Fig. 4
From: Targeting FTO induces colorectal cancer ferroptotic cell death by decreasing SLC7A11/GPX4 expression
FTO enhances the expression of SLC7A11 and GPX4 in CRC cells. (A)The total RNA were extracted from FTO knockdown or vector control CRC cells, and then mRNA was reversely transcribed into cDNA. The expression of SLC7A11 and GPX4 mRNA were examined by qRT-PCR. (B) The lysates were collected from FTO knockdown or vector control CRC cells for western blotting to examine the expression of FTO, SLC7A11, and GPX4. (C) FTO knockdown HCT116 and HCT8 cells with or without exogenous expression of FTO. The lysates were collected for western blotting to examine the expression of FTO, SLC7A11, and GPX4. (D) The relative abundance of m6A sites along SLC7A11 mRNA in FTO knockdown cells and control cells, as detected by m6A-seq. (E) The m6A modification levels on SLC7A11/GPX4 were examined by the MeRIP-qPCR in FTO knockdown cells and control CRC cells. (F) The total RNA were harvested from METTL3 or METTL14 knockdown and control cells, then the total RNA for dot blotting assay to determine the m6A levels. (G) The m6A modification levels on SLC7A11/GPX4 were examined by the MeRIP-qPCR in METTL3 or METTL14 knockdown cells and control cells (H) The lysates were collected from FTO knockdown or vector control CRC cells with or without knockdown METTL3 for western blotting to examine the expression of FTO, METTL3, SLC7A11, and GPX4. (I) Schematic diagram of SLC7A11 mRNA and the predicted ‘m6A’ sites at CDS and 3’UTR are highlighted. (J) Measurement of the m6A modification on eleven m6A-site clusters of SLC7A11 by the MeRIP-qPCR. (K) RIP-qPCR analysis to screen the reader protein by binding SLC7A11 mRNA. (L) Immunoblotting of YTHDF2 in HCT116 and 293T cells was pull downed by biotinylated-SLC7A11 (site 3 and site11) and the biotinylated-SLC7A11 (site 3 and site11) without m6A motif mutation. (M) FTO knockdown or vector control CRC cells were treated with 5 µg/mL actinomycin D (Actd) as indicated and were subjected to qRT-PCR analysis for the mRNA stability of SLC7A11. (N) FTO knockdown or vector control CRC cells with or without knockdown YTHDF2, and then treated with 5 µg/mL actinomycin D (Actd) as indicated and were subjected to qRT-PCR analysis for the mRNA stability of SLC7A11. (O) CRC cells with or without knockdown YTHDF2. The lysates were collected for western blotting to examine the expression of FTO, YTHDF2, and SLC7A11. (P) Schematic diagram of SLC7A11 mRNA and the predicted ‘m6A’ sites at CDS and 3’UTR are highlighted. (Q)Measurement of the m6A modification on three m6A-site clusters of GPX4 by the MeRIP-qPCR. (R) RIP-qPCR analysis to screen the reader protein by binding SLC7A11 mRNA. (All error bars, mean values ± SEM, p values were determined by unpaired two-tailed Student’s t test of n = 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001)