Extended Data Fig. 5: FTO loss upregulates Wnt/β-catenin signaling in breast and prostate cancers.
a, Numbers of m6A peaks and related transcripts identified by m6A-seq in RNAi Ctrl and RNAi FTO SKBR3 cells (upper panel, two independent biological replicates). Scatterplot displaying differentially m6A-methylated peaks (lower panel, hyper and hypo = peaks with increased and decreased m6A in FTO-depleted cells, respectively). b-d, Visualization of the IP m6A signal in RNAi FTO and RNAi Ctrl SKBR3 cells at the DVL3, FZD1, SOX11, MARK2, CSNK1G2 (b), CTNNB1 (c), and WNT5A (d) transcripts. e, Representative western blot of β-catenin in RNAi FTO #2 and RNAi Ctrl #2 SKBR3 cells with β-actin as loading control (n = 3). f, Representative western blot of cytoplasmic and nuclear β-catenin in RNAi FTO and RNAi Ctrl SKBR3 cells (n = 3). Loading controls: β-Tubulin (cytoplasmic) and histone H3 (nuclear). Wnt3a treatment as positive control for nuclear translocation of β-catenin. g, Representative immunofluorescence staining of β-catenin (red) and DAPI (blue) in RNAi Ctrl and RNAi FTO SKBR3 (n = 3). Scale bars represent 20μm. h, Relative gene expression of Wnt/β-catenin targets measured by RT-qPCR in SKBR3 cells. Mean + SD from n = 6 independent replicates (except for n(TCF1) = 5), normalized to ACTB and SDHA. Two-tailed paired t-tests: PTCF1 < 0.0001; PAXIN2 = 0.0001; PNMYC=0.001. i, Western blot of total β-catenin in RNAi FTO vs. RNAi Ctrl from SKBR3 xenografted tumors in mice (n = 4 per group) with β-actin as loading control. j, Numbers of m6A peaks and related transcripts identified in three human breast cancer biopsies by m6A-seq (upper left panel). The m6A motif (upper right) retrieved from sample BC1 includes the consensus motif DRACH. Bar graph displaying the distribution of m6A peaks relatively to transcriptomic regions (lower panel, representative of sample BC1). k, m6A IP signal at transcripts related to the Wnt/β-catenin signaling (CSNK1G2, MARK2, AXIN1 and FZD1), in three human breast cancer biopsies.
