Fig 2: Hypermethylation of TRIM29 promoter leads to its deficiency in ESCA.A Heatmap of TRIM29 mRNA levels and ß-value (methylation level) of 12 CpG sites in TRIM29 promoter. Samples were ordered from top to bottom by the TRIM29 mRNA levels. Each row indicates one sample. Blue: low level; Red: high level. B, C Scatterplot showing the positive correlation between TRIM29 mRNA expression and average ß-value of CpG sites in TRIM29 promoter in TCGA ESCA and CCLE esophageal cancer cells. D–G Comparison of the average methylation level of TRIM29 promoter among different types of specimens in GSE26784, GSE52826, GSE81334, and GSE104707 datasets. H UCSC genome browser shows the location of TRIM29 gene and its upstream region. Twenty CpG sites within the CpG island of TRIM29 promotor region analyzed by BSP (bisulfite sequencing PCR) are indicated. I The methylation status of CpG sites in H were measured by BSP in HEEC, KYSE30, KYSE410, and KYSE450 cells. The methylated CpG sites were represented with filled circles, and the un-methylated CpG sites were represented with hollow circles. J, K TRIM29 protein and mRNA levels in HEEC, KYSE30, KYSE410, and KYSE450 were analyzed by WB and qRT–PCR. L Effect of 5-aza treatment on the TRIM29 levels in KYSE30.

Fig 3: TRIM29 deficiency regulates ZNF750 expression and ESCC progression through activation of the IL6-STAT3 signaling pathway.A, B GSEA analysis shows a negative correlation between TRIM29 mRNA levels and “HALLMARK IL6 JAK STAT3 SIGNALING” signature in TCGA ESCC and GSE21293. C, D Scatterplot of mRNA expression correlation between TRIM29 and IL6 in TCGA ESCC and GSE21293. E Western blot analysis of STAT3 and p-STAT3(Tyr705) protein levels after TRIM29 overexpression in KYSE30, and TRIM29 knockdown in KYSE410/KYSE450 cells. F Western blot analysis of protein levels in STAT3, p-STAT3(Tyr705), ZNF750, KLF4, KRT5, Snail, E-cadherin, N-cadherin, Vimentin, MMP2, and MMP9 in TRIM29-knockdown KYSE410/KYSE450 cells treated with increasing concentrations of STAT3 inhibitor cryptotanshinone (CTN) for 24 h. G, H The effect of cryptotanshinone on cell migration and invasion of TRIM29-knockdown KYSE410/KYSE450 cells was evaluated by wound-healing assay and matrigel-transwell assay, respectively. I Schematic representation of the study.

Fig 4: TRIM29 inhibits proliferation of ESCC cells.A, B GSEA analysis shows a negative correlation between TRIM29 mRNA levels and “HALLMARK MITOTIC SPINDLE” signature in TCGA ESCC and GSE21293. C, D GSEA analysis shows a negative correlation between TRIM29 mRNA levels and “HALLMARK G2M CHECKPOINT” signature in TCGA ESCC and GSE21293. E, F Scatterplot of mRNA expression correlation between TRIM29 and PCNA/MKI67/CDKN1A in TCGA ESCC and GSE21293. G Scatterplot of protein expression correlation between TRIM29 and PCNA/MKI67/CDKN1A in Liu2021 protein dataset. H Western blot analysis of p21 and Cyclin D protein levels after TRIM29 overexpression in KYSE30, and TRIM29 knockdown in KYSE410/KYSE450 cells. I–K The effect of TRIM29 expression on cell proliferation of KYSE30, KYSE410, and KYSE450 was evaluated by CCK8 assay. L–N Flow cytometric analysis of the effect of TRIM29 expression on cell cycle distribution in KYSE30, KYSE410, and KYSE450 cells. O–Q Representative images of colony formation assays in KYSE30 vs. TRIM29-OE KYSE30 cells (O), KYSE410 vs. TRIM29-KD KYSE410 cells (P), and KYSE450 vs. TRIM29-KD KYSE450 cells (Q), respectively. Con control, OE overexpression, KD knockdown.

Fig 5: TRIM29 is lowly expressed in ESCA and its precursor lesions.A Expression profiles of TRIM29 across TCGA cancer types were analyzed using the GEPIA (red dots: tumor specimens; green dots: normal specimens; red characters: expression increased in tumors, green characters: expression decreased in tumors). B Expression profiles of TRIM29 across multiple cancer types were analyzed in Oncomine (number: the number of datasets; red: expression increased in tumors; blue: expression decreased in tumors). C–T Comparison of TRIM29 mRNA levels among adjacent non-cancerous tissues, BE, ESCA, ESCC, and EAC in 18 datasets from GEO and Arrayexpress. U Comparison of TRIM29 protein levels between adjacent non-cancerous tissues and ESCC samples in “Liu2021” datasets. V The protein levels of TRIM29 in paired paracancerous and ESCC tissues were detected by western blot. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.