Fig 2: TH1L promotes colorectal cancer cell growth. (A–D) The cell growth curves of TH1L‐overexpressing DLD1 and HCT116 cells and TH1L‐knockdown HCT116 and RKO cells were determined through IncuCyte. The expression of TH1L was confirmed by western blotting. (E–L) The colony formation capacity of TH1L‐overexpressing DLD1 and HCT116 cells and TH1L‐knockdown HCT116 and RKO cells was determined by counting the colonies. Photographs of colonies are shown in (E–H). Quantitative analyses are shown in (I–L). All experiments were repeated three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (M–O) Knocking down TH1L suppresses xenograft tumour growth. Nude mice were injected on the left side with control cells and the right side with TH1L‐depletion (shTH1L) RKO cells using 1 × 107 cells. The xenograft tumours were measured over times and dissected at day 42. The final tumour volumes (M) and quantification of tumour weight (N) and tumour size (O) were shown, respectively (n = 3).

Fig 3: TH1L regulates CCL20 expression in CRC cells. (A) Volcano maps displaying fold changes in gene expression in TH1L‐overexpressing DLD1 cells compared with negative control cells. (B, C) The mRNA levels of CCL20 were analysed in the tumours compared with their paired adjacent normal tissues from the TCGA database (shown in B) and GSE44076 (shown in C). (D) CCL20 protein levels were analysed in tumour tissues compared with normal tissues from the CPTAC database. (E) Analysis of the correlation between TH1L and CCL20 expression levels in CRC tissues in GSE44076. The expression of CCL20 was determined by qRT–PCR (F–I), Western blot (J, K) and ELISA (L, M) after TH1L was overexpressed and knocked down in CRC cells. All experiments were repeated three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig 4: TH1L promotes colorectal cancer cell migration. (A–H) The capacity of cell migration was assessed by wound healing assay. Photographs of representative wound healing are shown in (A–D). Quantitative analyses of the wound healing percentage are shown in (E–H). (I–P) Cell invasion was determined by transwell assay. Photographs of representative cancer cells that migrated to the bottom side of the membrane are shown in (I–L). (M–P) display the number of invaded cells. (Q) The protein expression of E‐cadherin and N‐cadherin was assessed by Western blotting when TH1L was overexpressed. (R) Bar chart of the banding grayscale value of (Q). (S) The Vimentin protein level was assessed by Western blotting when TH1L was overexpressed. (T) Bar chart of the banding grayscale value of (S). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig 5: TH1L promotes the activation of the NF‐κB signalling pathway. (A) Results of hallmark signalling pathway enrichment analysis. The size of the dots represents the number of genes in different hallmark pathways, and the colours of the dots indicate the change in the adjusted p value. (B, C) The activity of the NF‐κB signalling pathway in HEK 293 T cells was confirmed by luciferase reporter assay after TH1L overexpression (shown in B) or knockdown (shown in C). (D) The protein expression of TAK1, IKKβ and p65 was detected in TH1L‐overexpressing DLD1 and HCT116 cells and TH1L‐knockdown RKO and HCT116 cells by western blotting. GAPDH was used as the loading control. (E, F) The mRNA levels of NF‐κB signalling pathway target genes were assessed by qRT–PCR when TH1L was overexpressed and knocked down. (G–J) Bar charts of the banding grayscale value of (D). All experiments were repeated three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.