Fig. 5. Binding to m⁶A was necessary for NKAP to promote SLC7A11 expression.

A m⁶A northwestern blot assay was performed using an equal amount of RNA (1 μg) isolated from each group. Briefly, the cells were treated with 40 μM cycloleucine for 24 h until the change of culture media. B Western blot assay showed SLC7A11 protein expression regulated by NKAP knockdown and 40 μM cycloleucine for 24 h. C SLC7A11 protein expression regulated by NKAP overexpression and 40 μM cycloleucine for 24 h. D SLC7A11 protein expression is regulated by NKAP knockdown and METTL3 knockdown. E SLC7A11 protein expression regulated by NKAP overexpression and METTL3 knockdown. F Similar m⁶A peak density in the 5ʹ-UTR, CDS, and 3ʹ-UTR regions in the scramble and shNKAP groups. G Enrichment of reads near the transcription start site (TSS) and transcription end site (TES). H HOMER identified the m⁶A consensus motif in the U87MG cells as “RGACH”. I MEME SUIT analyzed NKAP footprints as the “RGAC” motif based on the NKAP-iCLIP public data. J FLAG-NKAP-RIP revealed that enrichment of SLC7A11 mRNA significantly decreased in the cycloleucine-treated and METTL3 knockdown groups. K m⁶A-RIP showed no significant difference in SLC7A11 mRNA enrichment between the scramble and shNKAP groups. L Co-IP analysis of the interaction between m⁶A and Flag-NKAP in U87MG cells. M m⁶A-seq showed that the m⁶A peak in the SLC7A11 transcript ENST00000613322 had an overlap with the predicted NKAP binding region. GAPDH was used as an internal control in western blot. All bars show mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.