Extended Data Fig. 4: GABABR activation dampens GSK-3β activity to stabilize β-catenin.
From: Cancer-cell-derived GABA promotes β-catenin-mediated tumour growth and immunosuppression
a, Antagonists and agonists for GABAAR or GABABR used in this study. b, Western blot analysis of β-catenin expression in LLC-ova cells treated with bicuculline or 2-OH-saclofen at concentrations of 0, 10, 50, and 100 μM. c, d, Western blot analysis of β-catenin, p-GSK-3β (S9), and GSK-3β levels in HT29 (c) and LLC-ova (d) cells stably expressing GAD1/Gad1 shRNAs (sh1 and sh2) or NTC, followed by treatment with muscimol (50 μM) or baclofen (50 μM) for 48 hours. e, f, MC38 (e) or H520 (f) cells were subcutaneously injected into nude mice. Intratumoral injection of saline, 3-MPA or 2-OH-saclofen was initiated on day 15 (n = 5 per group, biological replicates). Tumor masses were weighed at the end of the experiment. g, Western blot analysis of p-GSK-3β (S9), GSK-3β, and β-catenin levels in HT29 cells stably expressing GAD1 shRNAs (sh1 and sh2) or NTC, followed by treatment with or without LiCl (20 mM) for 48 hours. β-actin served as loading control in western blot analysis. Western blot data shown in (b-d, g) are repeated independently at least two times with similar results. Data are presented as the mean ± SD (e, f left) and mean ± SEM (e, f right). P-values were calculated by two-way ANOVA (e, f left) and two-tailed Student’s t test (e, f right). *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. Exact P values can be found in the Source Data.
