Fig. 4. FTO affects stem-like properties through m⁶A_m demethylase activity.

a FTO silencing increases m⁶A_m level rather than m⁶A. LC-MS/MS mRNA quantification of m⁶A_m/A and m⁶A/A ratios in either CRC1 sh-FTO or sh-CTL and SW620 sh-FTO or sh-CTL cell line. Bar plot represents mean ± S.E.M of at least three biological replicates. **p-value < 0.01, *p-value < 0.05, ns not significant. Two-sided unpaired T-test. b m⁶A_m tends to be increased in CTC lines but not m⁶A. LC-MS/MS analysis of mRNA from patient derived cell lines (same as in Fig. 3). m⁶A_m/A and m⁶A/A were evaluated. Graphs represent mean ± S.E.M of m⁶A_m/A or m⁶A/A level for each cell line or group of cell lines. Three biological replicates. c FTO overexpression decreases m⁶A_m rather than m⁶A in CTC lines. mRNA quantification of m⁶A_m/A and m⁶A/A after FTO overexpression in CTC44 line. Graphs represent mean± S.E.M of at least three biological replicates. ***p-value < 0.001, *p-value < 0.05, ns not significant. Two-sided unpaired T-test. d Effectors of m⁶A_m modification. e PCIF1 silencing decreases colonosphere formation. Sphere forming ability of CTC44 line after silencing of the m⁶A_m writer PCIF1. Bar plot represents mean ± S.E.M of three experiments. ***p-value < 0.001, *p-value < 0.05. Two-sided unpaired T-test. f PCIF1 silencing rescues sphere forming ability in CRC1 sh-FTO cell line. PCIF1 was depleted by siRNA treatment in sh-FTO cell line and sphere formation assay was performed. Results are mean ± S.E.M of three independent experiments. *p-value < 0.05, ns not significant. One-way Anova followed by multiple comparisons. g PCIF1 silencing rescues chemosensitivity in CRC1 sh-FTO cell line. FIRI toxicity was measured with or without siRNA-mediated PCIF1 silencing in sh-FTO cell line. Cell viability curve of one representative experiment is shown (left). Results are expresses as mean ± S.E.M (barplots) of four independent experiments (right). *p-value < 0.05, ns not significant. One-way anova followed by multiple comparisons.