Fig. 4: FTO affects stem-like properties through m6Am demethylase activity. | Nature Communications

Fig. 4: FTO affects stem-like properties through m6Am demethylase activity.

From: FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell

Fig. 4

a FTO silencing increases m6Am level rather than m6A. LC-MS/MS mRNA quantification of m6Am/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 m6Am 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). m6Am/A and m6A/A were evaluated. Graphs represent mean ± S.E.M of m6Am/A or m6A/A level for each cell line or group of cell lines. Three biological replicates. c FTO overexpression decreases m6Am rather than m6A in CTC lines. mRNA quantification of m6Am/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 m6Am modification. e PCIF1 silencing decreases colonosphere formation. Sphere forming ability of CTC44 line after silencing of the m6Am 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.

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