Fig. 6

Identification of Mupirocin as a novel inhibitor of FTO and regulates CRC ferroptosis and tumor growth. (A) The flowchart of the pipeline to identify FTO inhibitors based on virtual screening. (B) HCT116 cells were treated seven compounds, and then the total RNA were harvested for dot blotting assay to determine the m⁶A levels. The optical density of blotting bands was quantified by Image J software and normalized to control. (C) The total RNA were harvested from HCT116 cells, and then incubated with FTO protein from HCT116 cells by flag pull-down in kinase buffer with or without Mupirocin. The enzymatic activity of FTO was anlysis by dot blotting assay. (D) The total RNA were harvested from HCT116 cells, and then incubated with recombinant FTO protein in kinase buffer with or without Mupirocin. The enzymatic activity of FTO was anlysis by ELISA. (E) The binding model of Mupirocin in FTO catalytic pocket. (F) The affinity of Mupirocin (0, 3.125, 6.25, 12.5, 25 µM, 50 µM, 100µM,  and 200 µM) for FTO was determined using SPR. (G) Thermal shift analysis for the affinity of Mupirocin for FTO, and then anlyzed by western blotting. (H) The in situ pull-down assay in CRC cells was performed to identifying the interaction between Mupirocin probe and FTO proteins. (I-J) The MDA concentration or GSH/GSSG ratio were detected using assay kits in CRC cells treated with the indicated doses of Mupirocin for 72 h. (K-L) The MDA concentration or GSH/GSSG ratio were detected using assay kits in FTO knockdown CRC cells treated with the indicated doses of Mupirocin for 72 h. (M) The expression of FTO, SLC7A11, and GPX4 were examined by western blotting in FTO knockdown CRC cells treated with the indicated doses of Mupirocin for 72 h. (N) CRC cells treated with 12.5 µM, 25 µM, and 50 µM of Mupirocin, and then harvested cells for counting cell number at indicated day 1, day 2, day 3, and day 4 to determine the cell proliferation. (O) CRC organoids treated with 100 µM and 200 µM of Mupirocin for 7 days, and then the pictures were taken for examining the effect of Mupirocin on growth of CRC organoids. Representative images of organoids treated with the indicated doses of Mupirocin (scale bar = 50 μm). (P) Tumor growth was compared between xenograft nude mice bearing with CRC PDX, whch IP injection with 25 mg/kg and 50 mg/kg Mupirocin (n = 4). Tumor volume was calculated for each group at the indicated times. (Q) All tumors from nude mouse were shown. (R) Tumor mass of xenograft nude micewith PDX tumor treated with Mupirocin. (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).