtype (C57BL/6) adult mouse to generate injury region (marked by dotted white line) with two migrating endothelial fronts. Note the presence of Notch1 (marked by white arrows) at the downstream pole of the cell regardless of migration direction (at least 40 cells at the wound margins were evaluated in three distinct wounded animals). Scale bars = 20 µm

Fig 4: Biphasic modulation of insulin signaling promoted highly efficient HSPC differentiation. a Schematic stepwise induction of hematopoietic progenitors from hPSCs. b Representative images of stages I–III of hematopoietic differentiation. “Grape-like” clusters at differentiation day 6 indicated by arrows. Scale bars, 50 μm. c Time-lapse images of day 6 differentiating cells. Cells undergoing endothelial-to-hematopoietic transition indicated by arrowheads. Scale bars, 50 μm. d Representative immunostaining images of day 8 (i–iii) and day 13 (iv) cells for CD34 and VE-cadherin (both green), and CD43, CD31 and CD45 (all red). (i–iii) Grape-like clusters coexpressed CD43, CD34, VE-CAD and CD31(white arrow). (iv) CD45+CD43low and CD45lowCD43+ indicated by arrows and arrowheads respectively. Scale bars, 50 μm. e Strategy for biphasic insulin treatment. Cells differentiated in B27 without insulin (B) or B27 with insulin (BI). Insulin was added at different stage as indicated. f Representative flow cytometry results of surface markers CD31, CD43 and CD34 at day 5, day 6 and day 8. N > 3 independent biological replicates. g Bar graph of percentage of CD31+CD34+, CD31+CD34− and CD31−CD34+ cells in B + B and B + BI groups on day 8. N > 3 independent biological replicates. h Bar graph of percentage of CD43+CD34+, CD43+CD34− and CD43−CD34+ cells in B + B and B + BI group on day 8. N > 3 independent biological replicates. HE hemogenic endothelium, hPSC human pluripotent stem cell, HSPC hematopoietic stem/progenitor cell, ME mesoderm

Fig 5: Transcriptome of siNOTCH1 indicates changes in cell cycle, inflammatory genes, and regulators of calcium. a Cultures of HAECs treated with siNOTCH1 and siControl were analyzed for transcriptional changes by microarray. Heatmap to display differentially expressed genes (=0.5 (log2) upregulated or = -0.5 (log2) downregulated with P < 0.001) marking NOTCH1 target genes and upregulated cell cycle, inflammatory genes and regulators of intracellular calcium. (b–d) Volcano plot visualization of the differentially expressed genes between siNOTCH1 and siControl. Colors indicate as follows: gray—no differential expression, black—genes whose fold change is either =0.50 or =1.5, red—genes known to contribute to the regulation of cell cycle, orange—genes involved in inflammation, and green—genes known to be regulators of intracellular calcium. (e, f) Confocal images of HAECs exposed to flow for 48 h in the presence of ?-secretase inhibitor DAPT or vehicle and stained for F-actin, VECAD, and NFAT2. Note the presence of nuclear NFAT2 and gaps between cells only in DAPT-treated monolayers (marked by white arrows). Scale bars = 20 µm