Fig. 6

LINC01605 binds to METTL3 to promote the m6A modification of SPTBN2. A microarray analysis of genes differentially expressed in CC cells with knockdown of LINC01605. B SPTBN2 protein expression in LoVo and Caco-2 cells by western blot assay. C m6A modification sites of SPTBN2 mRNA predicted using SRAMP website. D m6A modification levels of SPTBN2 in LoVo and Caco-2 cells with sh-LINC01605 analyzed using MeRIP-seq. E the binding of LINC01605 to SPTBN2 mRNA verified using RNA pull-down assay. F regulatory-associated RBP bound to LINC01605 predicted using Starbase website. G proteins that bind to LINC1605 examined using RIP assays. H Cy3-label LINC01605 with anti-METTL3 for fluorescence co-localization assay to detect subcellular localization of LINC01605 with METTL3 in LoVo and Caco-2 cells. I diagrammatic sketch presented the construction of METTL3-WT and METTL3-MUT (up). diagrammatic sketch presented that the fragment of wild-type SPTBN2 CDS (SPTBN2) containing predicted METTL3 target sites was cloned into pGL3 vector with Firefly luciferase reporter genes (F-Luc) (down-left). The luciferase activity measurement in Lovo and Caco-2 cells (down-right). J the binding relationship between METTL3 and SPTBN2 mRNA measured using RIP assays. K detection of m6A modification levels of SPTBN2 mRNA in LoVo or Caco-2 cells by MeRIP-qPCR. L the shRNAs targeting METTL3 were transfected into LoVo or Caco-2 cells, and the protein expression of METTL3 and SPTBN2 in the cells were detected by western blot. Error bars represent means ± SD for three independent experiments (**p < 0.01, two-way ANOVA, followed by Tukey’s multiple comparison)