Journal: Molecular cell

Article Title: Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases m 6 A Methylation

doi: 10.1016/j.molcel.2020.10.026

Figure Lengend Snippet: (A) Comparison of METTL3 and WTAP protein levels in mESCs and A375 stable transfectants by immunoblotting (IB).

Article Snippet: Mouse METTL3 (MR209093), mouse WTAP (MR216877), and human USP5 (RC224191) were purchased from OriGene.

Techniques: Western Blot

Journal: Molecular cell

Article Title: Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases m 6 A Methylation

doi: 10.1016/j.molcel.2020.10.026

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse METTL3 (MR209093), mouse WTAP (MR216877), and human USP5 (RC224191) were purchased from OriGene.

Techniques: Recombinant, Luciferase, Mutagenesis, Modification, Multiplex Assay, CRISPR, Generated, Software

Journal: Cellular & Molecular Biology Letters

Article Title: WTAP-mediated N 6 -methyladenosine modification of NLRP3 mRNA in kidney injury of diabetic nephropathy

doi: 10.1186/s11658-022-00350-8

Figure Lengend Snippet: WTAP is highly expressed in DN tissues and HG-induced HK-2 cells. A m 6 A levels in control ( n = 10) and DN tissues ( n = 63) measured by ELISA. B , C mRNA expression of METTL3 and WTAP in the control ( n = 10) and DN tissues ( n = 63) measured by real-time (RT)-qPCR. D , E Pearson correlation scatter plots ( n = 63). F , G Protein expression of WTAP in the control ( n = 10) and DN tissues ( n = 63) measured by immunohistochemical staining. Scale bar, 50 μm. ( H – J ) mRNA and protein expression of WTAP in NG- or HG-induced HK-2 cells measured by RT-qPCR and western blot ( n = 3). Unpaired Student’s t -test was used for the analysis between two groups, and one-way analysis of variance was used to analyze the data among multiple groups, followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with control or 0 h

Article Snippet: The human WTAP shRNAs and mouse WTAP shRNAs used were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: WTAP-mediated N 6 -methyladenosine modification of NLRP3 mRNA in kidney injury of diabetic nephropathy

doi: 10.1186/s11658-022-00350-8

Figure Lengend Snippet: Knockdown of WTAP inhibits high glucose (HG)-induced cell pyroptosis and pro-inflammatory factor release in HK-2 cells. A Cell viability; ( B , C ) cell pyroptosis; ( D ) LDH activity; E , F expression of WTAP, NLRP3, pro-caspase-1, active caspase-1, GSDMD, and GSDMD-N; and ( G , H ) the release of IL-18, IL-1β, TNF-α, and IL-6 in HK-2 cells treated with NG or HG with or without WTAP knockdown ( n = 3). One-way analysis of variance was used to analyze the data among multiple groups, followed by Tukey’s post hoc test. *** P < 0.001 compared with NG + shNC. ## P < 0.01, ### P < 0.001 compared with HG + shNC

Article Snippet: The human WTAP shRNAs and mouse WTAP shRNAs used were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Knockdown, Activity Assay, Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: WTAP-mediated N 6 -methyladenosine modification of NLRP3 mRNA in kidney injury of diabetic nephropathy

doi: 10.1186/s11658-022-00350-8

Figure Lengend Snippet: WTAP overexpression promotes cell pyroptosis and pro-inflammatory factor release in HK-2 cells. A Cell viability; ( B , C ) cell pyroptosis; ( D ) LDH activity; ( E , F ) expression of WTAP, NLRP3, pro-caspase-1, active caspase-1, GSDMD and GSDMD-N; and ( G , H ) the release of IL-18, IL-1β, TNF-α, and IL-6 in HK-2 cells with or without WTAP overexpression ( n = 3). Unpaired Student’s t -test was used for the analysis between two groups. ** P < 0.01, *** P < 0.001 compared with vector

Article Snippet: The human WTAP shRNAs and mouse WTAP shRNAs used were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Activity Assay, Expressing, Plasmid Preparation

Journal: Cellular & Molecular Biology Letters

Article Title: WTAP-mediated N 6 -methyladenosine modification of NLRP3 mRNA in kidney injury of diabetic nephropathy

