Figure EV3. Induction of Annexin A1 mRNA expression in HT29 cells by F. nucleatum 25586 and effect of F. nucleatum 12230 on E‐cadherin mRNA expression in SB, 10C, HCT116 and DLD1 cells.

1. Statistical analysis of associations between exposure to F. nucleatum and up‐regulation of ANXA1 mRNA expression levels in colon cancer cell HT29. The ANXA1 mRNA levels in HT29 cells were analyzed in an RNA‐sequencing (RNA‐seq) dataset publicly available from the NCBI‐GEO online repository (GSE90944) and containing global gene‐expression measurements from HT29 cells, both at baseline and following incubation with F. nucleatum ATCC25586 in triplicates 14. The distribution of ANXA1 mRNA expression levels in the two sample groups (baseline versus infected) was visualized using boxplots, using the log₂ of their TPM (transcripts per million) expression values as a metric. Individual data points were represented as circles, and box‐plots were drawn to span the inter‐quartile range (from the 25^th to the 75^th percentile) with an internal band to identify the median. Differences in mean log₂ TPM values between HT29 cells at baseline (n = 3) and following incubation with F. nucleatum (n = 3) were tested for statistical significance using a two‐tailed t‐test for continuous variables. The analysis revealed that HT29 cells exposed to F. nucleatum were characterized by increased levels of ANXA1 mRNA expression, as compared to HT29 cells at baseline (P = 0.01).
2. Real‐time qPCR analysis of E‐cadherin (CDH1) mRNA levels in SB, 10C, HCT116, and DLD1 cells following incubation with F. nucleatum 12230 (Fn) and fadA‐deletion mutant US1 (US1) for indicated time periods. All results were normalized to those of the untreated cells. Data are mean values ± SEM of three independent experiments each performed in triplicates.