Journal: Molecular Cancer

Article Title: METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer

doi: 10.1186/s12943-023-01809-8

Figure Lengend Snippet: METTL1 is upregulated in prostate cancer. A A schematic overview of the research workflow used to identify altered expression of RMPs associated with PCa. B Heatmap of average Z-scores of mRNA expression values in human primary (P) and metastatic (M) PCa samples compared to healthy tissue for significant differentially expressed RNA-modifying enzymes reveals METTL1 as the most overexpressed RMPs in PCa. Data from are from Grasso et al . ( n = N: 12, P: 49, M: 27); Taylor et al. ( n = N: 29, P: 131, M: 19); Varambally et al . ( n = N: 6, P: 7, M: 6); Lapointe et al . ( n = N: 9, P: 13, M: 4); Tomlins et al. ( n = N: 23, P: 32, M: 20) (left panel) datasets. Z-score averages for all datasets are also shown as “Aver”. Z-score values are indicated using numeric values. Grey Z-score values indicate no significant p -value. The right heatmap shows the average log 2 fold change in mRNA expression values in Pten-cKO mice with prostate intraepithelial neoplasia (PIN) and invasive prostate carcinoma (Inv) compared to normal prostate tissue (right panel) ( n = 4). C METTL1 and WDR4 expression are increased in primary (PT) and metastatic tumours (M) compared to normal tissues (N). Data are from Grasso et al. , Taylor et al . , and Varambally et al . datasets. Log 2 -normalised gene expression values are shown. D High expression of METTL1 but not WDR4 is associated with poor patient prognosis. Kaplan–Meier curves representing biochemical recurrence-free survival (DFS) of patient groups selected according to gene expression, data from the Cambridge, Stockholm and Taylor cohorts [ , ]. Data were retrieved from the camcAPP and cBioportal. E Western blot from benign prostatic hyperplasia (BPH) ( n = 7) and PCa patient samples ( n = 14) (upper panel) and correlation analysis between METTL1 and WDR4 expression, AR and phospho-S6K (right panels). Statistical tests: ANOVA test ( B, C ), and log-rank Cox test ( D ). Data are represented as mean ± standard deviation (SD). Student’s t -test and Spearman’s correlation test ( E )

Article Snippet: TaqMan probes Hs01096146_m1 and Hs02758991_g1 were used for human METTL1 and GAPDH amplification, respectively, and Mm99999915_g1 was used for mouse GAPDH amplification.

Techniques: Expressing, Gene Expression, Western Blot, Standard Deviation

Journal: Molecular Cancer

Article Title: METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer

doi: 10.1186/s12943-023-01809-8

Figure Lengend Snippet: PI3K-AKT-mTORC pathway mediate upregulation of METTL1 expression in PCa. A Correlation analysis between METTL1 and PTEN expression in human primary prostate tumours expression datasets. Plotted values correspond to the log 2 -normalised gene expression values for each patient in the indicated dataset. The black line represents linear regression, grey area indicates the limits of the confidence intervals. Pearson’s correlation coefficient (R) and p -values are indicated. Grasso n = 88; Taylor n = 183; TCGA n = 497. B, C METTL1 expression is regulated downstream of AKT signalling. Western blot ( B ) and RT-qPCR ( C ) analyses of METTL1 expression upon PI3K pathway inhibition in DU145 cells. DMSO as vehicle (Veh), BKM-120 (BKM) as pan-PI3K inhibitor, MK2206 (MK) as AKT inhibitor, rapamycin (RAPA) as mTORC1 inhibitor, and Torin (TOR) as mTORC1/2 inhibitor. For western blotting, cells were treated for 48 h, and for RT-qPCR, for 8 h. Means ± SD are shown ( n = 2) ( B ) and ( n = 6) ( C ). D Stratification of patients with a worse prognosis according to high METTL1 levels and low PTEN expression. Kaplan–Meier curves representing the disease-free survival (DFS) of patient groups selected according to Q1 (Pten L ) and Q4 (Pten H ) quartile expression of PTEN , and METTL1 high (Met H , log 2 -normalised expression > 8.72) and METTL1 low (Met L , log 2 -normalised expression < 8.72) in recurrent and disease-free (DF) tumour samples from the TCGA dataset. E–G Mettl1 is highly expressed in mouse prostate tumours. Mettl1 expression analysis in Pten-KO mice at 3 and 6 months of age compared to wild-type mice (WT) at the same ages by western blot ( E, F ) and RT-qPCR ( G ). * in ( E ) indicates an unspecific band. Means ± SD are shown for three ( F ) and five replicates ( G ). H, J Increased 7-guanine tRNA methylation in RNA from prostate tumours compared with that in normal prostate. North-dot blot of m 7 G levels in long RNAs (> 200 nucleotides long RNAs) and tRNAs extracted from prostatic tissue of Pten-KO mice with intraepithelial prostatic neoplasia (at 3 months of age) or invasive tumour (at 6 months), and wild-type (WT) mice of the same age. Methylene blue staining was used as the loading control ( H , bottom panel). Means ± SD are represented ( n = 4) ( J ). I, K Increased 7-guanine tRNA methylation in the urine of mice bearing PCa tumours. North-dot blot of m. 7 G levels from the urines of Pten-KO mice with invasive tumour (at 6 months), and wild-type (WT) mice of the same age. Means ± SD are shown (n > 3). L Representative images of immunostained sections for Mettl1 and markers for luminal (AR), and basal (K14) cells in Pten-KO prostates (anterior lobes) at initiation (3 m) and in invasive carcinoma (6 m) and in age-matched wild-type (WT) prostates . Scale bars represent 50 μm. Statistical tests: one-tailed Student’s t-test ( C ), log-rank test ( D ), Mann–Whitney test ( F, G, J, K ). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: TaqMan probes Hs01096146_m1 and Hs02758991_g1 were used for human METTL1 and GAPDH amplification, respectively, and Mm99999915_g1 was used for mouse GAPDH amplification.

