Fig. 3: COL12A1 is a crucial molecule in the anti-tumor effects of caffeine.

Co-culture experiments of Jurkat cells treated with caffeine (0, 0.1, 1 mM). a Flow cytometry analysis the IFN-γ and PD-1 expression and b qPCR analysis of the mRNA expression of GZMB, IFNG, and PD-1, LAG3 in Jurkat cells (Representative data from n = 3 independent experiments). Co-culture experiments of Jurkat and HCT116 cells treated with caffeine (0, 0.1, 1 mM). c Flow cytometry analysis of the IFN-γ and PD-1 expression and d qPCR analysis of the mRNA expression of GZMB, IFNG, and PD-1, LAG3 in Jurkat cells (Representative data from n = 3 independent experiments). e Volcano plot and f Enrichment analysis of differentially expressed genes between caffeine-treated and control groups in HCT116 cells. g Venn diagram identifying COL12A1 as a gene differentially expressed in both RNA-Seq, TCGA datasets and correlated with survival outcomes. h qPCR analysis of COL12A1 mRNA expression and i western blot analysis of COL12A1 expression in paired normal and tumor tissues (n = 12). The samples derive from the same experiment, but different gels for COL12A1, GAPDH were processed in parallel. The quantification provided under the blots is for the representative blot from n = 3 independent experiments. j Immunohistochemistry staining of COL12A1 and CD8 in colorectal cancer tissue microarray (TMA) samples, scale bars, 100 μm, 50 μm. Quantification of COL12A1 expression in tumors (n = 202, Min = 1, Max = 12, Median=6, Lower Whisker = 1, Upper Whisker=12, Q1 = 4, Q3 = 9, IQR = 5) compared to normal tissues (n = 65, Min = 0, Max = 12, Median = 4, Lower Whisker = 0, Upper Whisker = 12, Q1 = 2, Q3 = 6, IQR = 4). k OS plot and l Cox regression analysis the prognostic significance of COL12A1 expression and other clinical variables based on TMA dataset (n = 202). m Representative images of immunofluorescence co-staining for COL12A1 (red) and CD8 (green) with TMA samples; statistical plot of the number of CD8⁺ T cells (COL12A1-high, n = 60, Min =12, Max = 257, Median = 78.5, Lower Whisker = 12, Upper Whisker = 257, Q1 = 46, Q3 = 143.25, IQR = 97.25; COL12A1-low, n = 60, Min = 45, Max = 402, Median = 168.5, Lower Whisker = 45, Upper Whisker = 402, Q1 = 129, Q3 = 235, IQR = 106). Scale bars, 100 μm. Data error bars are mean ± SD. The statistical analysis of h is Student’s two-tailed paired t-test, k is Log-rank test, the others are Student’s two-tailed unpaired t-test. The images of co-culture elements were drawn by Figdraw. Source data is provided as a Source Data file.