Fig. 2
IGF2BP2 enhances SLC7A5 mRNA stability through an m6A-dependent mechanism in lung cancer cells. A Left panel, Venn diagrams showing numbers of down-regulated genes in RNA-seq and target genes in GSE136433 and GSE90639 datasets. Right panel, the bar chart showing the overlapped 10 genes ranking according to Log2(Fold Change) value. B Volcano plot showing the differentially expressed genes from RNA-seq. C The expression of IGF2BP2 was positively correlated with SLC7A5 in TCGA LUAD database. D H1299 and A549 cells were infected with indicated lentivirus. After puromycin selection, cells were harvested for Western blotting and RT-qPCR analyses. n = 3, one-way ANOVA. Data are presented as Mean ± SD. E A549 cells were infected with indicated lentivirus. After puromycin selection, cells were treated with different concentrations of DAA (0, 20, and 50 μM), and were harvested for Western blotting and RT-qPCR analyses. n = 3, one-way ANOVA. Data are presented as Mean ± SD. F and G RIP-qPCR using IGF2BP2 antibody and MeRIP-qPCR analyses in A549 cells infected with shControl or shMETTL3. n = 3, one-way ANOVA. Data are presented as Mean ± SD. H mRNA stability assay in A549 cells infected with shControl and shIGF2BP2. n = 3. Data are presented as Mean ± SD. I The predicted m6A sites in SLC7A5 mRNA by SRAMP tool. CDS, coding sequence. J Luciferase reporter assay using luciferase reporters expressing wild-type (WT) or mutant (MUT) SLC7A5 3’UTRs in H1299 and A549 cells infected with shControl or shIGF2BP2. n = 3, unpaired t test. Data are presented as Mean ± SD. K Western blotting analysis of IGF2BP2 after RNA pull-down assay with cell lysate (Ly.), full length biotinylated SLC7A5 (1#), the SLC7A5 3’UTR region without or with m6A sites mutation (2#, 3#), and beads only (NC) in A549 cells. ns, not significant, P > 0.05; **, P < 0.01; ***, P < 0.001