Extended Data Fig. 9. Use of anti-NOTCH1 antibody to inhibit NOTCH1 signaling in vivo.
a. Protocol for b-e. C57Bl/6 wild type mice were treated with anti-NRR1.1E3 or control antibody (CTRL) for three days before tissue collection. b, c. Immune Capillary Electrophoresis was performed on peeled esophageal epithelia of mice in a. Visual representation of cleaved transmembrane and intracellular regions of NOTCH1 (NTM1 + NICD1, top panel, Extended data Fig. 1a) and of NOTCH2 (NTM2 + NICD2, bottom panel), and α-Tubulin proteins. Dashed lines indicate image cropping (b). Proteins expression relative α-Tubulin. Mean ± SEM, each dot represents a mouse, n = 3 mice. Two-tailed unpaired Student’s t-test (c). d. Representative images of staining for NICD1 (magenta), KRT14 (green) and nuclei (blue) in sectioned epithelium of 3 mice treated with control or anti-NRR1.1E3 antibodies. Scale bars, 25 µm. e. RT-qPCR for markers of Notch1 loss of function identified by bulk RNA-seq analysis (Extended data Fig. 6d, Supplementary Table 12) relative to Gapdh transcript in control and anti-NRR1.1E3 treated samples. Mean ± SEM, each dot represents a mouse, n = 3. Two-tailed unpaired Student’s t-test. f. Protocol. YFPCreNotch1flox/flox mice were induced at clonal density. One week later, mice were treated with anti-NRR1.1E3 or control antibodies for 3 weeks. g. Principle of assay shown in f, If anti-NRR1.1E3 treatment blocks NOTCH1 signaling, all cells have equal fitness and expansion of Notch1−/− clones is halted21. h. Representative images of NOTCH1 negative clones in EDTA peeled esophageal epithelia treated with control or anti-NRR1.1E3 antibody from 3 mice. Scale bars, 50 µm. i. Projected area of clones negative for NOTCH1 staining. Mean ± SEM, each dot represents a clone. Number of mice analyzed is in brackets. One-way ANOVA; Tukey’s multiple comparisons test, adjusted p-values versus control antibody. AU, arbitrary unit. SEM, standard error of mean. LO, loading. See, Supplementary table 25.