doi: 10.1186/s11658-022-00350-8

Figure Lengend Snippet: NLRP3 acts as a target of WTAP in high glucose (HG)-induced HK-2 cells. A – G HK-2 cells treated with NG or HG with or without WTAP knockdown or overexpression ( n = 3). A m 6 A levels measured by ELISA; B , C RNA immunoprecipitation (RIP) using anti-m 6 A antibody and RT-qPCR analysis of NLRP3 5′UTR and 3′UTR m 6 A levels; D , E relative mRNA and protein expression of WTAP and NLRP3 detected by RT-qPCR and western blot; F , G luciferase activity of NLRP3 5′UTR and 3′UTR; H NLRP3 mRNA level quantified by RT-qPCR in HK-2 cells with or without IGF2BP knockdown ( n = 3); I half-life of the NLRP3 transcript measured by RT-qPCR in HK-2 cells with or without IGF2BP1 knockdown ( n = 3). J – K The binding of IGF2BP1 to NLRP3 mRNA measured by RIP and RT-qPCR ( n = 3). Unpaired Student’s t -test was used for the analysis between two groups, and one-way analysis of variance was used to analyze the data among multiple groups, followed by Tukey’s post hoc test. ** P < 0.01, *** P < 0.001 compared with NG + shNC + vector or siNC. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with HG + shNC + vector

Article Snippet: The human WTAP shRNAs and mouse WTAP shRNAs used were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Knockdown, Over Expression, Enzyme-linked Immunosorbent Assay, RNA Immunoprecipitation, Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Activity Assay, Binding Assay, Plasmid Preparation

Journal: Cellular & Molecular Biology Letters

Article Title: WTAP-mediated N 6 -methyladenosine modification of NLRP3 mRNA in kidney injury of diabetic nephropathy

doi: 10.1186/s11658-022-00350-8

Figure Lengend Snippet: WTAP overexpression promotes cell pyroptosis and pro-inflammatory factor release in HK-2 cells by targeting NLRP3. A , B Cell pyroptosis; C lactate dehydrogenase activity; D , E expression of NLRP3, pro-caspase-1, active caspase-1, GSDMD, and GSDMD-N; ( F , G ) release of IL-18, IL-1β, TNF-α, and IL-6 in HK-2 cells with or without WTAP overexpression and NLRP3 knockdown ( n = 3). *** P < 0.001 compared with vector + siNC. One-way analysis of variance was used to analyze the data among multiple groups, followed by Tukey’s post hoc test. ## P < 0.01, ### P < 0.001 compared with WTAP-OE + siNC

Article Snippet: The human WTAP shRNAs and mouse WTAP shRNAs used were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Activity Assay, Expressing, Knockdown, Plasmid Preparation

Journal: Cellular & Molecular Biology Letters

Article Title: WTAP-mediated N 6 -methyladenosine modification of NLRP3 mRNA in kidney injury of diabetic nephropathy

doi: 10.1186/s11658-022-00350-8

Figure Lengend Snippet: WTAP knockdown inhibits cell pyroptosis and pro-inflammatory factor release in db/db mice. A H&E and Masson’s trichrome staining of mouse kidney tissues ( n = 6). Scale bar, 50 μm. B – D Serum creatinine, serum blood urea nitrogen, and urine protein levels ( n = 6). ( E ) m 6 A levels measured by ELISA in primary tubular epithelial cells ( n = 6). F RIP and RT-qPCR analysis of NLRP3 m 6 A levels in primary tubular epithelial cells ( n = 6). G , H Expression of WTAP, NLRP3, pro-caspase-1, active caspase-1, GSDMD, and GSDMD-N in primary tubular epithelial cells ( n = 3). ( I , J ) Serum levels of IL-18, IL-1β, TNF-α, and IL-6 ( n = 6). K Pearson correlation scatter plots ( n = 63). One-way analysis of variance was used to analyze the data among multiple groups, followed by Tukey’s post hoc test. *** P < 0.001 compared with db/m + shNC. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with db/db + shNC

Article Snippet: The human WTAP shRNAs and mouse WTAP shRNAs used were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Knockdown, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: WTAP-mediated N 6 -methyladenosine modification of NLRP3 mRNA in kidney injury of diabetic nephropathy

doi: 10.1186/s11658-022-00350-8

Figure Lengend Snippet: Histone acetyltransferase p300 promotes the transcription of the WTAP promoter through H3K27 acetylation. HG-induced HK-2 cells were treated with 10 μM histone acetyltransferase p300 inhibitor (C646). A – C WTAP expression and H3K27 acetylation (H3K27ac). D H3K27ac at WTAP promoter region. E Luciferase activity of WTAP promoter in H3 wild type (wtH3) or K27R-mutant (K27R) HK-2 cells treated with 10 μM C646 ( n = 3). F Schematic diagram of the relationship between WTAP, m 6 A modification, HK-2 cell pyroptosis, and inflammation. One-way analysis of variance was used to analyze the data among multiple groups, followed by Tukey’s post hoc test. * P < 0.05, *** P < 0.001 compared with 0 h

Article Snippet: The human WTAP shRNAs and mouse WTAP shRNAs used were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Techniques: Expressing, Luciferase, Activity Assay, Mutagenesis, Modification