Fig. 1: TRIM24 is highly expressed in CRC tissues and plays a critical role in the proliferation of CRC cells.

A The mRNA levels of TRIM24 in normal colon tissues (n = 51) and CRC tissues (n = 622) from TCGA-COAD. Two-tailed Student’s t-test was used. The mRNA analysis of TRIM24 was carried out by BEST. B The Kaplan–Meier survival curves based on the expression of TRIM24 showed CRC patients with high expression of TRIM24 had a shorter disease-free survival. The Kaplan–Meier survival analysis was performed by BEST and the cut-off value was set to “median”. “Number at risk” means the number of patients exposed to outcome risk at each point on the curve. C The protein abundance of TRIM24 in normal colon tissues (n = 93) and CRC tissues (n = 95) derived from CPTAC database. Two-tailed Student’s t-test was used. (https://proteomics.cancer.gov/programs/cptac). Boxplots in (A) and (C) showing 0th, 25th, 50th,75th, and 100th centiles. Outliers were defined as values larger than 1.5 × lQR (Interquartile Range) + 75th centile. D Left: Representative immunoblots of TRIM24 in colonic epithelial cells isolated from normal (N) or tumor (T) tissues derived from patients with colorectal adenocarcinoma (n = 12), with Actin as a loading control. Right: The comparison between the expression levels of TRIM24 in tumor tissues and paired adjacent normal tissue from CRC patients in our facility, with relative TRIM24 protein levels normalized to Actin. Data were expressed as two-tailed Student’s t-test. E Normal human colonic epithelial cell line FHC and human CRC cell lines were harvested and immunoblotted for TRIM24. Shown are the representative results from three independent experiments. F After 24 h transfection with siNC or two small interfering RNAs targeting distinct sequences of TRIM24 (siTRIM24-1/2), DLD1 or SW620 cells from each group were evenly spread in dishes. After 1–2 weeks, the colonies were stained and photographed for colony formation assays. Experiments were conducted independently three times, consistently producing similar results. G Left: After 72 h transfection with siNC or siTRIM24-1/2, DLD1 cells were collected and incubated with PI. The cell cycle analysis was performed by flow cytometry. Right: Graph representing percentage cells in each phase of cell cycle. Experiments were conducted independently three times, consistently producing similar results. H Left: After 72 h transfection with siNC or siTRIM24-1/2, SW620 cells were collected and incubated with FITC-CD44. The proportion of CD44-positive cells was analyzed by flow cytometry. Right: Quantification of CD44 positive cells in the indicated groups. n  = 3 biologically independent samples per group. Data were expressed as means ± SEM, two-tailed Student’s t-test. I In subcutaneous model, DLD1-WT (wild-type DLD1) cells were subcutaneously inoculated into the left flank area of five-week-old male BALB/c nude mice with the same amount of DLD1-KO-1 (TRIM24 knockout DLD1 clone-1) cells into the right flank (n = 11 mice per group). Tumor size was measured every 3 days from 8 days post-inoculation until day 25. Data were expressed as means ± SEM, two-tailed Student’s t-test. J Representative pictures of tumors dissected from mice in (I) on day 25 post-inoculation. K Left: Tumor tissues collected from mice in (I) were subjected to PCNA staining, with nuclei counterstained by hematoxylin. Scale bar, 50 µm. Right: PCNA staining intensity was quantified in randomly selected fields (WT group n = 6; KO-1 group n = 5) using ImageJ with the H-DAB color deconvolution vector, and relative intensity was calculated after normalization to the WT group. Data were expressed as means ± SEM, two-tailed Student’s t-test. L Left: The representative images of CRC patient-derived organoids at 7 days post transfect with siNC or siTRIM24-1. Scale bar, 100 µm. Right: Quantification of the size of organoids in each indicated group. n  = 10 biologically independent samples per group. Data were expressed as means ± SEM, two-tailed Student’s t-test.