rabbit anti mettl1  (Cell Signaling Technology Inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: m7G Methyltransferase METTL1 Promotes Post-ischemic Angiogenesis via Promoting VEGFA mRNA Translation

doi: 10.3389/fcell.2021.642080

Figure Lengend Snippet: m7G and METTL1 are decreased in ischemic gastrocnemius. (A) Quantitative LC-MS/MS analysis of m7G/G of mRNA from the tissues post-ischemia. (B) Quantification of METTL1 and WDR4 mRNA expression and β-actin was used as a loading control. (C) Representative blot image (left panel) and quantitative analysis (right panel) of METTL1 protein expression upon ischemia or non-ischemia. β-actin was used as a loading control. N = 6 and all data are presented as the mean ± SD. * P < 0.05; ** P < 0.01 and ns indicate no significance.

Article Snippet: Antibodies used in this study for Western blot are listed as follows: Mouse anti-METTL1 (1:1,000 dilution, #11525-MM05, Sino Biological), Rabbit anti-METTL1 (1:1,000 dilution, #ab157097, Sino Biological), Mouse anti-VEGFA (1:500, ab1316, Abcam), Goat anti-mouse lgG-HRP (1:3,000 dilution, sc-2005; Santa Cruz), Goat anti-rabbit lgG-HRP (1:5,000, #7074, CST), HRP-linked mouse anti-β-Actin (1:4,000 dilution, #ab20272, Abcam).

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: m7G Methyltransferase METTL1 Promotes Post-ischemic Angiogenesis via Promoting VEGFA mRNA Translation

doi: 10.3389/fcell.2021.642080

Figure Lengend Snippet: Improved blood flow recovery and increased angiogenesis with enhanced m7G upon METTL1 overexpression post-ischemic injury. (A) Schematic illustration of hind-limb ischemia design with METTL1 overexpression. The blood flow recovery was scanned at indicated time points. Twenty-one days post-ischemia, samples were collected for subsequent experiments. (B) Quantification of RT-qPCR data for METTL1 mRNA expression. β-actin was used as a loading control. (C) Representative blot image showing METTL1 protein expression. Protein expression was normalized to β-actin. (D) Quantitative LC-MS/MS analysis of mRNA m7G levels. (E) Representative Laser Doppler images of blood flow recovery scan. (F) Quantitative analysis of blood flow recovery data. (G) Representative frozen section immunofluorescence images of the angiogenesis markers CD31 and α-SMA expression. (H) Quantitative analysis of capillary density (CD31/DAPI) and small artery density (α-SMA/mm 2 ). N = 6 and all data are presented as the mean ± SD. * P < 0.05; ** P < 0.01 and ns indicate no significance compared to the OE-Ctrl.

Article Snippet: Antibodies used in this study for Western blot are listed as follows: Mouse anti-METTL1 (1:1,000 dilution, #11525-MM05, Sino Biological), Rabbit anti-METTL1 (1:1,000 dilution, #ab157097, Sino Biological), Mouse anti-VEGFA (1:500, ab1316, Abcam), Goat anti-mouse lgG-HRP (1:3,000 dilution, sc-2005; Santa Cruz), Goat anti-rabbit lgG-HRP (1:5,000, #7074, CST), HRP-linked mouse anti-β-Actin (1:4,000 dilution, #ab20272, Abcam).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Control, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: m7G Methyltransferase METTL1 Promotes Post-ischemic Angiogenesis via Promoting VEGFA mRNA Translation

doi: 10.3389/fcell.2021.642080

Figure Lengend Snippet: Increased HUVECs angiogenesis with enhanced m7G upon METTL1 overexpression post-hypoxic injury. (A) Quantification of HUVECs viability in different hypoxic time points without FBS supplemented in the culture medium. (B) Quantitative LC-MS/MS analysis of HUVECs mRNA m7G post-hypoxia. (C) Quantification of RT-qPCR data for HUVECs METTL1 and WDR4 mRNA expression post-hypoxia. (D) Representative blot image showing METTL1 protein expression post-hypoxia. (E) Representative blot image showing METTL1 protein overexpression efficacy post-hypoxia. (F) Quantitative LC-MS/MS analysis of mRNA m7G levels upon METTL1 overexpression post-hypoxia. (G) Quantification of HUVECs viability upon METTL1 overexpression post-hypoxia. (H) Representative EdU staining (left panel) and quantitative analysis (right panel) of relative EdU positive HUVECs proportion (EdU + /DAPI) upon METTL1 overexpression post-hypoxia. (I) Representative scratch closure images (left panel) and quantitative analysis (right panel) of relative scratch area upon METTL1 overexpression post-hypoxia. (J) Representative transwell images (left panel) and quantitative analysis (right panel) of relative migrated HUVECs numbers upon METTL1 overexpression post-hypoxia. (K) Representative tube formation images (left panel) and quantitative analysis (right panel) of relative tube formation upon METTL1 overexpression post-hypoxia. All experiments were from 4 independent replicates, and data are presented as the mean ± SD. * P < 0.05; ** P < 0.01 and ns indicate no significance.

Article Snippet: Antibodies used in this study for Western blot are listed as follows: Mouse anti-METTL1 (1:1,000 dilution, #11525-MM05, Sino Biological), Rabbit anti-METTL1 (1:1,000 dilution, #ab157097, Sino Biological), Mouse anti-VEGFA (1:500, ab1316, Abcam), Goat anti-mouse lgG-HRP (1:3,000 dilution, sc-2005; Santa Cruz), Goat anti-rabbit lgG-HRP (1:5,000, #7074, CST), HRP-linked mouse anti-β-Actin (1:4,000 dilution, #ab20272, Abcam).

Techniques: Over Expression, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR, Expressing, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: m7G Methyltransferase METTL1 Promotes Post-ischemic Angiogenesis via Promoting VEGFA mRNA Translation

doi: 10.3389/fcell.2021.642080

Figure Lengend Snippet: METTL1 promoted HUVECs angiogenesis via increasing VEGFA translation. (A) Quantification of MeRIP-qPCR shows the m7G fold enrichment of VEGFA mRNA. (B) Quantification of relative METTL1 and VEGFA mRNA expression. (C) Representative Western blot images of VEGFA protein expression. (D) Quantification of VEGFA concentration in the supernatant of HUVECs culture media. (E) Representative EdU staining images (left panel) and quantitative analysis (right panel) of EdU positive HUVECs proportion. (F) Representative scratch closure images (upper panel) and quantitative analysis (lower panel) of relative scratch area alteration. (G) Representative transwell images (upper panel) and quantitative analysis (lower panel) of relative migrated HUVECs numbers. (H) Representative tube formation images (upper panel) and quantitative analysis (lower panel) of relative tube formation. All experiments were from 4 independent replicates, and data are presented as the mean ± SD. * P < 0.05; ** P < 0.01 and ns indicate no significance.

Article Snippet: Antibodies used in this study for Western blot are listed as follows: Mouse anti-METTL1 (1:1,000 dilution, #11525-MM05, Sino Biological), Rabbit anti-METTL1 (1:1,000 dilution, #ab157097, Sino Biological), Mouse anti-VEGFA (1:500, ab1316, Abcam), Goat anti-mouse lgG-HRP (1:3,000 dilution, sc-2005; Santa Cruz), Goat anti-rabbit lgG-HRP (1:5,000, #7074, CST), HRP-linked mouse anti-β-Actin (1:4,000 dilution, #ab20272, Abcam).