Techniques: Expressing, Gene Expression, Western Blot, Quantitative RT-PCR, Inhibition, Methylation, Dot Blot, Staining, Control, One-tailed Test, MANN-WHITNEY

Journal: Molecular Cancer

Article Title: METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer

doi: 10.1186/s12943-023-01809-8

Figure Lengend Snippet: METTL1 preferentially methylates tRNAs. A tRNAs are the most common RNA species bound to METTL1. Upper panel: Boxplot representing the median Log 2 fold change of reads per million (RPM) per transcript bound to METTL1 in PC3 cells. Lower panel: The bar plot shows the total number of unique genes bound to METTL1. B Validation of the lack of METTL1 expression and m 7 G tRNA methylation in PC3 METTL1 KO cell lines. Western blot of METTL1 (upper panel) and north-dot blot of m 7 G levels (lower panel). Proteins, long RNAs (> 200 nucleotides), and tRNAs (< 200 nucleotides) were extracted from three independent PC3 METTL1 KO clones and three control clones. Parental PC3 cells are shown. C LC–MS analysis of m 7 G levels in tRNAs isolated from PC3 METTL1 KO , control, and parental PC3 cells validate the absence of m 7 G in tRNAs extracted from METTL1 KO cells. Means ± SD are represented ( n = 3). D Normalised cleavage signals for the tRNAs AlaAGC and PheGAA in PC3 WT and METTL1 KO cells. Letters on the right show the bases where methylation occurs. E Heatmap showing normalised cleavage values for all tRNAs with guanosines at position 46 in PC3 WT and METTL1 KO cells ( n = 2 for each genotype). F tRNA secondary structure showing METTL1-methylated guanines (red circles) in the variable loop. G Graphical summary of tRNA isoacceptors methylated by METTL1 (red) and non-methylated or non-transcribed (grey). Statistical tests: two-tailed Student’s t-test ( C ). **** p < 0.0001

Article Snippet: TaqMan probes Hs01096146_m1 and Hs02758991_g1 were used for human METTL1 and GAPDH amplification, respectively, and Mm99999915_g1 was used for mouse GAPDH amplification.

Techniques: Biomarker Discovery, Expressing, Methylation, Western Blot, Dot Blot, Clone Assay, Control, Liquid Chromatography with Mass Spectroscopy, Isolation, Two Tailed Test

Journal: Molecular Cancer

Article Title: METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer

doi: 10.1186/s12943-023-01809-8

Figure Lengend Snippet: The lack of m 7 G methylation in tRNAs leads to 5'tRNA fragment accumulation. A Heatmap showing no differences in the levels of mature tRNAs (log 2 normalised reads; RPKM) measured using tRNA-seq data. Rows represent individual tRNA isoacceptors and columns represent independent replicates. tRNA isoacceptor expression levels are row-scaled (each row is normalised to their mean expression and standard variation). B The top pie chart represents the percentage of all abundant tRNA fragments identified in PC3 METTL1 KO cells compared to that in WT cells. The bottom pie chart represents the percentage of differentially express tRNA fragments: fragments with log 2 fold change (FC) > 2 and p value < 0.05 in PC3 METTL1 KO cells versus WT cells. The pie charts show that while 5'tRNA fragments are very abundant in KO cells, 5'TOGs are significantly overexpressed in KO cells compared to WT cells. 3'tRFS: 3' tRNA fragments; Int-tRFs: internal tRFs; 5'tRNA: 5'tRNA fragments > 18 and < 35 nucleotides; 5'-halves: 5'tRNA fragments > 35 nucleotides; 5'TOGs: 5'tRNA fragments > 18 and < 35 nucleotides, with 5′ terminal oligoguanine. C The boxplot shows the log 2 fold change (FC) of all fragments (not just the differentially expressed tRNA fragments) in PC3 METTL1 KO versus WT cells. D Size and abundance (density) of 5'TOGs Cys-derived (5G) or Ala-derived (4G) fragments differentially expressed (log 2 FC > 1.5, > 18 nt, and p < 0.05) in PC3 METTL1 KO cells versus WT cells. E Summary of 5'TOGs formed in PC3 METTL1 KO cells. F Increased tRNA fragmentation was observed by northern blot detection of Cys-derived 5’TOGs in PC3 METTL1 KO vs. WT cells (2 technical replicates of 2 biological replicates are represented) (left panel). The boxplot of the right shows quantification of densities of Cys-derived 5'TOGs formed versus full-length tRNAs. G, H Cys-derived 5'TOG fragments are induced by stress. Northern blot detection of Cys-derived 5'TOG in PC3 and DU145 METTL1 KO and WT cells unexposed (0 h), or after 2 and 8 h of oxidative stress exposure. The boxplots of the right show the fraction of Cys-derived 5'tRFs formed normalised to the fraction of full length tRNAs. Values are from two biological replicates, shown in this figure and in Supplementary Fig. G and J. The loading control of tRNA is shown in the bottom panel as red-safe staining ( F–H ). Bands corresponding to full length tRNAs are indicated with stars and 5'tRNA fragments are indicated with arrows. Statistical tests: one-tailed Student’s t-test. ** p < 0.01 ( F–H )

Article Snippet: TaqMan probes Hs01096146_m1 and Hs02758991_g1 were used for human METTL1 and GAPDH amplification, respectively, and Mm99999915_g1 was used for mouse GAPDH amplification.

