Fig 2: Inhibition of STAT3 signalling mediates the effects of reduced m6A methylation on osteosarcoma cell proliferation and apoptosis. a. The efficiency of SOCS3 down-regulation was validated by qRT-PCR. b. Down-regulation of SOCS3 increased the expression of p-STAT3 in ALKBH5 overexpression cells. c. SOCS3 knockdown rescued the proliferation inhibition. d and e Elevation of cell apoptosis was reversed by SOCS3 knockdown. f. SOCS3 knockdown rescued the cycle arrest promotion caused by up-regulation of ALKBH5. The data represent the mean± S.D. of three independent experiments. ANOVA or t-tests was used for statistical analysis. *P<0.05 or **P<0.01 indicates a significant difference between the indicated groups.

Fig 3: Overexpression of ALKBH5 suppresses human osteosarcoma growth in vivo. a. Overexpression of ALKBH5 effectively inhibited human osteosarcoma subcutaneous tumor growth in nude mice (n = 12 per group). b. The size of tumor was monitored every 5 days. c. Representative images of IHC staining, showing ALKBH5, SOCS3, and STAT3 in two subcutaneous tumor models. d. The tumor growth was effectively inhibited in YTHDF2 down-regulated group. However, ALKBH5 had no significant effect on tumor growth in YTHDF2 deficiency group (n = 4 per group). The data represent the mean± S.D. of three independent experiments. ANOVA test was used for statistical analysis. *P<0.05 or **P<0.01 indicates a significant difference between the indicated groups.

Fig 4: RNA sequencing identifies transcripts with altered methylation in osteosarcoma. a. Volcano plots for ALKBH5 overexpression KHOS cells versus controls. Significantly upregulated (red) and downregulated (green) transcripts are shown (|logFC| > 1, P < 0.001, FDR < 0.01). b. Gene set enrichment analysis (GSEA) validated enrichment of cell cycle, apoptosis, and tumor-related pathway. c. A heatmap showing changes in representative cell proliferation, cell cycle and apoptosis related genes and pathways, including STAT3. d. By western blot, it was found that downstream targets of STAT3 (CyclinD1,c-Myc and bcl-2)showed decreased activation in the cells relative to control.

Fig 5: ALKBH5 expression in osteosarcoma and its relationship to patient survival. a. High ALKBH5 tumor tissues exhibited lower total m6A methylation levels compared to low ALKBH5 group (t-test p = 0.0035, n = 41 per group). b. Representative images showing low or high expression of ALKBH5 by IHC staining. c. Kaplan-Meier survival analysis showed that the overall survival time of patients with low ALKBH5 expression was significantly shorter than that of patients with high ALKBH5 expression (log-rank p = 0.0283,n = 41 per group). d. Representative western blot showed the expression of ALKBH5 was markedly decreased in osteosarcoma (OS) compared with the normal bone (N). The data represent the mean± S.D. *P < 0.05 or **P < 0.01 indicates a significant difference between the indicated groups.