BUSCADOR RECOLECTA

Additional file 5: Supplementary Figure S5. 5’TOGs bind to translation initiation factors. A) No differences in protein expression of translation initiation complex factors were detected by western blotting in PC3 parental, WT, and METTL1 KO cells. The bottom boxplot shows the normalised protein level quantification, and the dotted lines indicate the mean protein levels in WT cells. B) Global protein synthesis rate measured by OP-puromycin (OP-puro) incorporation in PC3 METTL1 KO cells compared to the control (WT) after NaAsO2 exposure at the indicated time points. Fluorescence was normalised to cell size (FSC). n = 3; mean ± SD. C) Representative western blot showing translation initiation factors pulled down by m7G-cap sepharose beads in PC3 METTL1 KO vs. WT cell extracts. D) Representative western blot showing translation initiation factors pulled with synthetic biotinylated-5'TOG in PC3 WT cells transfected with biotinylated-5'TOGs (5'TOG), anti-TOG (ANT) or scramble RNA (-). E) Representative western blot showing mRNA-cap-bound translation initiation factors after 5'TOG (TOG), anti-TOG (ANT), or scramble control (Ctrl) transfection in PC3 WT and METTL1 KO cells. Quantification of western blots are shown in Fig. 5 (C, D, E). Statistical tests: One-tailed Student’s t-test (A), and multiple t-test (B); ***p < 0.001,, Consejo Superior de Investigaciones Científicas (España), Peer reviewed

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

Additional file 7: Supplementary Figure S7. Translational and transcriptional characterisation of METTL1 KO cells. A) Differential mRNA expression ranked in a volcano plot according to their statistical P-value (-Log10 pV) and their relative abundance ratio (Log2 FC) between PC3 WT and METTL1 KO cells. Coloured dots represent statistically significant (p < 0.05) upregulated (red) and downregulated (blue) mRNAs levels. n = 3. B) GAPDH and KIF20A were not differentially translated in PC3 METTL1 KO cells. Percentage per fraction of mRNA abundance of the indicated genes found in polysomes of WT and METTL1 KO cell lysates. n = 3, mean ± SEM. The boxplot shows the fold change (FC) of mRNA abundance in polysome fractions of METTL1 KO versus WT cells. C) Global protein expression differences ranked in a volcano plot of PC3 METTL1 KO versus WT cells. D) Gene Ontology (GO) category enrichment of biological processes in significantly (p < 0.05) upregulated (UP) or downregulated (Down) proteins identified in PC3 METTL1 KO cells compared with WT cells. E) Western blots of PC3 WT and METTL1 KO cells transfected with synthetic 5’TOG RNAs (TOG), synthetic anti-TOG RNAs (ANT), or scramble RNAs (Ct). High (h) and low (l) western blot exposures are shown for pSTAT1, total STAT1 and IRF9. The bottom bar plots indicate the expression of each protein relative to GAPDH. Mean ± SEM, n = 3. F) Immunohistochemical staining for pSTAT1 in PC3 METTL1 KO and WT cell-derived xenografts. Scale bar represents 50 μm. G, H) Increased mRNA expression levels of ISGs genes in METTL1 KO PC3 (G) and METTL1-silenced 22Rv1 cells (H) cells. Mean ± SD, n = 3. I) Western blot of IRF9 and METTL1 protein levels in METTL1-silenced 22Rv1 cells compared to control cells (SCR). Bottom bar plots show quantification of IRF9 expression normalised to GAPDH. Mean ± SD, n = 3. J) Correlation analysis between METTL1 expression and ISG genes in three human PCa expression datasets reflects an inverse expression correlation. Correlations with p-value <  = 0.05 and |R|> = 0.2 are indicated with (*). Statistical tests: One-tailed (B, G-I) and two-tailed Student’s t-test and Pearson correlation test (J) were performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001., Peer reviewed

Additional file 8: Supplementary Figure S8. Immune cell characterisation and correlation with METTL1 expression in human PCa. A) Complete cytokine array analysis of PC3 METTL1 KO compared to WT cells’ c.m. shows differential expression of inflammatory cytokines. Mean ± SD of log2 fold change, n = 4. B) tSNE analysis of the expression markers of M1-, M2-like and Mø macrophages exposed to PC3 METTL1 KO and WT cells’ c.m. of a second biological replicate (KO2 and WT2). C) Phagocytic capacity of macrophages exposed to PC3 METTL1 KO and WT cells’ c.m. Mean ± SEM, n = 3. D) Polarisation of primary peripheral blood monocytes (PMBCs) exposed to PC3 METTL1 KO and WT cells’ c.m. The upper panel shows a schematic of the workflow used. The lower panels show the quantification of the percentage of positive cells. n = 3 technical replicates of 2 biological replicates. E) Activation of T cells from a second blood donor and co-culture with macrophages exposed to PC3 WT and METTL1 KO cells’ c.m. T cells were stained with CFSE on day 0 and measured on day 3. Means ± SD, n = 3 for two biological replicates. F) Representative immunohistochemical staining for METTL1 and CD86 (M1-like macrophages), CD163 (M2-likemacrophages), and CD8 T cells in human PCa biopsies. The scale bar represents 50 μm. G, H) METTL1 mRNA expression inversely correlates with M1-like macrophage infiltration using the CIBERSORT deconvolution algorithm with TCGA and Taylor datasets (G), and TAM infiltration analysed using TIMER (H). Statistical tests: One-tailed t-test: *p < 0.05, **p < 0.01, ***p < 0.001 (D, E), Pearson’s correlation (r), p-value (pV), and linear regression with 95% confidence (bands) are shown (G, H)., Consejo Superior de Investigaciones Científicas (España), Peer reviewed

