a Actors of m6A modification targeted by the siRNA screening. The writer complex METTL3—METTL14—WTAP deposits m6A while ALKBH5 and FTO erases m6A. Both YTHDF1 and YTHDF2 are readers of m6A modification. b Sphere forming assay. siRNA-transfected cells were seeded at low density (1 cell/µL) in non-adherent conditions and serum-deprived medium. This type of suspension culture allows the survival and growth of stem-like/progenitor cells. Following 7 days of culture, the number of spheres correlates with the initial number of CSC. c FTO silencing increases the sphere forming potential of CRC1 cell line. Colonosphere formation was quantified following knockdown of the main m6A actors. Results are expressed in fold change compared to si-CTL. n = 3 biological replicates. Mean ± S.E.M, **p-value < 0.01. Two-sided unpaired T-test. d FTO silencing increases the sphere forming potential in four CRC cell lines. Colonospheres quantification after silencing of FTO by two distinct siRNA, in four cell lines derived from primary tumors or metastasis. Results are expressed in fold change compared to si-CTL (n = 3 biological replicates). Mean ± S.E.M, *p-value < 0.05; **p-value < 0.01. Two-sided unpaired T-test. e Expression of CSC biomarkers. Level of ALDH activity as well as cell-surface expression of CD44 and CD44v6 were evaluated by flow cytometry in CRC1-sh-FTO cell line vs. sh-CTL. Graphs show one representative biological replicate (n = 3). Bar plots show quantification of the number of ALDH, CD44, and CD44v6 positive cells from three biological replicates. Mean ± S.E.M, *p-value < 0.05, **p-value < 0.01. Two-sided unpaired T-test. f In vivo tumor initiation assay. Either 1000, 500, or 100 CRC1 sh-FTO/sh-CTL cells were subcutaneously injected into nude mice. After 7 weeks, the number of tumor-bearing mice (tumor > 100 mm3) was evaluated. g FTO silencing increases tumor initiation in vivo. Picture represents the number of tumor bearing mice for each group (five mice per group) after injection of sh-FTO or sh-CTL CRC1 cell line. Bar plot represents the quantification of tumor initiation (T.I) frequency obtained by ELDA software. ***p-value < 0.001, two-sided unpaired T-test.