Additional file 6: Supplementary Figure S6. METTL1 mediated methylation promotes cell proliferation, and tumour growth in vivo. A) Proliferation of DU145 METTL1 KO and WT cell clones. Mean ± SD, n = 6. The thick dotted line represents the average growth of the WT and KO cells. B) Proliferation of METTL1-silenced 22Rv1 cells. Mean ± SD, n = 3. C) Cell cycle analysis of PC3 METTL1 KO, WT, and parental cells showing decreased cell cycle progression in METTL1 KO cells. Mean ± SD, n = 3. The dotted lines indicate the mean of three biological replicates. D) Representative images of BrdU staining of three clones of PC3 METTL1 KO and WT cells. Quantification is shown in Fig. 5. E) Schematic representation of generation of single-cell-derived spheroids, and representative images of single-cell-derived spheroids (lower panel). Quantification is shown in Fig. 5. F) METTL1 depletion affects tumour growth in vivo. Immunohistochemical staining for METTL1, Ki67, and cleaved caspase 3 (Cl-Casp3) in PC3 METTL1 KO and WT cell-derived xenografts. Right charts show the quantification of Ki67 + and Cl-Casp3 + cells per microscopic field. Mean ± SD, n = 5, and ten images per biological replicate. G-I) Protein expression (G) and m7G methylation levels (H, I) of a second PC3 METTL1 KO cell clone (KO2) ectopically expressing an HA-tagged wild-type (WT) or catalytic dead mutant (AFPA) version of METTL1. METTL1 KO2 cells were infected with the empty vector (eV) as a control. Methylene blue staining was used as the loading control (H, bottom panel). Mean ± SD, n = 3 (H). J, K) Proliferation (J) and spheroid formation capacity (K) of PC3 METTL1 KO2 cells re-expressing METTL1 (WT) or catalytic dead mutant (AFPA) compared with METTL1 KO2 (eV). Mean ± SD, n = 6. L) WDR4 mRNA expression and cell growth of PC3 cells expressing doxycycline-inducible SCR shRNA or two shRNA against WDR4 with or without doxycycline induction. Mean ± SD are represented (n = 6). M) WDR4 mRNA expression and tumour growth of xenografts of cells expressing doxycycline-inducible SCR shRNA or a shRNA against WDR4 with or without doxycycline induction. Mean ± SEM are represented (n = 10 for shWD2, n = 5 for SCR, for each Dox condition). Scale bar represents 100 μm (D), 50 μm (F). Statistical tests: Two-way ANOVA (A, I, J), one-way ANOVA (C), and one-tailed Student’s t-test (B, F, K, L, M). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Consejo Superior de Investigaciones Científicas (España)

Peer reviewed