Figure 3. DDIT4 represents a major m⁶A-decorated gene in macrophages.

Splenic macrophages (CD11b⁺F4/80⁺ cells) were sorted from 10-week-old KO and WT littermates. Total RNA was purified and applied to RNA-seq and m⁶A-microarray analysis.

(A) KEGG analysis of all the differentially expressed genes of KO versus WT with significance (p < 0.05).

(B) Venn diagram analysis of populations of mRNA with significant changes in expression levels or m⁶A decoration in KO versus WT.

(C) Heatmap of 37 genes with significant changes in expression levels and m⁶A decoration in KO versus WT.

(D) STRING network analysis of DDIT4-associated gene signatures with pre-enrichment from KEGG and further validated pathways from (A).

(E) Gene Ontology (GO) analysis of DDIT4-associated gene signatures from (D).

(F) Percentage of m⁶A methylation of mRNA in KO versus WT.

(G and H) MeRIP-m⁶A using mRNA from thapsigargin (thap)-treated macrophages from KO and WT control (G) and liver tissue from aged KO and WT control (H). DDIT4 transcripts quantified by qRT-PCR are shown as percentage of input RNA (n = 3).

(I) Fitted exponential decay curve of Ddit4 mRNA expression after actinomycin D (2.5 μM) treatment. The residual mRNAs were normalized to t = 0.

(J) mRNA level of DDIT4 gene expression in KO from splenic macrophages, KCs, ATMs, and BMDMs was confirmed by qRT-PCR. The gene expression level was normalized to β-actin. Data represent mean ± SD (n = 10–15). Two-tailed Student’s t test or two-way ANOVA: *p < 0.05.