Fig 2: MCT1 K473 tri-methylation is positively related to CRC and has prognostic significance in CRC patients. a) MCT1 K473 tri-methylation IHC staining score was detected in tumor and adjacent tissues, n = 79. Student's two-tailed t-test, p < 0.001. b) Representative image of IHC staining for SETDB1, MCT1 K473 tri-methylation, and CD206 in colorectal cancer. Black scale bar, 50 µm . c) Scatter plot of the IHC staining scores for SETDB1, MCT1 K473 tri-methylation, and CD206 in CRC, n = 80. All p and r values were calculated with Spearman's r test. c) Quantitative IHC staining score showing the correlation of SETDB1 and MCT1 K473 tri-methylation. Chi-square test, p < 0.0001. d) Correlation between SETDB1 and MCT1 K473 tri-methylation expression was determined by Pearson correlation coefficient test, p < 0.0001. e) Kaplan–Meier analysis of overall survival in a set of 80 colorectal cancer patients according to MCT1 K473 tri-methylation expression. Log-rank test, p = 0.0310. f,g) Quantitative IHC staining score showing the correlation between MCT1 K473 tri-methylation and TNM stage or N stage using microarray of colorectal cancer specimen. Chi-square test, p = 0.0155 and p = 0.0352, respectively. h) The working model of SETDB1-mediated MCT1 K473 tri-methylation.

Fig 3: MCT1 K473 tri-methylation is required for SETDB1-mediated tumor glycolysis and M2-like polarization of TAMs. a) Lactate production was measured in the conditioned medium (CM) of SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag-SETDB1, n = 3. b) Glucose uptake was measured in SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag-SETDB1, n = 3. c,d) Bioenergetic analysis was performed with the Seahorse XF24 analyzer platform. ECAR of SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag-SETDB1 was measured and calculated, n = 3. e) WCL collected from SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag-SETDB1, followed by IB assay. f) The relative mRNA levels of VEGF and ARG1 macrophage markers in BMDMs were treated with indicated CM, n = 3. g) A syngeneic tumor model was performed by injecting MCT1-3'UTR CT26 cells stably expressing MCT1 WT or MCT1 K473R cells and overexpression Vector or SETDB1 into BALB/c mice. n = 5 mice. h,i) Quantification of tumor weight and volume of tumors generated in (g). j,k) Flow cytometry analysis of macrophage polarization of tumors generated in (g). All immunoblots were performed three times, independently, with similar results. Data are represented as mean ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns means no significant, by a,b,h,k) one-way ANOVA with Tukey's test and d,f,i) two-way ANOVA with Tukey's test.

Fig 4: MCT1 K473 tri-methylation blocks Tollip-mediated autophagic degradation of MCT1. a) The degradation of MCT1 was detected in SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R cells treated with CHX (100 µg mL-1) for 0, 4, 8, or 12 h by CHX-chase assay. b) Quantification of relative protein level of MCT1 in (a). c) HEK293T cells were transfected with HA-MCT1 and indicated Flag-tagged cargo receptors, WCL were collected for IP with anti-Flag beads, followed by IB analysis. d) The degradation of MCT1 was evaluated by CHX-chase assay in Tollip WT or Tollip KO SW480 cells. e) Quantification of the relative protein level of MCT1 in (d). f) WCL collected from SETDB1 knockdown SW480 and HT29 cells were subjected to IP assay with anti-MCT1 antibody, followed by IB analysis. g) WCL collected from SW480 cells transfected with Vector, Flag-SETDB1 (WT), and Flag-SETDB1 (H1224K) plasmids were subjected to IP assay with anti-MCT1 antibody, followed by IB analysis. h) HEK293T cells were co-transfected with Flag-Tollip and Vector, HA-MCT1 WT or HA-MCT1 K473R, and WCL was collected for IP with anti-HA beads, followed by IB analysis. i) The protein level of MCT1 in Tollip WT and Tollip KO SW480 cells treated with DMSO or Mithramycin A (100 × 10-9 m, 24 h). All immunoblots were performed three times, independently, with similar results. a,b) Data are represented as mean ± s.d. ****p < 0.0001, by two-way ANOVA with Tukey's test.

Fig 5: SETDB1 interacts with MCT1 and enhances its expression. a,b) Whole cell lysates (WCL) of SW480 and HT29 cells were collected for IP with anti-MCT1 or anti-SETDB1 antibody, followed by immunoblots (IB) analysis. c) The co-localization of MCT1 and SETDB1 was identified by IF analysis using anti-MCT1 and anti-SETDB1 antibodies in SW480 cells. White scale bars, 5 µm. d) In vitro binding assay was performed. Purified Flag-SETDB1 was incubated with His-MCT1(444-500aa) and pulled down using anti-Flag beads, followed by IB analysis. e–h) Protein and mRNA expression of MCT1 were detected by IB assays and qRT-PCR assays in SW480 and HT29 cells silenced with control (shNC) or SETDB1 shRNA (#1 and #2). i,j) Protein and mRNA expression of MCT1 were measured by IB assays and qRT-PCR assays in SW480 cells transfected with Vector, Flag-SETDB1 (WT), and Flag-SETDB1 (H1224K) plasmids. k,l) Protein and mRNA expression of MCT1 were determined by IB assays and qRT-PCR assays in SW480 cells treated with Mithramycin A at the indicated concentrations. m) Colorectal tumors and paired normal tissues were extracted and subjected to detection of MCT1 and SETDB1 protein expression by IB analysis. All immunoblots are performed three times, independently, with similar results. f,h,j,l) Data are represented as mean ± s.d. ns means no significant, by one-way analysis of variance (ANOVA) with Tukey's test.