Extended Data Fig. 3: Tumor cell-derived GABA induces β-catenin signaling to support proliferation.
From: Cancer-cell-derived GABA promotes β-catenin-mediated tumour growth and immunosuppression
a, Growth curves of H1650, HCC827, HCT116, SW480 and LLC-ova cells stably expressing GAD1/Gad1 shRNAs (sh1 and sh2) or NTC (n = 3 per group). b, Cell numbers were examined in LLC-ova (left) and MC38 (right) cells stably expressing Gad1 shRNAs or NTC in the absence or presence of 10 μM or 100 μM GABA for 3 days (n = 3 per group). c, HT29 cells stably expressing GAD1 shRNAs or NTC were subcutaneously injected into nude mice (n = 5 per group). Intratumoral injection of saline or saline containing GABA was initiated on day 13. d, Cellular ATP levels in H520, HT29, LLC-ova and MC38 cells stably expressing GAD1/Gad1 shRNAs (sh1 and sh2) or NTC were determined by ATP assay kit (n = 3 per group). e, Western blot analysis of β-catenin expression in NHBE cells obtained from three independent donors and the indicated NSCLC cell lines. f, Western blot analysis of β-catenin expression in the indicated normal human colon cell lines and COAD cell lines. g, Quantitative real-time PCR analysis of CTNNB1/Ctnnb1 expression in the indicated cancer cell lines stably expressing GAD1/Gad1 shRNAs (sh1 and sh2) or NTC (n = 3 per group). h, Western blot analysis of β-catenin and cyclin D1 expression in LLC-ova, LG1233, and MC38 cells stably expressing Gad1 shRNAs (sh1 and sh2) or NTC. i, Quantitative real-time PCR analysis of CCND1, VEGFA, CMYC, and LEF1 expression in H1650 cells stably expressing GAD1 shRNAs (sh1 and sh2) or NTC (n = 3 per group). j, Quantitative real-time PCR analysis of Atf3, Vegf, Lef1, and Ccnd1 expression in LLC-ova, LG1233, and MC38 cells stably expressing Gad1 shRNAs (sh1 and sh2) or NTC (n = 3 per group). k, Western blot analysis of GAD1, β-catenin, and cyclin D1 expression in LLC-ova (left) and MC38 (right) cells stably expressing Gad1 shRNAs (sh1 and sh2) or NTC, with subsequent GABA (50 μM) treatment for 48 hours. l, LLC-ova and MC38 cells stably expressing Gad1 shRNAs or NTC were further infected with virus expressing mutant β-catenin (∆GSK) or empty vector (n = 3 per group). Cell numbers were examined on day 3 after cells were seeded. β-actin served as loading control in western blot analysis. Western blot data shown in (e, f, h, k) are repeated independently at least two times with similar results. n indicates the number of biological (a-d, l) or technical (g, i, j) replicates. Data are presented as the mean ± SD. P-values were calculated by two-tailed Student’s t test (a, b, d, g, i, j, l) and two-way ANOVA (c). *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. Exact P values can be found in the Source Data.
