Fig 2: TRIM24 directly activates c-Met to support metaplastic carcinosarcoma cell viability.a qRT-PCR validation of cMET RNA expression in TRIM24 overexpressing tumors compared to MMTV-CreTg/0 mammary gland (03) (n = 2 technical replicates for each tumor samples). b Snapshot of Integrated Genome Viewer (IGV) showing tracks and peaks of ATAC-Seq-determined chromatin accessibility of MMTV-Cre spontaneous tumor cell line 823 (blue) and TRIM24 overexpressing primary metaplastic carcinosarcoma cell line 897 (red). The scales of the tracks are adjusted to 168. The color bar below the tracks shows positions of primers used to assess TRIM24 interactions with cMET chromatin. c Chromatin immunoprecipitation (ChIP) PCR showing enrichment of TRIM24 on Met promoter to validate ATAC-Seq on 823 and 897 cell lines. The color of each bars indicates primers corresponding to arrows shown in B (n = 3 biological replicates). (*p = 0.0209, **p = 0.0014, ***p = 0.0004). d Cell viability assay on 4T1 (mouse TNBC cell line- non-metaplastic) and TRIM24 overexpressing primary cell lines (567, 64, and 897) upon treatment with Crizotinib, an FDA-approved ATP-competitive selective small molecule inhibitor of c-Met and ALK receptor. Percent cell viability is measured using a luminescence reader; values are normalized to DMSO, a vehicle control (n = 3 biological replicates). Data represented as mean with SD in a, and mean ± SEM in c, d. p-values are calculated in c based on two-way ANOVA for multiple comparisons using Tukey method and in d using two-way ANOVA for multiple comparisons using Holm–Sidak method. (***p < 0.001, ****p < 0.0001).

Fig 3: R193/K195 sites are necessary for TRIM24 to bind with and recruit STAT3 to chromatin. a Schematics of TRIM24WT and various TRIM24 deletion constructs. b, c STAT3 interacts with TRIM24 through its BBOX1 domain with amino acid residues 131–205. TRIM24WT or the indicated TRIM24 mutants were transfected into U87 GBM cells. d Re-expression of shRNA-resistant Flag-tagged TRIM24WT*, but not TRIM24 R193A/K195A* mutant or an empty vector control (EV), resulted in binding between TRIM24 and STAT3 in LN229/vIII/shT24 and U87/vIII/shT24 GBM cells. e Effects of re-expression of shRNA-resistant Flag-tagged TRIM24WT*, TRIM24 R193A/K195A* mutant, or an empty vector control (EV), on STAT3 phosphorylation and ID1 protein expression. f qRT-PCR analysis of effects of re-expression of shRNA-resistant TRIM24WT*, TRIM24 R193A/K195A* mutant or an empty vector control (EV) on ID1 mRNA expression. g–i Effects of re-expression of shRNA-resistant TRIM24WT*, TRIM24 R193A/K195A* mutant or an empty vector control (EV) on cell proliferation g, colony formation in soft agar h, and cell migration i. j ChIP-qRT-PCR analyses of effects of re-expression of shRNA-resistant TRIM24WT*, TRIM24 R193A/K195A* mutant or an empty vector control (EV) on the binding of TRIM24, STAT3 and H3K23ac with the ID1 promoter. Error bars, s.d. Data represent two or three independent experiments with similar results. ***P < 0.001, paired two-way Student’s t-test, compared with EGFRvIII cells treated with shC/EV

Fig 4: Expression levels of both TRIM24 protein and mRNA were significantly lower in ESCC tissues than in the matched adjacent non-cancerous tissues (NCET) of the same patients(A) Comparison of the relative expression levels of TRIM24 mRNA in 42 pairs of ESCC and NCET samples, which were examined by RT-PCR (mean ± SD, Paired t test, P< 0.001). (B) Comparison of the expression levels of TRIM24 protein in 19 pairs of ESCCs and the matched NCETs, which were detected by Western blot. (C) Protein levels of TRIM24 in eight representative ESCC tissues (T) and paired adjacent NCET (N) were analyzed by Western blotting.

Fig 5: TRIM24 and RARa protein expression in ESCCs and adjacent NCETs(A) Representative immunohistochemistry images for TRIM24 (upper panel) and RARa (lower panel) protein expression were presented. Upper panel: The first image represents the high expression of TRIM24 in an adjacent NCET, and then weak, moderate and strong expressions of TRIM24 protein in ESCC tissues. Lower panel: The first image represents the very weak expression of RARa in adjacent non-cancerous tissues, and then weak, moderate and strong expressions of RARa in ESCC tissues. All the images were shot at 200x magnification. (B) and (C) TRIM24 and RARa protein expression levels were compared between ESCC and adjacent NCET specimens. Statistical analyses were performed by Paired-Samples t-test.