Journal: Heliyon
Article Title: FLOT1, stabilized by WTAP/IGF2BP2 mediated N6-methyladenosine modification, predicts poor prognosis and promotes growth and invasion in gliomas
doi: 10.1016/j.heliyon.2023.e16280
Figure Lengend Snippet: WTAP and IGF2BP2 mediates the M6A modification of FLOT1 and maintains its stability in gliomas. (A) MeRIP-qPCR indicating the m6A enrichment of FLOT1 transcripts in U251 and T98G cells. (B) RT-qPCR and (C) WB results indicating the effects of WTAP depletion on the level of FLOT1 transcript and protein in U251 and T98G cells. (D) and (E) RT-qPCR indicating the stability of FLOT1 mRNA in U251 and T98G cells upon actinomycin D (10 μg/mL) treatment. (F) Relative luciferase activity of U251 and T98G cells transfected with siNC and siWTAP. (G) RT-qPCR and (H) WB results indicating the effects of IGF2BP2 depletion on the level of FLOT1 transcript and protein in U251 and T98G cells. (I) and (J) RT-qPCR indicating the stability of FLOT1 mRNA in U251 and T98G cells upon actinomycin D (10 μg/mL) treatment. (K) Relative luciferase activity of U251 and T98G cells transfected with siNC and siWTAP. (L) RIP-qPCR indicating the enrichment of FLOT1 transcripts by IGF2BP2 immunoprecipitation in U251 and T98G cells. (M) RNA pulldown assay showing the direct interaction of FLOT1 transcripts with the IGF2BP2 protein. NS stands for no significant difference. * , P < 0.05; ** , P < 0.01; *** , P < 0.001; ns , no significant difference.
Article Snippet: The membranes were blocked with 5% non-fat milk and incubated with the appropriate primary antibodies, including rabbit anti -FLOT1 (Abcam; Cambridge, UK), rabbit anti -IGF2BP2, mouse anti -WTAP (Proteintech; IL, USA), and rabbit anti -GAPDH (Bioworld; Nanjing, China).
Techniques: Modification, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Immunoprecipitation
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