Fig. 2
From: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma
Notch1 promotes ESCC tumorigenesis. a Schematic of 8×CSL-GFP reporter. Notch activation permits GFP reporter expression via concatemeric CSL-binding sites. Constitutively active UBC promoter drives tRFP expression concurrently. Cells without tRFP represent host-derived stromal cells. b Representative flow cytometry scatter plots determining 8×CSL-mediated GFP reporter activation, indicating Notch active population of tRFP-labeled ESCC cells in TE11 and EN60 xenograft tumors. On average, 43.5 ± 0.5% s.d. of EN60 and 11.2 ± 6.5% s.d. of TE11 cells comprised the live GFPPos/tRFPPos fraction across three independent tumors. c, d Tumor growth curves in immunodeficient mice carrying TE11 tumors of indicated genotypes. Upon xenograft transplantation, mice were treated with DOX to induce ICN1 or NOTCH1-directed shRNA. Tumor growth was monitored for indicated time periods. In c, *P < 0.01 vs. zeo and DOX (−), # P < 0.05 vs. zeo and DOX (+), n = 6–8 per group. In d, *P < 0.05 vs. DOX (−); n = 6 per group. e TE11 cells with CRISPR/Cas9-mediated NOTCH1 deletion. Immunoblotting confirms ICN1 loss in NOTCH1-deleted TE11 (N1Δ), but not non-targeted control TE11 (N1WT) cells. Bar graph shows tumor formation rate in immunodeficient mice 7 weeks after xenograft transplantation. *P < 0.05 vs. N1WT, n = 8 per group. f Esophageal keratinocytes isolated from 4NQO-induced IEN (dysplasia) lesions or ESCC tumors of Notch1 loxP/loxP mice were utilized to generate 3D esophageal organoids in the presence or absence of ex vivo Cre-mediated recombination. qRT-PCR analysis confirms inhibition of Notch1 gene expression upon ex vivo Cre-mediated recombination in ESCC organoids. *P < 0.000 vs. Cre (−), n = 3. Organoid formation rate was evaluated at day 14 post-plating and is represented as relative to Cre (−) for IEN and ESCC. *P < 0.000 vs. Cre (−) IEN; # P < 0.001 vs. Cre (−) ESCC. Line graphs represent mean ± s.e.m. in c, d. Bar diagrams represent mean ± s.d. in f. At least two independent replicates were performed for all experiments. Repeated measures ANOVA with Tukey’s post hoc test were used for multiple comparisons in c, d. Chi square with Fisher’s exact test was used for percentage comparisons in e. Student’s t-test was used for paired data comparisons in f