Techniques: Methylation, Expressing, Derivative Assay, Northern Blot, Control, Staining, One-tailed Test

Journal: Molecular Cancer

Article Title: METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer

doi: 10.1186/s12943-023-01809-8

Figure Lengend Snippet: METTL1 downregulation suppresses protein synthesis, proliferation and tumour growth in vivo. A Global protein synthesis rate measured by flow cytometry analysis of OP-puromycin (OP-puro) incorporation reflects reduced protein synthesis in PC3 METTL1 KO cells compared to the control (WT). Fluorescence was normalised to cell size (FSC) in WT and METTL1 KO cells. Two biological and three technical replicates and the mean ± SD are shown. B Translation initiation and regulatory factors are displaced from the cap of mRNAs in METTL1 KO cells. Log 2 fold change (FC) binding of the indicated translation initiation and regulatory factors to m 7 G-cap-coated sepharose beads in PC3 METTL1 KO vs. WT cells. Densitometry data were normalised to the input. Mean ± SEM, n = 3. C Anti-TOG RNAs block the 5'TOG-dependent displacement of translation initiation factors in vitro. Log 2 FC of translation initiation factors bound to synthetic biotinylated-5'TOG in PC3 WT cells transfected with 5'TOG + Anti-TOGs (ANT) compared to PC3 WT cells transfected with 5'TOG + scramble RNAs (TOG). Densitometry data were normalised to the input. Mean ± SEM, n = 4. D Displacement of translation initiation factors from mRNA caps is TOG-dependent and can be reversed by expressing anti-TOG RNAs. Log 2 fold change (FC) of m 7 G-cap-bound translation initiation factors in PC3 WT and METTL1 KO cells transfected with biotinylated-5'TOG (TOG) or anti-TOG RNA (ANT) versus cells transfected with scramble RNA oligonucleotides. Densitometry data were normalised to the input. Mean ± SEM, n = 3. Original wester blots are shown in supplementary figure ( B, C, D ). E Growth curves of PC3 METTL1 KO , WT, and parental cells (PC3). Mean ± SD, n = 3. The dotted line represents the average growth of WT and parental or KO cells. F Reduced cell division rate, as measured by BrdU incorporation. G METTL1 depletion increased apoptosis in PC3 METTL1 KO cells. Flow cytometry analysis of Annexin V staining. Mean ± SD, n = 3. H Reduced spheroid formation capacity in PC3 METTL1 KO cells. Means ± SD, n = 3. The dotted line represents the average values of all WT or KO cells. I Tumour growth in xenografted PC3 METTL1 KO and WT cells in athymic nude mice reflects impaired tumour formation in the absence of METTL1 . Mean ± SEM, n = 10. J-L Protein expression ( J ) and m 7 G methylation levels of tRNAs ( K, L ) of PC3 METTL1 KO cells ectopically expressing a doxycycline-inducible HA-tagged wild-type (WT) or a catalytic dead mutant (AFPA) version of METTL1. PC3 METTL1 KO cells were infected with an empty vector (eV) as a control. Methylene blue staining was used as the loading control ( L , bottom panel). Mean ± SD, n = 3 ( L ). M, N Proliferation ( M ) and spheroid formation capacity ( N ) were dependent on METTL1 catalytic activity. PC3 METTL1 KO cells re-expressing METTL1 (WT) or catalytic dead mutant (AFPA) compared to METTL1 KO cells infected with empty vector (eV). Mean ± SD, n = 6. O, P 5'TOG transfection induces apoptosis and reduces cell proliferation. Percentage of apoptotic ( O ) and growth rates ( P ) of PC3 METTL1 KO and WT cells after transfection with synthetic 5'TOGs (TOG) or anti-TOG (ANT) RNAs. Controls (Cont) were transfected with scramble RNAs. Mean ± SD, n = 6 ( O ), n ≥ 10 ( P ). Statistical tests: Two-way ANOVA ( E, I, L ), one-way ANOVA ( F, G, H ), and one-tailed Student’s t-test ( A-D, M-P ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: TaqMan probes Hs01096146_m1 and Hs02758991_g1 were used for human METTL1 and GAPDH amplification, respectively, and Mm99999915_g1 was used for mouse GAPDH amplification.

Techniques: In Vivo, Flow Cytometry, Control, Fluorescence, Binding Assay, Blocking Assay, In Vitro, Transfection, Expressing, BrdU Incorporation Assay, Staining, Methylation, Mutagenesis, Infection, Plasmid Preparation, Activity Assay, One-tailed Test

