Fig. 3: Intensified RNA m⁶A methylation in ESCC elicited by the methionine-SAM-METTL3 cascade.

A Dot blot assay comparing RNA m⁶A content between clinical ESCC tissues and paired NATs from validation cohort 2 (n = 48). B Quantification of RNA m⁶A levels of paired tissues described in A, with p value calculated using paired Wilcoxon rank-sum test. Impact of methionine removal from culture medium on RNA m⁶A abundance in ESCC cell lines (C) and primary fresh ESCC tissues from patients (D) after 24 h of in vitro culture. E Recovery of intracellular RNA m⁶A content in the absence of methionine through supplementation with 100 μM SAM for 24 h. F Proteomic analysis of the expression of three writer enzymes and two cofactors in the RNA m⁶A machinery between clinical ESCC tissues and paired NATs from discovery cohort. G, H Influence of METTL3 abrogation on the intracellular RNA m⁶A intensity in ESCC cells in the presence of methionine. I Influence of STM2457 treatment on the intracellular RNA m⁶A abundance in ESCC cells in the presence of methionine. Comparison of METTL3 expression between ESCC tissues and NATs from validation cohort 1 (J and K) and validation cohort 2 (L and M). Representative IHC images (J and L) and statistical violin-box-scatter plots (K and M) are presented, with p values calculated using Wilcoxon rank-sum test. NC non-target control.