Research Paper Volume 13, Issue 3 pp 4045—4062

ALKBH5-mediated m6A demethylation of FOXM1 mRNA promotes progression of uveal melanoma

ALKBH5 promoted UM cell migration, invasion, and EMT. (A) After ALKBH5 knockdown plasmid transfection for 24 hours, the transwell migration assay and Matrigel invasion assay were used to determine cell migration and invasion ability, respectively, in C918 and MuM-2B cells. (B) After transfection with shRNA-targeted ALKBH5, the expression of MMP2, MMP7, and MMP9 was detected using western blot. (C) Knockdown of ALKBH5 decreased mesenchymal markers (N-cadherin, vimentin, Snail, Slug, and β-catenin) and increased epithelial marker (E-cadherin) in UM cells. (D) The ALKBH5 overexpression efficiency was verified at the protein level in UM cells by western blot assay. (E) Upregulation of ALKBH5 increased cell migration and invasion abilities in UM cells. (F) Western blot assay was used to detect the ALKBH5 protein level by transfecting UM cells with wild-type or catalytic inactive mutation plasmid of ALKBH5. (G) Compared with ALKBH5 wild-type plasmid transfection, catalytic inactive mutation of ALKBH5 decreased the migration and invasion of UM cells. (H) Overexpression of ALKBH5 increased the expression of EMT markers. (I) Loss of ALKBH5 catalytic activity suppressed the expression of EMT-related proteins in UM cells. Mean ± SEM, t-test, *P P P

Figure 4. ALKBH5 promoted UM cell migration, invasion, and EMT. (A) After ALKBH5 knockdown plasmid transfection for 24 hours, the transwell migration assay and Matrigel invasion assay were used to determine cell migration and invasion ability, respectively, in C918 and MuM-2B cells. (B) After transfection with shRNA-targeted ALKBH5, the expression of MMP2, MMP7, and MMP9 was detected using western blot. (C) Knockdown of ALKBH5 decreased mesenchymal markers (N-cadherin, vimentin, Snail, Slug, and β-catenin) and increased epithelial marker (E-cadherin) in UM cells. (D) The ALKBH5 overexpression efficiency was verified at the protein level in UM cells by western blot assay. (E) Upregulation of ALKBH5 increased cell migration and invasion abilities in UM cells. (F) Western blot assay was used to detect the ALKBH5 protein level by transfecting UM cells with wild-type or catalytic inactive mutation plasmid of ALKBH5. (G) Compared with ALKBH5 wild-type plasmid transfection, catalytic inactive mutation of ALKBH5 decreased the migration and invasion of UM cells. (H) Overexpression of ALKBH5 increased the expression of EMT markers. (I) Loss of ALKBH5 catalytic activity suppressed the expression of EMT-related proteins in UM cells. Mean ± SEM, t-test, *P < 0.05, **P < 0.01, ***P < 0.001.