Journal: Molecular Cancer

Article Title: METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer

doi: 10.1186/s12943-023-01809-8

Figure Lengend Snippet: Loss of m 7 G tRNA methylation results in distinct translational programmes. A Schematic overview of nascent polypeptide OP-puro-labelling and enrichment followed by LC–MS/MS peptide identification and quantification analysis (upper panel). Lower panel shows common differentially expressed nascent proteins ranked in a volcano plot according to their statistical p -value (-Log 10 pV) and their relative abundance ratio (Log 2 FC) between four replicates of PC3 WT and METTL1 KO cells. Coloured dots represent statistically ( p -value < 0.05) upregulated (red) and downregulated (blue) proteins in METTL1 KO cells. B Gene Ontology (GO) category enrichment of biological processes in significantly ( p -value < 0.05) upregulated (UP, FC > 1) or downregulated (Down, FC < 1) nascent proteins in METTL1 KO cells compared to WT cells. Categories were ranked according to their statistical P -value (-Log 10 pV) and fold enrichment of genes found for each category. C No correlation between differentially expressed proteins (protein log 2 FC) and their mRNA (protein log 2 FC) was observed in PC3 WT vs. METTL1 KO cells. Coloured dots represent significant ( p -value < 0.05) differentially expressed proteins (blue), mRNAs (yellow) or both (red) for each gene. D Representative polysome profile in WT (grey line) and METTL1 KO PC3 cells (red line) (left panel) shows reduced translation in METTL1 KO cells. The fraction of the abundance of each mRNA in each polysome fraction is shown with respect to the content in all fractions, reflecting increased translation of IRF9 and ISG15 in METTL1 KO cells. Mean ± SEM, n = 3. The right boxplot represents the fold change (FC) of mRNA content in the polysome fraction relative to non-polysome fractions. E Increased IRF9, ISG15 and STAT1 protein expression observed in PC3 METTL1 KO cells is 5'TOG-dependent. Protein expression levels are represented as log 2 fold change for WT (grey bars) transfected with 5'TOG RNA (TOG) versus scramble control RNA (Ct), and METTL1 KO cells (red bars) transfected with anti-TOG RNAs (ANT) versus scramble control RNAs (Ct). Mean ± SEM, n = 3. Original western blots are shown in supplementary figure . F, G STAT1-dependence of apoptosis ( F ) and growth rates ( G ) of METTL1 KO and WT PC3 cells. KO cells were transfected with siScramble or siSTAT1. Mean ± SD, n = 6 ( F, G ). Statistical tests: One-tailed Student’s t-test was used ( E–G ). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: TaqMan probes Hs01096146_m1 and Hs02758991_g1 were used for human METTL1 and GAPDH amplification, respectively, and Mm99999915_g1 was used for mouse GAPDH amplification.

Techniques: Methylation, Liquid Chromatography with Mass Spectroscopy, Expressing, Transfection, Control, Western Blot, One-tailed Test

Journal: Molecular Cancer

Article Title: METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer

doi: 10.1186/s12943-023-01809-8

Figure Lengend Snippet: METTL1 low expression in PCa is associated with increased cytotoxic infiltration and good response to ICB treatment. A Cytokine content in PC3 METTL1 KO compared to WT cell-conditioned media shows upregulation of pro-inflammatory (red) and downregulation of anti-inflammatory (blue) cytokines in METTL1 KO cells. The mean ± SD log 2 fold change is shown ( n = 4). The full expression array is shown in supplementary figure . B The upper panel shows a schematic overview of the workflow followed to analyse M1- or M2-like endotype polarisation of THP-1-derived Mø macrophages exposed to METTL1 KO and WT cell-conditioned medium (c.m.). The lower panel shows the T-distributed stochastic neighbour embedding (tSNE) analysis of macrophage polarisation ( n = 3). C Proliferation of human peripheral blood CD3+ T cells co-cultured with primary Mø macrophages exposed to PC3 WT or METTL1 KO cells ’ c.m. (n = three technical, two biological replicates). D Migration of human peripheral blood CD3+ T cells towards primary macrophages exposed to PC3 WT or METTL1 KO cells ’ c.m . (n = three technical, two biological replicates). E Correlation between METTL1 expression and the immune cell infiltrates of M1-like macrophages (CD86), M2-like macrophages (CD163), and CD8+ T cells in human PCa samples. Immunostainings are shown in supplementary figure . F Prostate tumour volume from Pten-KO/Mettl1 + / + and Pten-KO/Mettl1 flox/flox five-month-old mice reflects reduced tumour burden after conditional Mettl1 deletion . Ventral (V), dorsal (D) and anterior (A) lobes. Mean ± SD, n ≥ 5. G Conditional 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 flox/flox (fl/fl) five-month-old mice . Mean ± SD, n ≥ 5, > 10 images per biological replicate. H Fold change of cytokines content in Pten-KO/Mettl1 flox/flox versus Pten-KO/Mettl1 + / + tumours ( n = 3). I Significant decrease in tumour volume (fold change: FC) in Pten-KO/Mettl1 flox/flox (fl/fl) mice treated with anti-PD1 + anti-CTLA4 antibodies compared to untreated controls (IgG). Pten-KO/Mettl1 + / + (+ / +) mice tumour volume did not change after anti-PD1 + anti-CTLA4 treatment. Mean ± SD, n ≥ 6. J METTL1 mRNA expression levels in anti-PD1 responders and non-responders in clinical trials of breast cancer, ovarian cancer, colorectal cancer, and glioblastoma ( n = 484). The data were retrieved from the ROC plotter. Statistical tests: Pearson’s correlation (r), p -value (pV), and linear regression with 95% confidence (bands) are shown ( E) . One-tailed Student’s t-test ( A, C, D, G ) , Mann–Whitney test ( F, I, J ), * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: TaqMan probes Hs01096146_m1 and Hs02758991_g1 were used for human METTL1 and GAPDH amplification, respectively, and Mm99999915_g1 was used for mouse GAPDH amplification.

Techniques: Expressing, Derivative Assay, Cell Culture, Migration, Staining, Clinical Proteomics, One-tailed Test, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: m 7 G tRNA modification and its catalyzing enzyme components METTL1 and WDR4 are elevated in HCC. (A) The percentage of m 7 G tRNA modification and other tRNA modifications in four pairs of HCC tissues and corresponding normal liver tissues identified by liquid chromatography‐coupled mass spectrometry. Paired Student's t test was used ( n = 4). (B) Northwestern blot of m 7 G tRNA modification in four pairs of HCC tissues and corresponding normal liver tissues. U6 northern blot serves as a loading control. (C) Western blot of METTL1 and WDR4 in six pairs of HCC tissues and corresponding peritumoural tissues. GAPDH serves as a loading control. (D) Western blot of METTL1 and WDR4 in HCC cell lines. A normal liver cell line THLE‐2 was used as a normal control and GAPDH serves as a loading control. (E, F) Representative images of METTL1 (E) and WDR4 (F) IHC staining in HCC specimens. The mean density of IHC staining less than median was defined as low, while more than median was defined as high. Scale bar, 250 μm. (G, H) Quantification of METTL1 (G) and WDR4 (H) IHC staining intensity in HCC specimens. Paired Student's t test was used ( n = 48). (I, J) qRT‐PCR analysis of METTL1 (I) and WDR4 (J) mRNA expression in HCC specimens. Wilcoxon signed rank test was used ( n = 57). Data presented as mean ± SD. * p < .05, ** p < .01, *** p < .001 by Student's t test or the Mann–Whitney U test unless specified. Abbreviations: IHC, immunohistochemistry; I, inosine; i 6 A, N 6 ‐isopentenyladenosine; P, peri‐tumour tissue; Peri, peri‐tumour tissue; Q, queuosine; T, tumour tissue; t 6 A, N 6 ‐threonylcarbamoyladenosine; Um, 2′‐O‐methyluridine

