Journal: BMC Cancer

Article Title: N6-methyladenosine RNA modification (m6A) is of prognostic value in HPV-dependent vulvar squamous cell carcinoma

doi: 10.1186/s12885-022-10010-x

Figure Lengend Snippet: Summary of the analyzed m6A proteins as indicated and their correlation with overall survival (indicated as %alive) for the entire study cohort, HPV-independent, and HPV-dependent VSCC. The HPV-status was not available for 24 patients. Samples were grouped according to high and low expression based on the staining intensities. p -values for the group comparisons are based on log-rank tests (significance threshold p < 0.5). q -values are based on multiple hypotheses testing using the method of Benjamini and Hochberg with a significance threshold of q < 0.1

Article Snippet: Immunostaining of METTL3, METTL4, METTL14, WTAP, KIAA1429, FTO, ALKBH5, HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1,YTHDF2, and YTHDF3 was performed on the TMAs using an automated staining system (BenchMark ULTRA; Ventana Medical Systems) which performed deparaffinization, pretreatment with cell conditioning buffer (CC1 buffer, pH8), and incubation with primary antibodies (FTO (1:50; Atlas Antibodies #HPA041086), ALKBH5 (1:200; Novus #NBP1-82,188), METTL3 (1:1000; Biorbyt #orb374082), METTL4 (1:40; Atlas Antibodies #HPA040061), METTL14 (1:100; Atlas Antibodies #HPA038002), WTAP (1:100; Atlas Antibodies #HPA010550), KIAA1429 (1:25; Atlas Antibodies #HPA031530), HNRNPC (1:25; Atlas Antibodies #HPA051075), HNRNPA2B1 (1:100; Atlas Antibodies #HPA001666), YTHDC1 (1:25; Atlas Antibodies #HPA036462), YTHDF1 (1:10; Biorbyt #orb179018), YTHDF2 (1:200; Biorbyt #orb39199), YTHDF3 (1:200; Biorbyt #orb374095) at 4 °C overnight.

Techniques: Expressing, Staining

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: Increase in the nuclear 6mA levels through nuclear activation of METTL4 expression under hypoxia. a An increase in nuclear 6mA levels was observed in BFTC909 and FADU cells under hypoxia (see “Methods”). The collected results were summarized as the ratios of 6mA/dA in the bar graph (lower panel). Corresponding 6mA dot blots with methyl blue loading controls are shown together with bar graphs. N, normoxia; H, hypoxia. Normoxic condition was used as a control. b Immunofluorescence staining shows the increased nuclear staining of 6mA in FADU cells under hypoxia. Staining of cell nuclei by DAPI and mitochondria by MitoTracker was used as controls. Bar graph indicated the percentage of cells containing nuclear 6mA signals. c Detected 6mA and dA in METTL4-induced gDNAs were verified by product ion conformation spectra (PICS) fit to the spectrum generated from each standard. Parental ion of 6mA was m/z 266 with major daughter ion m/z 150. Parental ion of dA was m/z 252 with major daughter ion m/z 136. d Western blot analysis shows the more prominent nuclear activation of METTL4 levels through nuclear fractionation in two different cell lines. Histone H3 and GAPDH was used as a nuclear and cytoplasmic control, respectively. e Immunofluorescence staining shows the increased nuclear METTL4 expression in cells under hypoxia in BFTC909 and FADU cells. Mitotracker: mitochondria DNA. Cell nuclei were stained by DAPI. f Knockdown of METTL4 abolished the increase in nuclear 6mA levels induced by hypoxia in BFTC909 and FADU cells. Corresponding 6mA dot blots are shown. g In vitro DNA methylation assays show an increase in the 6mA levels by incubating METTL4 with genomic DNAs from BFTC909 or FADU cells. The METTL4 mutant and incubation without SAM were used as controls. Corresponding 6mA dot blots are shown. N, normoxia; H, hypoxia. Normoxic condition was used as a control. The asterisk (*) indicated statistical significance ( P <0.05) between experimental and control groups

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: Activation Assay, Expressing, Control, Immunofluorescence, Staining, Generated, Western Blot, Fractionation, Knockdown, In Vitro, DNA Methylation Assay, Mutagenesis, Incubation

