Fig. 6

Inosine, but not guanosine, activates PPARγ signaling pathway in human colon epithelial cells. a, b HT29 cells were treated with inosine (0.5, 1, 2, and 4 mM) or guanosine (0.5, 1, 2, and 4 mM) for 24 h and cell viability analysis was measured by CCK-8 assay. c, d HT29 cells were treated with inosine (0.5, 1, 2, and 4 mM) or guanosine (0.5, 1, 2, and 4 mM) or 10 μM rosiglitazone (Rosi) for 24 h and PPARγ activity was measured by luciferase reporter gene assay. e, f HT29 and Caco2 cells were treated with inosine (2 and 4 mM) for 24h and the expression of genes in the PPARγ signaling pathway was examined by real-time PCR assay. g, h HT29 and Caco2 cells were treated with inosine (2 and 4 mM) for 24h and the protein level of PPARγ was examined by western blot. i HT29 and Caco2 cells were treated with inosine (2 and 4 mM) for 24h and the protein level of PPARγ (green) was examined by immunofluorescence analysis. Nuclei were stained with DAPI (blue). Scale bar = 20 μm. j, k HT29 and Caco2 cells were treated with inosine (2 and 4 mM) for 24 h and PPARγ was knocked down using siRNA. The protein levels of PPARγ and Muc2 were examined by western blot. l, m HT29 and Caco2 cells were treated with inosine (2 and 4 mM) for 24h and A_2AR was knocked down using siRNA. The protein levels of A_2AR, PPARγ, and Muc2 were examined by western blot. Scrambled (Sc) siRNA transfections were used as controls. Immunoblots were quantified using ImageJ software. Data are pooled from three independent experiments (a–f). Data are representative of two independent experiments (g-m). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical analysis was performed using Student’s t test