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Modification, Liquid Chromatography, Mass Spectrometry, Northern Blot, Western Blot, Immunohistochemistry, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: Inhibition of METTL1 impairs HCC progression in vitro and in xenograft model. (A) The knockdown effect of METTL1 in MHCC97H cells was confirmed by western blot. (B) The downregulation of m 7 G tRNA modification was confirmed by northwestern blot. (C) CCK‐8 assay of METTL1 knockdown and control MHCC97H cells. Data presented as mean ± SD (six technical replicates). (D) Representative images and quantification of clone formation in METTL1 depleted and control MHCC97H cells. Data presented as mean ± SD (three technical replicates). (E) Representative images and quantification of cell apoptosis assays in MHCC97H cells with or without METTL1 knockdown. Data presented as mean ± SD (three technical replicates). (F) Cell cycle analysis and quantification of METTL1 depleted and control MHCC97H cells. Data presented as mean ± SD (three technical replicates). (G) Representative images and quantification of migration in METTL1 depleted and control MHCC97H cells. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (H) Representative images and quantification of invasion in METTL1 depleted and control MHCC97H cells. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (I) Validation of the rescue of METTL1 by western blot in MHCC97H cells. siMETTL1‐1 was used. (J) CCK‐8 assay of METTL1 knockdown MHCC97H cells with rescue expression of wild‐type METTL1 or the mutant. (K) Growth of subcutaneous transplanted tumours in NC and shMETTL1 group. Tumour sizes were measured every 4 days. Data presented as mean ± SEM ( n = 9). shMETTL1‐2 was used. (L) Overview of subcutaneous transplanted tumours in NC and shMETTL1 group. (M) Tumour weights formed in NC and shMETTL1 group at the time of sacrifice. Data presented as mean ± SEM ( n = 9). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. All the in vitro assays were biologically repeated for three times. Abbreviations: Mut, mutant METTL1; NC, negative control; sh1, shMETTL1‐1; sh2, shMETTL1‐2; shM1, shMETTL1; si1, siMETTL1‐1; WT, wild‐type METTL1

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Inhibition, In Vitro, Western Blot, Modification, CCK-8 Assay, Cell Cycle Assay, Migration, Expressing, Mutagenesis, MANN-WHITNEY, Negative Control

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: METTL1 regulates m 7 G tRNA methylome, tRNA expression and global mRNA translation. (A) Flowchart of m 7 G TRAC‐Seq. (B) List of m 7 G‐modified tRNA identified in MHCC97H cells. (C) Sequence motif in the m 7 G sites identified by TRAC‐seq. (D) Representative images of cleavage scores of indicated tRNA in MHCC97H cells with or without METTL1 knockdown. (E) Global m 7 G tRNA methylation level of MHCC97H cells with or without METTL1 knockdown. Wilcoxon signed‐rank test was used. (F) Expression level of m 7 G‐modified and non‐m 7 G‐modified tRNAs revealed by TRAC‐seq. Fold change was calculated as the ratio of tRNA expression level of shMETTL1 group to the control group. (G) Validation of the downregulated expression of m 7 G‐modified tRNAs upon METTL1 depletion by m 7 G methylated tRNA immunoprecipitation qPCR. Relative expression of specific tRNA was obtained using input samples and U6 served as an internal control. The shMETTL1‐2 was used in this experiment (three technical replicates). (H) Polysome profiling of Huh7 and MHCC97H with or without METTL1 knockdown. shMETTL1‐2 was used in this experiment. (I) Global translation of SNU‐449 cells with overexpression of wild‐type or mutant METTL1. Coomassie brilliant blue staining of the gel was used as control. (J) Puromycin intake assay of siMETTL1 MHCC97H cells rescued by wild‐type METTL1 or its catalytic dead mutant. Coomassie brilliant blue staining of the gel was used as control. Quantitation of the bands was showed (three biological replicates). siMETTL1‐1 was used. Data presented as mean ± SD. * p < .05, ** p < .01, *** p < .001 by Student's t test or the Mann–Whitney U test unless specified. Abbreviations: Mut, mutant METTL1; NC, negative control; shM1, shMETTL1; si1, siMETTL1‐1; WT, wild‐type METTL1

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Expressing, Modification, Sequencing, Methylation, Immunoprecipitation, Over Expression, Mutagenesis, Staining, Quantitation Assay, MANN-WHITNEY, Negative Control