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: METTL4 is essential in hypoxia-induced EMT and in vitro / in vivo metastatic activity. a Overexpression of METTL4 induced EMT in BFTC909 and FADU cells by Western blot analysis. The cell clone transfected with the control vector was used as a control. b Knockdown of METTL4 reversed the expression of EMT markers regulated by hypoxia in BFTC909 and FADU cells. c Overexpression of METTL4 induced the expression of a set of EMT transcriptional regulators and knockdown of METTL4 abolished the activation of these EMT regulators induced by hypoxia. d Overexpression of METTL4 induced increased numbers of metastatic lung nodules in mice in tail vein and orthotopic implantation experiments. Representative gross anatomy and histology are shown on the left; measurement of metastatic lung nodules is shown on the right. e Knockdown of METTL4 significantly decreased the increased metastatic lung nodules in mice injected with cells overexpressing a HIF-1α constitutively active mutant. Representative gross anatomy and histology are shown on the left; measurement of metastatic lung nodules is shown on the right. f Hypoxic tumor cells sorted from xenografted tumors from BFTC909 and FADU cells show the increased HIF-1α and METTL4 protein levels together with increased HIF-1α target gene expression. Glut1 activation was used as a positive control. N, normoxia; H, hypoxia. Normoxic cells were used as a control. g Hypoxic tumor cells sorted from xenografted tumors from BFTC909 and FADU cells show an increase in the 6mA levels. Corresponding 6mA dot blots with methyl blue loading controls are shown together with bar graphs. N, normoxia; H, hypoxia. h Immunofluorescence staining shows the co-staining of 6mA and METTL4 in xenografted tumors from BFTC909, FADU, and KTCC28M cells. Green fluorescence: 6mA staining (RNase treatment); red fluorescence: METTL4 staining. Cell nuclei were stained by DAPI. H, highly colocalized area; L, less colocalized area. The asterisk (*) indicated statistical significance ( P <0.05) between experimental and control groups

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: In Vitro, In Vivo, Activity Assay, Over Expression, Western Blot, Transfection, Control, Plasmid Preparation, Knockdown, Expressing, Activation Assay, Injection, Mutagenesis, Targeted Gene Expression, Positive Control, Immunofluorescence, Staining, Fluorescence

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: The essential role of the enzymatic activity of METTL4 in hypoxia-induced phenotypes and clinical implications. a Mutation of the enzymatic site of METTL4 by a prime-cutting CRISPR-Cas9 approach in BFTC909 cells abolished the induction of EMT by hypoxia and significantly decreased the RNA expression of RP11-390F4.3 and Glut1 . The induction of 6mA levels was abolished in enzymatically inactive METTL4 mutant BFTC909 cells. N, normoxia; H, hypoxia. The normoxic condition for METTL4 wild type BFTC909 cells was used as a control. A corresponding 6mA dot blot with methyl blue loading control is shown together with the bar graph. The asterisk (*) indicated statistical significance ( P <0.05) between experimental and control groups. b Immunofluorescence staining shows the abolishment of EMT induction by hypoxia in the enzymatically inactive METTL4 mutant FADU and BFTC909 cells. Green fluorescence represented staining of E-cadherin; red fluorescence represented staining of vimentin. Cell nuclei were stained by DAPI. N, normoxia; H, hypoxia. The normoxic condition for METTL4 wild type BFTC909 and FADU cells were used as a control. c Increased 6mA levels in UTUC, but not in bladder cancer (BC), patient samples. d Increased METTL4, 6mA, and HIF-1α levels by immunohistochemistry staining in the tumor part (T) (vs. the normal part (N)) of UTUC patient samples are shown by H-score measurement. The error bars represented the standard deviation (SD). Student’s t test was used to compare two groups of independent samples. e A representative case of immunohistochemistry staining of UTUC patient samples using antibodies against METTL4, 6mA, and HIF-1α between normal and tumor tissues. f Co-expression of METTL4 and 6mA predicted a poor prognosis of UTUC patients in either overall survival or disease-free survival by Kaplan-Meier analysis. Subgroup analysis of overall survival and disease-free survival of UTUC cases according to the expression profile of METTL4 low/6mA low (Group 1), METTL4 high/6mA high (Group 2), and others (Group 3) in tumors. P values of the comparison between each group are shown

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: Activity Assay, Mutagenesis, CRISPR, RNA Expression, Control, Dot Blot, Immunofluorescence, Staining, Fluorescence, Immunohistochemistry, Standard Deviation, Expressing, Comparison

