Fig. 6: COL12A1 regulates Kyn production via MAPK-IDO1 axis.
From: Caffeine enhances antitumor T-cell activity by suppressing kynurenine pathway in colorectal cancer
a Schematic of tryptophan-kynurenine pathway. b Western blot and c qPCR analysis of the expression of IDO1, IDO2, and TDO2 in CRC cells with COL12A1 overexpression (Ad-COL12A1) and knockdown (sh-COL12A1) (Representative data from n = 3 independent experiments). The samples derive from the same experiment but different gels for IDO1, IDO2, another for TDO2 and another for GAPDH were processed in parallel. d Volcano plot, e Enrichment analysis and f KEGG analysis in Ad-COL12A1 MC38 cells compared to control. g Western blot analysis of ERK1/2, JNK1/2/3, p38, and IDO1 in CRC cells with different COL12A1 expression. The samples derive from the same experiment, but different gels for IDO1, p-ERK1/2, another for ERK1/2, another for p-JNK1/2/3, another for JNK1/2/3, another for p-P38, another for P38 and another for GAPDH were processed in parallel. h Representative fluorescence images and quantitative analysis of p-ERK (green) in Ad-COL12A1 HCT116 cells (Representative images from n = 5 independent experiments). Scale bars, 20 μm. i Western blot analysis the expression of IDO1 and ERK1/2 levels and j qPCR analysis the expression of IDO1 in COL12A1-OE CRC cells treated with U0126 (5 μM) (Representative data from n = 3 independent experiments). The samples derive from the same experiment but different gels for IDO1, p-ERK1/2, another for ERK1/2 and another for GAPDH were processed in parallel. k Schematic depicting the treatment of caffeine or U0126 in subcutaneous COL12A1-OE MC38 tumor-bearing C57BL/6 mice (n = 6 per group). Scale bars, 1 cm. Quantification of the tumor growth curve and tumor weight. l Flow cytometry analysis of CD8+ T cells and GZMB, PD-1, LAG3 expression (n = 4) in tumor of Fig. 6k. m ELISA analysis of kynurenine (Kyn) levels in COL12A1-OE CRC cells treated with caffeine (1 mM) or U0126 (5 μM) (Representative data from n = 3 independent experiments). n Flow cytometry analysis the GZMB, PD-1, Ki67 expression in CD8+ T cells of splenocytes co-cultured with COL12A1-OE MC38 cells treated with caffeine (1 mM) or U0126 (5 μM) (n = 4 technical replicates, Representative data from n = 3 independent experiments). Data error bars are mean ± SD. The statistical analysis of the tumor growth curve is Two-way ANOVA, the others are Student’s two-tailed unpaired t-test. Source data is provided as a Source Data file.
