Cardiac Protein and Gene Expression Analysis

Western blotting was performed from neonatal rat cardiomyocytes using standard procedures. Antibodies used were METTL3 (Bethyl Laboratories), MAP3K6 (Novus Biologicals), MAP4K5 (Thermo Scientific Pierce), MAPK14 (Cell Signaling Technology), and GAPDH (Fitzgerald Industries). Masson trichrome staining was performed from histological sections generated from paraffin-embedded hearts. Immunostaining was performed using α-actinin antibodies (Sigma-Aldrich), and cell area was quantified using CellProfiler following published methods.^{24 (link)} Detection of the cell membrane was performed using fluorescein isothiocyanate–conjugated wheat germ agglutinin (Sigma-Aldrich). The cross-sectional area was measured using ImageJ. RNA was extracted from neonatal rat cardiomyocytes or mouse hearts using Trizol, and reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Selected genes and m6A peaks were analyzed by real-time polymerase chain reaction using SYBR green (Applied Biosystems). Quantified mRNA expression was normalized to Rpl7 (ribosomal protein L7) and expressed relative to controls.

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Dorn L.E., Lasman L., Chen J., Xu X., Hund T.J., Medvedovic M., Hanna J.H., van Berlo J.H, & Accornero F. (2018). The N6-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy. Circulation, 139(4), 533-545.

Publication 2018

METTL3 MAP3K6 MAP4K5 MAPK14 GAPDH α-actinin antibodies fluorescein isothiocyanate–conjugated wheat germ agglutinin High Capacity cDNA Reverse Transcription kit SYBR green

Actinin Antibodies Biologicals Cardiomyocytes Cdna Cell Cell membrane Embedded paraffin Fluorescein isothiocyanate wheat germ agglutinin Gapdh Genes Hearts Mapk14 Mettl3 Mouse Mrna Neonatal Novus Real time polymerase chain reaction Reverse transcription Ribosomal protein Sybr green Trizol

Corresponding Organization :

Other organizations : The Ohio State University, Weizmann Institute of Science, University of Cincinnati, Minneapolis Heart Institute Foundation, University of Minnesota

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Variable analysis

independent variables

Western blotting performed from neonatal rat cardiomyocytes
Masson trichrome staining performed from histological sections generated from paraffin-embedded hearts
Immunostaining performed using α-actinin antibodies
Detection of the cell membrane performed using fluorescein isothiocyanate–conjugated wheat germ agglutinin
RNA extracted from neonatal rat cardiomyocytes or mouse hearts
Reverse transcription performed using the High Capacity cDNA Reverse Transcription kit
Selected genes and m6A peaks analyzed by real-time polymerase chain reaction using SYBR green

dependent variables

Cell area quantified using CellProfiler
Cross-sectional area measured using ImageJ
Quantified mRNA expression normalized to Rpl7 and expressed relative to controls

control variables

Standard procedures used for Western blotting
Published methods used for cell area quantification
Rpl7 used as a reference gene for mRNA expression normalization

positive controls

GAPDH used as a positive control for Western blotting

negative controls

No negative controls explicitly mentioned

Annotations

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