decr1  (Bioss)

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Therapeutic Targeting of Decr1 Ameliorates Cardiomyopathy by Suppressing Mitochondrial Fatty Acid Oxidation in Diabetic Mice

doi: 10.1002/jcsm.13761

Figure Lengend Snippet: The transcriptional level of Decr1 is upregulated in DCM. (a) Volcano plot showing the differentially expressed genes from GSE155377 database, GSE161827 database, GSE161931 database, and GSE173384 database. (b) Vein diagram showing the upregulated genes from GSE155377 database, GSE161827 database, GSE161931 database, and GSE173384 database. (c) Vein diagram showing the downregulated genes from GSE155377 database, GSE161827 database, GSE161931 database, and GSE173384 database. (d) Seven genes upregulated and downregulated in all four databases. (e) Relative mRNA level of Cpxm2, Oxct1, Cpt1, Decr1, Ucp2, Acot1, and Acot2 in the heart from control mice and T2D mice. Data were calculated as means ± SD. * p < 0.05 vs. Control (Con).

Article Snippet: Regarding DECR1 expression in cardiomyocyte clusters, we observed elevated DECR1 levels in clusters 0 and 1 from HUMAN PROTEIN ATLAS.

Techniques: Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Therapeutic Targeting of Decr1 Ameliorates Cardiomyopathy by Suppressing Mitochondrial Fatty Acid Oxidation in Diabetic Mice

doi: 10.1002/jcsm.13761

Figure Lengend Snippet: Cardiac‐specific knockdown of Decr1 ameliorates DCM in T2D mice. (a) The flow chart of animal experiments. (b) Serum LDH and CK‐MB level. (c) Cardiomyocyte size measured by WGA staining. At least 100 cells measured in different visual fields from 6 samples per group. Each point on each column represents the average area of 100 myocardial cells in each sample. The quantification of average cross‐sectional area of cardiomyocytes compared with control in the indicated groups was shown in the bar graph. (d) The quantification of DHE staining. (e) Representative images of H&E staining, WGA staining, and DHE staining of left ventricle myocardium. (f) Relative mRNA level of Nppa , Nppb , Myh7 , Col1a1 , and Tgfb1 . Data were calculated as means ± SD. * p < 0.05 vs. ShCon. † p < 0.05 vs. T2D + ShCon.

Article Snippet: Regarding DECR1 expression in cardiomyocyte clusters, we observed elevated DECR1 levels in clusters 0 and 1 from HUMAN PROTEIN ATLAS.

Techniques: Knockdown, Staining, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Therapeutic Targeting of Decr1 Ameliorates Cardiomyopathy by Suppressing Mitochondrial Fatty Acid Oxidation in Diabetic Mice

doi: 10.1002/jcsm.13761

Figure Lengend Snippet: Downregulation of Decr1 protects cardiomyocytes against HG/HP‐induced injury. (a) Relative mRNA level of Nppa , Nppb , Myh7 . (b) Cardiomyocyte size measured by α‐actinin staining. (c) Representative images of α‐actinin staining, TUNEL staining, DHE staining, mitochondrial ROS levels, and JC‐1 staining. (d) The quantification of TUNEL staining. (e) The quantification of DHE staining. (f) The quantification of mitochondrial ROS levels. Data were calculated as means ± SD. * p < 0.05 vs. Con siRNA. † p < 0.05 vs. HG/HP + Con siRNA.

Article Snippet: Regarding DECR1 expression in cardiomyocyte clusters, we observed elevated DECR1 levels in clusters 0 and 1 from HUMAN PROTEIN ATLAS.

Techniques: Staining, TUNEL Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Therapeutic Targeting of Decr1 Ameliorates Cardiomyopathy by Suppressing Mitochondrial Fatty Acid Oxidation in Diabetic Mice

doi: 10.1002/jcsm.13761

Figure Lengend Snippet: RNA sequencing revealed that Decr1 upregulated PDK4 in cardiomyocytes. (a) Heatmap showing the differentially expressed genes in the heart after Decr1 overexpression. (b, c) Validation of top 18 upregulated gene mRNA level. (d) The protein expression of Decr1 in diabetic heart and HG/HP‐incubated cardiomyocytes. (e) Relative mRNA level of Nppa , Nppb , Myh7 in cardiomyocytes after knockdown of PDK4. (f) The protein expression of β‐MyHc, IL‐1β, cleaved caspase‐3 in cardiomyocytes after knockdown of PDK4. Data were calculated as means ± SD. * p < 0.05 vs. Vector or Con siRNA. † p < 0.05 vs. HG/HP + Con siRNA.

Article Snippet: Regarding DECR1 expression in cardiomyocyte clusters, we observed elevated DECR1 levels in clusters 0 and 1 from HUMAN PROTEIN ATLAS.

Techniques: RNA Sequencing, Over Expression, Biomarker Discovery, Expressing, Incubation, Knockdown, Plasmid Preparation

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Therapeutic Targeting of Decr1 Ameliorates Cardiomyopathy by Suppressing Mitochondrial Fatty Acid Oxidation in Diabetic Mice

doi: 10.1002/jcsm.13761

Figure Lengend Snippet: Overexpression of PDK4 eliminated the benefits of Decr1 deficiency in DCM. (a) The flow chart of animal experiments. (b) Serum LDH level and CK‐MB level. (c) Cardiomyocyte size measured by WGA staining. At least 100 cells measured in different visual fields from six samples per group. Each point on each column represents the average area of 100 myocardial cells in each sample. The quantification of average cross‐sectional area of cardiomyocytes compared with control in the indicated groups was shown in the bar graph. (d) The quantification of DHE staining. (e) Representative images of H&E staining, WGA staining, and DHE staining of left ventricle myocardium. (f) Relative mRNA level of Nppa , Nppb , Myh7 , Col1a1 , and Tgfb1 . Data were calculated as means ± SD. * p < 0.05 vs. T2D. † p < 0.05 vs. T2D + ShDecr1.