Techniques: Expressing, Western Blot, Concentration Assay, Staining

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 expression is upregulated in LUAD, and its increased expression is associated with unfavorable OS and FP. (A) METTL1 expression profile in LUAD tissues (n=483) and normal lung tissues (n=59) from TCGA database was analyzed using Gene Expression Profiling Interactive Analysis software. METTL1 expression was significantly upregulated in LUAD tissues. (B) METTL1 expression was significantly higher in different LUAD stages compared with normal tissues. No significant differences were observed among the different tumor stages. The METTL1 expression profile in different LUAD stages (stage I, n=277; stage II, n=125; stage III, n=85 and stage IV, n=28) and normal lung tissues (n=59) from TCGA database was analyzed using UALCAN software. (C) The METTL1 expression profile in LUAD tissues (n=58) and normal lung tissues (n=49) from the GSE10072 dataset in the GEO database was analyzed using GraphPad Prism 5.0 software. METTL1 expression was significantly upregulated in LUAD tissues. (D) METTL1 expression was significantly higher in different LUAD stages compared with normal tissues. No significant differences were observed among the different stages. The METTL1 expression profile in different stages (stage I, n=22; stage II, n=21; stage III, n=12 and stage IV, n=3) and normal lung tissues (n=49) from the GSE10072 dataset in the GEO database was analyzed using GraphPad Prism 5.0 software. (E) METTL1 expression was upregulated in LUAD tissues. Representative images of immunohistochemistry staining from the Human Protein Atlas database. Scale bar at low magnification, 200 µM; high magnification, 50 µM. HPA Patients’ ID, negative: 2268, medium: 3052, low: 2403. (F) High METTL1 expression was associated with unfavorable OS. The OS curve of 719 patients was plotted [cut-off value, 347; HR=1.7 (1.35–2.15), 95% CI; log-rank P=6×10 −6 −05]. (G) High METTL1 expression was associated with unfavorable FP. The FP curve of 461 patients was plotted [cut-off value, 324; HR=2.26 (1.65–3.1), 95% CI; log-rank P=2.2×10 −7 ]. The online Kaplan Meier Plotter software was used to construct the OS and FP graphs. *P<0.05, ***P<0.001. METTL1, methyltransferase-like 1; LUAD, lung adenocarcinoma; OS, overall survival; FP, first progression; TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus; HR, hazard ratio; CI, confidence interval.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing, Gene Expression, Software, Immunohistochemistry, Staining, Construct

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: Association between METTL1 expression and the clinicopathological characteristics of patients with lung adenocarcinoma (n=517).

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: Cox regression multivariate analysis of METTL1 on OS and FP of patients with lung adenocarcinoma.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques:

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 promotes colony formation and A549 cell proliferation. (A) METTL1 expression was increased in A549 cells. (B) Western blot analysis demonstrated that METTL1 protein was upregulated and downregulated in the overexpression and knockdown experiments, respectively. Overexpression of METTL1 promoted A549 and H1993 cell (C) proliferation and (D) colony formation. METTL1 silencing attenuated A549 and H1993 cell (C) proliferation and (D) colony formation. *P<0.05, **P<0.01, ***P<0.001. METTL1, methyltransferase-like 1; si, small interfering; OD, optical density.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing, Western Blot, Over Expression, Knockdown

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 inhibits autophagy in A549 cells. (A) METTL1 overexpression inhibited the conversion of LC3-I to LC3-II and the degradation of p62/SQSTM1, suggesting that METTL1 overexpression may inhibit autophagy. (B) METTL1 knockdown enhanced the conversion of LC3-I to LC3-II and the degradation of p62/SQSTM1, suggesting that METTL1 knockdown may promote autophagy. (C) METTL1 knockdown increased both the size and number of GFP-LC3 puncta in HCC827/GFP-LC3 cells, suggesting that METTL1 knockdown may promote autophagy. *P<0.05, **P<0.01, ***P<0.001. METTL1, methyltransferase-like 1; SQSTM1, sequestosome 1; si, small interfering.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Over Expression, Knockdown

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 activates the AKT/mTORC1 signaling pathway. GSEA analysis of the TCGA-LUAD dataset revealed that the gene sets (A) HALLMARK_MTORC1_SIGNALING and (B) HALLMARK_PI3K_AKT_MTOR_SIGNALING were enriched in the high-METTL1 expression group. (C) DAVID analysis of the GSE112180 dataset indicated that the gene cluster of the PI3K/AKT signaling pathway was enriched in the high-METTL1 expression group (D) METTL1 overexpression increased the protein levels of p-AKT and p-S6K, the effects of which were reversed following METTL1 silencing. Total protein levels of AKT and S6K remained unchanged compared with the control group. *P<0.05. METTL1, methyltransferase-like 1; p-AKT, phosphorylated protein kinase B; mTORC1, mechanistic target of rapamycin complex 1; GSEA, Gene Set Enrichment Analysis; TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; DAVID, Database for Annotation, Visualization and Integrated Discovery; PI3K, phosphatidylinositol 3-kinase; si, small interfering.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing, Over Expression, Control