Additional file 9: Supplementary Figure S9. Characterization of Mettl1 deletion in murine PCa. A) Schematic overview of Mettl1 systemic knockout and Mettl1flox/flox mouse model generation. B) mRNA levels of Mettl1 in prostatic tissue from five-month-old Pten-KO/Mettl1+/+, Pten-KO/Mettl1+/-, and Pten-KO/Mettl1fl/fl mice. Mean ± SD, n=3. C) 5’tRNA fragment accumulation in Pten-KO/Mettl1+/+, Pten-KO/Mettl1+/- and Pten-KO/Mettl1fl/fl tumours. The right bar chart shows means ± SD, n ≥5. D) Haematoxylin and eosin staining of Pten-KO/Mettl1+/+, Pten-KO/Mettl1+/-, and Pten-KO/Mettl1fl/fl tumour sections from the ventral, dorsal, and anterior lobes. E) Representative images of immunostained sections for METTL1 and markers of proliferation (Ki67), M1-like (iNOS) and M2-like (CD68, Arg1) macrophages, and CD8+ T cells (CD8) from Pten-KO/Mettl1+/+, Pten-KO/Mettl1+/-, and Pten-KO/Mettl1fl/fl prostate tumours. Arrows indicate positive cells. F) Prostate tumour volume from Pten-KO/Mettl1+/+ and Pten-KO/Mettl1+/- five-month-old mice reflects reduced tumour burden after Mettl1 deletion. Ventral (V), dorsal (D) and anterior (A) lobes. Mean ± SD, n≥3. G) Systemic deletion of Mettl1 resulted in reduced tumour proliferation (Ki67+ cells) and increased immune infiltration of iNOS+ (M1-like) macrophages and CD8+ T cells. Staining of tumours from Pten-KO/Mettl1+/+ and Pten-KO/Mettl1+/- five-month-old mice. Mean ± SD, n≥3, >10 images per biological replicate. H) Tumour growth of allografts derived from Pten-KO/Mettl1+/+ and Pten-KO/Mettl1flox/flox tumour cells injected into NSG male mice reflects not significant tumour formation reduction in the absence of Mettl1. Mean ± SEM, n=9. I) Prostate lobe volumes in Pten-KO/Mettl1flox/flox (fl/fl) mice and Pten-KO/Mettl1+/+ (+/+) mice treated with anti-CD8 antibodies or IgG control. Mean ± SD, (n≥6). J) Prostate lobe volumes in Pten-KO/Mettl1flox/flox (fl/fl) mice and Pten-KO/Mettl1+/+ (+/+) mice treated with anti-PD1+anti-CTLA4 antibodies or IgG control. Normal prostate lobe volumes (from wild-type mice) are also shown. The bar chart on the left shows the volume in separated lobes. The bar chart on the right shows the sum of the volume of all lobes. Mean ± SD, (fl/fl and +/+ n≥6; Normal n=5). Scale bars represent 10 mm (D), 50 μm (E). Statistical tests: One-tailed Student’s t-test (G), Mann-Whitney test (C, F, H, I, J), *p<0.05, **p<0.01, ***p<0.001., Consejo Superior de Investigaciones Científicas (España), Peer reviewed

Additional file 11: Supplementary Table S2. METTL1 bound RNAs in PC3 cells., Consejo Superior de Investigaciones Científicas (España), Peer reviewed

Additional file 12: Supplementary Table S3. METTL1 bound RNAs in HEK293T cells., Consejo Superior de Investigaciones Científicas (España), Peer reviewed

5 tables, Supplementary Material for the article https://doi.org/10.3390/microorganisms8081121, Table S1. Basic biochemical composition of fish peptones (mean values±confidence intervals).-- Table S2. Composition of culture media for marine probiotic bacteria (g/L).-- Table S3. Numerical values and confidence intervals for parameters obtained from experimental data of Pseudomonas fluorescens (Pf) growths modelled by logistic equation (1-4).-- Table S4. Continued table 3.-- Table S5. Costs of production of biomass and viable cells in the commercial marine medium and in the media formulated with fish peptones and seawater, Peer reviewed

Additional file 15: Supplementary Table S6. Differentially expressed proteins in WT and METTL1 KO PC3 cells., Consejo Superior de Investigaciones Científicas (España), Peer reviewed

Additional file 16: Supplementary Table S7. Differentially expressed mRNAs in WT vs METTL1 KO PC3 cells., Peer reviewed