H3K18 La-METTL5-CCT2 signaling axis promotes colorectal cancer progression by inhibiting pyroptosis

Figure 3 METTL5 promotes CCT2 expression in an m6A-dependent manner. A: Total RNA from SW480 cells before and after METTL5 knockdown subjected to transcriptome sequencing. Volcano plot to visualize the global changes in mRNA expression; genes with |logFC| > 2 and adj.P.Val < 0.01 are labeled; B: SW480 cells with stable METTL5 knockdown were analyzed via quantitative polymerase chain reaction for mRNA expression of RANBP1, ANXA3, PSAT1, CCT2, and EMC1; C: RNA immunoprecipitation in SW480 cells using a METTL5 antibody, with an IgG antibody as the negative control. The enrichment of METTL5 protein on CCT2 mRNA (left panel). The enrichment difference (right panel); D: SW480 cells cultured in normal medium (con) or medium containing 5 mmol/L sodium lactate for 24 hours. MeRIP using an m6A antibody, with an IgG antibody as the negative control. Quantitative polymerase chain reaction to analyze the level of m6A modification on CCT2 mRNA; E: METTL5-knockdown and control SK-Hep1 cells treated with cycloheximide at a final concentration of 100 μM for 0, 1, 2, and 4 hours. Western blotting was used to detect METTL5 protein expression, and grayscale value quantitative analysis was performed; F: SW480 cells with non-targeted knockdown (sh-NC) or METTL5 knockdown (sh-METTL5) were stably transfected with oe-NC or CCT2-overexpressing plasmid (oe-CCT2), respectively. These cells (1 × 10⁶ cells/mouse) were subcutaneously inoculated into BALB/c nude mice to establish a cell line-derived xenograft model. Tumor photograph (left panel), tumor weight (middle panel, n = 6), and tumor volume plot (right panel); G: Immunohistochemistry to detect the expression levels of Ki67, METTL5, and CCT2 in xenograft tumors from different treatment groups (as in F), followed by quantitative analysis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001.