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Fig. 6 PF-502 mediates Notch S3 cleavage and activates the Notch signaling pathway in endothelial cells (A) KEGG analysis of the upregulated genes (738) in HUVECs after being treated with PF-502 (0.15 ?M for 12 h) highlights the Notch signaling pathway (p < 0.05) (red arrow). (B) Heatmap depicting differentially expressed genes in HUVECs treated with or without PF-502 (0.15 ?M for 12 h). (C) RT-PCR analysis of the notch-targeted transcripts DLL4, Notch1, Notch4, Hes1 and Hey2 in HUVECs with or without PF-502 (0.15 ?M) treatment for 12 h. Data are expressed as means +SEM. ???p < 0.001, ??p < 0.01, ?p < 0.05. Student?s t test. (D) The left panel shows the western blotting (WB) results of (transmembrane Notch (NTM and Notch intracellular domain (NICD) in HUVECs treated with or without PF-502 (0.15 ?M) for 24 h. The right panel presents a graph indicating the relative expression levels of NTM and NICD in HUVECs after PF-502 (0.15 ?M) treatment for 24 h (n = 3). (E) Maximum intensity projection of the tumor vasculature of the xenografts on 3 dpi on kdrl:EGFP transgenic zebrafish that were treated with ?-secretase inhibitor LY411575 (GSI) (10 ?M for 72 h), PF-502(1 ?M for 72 h) and combined. The areas in the dotted boxes are magnified below showing vasculature signaling in the gray channel. Scale bars: 50 ?m. (F) Percentages of lumenized vessels in xenografts with or without GSI and PF-502 treatment (1 ?M for 72 h) (n = 5?7). Data are expressed as means +SEM. ????p < 0.0001. Student?s t test. (G) WB of NICD in HUVECs with or without drug treatment. LY411575(10 ?M for 72 h), PF-502(0.15 ?M for 72 h) and combined. (H) Maximum intensity projection of the tumor vasculature of the xenografts on 3 dpi on kdrl:mCherry transgenic zebrafish with indicated notch mutations. Zebrafish were all treated with PF-502 (1 ?M for 72 h). Scale bars: 20 ?m. (I) Graph showing the percentage of lumenized blood vessels in xenografts at 3 dpi on kdrl:mCherry transgenic zebrafish with the indicated notch mutations treated with PF-502 (1 ?M for 72 h). Data are expressed as means +SEM. ????p < 0.0001, ?p < 0.05. Student?s t test.