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: Analysis of RNA-seq and 6mA-ChIP-exo-seq datasets shows hypoxia/METTL4 co-regulated genes and 6mA signals-regulated genes. a The expression heatmap of EMT-related genes in FADU and BFTC909 cells, whose expression increased under hypoxia and decreased in the hypoxic status undergoing METTL4 knockdown. b Venn diagram shows the overlapping set of genes ( n =220) co-regulated by hypoxia and METTL4 through overlapping of upregulated genes under hypoxia and downregulated genes in the hypoxic status undergoing METTL4 knockdown in both FADU and BFTC909 cells. c Pie chart shows the different percentage of hypoxia and METTL4 co-regulated genes according to the classification of gene feature (protein coding, 75%; lncRNA, 15%; others, 10%). d KEGG analysis shows the top 10 enriched pathways in the class of protein-coding genes co-regulated by hypoxia and METTL4. e Heatmap shows the expression of top ten lncRNAs co-regulated by hypoxia and METTL4 using RNA-seq datasets from FADU cells. f Western blot analysis shows that overexpression of lncRNA RP11-390F4.3 activated a set of EMT transcriptional regulators. g Pie chart shows the annotation of hypoxia-induced/METTL4 dependent gain-of-6mA regions located in different genomic regions. h LncRNA RP11-390F4.3 was used as an example of hypoxia-induced/METTL4-dependent 6mA regulated gene that contained hypoxia-induced/METTL4-dependent 6mA signals on its promoter region. Different 6mA motifs were calculated by HOMER. Only motif-10 was indicated on the lncRNA RP11-390F4.3 promoter. A magnified window around the motif-10 area from 3 gene tracks is shown. Y -axis denotes the scale of the number of reads

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: RNA Sequencing Assay, Expressing, Knockdown, Western Blot, Over Expression

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: Characterizations of lncRNA RP11-390F4.3 using different in vitro and in vivo metastatic assays. a Immunofluorescence staining shows the nuclear localization of lncRNA RP11-390F4.3. Cell nuclei were stained by DAPI. b Measurement of the copy number of lncRNA RP11-390F4.3 in FADU cells (normoxia vs. hypoxia). Titration standard curve was used for measurement of the copy number of lncRNA RP11-390F4.3 per 500,000 cells. The red point represents the qRT-PCR value from a standard sample of 500,000 FADU cells under hypoxia. c Knockdown of lncRNA RP11-390F4.3 significantly decreased the induction of various HIF-1α target genes using qRT-PCR analysis. Knockdown using the scrambled control siRNA was used as a control. N, normoxia; H, hypoxia. The asterisk (*) indicated statistical significance ( P <0.05) between experimental and control clones ( n =3). d qChIRP assays show the significantly decreased lncRNA RP11-390F4.3 binding to the promoter regions of EMT regulators and Glut1 gene in hypoxic FADU cells under knockdown of lncRNA RP11-390F4.3 vs. the hypoxic control knockdown cells. e Heatmap analysis of the EMT transcription regulator genes induced by lncRNA RP11-390F4.3 or METTL4 overexpression using RNA-seq datasets. f Western blot analysis shows that knockdown of lncRNA RP11-390F4.3 abolished the EMT phenotypes induced by METTL4 overexpression in FADU and BFTC909 cells. g Overexpression of lncRNA RP11-390F4.3 in BFTC909 cells significantly increased metastatic lung nodules in mice after injection of these cells into mice. Representative gross anatomy and histology are shown on the left, and measurement of metastatic lung nodules is shown on the right. BFTC control-transfected clone was used as a control. h Knockdown of lncRNA RP11-390F4.3 in BFTC909 cells overexpressing METTL4 significantly decreased the metastatic lung nodules in mice. Representative gross anatomy and histology are shown on the left, and measurement of metastatic lung nodules is shown on the right. BFTC control-transfected clone was used as a control. The asterisk (*) indicates statistical significance ( P <0.05) between experimental and control groups

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: In Vitro, In Vivo, Immunofluorescence, Staining, Titration, Quantitative RT-PCR, Knockdown, Control, Clone Assay, Binding Assay, Over Expression, RNA Sequencing Assay, Western Blot, Injection, Transfection

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: Analysis of overlapping datasets regulated by METTL4/6mA, lncRNA RP11-390F4.3 , and HIF-1α, and a summary model. a Venn diagram shows the number of genes that was co-regulated by METTL4/6mA, lncRNA RP11-390F4.3 , and HIF-1α. b KEGG analysis of the 92 co-regulated genes, in which glycolysis and HIF-1 signaling pathways are on the top of the list. c Categorization of the 92 genes shows that they mainly belonged to the main hypoxia-induced phenotypes (angiogenesis, stemness, cancer metabolism). d A model to summarize the results and the mechanisms from this report

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: