Figure 5.

Overexpression of ALKBH5 enhances SOCS3 mRNA stability via an m6A-YTHDF2-dependent mechanism. a. Dual-luciferase reporter showed that knockdown of YTHDF2 increased luciferase activity of reporter carrying wild-type 3′ UTR fragment of SOCS3, while these changes were abolished when the m6A sites were mutated. b. Bioinformatics analysis of osteosarcoma showed a negative correlation between YTHDF2 and SOCS3 (http://hgserver1.amc.nl)(n = 88). c. inhibition of YTHDF2 in osteosarcoma cells improved the expression of SOCS3 to a similar degree to overexpression of ALKBH5. d. Streptavidin RNA pull-down assay demonstrated that YTHDF2 bound the SOCS3 full-length transcripts in osteosarcoma cells. e. The SOCS3 mRNA half-lives were significantly increased upon ALKBH5 overexpression and YTHDF2 inhibition in osteosarcoma cells. f. There was no significant difference in SOCS3 mRNA half-lives between ALKBH5 overexpression and control cells with YTHDF2 deficiency. g. Representative images showing high or low expression of SOCS3,ALKBH5 and YTHDF2 in osteosarcoma tissues. h. Correlation between SOCS3 and ALKBH5 or YTHDF2 in osteosarcoma microarray specimens. The data represent the mean± S.D. of three independent experiments. ANOVA or t-tests was used for statistical analysis. **P<0.01 indicates a significant difference between the indicated groups.