Fig. 6

FTO mediates m6A demethylation of LINC00022 and promotes its up-regulation. (A) The colorimetric m6A detection assay showed that the overall m6A levels in ESCC cells were decreased compared with Het-1A, **p < 0.01; ***p < 0.001. (B) The relative overall m6A level in 20 pairs of ESCC tissues was tested by the colorimetric m6A detection kit, **p < 0.01. (C) MeRIP combined with specific qRT-PCR was utilized to detect the relative m6A enrichment at three sites of LINC00022 transcript in Het-1A, KYSE70 and KYSE150 cells. Three putative m6A modification sites (site1, site2, site3) of LINC00022 RNA analyzed by SRAMP and RMBase V2.0 were shown in Supplementary Fig. 10B. (D) The mRNA levels of FTO in 44 pairs of ESCC tumor tissues and adjacent normal tissues were determined by qRT-PCR. (E) Person’s Coefficient analysis showed the positive correlation between LINC00022 transcription and FTO mRNA in 44 cases of ESCC tumors. (F) The protein levels of FTO in Het-1A, TE1, KYSE70, KYSE150 and KYSE450 cell lines were examined by Western blot. (G) Person’s Coefficient analysis revealed the positive correlation between LINC00022 transcription and FTO protein in TE1, KYSE70, KYSE150 and KYSE450 cells. (H-I) Knockdown or over-expression of FTO resulted in a significant decrease or increase in LINC00022 expression in ESCC cells revealed by qRT-PCR, *p < 0.05; ***p < 0.001. (J-K) Knockdown or over-expression of FTO led to an increase or decrease in overall m6A levels of RNAs in ESCC cells analyzed by colorimetric m6A detection kit, *p < 0.05; **p < 0.01. (L) MeRIP combined with specific qRT-PCR uncovered that FTO over-expression led to a dramatic decrease in m6A enrichment (site2) of LINC00022 transcript in KYSE70 cells (upper panel, **p < 0.05). The qRT-PCR products were then examined by agarose gel electrophoresis (nether panel). (M-N) Cell cycle phase distribution of KYSE150 or KYSE70 following lentivirus-mediated knockdown or over-expression of FTO was detected by PI-staining flow cytometry, *p < 0.05; **p < 0.01; ns means no significance