Supplementary Figure S3 from Inhibition of Notch4 Using Novel Neutralizing Antibodies Reduces Tumor Growth in Murine Cancer Models by Targeting the Tumor Endothelium

A, Schematic of experimental design of Py8119 orthotopically implanted tumors in RiboTagEC mice. Female RiboTagEC mice with 75mg/kg of tamoxifen (20mg/mL) for 5 days by IP injection. Py8119 tumors were implanted at 8 weeks of age and then treated once tumors reached 50mm3 with twice weekly IV control IgG or 6-3-A6 for a total of 10 days (n=3). B, Growth curve of RiboTagEC treated with 25mg/kg of IV control IgG or 6-3-A6 twice weekly for 10 days (n=3). C, Heatmap of endothelial expressed genes segregated by total mRNA input and ribosomal IP mRNA from control IgG treated and 6-3-A6 treated Py8119 tumors. D, PCA plots of the ribosomal IP fraction from control IgG and 6-3-A6 treated tumors. Two samples from the control IgG and 6-3-A6 groups were identified as outliers and removed from analysis. E, Heatmap of downstream Notch regulated genes from endothelial IP of the control IgG or 6-3-A6 treated tumors and quantitative rt-PCR of the canonical Notch target gene Hes1 and Rnd1 amplified from 6-3-A6 treated and control RiboTag IP fractions (n=4). F, Heatmaps of genes associated with significantly enriched or downregulated signaling pathways involved in inflammation and extracellular matrix interactions. All heatmap data presented as Z-scores. Statistics by t-test for qPCR studies