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: m 7 G tRNA modification regulates HCC mRNA translation in a codon‐dependent manner. (A) Scatterplot of translation efficiency (TE) in the MHCC97 cells with or without METTL1 knockdown. TE was calculated as the ratio of the polyribosome signals to the input signals. (B) Scatterplot of mRNA expression in the MHCC97 cells with or without METTL1 knockdown. (C) Correlation of m 7 G‐related codons frequency and translation efficiency in MHCC97H. Pearson correlation analysis was used. (D) Correlation of m 7 G‐related codons frequency and translation ratio. Translation ratio was calculated as the ratio of the translation efficiency of shMETTL1 group to the control group. Pearson correlation analysis was used. (E) Translation ratio of mRNAs in low ( n = 2018) and high ( n = 2018) m 7 G‐related codon frequency groups. (F) Frequencies of m 7 G‐related codons in TE‐decreased genes ( n = 1720), TE‐increased genes ( n = 1195) and other genes ( n = 5159). (G) Pathway analysis using the TE‐decreased genes upon METTL1 knockdown. (H, I) Relative expression and translation efficiency (TE) of Cyclin A2, EGFR and VEGFA mRNA in METTL1 depleted and control MHCC97H (H) and Huh7 (I) cells. β‐Actin was used as an internal control. TE was calculated as the ratio of the polyribosome signals to input signals. RPS10 was used as a negative control. Data presented as mean ± SD (three technical replicates). (J) Western blot of Cyclin A2, EGFR, VEGFA, p‐Akt and p‐p44/42 MAPK in METTL1 depleted and control Huh7 and MHCC97H cells. GAPDH was used as a loading control. (K) Left, m 7 G tRNAs decoded‐codons frequency of TE down mRNAs identified by polyribosome‐mRNA‐seq. Right, the expression profile of m 7 G tRNAs in MHCC97H cells. (L) 5X AAG (Lys) codon sequences were inserted in the front of firefly luciferase coding region. Depletion of METTL1 resulted in decreased luciferase activity compared to that in the controls in Huh7 and MHCC97H cells. The control reporter without any insertion was used to normalize the translation differences. Data presented as mean ± SD (three technical replicates). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. Polyribosome‐mRNA‐seq was biologically repeated for three times. All the in vitro assays were biologically repeated for three times. Abbreviations: NC, negative control; sh1, shMETTL1‐1; sh2, shMETTL1‐2; TE, translation efficiency

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Modification, Expressing, Negative Control, Western Blot, Luciferase, Activity Assay, MANN-WHITNEY, In Vitro

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: Overexpression of LysCTT and EGFR rescues HCC malignant phenotype. (A) Validation of the LysCTT, METTL1, EGFR, Cyclin A2 expression and METTL1 depletion by northern and western blots in MHCC97H and Huh7 cells. (B) CCK‐8 assay of METTL1 knockdown MHCC97H and Huh7 cells with or without overexpression of LysCTT. Data presented as mean ± SD (six technical replicates). (C, D) Representative images and quantification of migration in METTL1‐knockdown MHCC97H (C) and Huh7 (D) cells with or without overexpression of LysCTT. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (E, F) Representative images and quantification of invasion in METTL1‐knockdown MHCC97H (E) and Huh7 (F) cells with or without overexpression of LysCTT. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (G) Validation of the EGFR overexpression and METTL1 depletion by western blot in MHCC97H cells. (H) CCK‐8 assay of METTL1 knockdown MHCC97H cells with or without overexpression of EGFR. Data presented as mean ± SD (six technical replicates). (I) Representative images and quantification of migration in METTL1‐knockdown MHCC97H cells with or without overexpression of EGFR. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (J) Representative images and quantification of invasion in METTL1‐knockdown MHCC97H cells with or without overexpression of EGFR. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. All the in vitro assays were biologically repeated for three times. Abbreviations: NC, negative control; oeEGFR, overexpression of EGFR; oeLysCTT, overexpression of LysCTT; siM1, siMETTL1‐1

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Over Expression, Expressing, Northern Blot, Western Blot, CCK-8 Assay, Migration, MANN-WHITNEY, In Vitro, Negative Control

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: Liver‐specific knockout of Mettl1 inhibits HCC tumourigenesis in vivo. (A) The schematic diagram of the construction of Mettl1 conditional liver‐specific knockout mice. (B) Representative image of livers harvested from Mettl1‐cKO and control group. (C) Comparison of ratio of liver weight to body weight between Mettl1‐cKO and control group. Data presented as mean ± SD ( n = 5). (D) Representative images of H&E staining of control and Mettl1‐cKO mouse livers. Scale bar: 100 μm. (E) IHC staining of Ki‐67 in control and Mettl1‐cKO mouse livers. Arrows point to Ki67‐positive cells. Scale bar:100 μm. Data presented as mean ± SD ( n = 5). (F) General view of hydrodynamics transfection experiment in Mettl1 conditional knockout mice and control. (G) Representative image of livers harvested from Mettl1‐cKO and control group. (H) Comparison of tumour burden between Mettl1‐cKO and control group by ratio of liver weight to body weight (LW/BW). Data presented as mean ± SD ( n = 9). (I) Representative images of H&E staining of control and Mettl1‐cKO mouse livers. Arrows point to tumour lesions. Scale bar: 250 μm. The number of tumour foci in these mouse livers was evaluated. Data presented as mean ± SD ( n = 9). (J) Left panel, representative images of IHC staining of Ki‐67. Arrows point to Ki67‐positive cells. Scale bar:100 μm. Right panel, the statistical analyses. Data presented as mean ± SD ( n = 9). (K) Western blot of Mettl1, EGFR, Cyclin A2 and AFP in tumour tissues from control group and Mettl1‐cKO group. β‐Actin was used as a loading control. Downregulation of m 7 G tRNA modification and LysCTT in Mettl1‐cKO group was confirmed by northwestern blot and northern blot. U6 was used as a loading control. (L) qRT‐PCR analysis of Cyclin A2 and EGFR mRNA levels in tumour tissues from control group and Mettl1‐cKO group. Data presented as mean ± SD ( n = 3). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. All the in vitro assays were biologically repeated for three times. Abbreviations: cKO, conditional knockout; Ctrl, control

