Extended Data Fig. 2: Depletion of FTO promotes breast cancer cell motility and metastases.
a-b, Real-time chemotactic invasion (a) and migration (b) of RNAi FTO (red, shRNA #1 and #2, respectively) and RNAi control SKBR3 cells (green). c, Wound scratch assay performed with RNAi Ctrl and RNAi FTO SKBR3 cells. Representative pictures (left) with quantification of wound closure (as percentage of the width at 0 h, right). Data from a single experiment, representative of n = 2 biologically independent experiments provided as Source Data. d, Cell viability by MTT assay (left, mean + SD, n = 5, two-tailed paired t-test, p = 0.37) and real-time proliferation (right) of FTO-depleted and control SKBR3 cells. e, Western blotting of FTO depletion and rescue by overexpression of wild-type FTO (FTO-WT) or a catalytically inactive FTO mutant (FTO-MUT) in SKBR3 cells (upper panel, n = 3 with β-actin as loading control) and real-time chemotactic migration (lower panel). f, Expression of FTO in PDX shown by immunochemistry. Representative image of tissue sections with scale bar of 250 µm (n = 3). g, Metabolism imaging with 18F-FDG PET of subiliac lymph nodes xenograft mice bearing RNAi Ctrl (black, n = 4) or RNAi FTO (red, n = 5) SKBR3 tumors. Representative images (9 weeks after inoculation) are shown (pathological uptake in LN encircled; physiological uptake in bladder (Bl)). The box defines the IQR split by the median, with whiskers extending to the most extreme values within 1.5×IQR beyond the box. P-value calculated by two-tailed t-test (P = 0.0061). h, Kaplan-Meier based on FTO expression and disease-specific survival in the breast cancer METABRIC cohort (n(high FTO) = 660, n(low FTO) = 661, logrank test, P = 7e-4). i, Forest plot showing association between FTO expression and distant-metastasis-free survival in breast cancer. Data shown as hazard ratio (log2), with 95% confidence interval, for both the aggregate KM Plot data set (‘overall’, presented in Fig. 2f) and individual cohorts. The number of samples (n) and P-values (calculated by two-sided logrank tests, in bold if P < 0.05) are indicated for each cohort. Data for (a), (b), (d), (e) are from n = 3 technical replicates (mean + SD) within a single experiment, representative of 3 biologically independent experiments (except for (e): 2 replicates). Independent repeats have been deposited on the Figshare repository (DOI: 10.6084/m9.figshare.14602965).
