ALKBH5 demethylates m 6 A but not m 6 A m in mRNA in <t>HEK293T</t> cells a , ALKBH5 expression does not decrease m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST–ALKBH5) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we did not observe a significant decrease of m 6 A m in ALKBH5-overexpressing cells, indicating that ALKBH5 does not convert m 6 A m to A m in vivo (representative images show n; n = 3 biologic al replic ates; me an ± s.e.m.). b , ALKBH5 knockdown does not increase m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we did not observe a significant increase of m 6 A m in ALKBH5-expressing cells, indicating that ALKBH5 does not convert m 6 A m to A m in vivo (repres entative images shown; n = 3 biological replicates; mean ± s.e.m.). c , ALKBH5 knockdown increases m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. We observed an approximately 30% increase of m 6 A upon ALKBH5 knockdown, indicating that ALKBH5 readily influences the levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.05). d , ALKBH5 expression decreases m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST-ALKBH5) was determined by 2D TLC. We observed a significant decrease of m 6 A upon ALKBH5 expression, indicating that SLKBH5 readily influences levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, ** P ≤ 0.01).
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