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Knock-Out, In Vivo, Staining, Immunohistochemistry, Transfection, Western Blot, Modification, Northern Blot, Quantitative RT-PCR, MANN-WHITNEY, In Vitro

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: Overexpression of METTL1 promotes HCC progression in vivo. (A) Representative image of livers harvested from Mettl1‐KI and control group. (B) Comparison of ratio of liver weight to body weight between Mettl1‐KI and control group. Data presented as mean ± SD ( n = 5). (C) Representative images of H&E staining of control and Mettl1‐KI mouse livers. Scale bar: 100 μm. (D) IHC staining of Ki‐67 in control and Mettl1‐KI mouse livers. Arrows point to Ki67‐positive cells. Scale bar: 50 μm. Data presented as mean ± SD ( n = 5). (E) General view of hydrodynamics transfection experiment. AKT and NRAS plasmids along with SB transposase were injected into control and Mettl1 knockin mice (Mettl1‐KI mice). The mice were sacrificed after 4 weeks. n = 6. (F) Representative image of livers harvested from Mettl1‐KI and control group. (G) Comparison of tumourigenic capacity between Mettl1‐KI and control group by ratio of liver weight to body weight (LW/BW). Data presented as mean ± SD ( n = 6). (H) Representative images of H&E staining of control and Mettl1‐KI mouse livers. Scale bar: 100 μm. The number of tumour foci in these mouse livers was evaluated. P, peri‐tumour; T, tumour. Data presented as mean ± SD ( n = 6). (I) IHC staining of Ki‐67. Scale bar: 50 μm. Arrows point to Ki67‐positive cells. Data presented as mean ± SD ( n = 6). (J) Western blot of Mettl1, EGFR, Cyclin A2 and AFP in tumour tissues from control group and Mettl1‐KI group. β‐Actin was used as a loading control. Upregulation of m 7 G tRNA modification and LysCTT in Mettl1‐KI group was confirmed by northwestern blot and northern blot. U6 was used as a loading control. (K) qRT‐PCR analysis of Cyclin A2 and EGFR mRNA levels in tumour tissues from control group and Mettl1‐KI group. Data presented as mean ± SD ( n = 3). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. All the in vitro assays were biologically repeated for three times. Abbreviations: Ctrl, control; KI, knockin

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Over Expression, In Vivo, Staining, Immunohistochemistry, Transfection, Injection, Knock-In, Western Blot, Modification, Northern Blot, Quantitative RT-PCR, MANN-WHITNEY, In Vitro

Journal: ACS Bio & Med Chem Au

Article Title: Small-Molecule Inhibitors of the m7G-RNA Writer METTL1

doi: 10.1021/acsbiomedchemau.3c00030

Figure Lengend Snippet: Crystal structures of the complex of METTL1 and (A) SAH and (B) sinefungin. The carbon atoms of SAH (PDB code 7OGJ ) and sinefungin (PDB code 7PL1 ) are in orange and those of the protein in white, while hydrogen bonds are represented as yellow dashed lines.

Article Snippet: The plasmid encoding the hexahistidine-tagged METTL1 Δ 1–31, 266–276 construct was a gift from Cheryl Arrowsmith (Addgene ID: 25264).

Techniques:

Journal: ACS Bio & Med Chem Au

Article Title: Small-Molecule Inhibitors of the m7G-RNA Writer METTL1

doi: 10.1021/acsbiomedchemau.3c00030

Figure Lengend Snippet: Time series of the root-mean-square deviation (RMSD) of the five compounds and SAH, which is used as a control, in the SAM binding pocket of METTL1. The RMSD was calculated between the adenine ring atoms of each compound and the adenine ring of SAH in the crystal structure (PDB code: 7OGJ ). For each compound, the eight independent MD runs are shown in different colors.

Article Snippet: The plasmid encoding the hexahistidine-tagged METTL1 Δ 1–31, 266–276 construct was a gift from Cheryl Arrowsmith (Addgene ID: 25264).

Techniques: Binding Assay

Journal: ACS Bio & Med Chem Au

Article Title: Small-Molecule Inhibitors of the m7G-RNA Writer METTL1

doi: 10.1021/acsbiomedchemau.3c00030

Figure Lengend Snippet: Predicted binding poses of compounds (A) 1 , (B) 2 , (C) 5 (S,S), and (D) 6 . The carbon atoms of the inhibitors are in orange and those of METTL1 in white, and hydrogen bonds and salt bridges are represented as yellow dashed lines. The binding mode of SAH (carbon atoms in green, from PDB entry 7OGJ ) is shown as a basis of comparison.

Article Snippet: The plasmid encoding the hexahistidine-tagged METTL1 Δ 1–31, 266–276 construct was a gift from Cheryl Arrowsmith (Addgene ID: 25264).

Techniques: Binding Assay, Comparison

Journal: Nucleic Acids Research

Article Title: METTL1 interacts with XPO5 to modulate pre-miRNA export

doi: 10.1093/nar/gkag037

Figure Lengend Snippet: APEX-based proximity labeling led to the identification of XPO5 as an interaction partner of METTL1. ( A ) A schematic illustration of the APEX labeling workflow. ( B ) Western blot analysis showing comparable APEX2 labeling efficiencies among METTL1-APEX, EGFP-APEX, and APEX-NLS. HEK293T cells were transfected with the respective plasmids for 24 h, treated with biotin phenol for 30 min, and subsequently exposed to H 2 O 2 for 1 min or left untreated. Biotinylated proteins were detected using streptavidin-horseradish peroxidase (SA-HRP). α-tubulin was used as a control to confirm equal protein loading. ( C ) Venn diagrams of proteins identified in the METTL1 proximal proteome. ( D ) Co-IP followed by western blot analysis in HEK293T cells showed a preferential interaction between overexpressed Flag-tagged METTL1 and endogenous XPO5. HEK293T cells were transfected with empty vector or Flag-tagged METTL1, followed by anti-Flag pull-down and western blot analysis. α-tubulin served as a loading control. XPO5 enrichment was quantified by normalizing the amount of immunoprecipitated XPO5 to its corresponding input level (mean ± SEM, *** P < .0001, unpaired t -test, n = 3).

Article Snippet: Antibodies recognizing human XPO5 (Proteintech, #28628-1-AP; 1:2000), V5 (Proteintech, #14440-1-AP; 1:2000), streptavidin (Thermo Scientific, #S911), METTL1 (Proteintech, #14994-1-AP; 1:1000), ERK (Santa Cruz, SC-514302; 1:100), and p-ERK (Cell Signaling, #4370T; 1:1000) were used as primary antibodies for western blot analysis.

Techniques: Labeling, Western Blot, Transfection, Control, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: METTL1 interacts with XPO5 to modulate pre-miRNA export

doi: 10.1093/nar/gkag037

Figure Lengend Snippet: METTL1 modulates the nucleocytoplasmic transport of pre-miRNAs through a mechanism that is independent of its catalytic activity. ( A, B ) RT-qPCR analysis of pre-miRNAs (top) and the corresponding cytoplasmic miRNAs (bottom) in the HEK293T cell lines. “ METTL1 −/− ” indicates HEK293T cells with METTL1 being knocked out; “ METTL1 −/− + WT” and “ METTL1 −/− + MT” represent METTL1 −/− cells reconstituted with WT METTL1 and its catalytically inactive mutant, respectively. All data were normalized to the mRNA level of GAPDH . The P -values were calculated using an unpaired, two-tailed Student’s t -test: ns, not significant ( P > .05); *, .01 ≤ P < .05; **, .001 ≤ P < .01; ***, P < .001.

Article Snippet: Antibodies recognizing human XPO5 (Proteintech, #28628-1-AP; 1:2000), V5 (Proteintech, #14440-1-AP; 1:2000), streptavidin (Thermo Scientific, #S911), METTL1 (Proteintech, #14994-1-AP; 1:1000), ERK (Santa Cruz, SC-514302; 1:100), and p-ERK (Cell Signaling, #4370T; 1:1000) were used as primary antibodies for western blot analysis.

Techniques: Activity Assay, Quantitative RT-PCR, Mutagenesis, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: METTL1 interacts with XPO5 to modulate pre-miRNA export

doi: 10.1093/nar/gkag037

Figure Lengend Snippet: METTL1 promotes nuclear retention of XPO5 through promoting ERK activation. ( A ) Western blot illustrating the XPO5 expression levels in the whole-cell lysates (WCL), as well as the cytoplasmic and nuclear fractions of control and METTL1 knockout cells. The result demonstrates a significant increase in cytosolic XPO5 and a significant decrease in nuclear XPO5 in METTL1 − /− cells, whereas total XPO5 remains unchanged. α-tubulin and GAPDH were used as loading controls for cytosolic fraction, and Lamin B1 as a loading control for the nuclear fraction. ( B ) Western blot analysis of ERK and p-ERK in HEK293T and METTL1 − /− cells. While total ERK expression remained unchanged, p-ERK levels were significantly reduced in the absence of METTL1. ( C ) Co-IP followed by western blot analysis showing the interaction between METTL1 and p-ERK. The P values were calculated using paired, two-tailed Student’s t -test: ns, not significant ( P > .05); *, .01 ≤ P < .05; **, .001 ≤ P < .01.

Article Snippet: Antibodies recognizing human XPO5 (Proteintech, #28628-1-AP; 1:2000), V5 (Proteintech, #14440-1-AP; 1:2000), streptavidin (Thermo Scientific, #S911), METTL1 (Proteintech, #14994-1-AP; 1:1000), ERK (Santa Cruz, SC-514302; 1:100), and p-ERK (Cell Signaling, #4370T; 1:1000) were used as primary antibodies for western blot analysis.

Techniques: Activation Assay, Western Blot, Expressing, Control, Knock-Out, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: METTL1 interacts with XPO5 to modulate pre-miRNA export

doi: 10.1093/nar/gkag037

Figure Lengend Snippet: ERK activation restores XPO5’s nuclear retention in METTL1 −/− cells. ( A ) Western blot analysis of nuclear XPO5 in METTL1 −/− cells transfected with Flag-MEK1 or Flag-MEKDD. ( B ) Western blot analysis showing p-ERK and XPO5 in HEK293T (labeled as “Ctrl”) and the isogenic METTL1 −/− cells. GAPDH was used as a loading control. ( C ) Subcellular fractionation analysis of XPO5 in cytosolic and nuclear compartments in HEK293T and METTL1 −/− cells expressing Flag-MEKDD. GAPDH and Lamin B1 were used as loading controls for the cytosolic and nuclear fractions, respectively. The P -values were calculated using a paired, two-tailed Student’s t -test: ns, not significant ( P > .05); *, .01 ≤ P < .05.

Article Snippet: Antibodies recognizing human XPO5 (Proteintech, #28628-1-AP; 1:2000), V5 (Proteintech, #14440-1-AP; 1:2000), streptavidin (Thermo Scientific, #S911), METTL1 (Proteintech, #14994-1-AP; 1:1000), ERK (Santa Cruz, SC-514302; 1:100), and p-ERK (Cell Signaling, #4370T; 1:1000) were used as primary antibodies for western blot analysis.

Techniques: Activation Assay, Western Blot, Transfection, Labeling, Control, Fractionation, Expressing, Two Tailed Test