Article Snippet: Regarding DECR1 expression in cardiomyocyte clusters, we observed elevated DECR1 levels in clusters 0 and 1 from HUMAN PROTEIN ATLAS.

Techniques: Over Expression, Staining, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Therapeutic Targeting of Decr1 Ameliorates Cardiomyopathy by Suppressing Mitochondrial Fatty Acid Oxidation in Diabetic Mice

doi: 10.1002/jcsm.13761

Figure Lengend Snippet: Interaction of Decr1 with PDK4 accelerated lipid oxidation and reduced glucose oxidation in DCM. (a) KEGG analysis of the differentially expressed genes in the heart after Decr1 overexpression. (b) GSEA analysis. (c) Helin diagram analysis. Ten significantly enriched GO entries and corresponding differentially expressed genes can be seen. The plot is divided into left and right sides, with the left side showing the top 10 genes with the highest|logFC|in each category of each GO entry, and the right side showing the 10 GO entries with the lowest p ‐value or q‐value selected. The middle line represents the correspondence between genes and entries. The outer heatmap represents the logFC value of the corresponding gene. (d) The protein expression of CD36 and CPT1. (e) Kinetic OCR responses of isolated cardiomyocytes to palmitate acid. (f) Calculated palmitate acid oxidation. Palmitate acid oxidation is calculated by the OCR increase by normalizing cell protein contents. Data were calculated as means ± SD. * p < 0.05 vs. Con siRNA or NG‐PA. † p < 0.05 vs. HG/HP + Con siRNA or HG/HP‐PA.

Article Snippet: Regarding DECR1 expression in cardiomyocyte clusters, we observed elevated DECR1 levels in clusters 0 and 1 from HUMAN PROTEIN ATLAS.

Techniques: Over Expression, Expressing, Isolation

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Therapeutic Targeting of Decr1 Ameliorates Cardiomyopathy by Suppressing Mitochondrial Fatty Acid Oxidation in Diabetic Mice

doi: 10.1002/jcsm.13761

Figure Lengend Snippet: PDK4 induced phosphorylation and mitochondrial translocation of HDAC3 that led to HADHA deacetylation. (a) HADHA deacetylation level in cardiomyocytes after Decr1. (b) Interaction of HADHA with HDAC3 in cardiomyocytes after Decr1. (c–f) The protein of HDAC3 in the mitochondria and non‐mitochondria, as well as HDAC3 phosphorylation after knockdown of Decr1 or PDK4. Data were calculated as means ± SD. * p < 0.05 vs. Con siRNA. † p < 0.05 vs. HG/HP + Con siRNA.

Article Snippet: Regarding DECR1 expression in cardiomyocyte clusters, we observed elevated DECR1 levels in clusters 0 and 1 from HUMAN PROTEIN ATLAS.

Techniques: Phospho-proteomics, Translocation Assay, Knockdown

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Therapeutic Targeting of Decr1 Ameliorates Cardiomyopathy by Suppressing Mitochondrial Fatty Acid Oxidation in Diabetic Mice

doi: 10.1002/jcsm.13761

Figure Lengend Snippet: Atranorin and Kurarinon prevented DCM in T2D mice by binding to and inhibiting Decr1. (a) Molecular docking showing the direct binding of Atranorin to Decr1, and Kurarinon to Decr1. (b) The flow chart of animal experiments. (c) Serum LDH level and CK‐MB level. (d) Cardiomyocyte size measured by WGA staining. At least 100 cells measured in different visual fields from six samples per group. Each point on each column represents the average area of 100 myocardial cells in each sample. The quantification of average cross‐sectional area of cardiomyocytes compared with control in the indicated groups was shown in the bar graph. (e) The quantification of DHE staining. (f) Representative images of H&E staining, WGA staining, and DHE staining of left ventricle myocardium. Data were calculated as means ± SD. * p < 0.05 vs. Control. † p < 0.05 vs. T2D.

Article Snippet: Regarding DECR1 expression in cardiomyocyte clusters, we observed elevated DECR1 levels in clusters 0 and 1 from HUMAN PROTEIN ATLAS.

Techniques: Binding Assay, Staining, Control

Journal: iScience

Article Title: WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation

doi: 10.1016/j.isci.2023.107931

Figure Lengend Snippet: Leptin receptor-deficient induced diabetic cardiac fibrosis, resulting in increased mitochondrial lipid oxidation and impaired heart function (A) Leptin receptor-deficient induced diabetes in mice and established an animal model of diabetic cardiac fibrosis. (B) Lipid accumulation was evaluated by oil red O staining of mice heart sections. Scale bars: 50 μm, n = 6. (C) Lipid accumulation was evaluated by BODIPY staining of mice heart sections. Count the quantification of stains by ImageJ software. Scale bars: 20 μm, n = 6. (D) The expression levels of FASN, ACLY, ACCα, SREBP-1, Decr1, CPT1a, ATGL, and HSL in cardiac tissue were detected by RT-qPCR. Results were normalized to the β-actin levels in each sample and expressed relative to the untreated control, n = 6. (E) The expression levels of FASN, ACLY, ACCα, SREBP-1, Decr1, CPT1a, ATGL, and HSL in cardiac tissue were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (F) The expression levels of Decr1, CPT1a, and ATGL in cardiac tissue were detected by immunohistochemistry. Results were normalized to expression relative to untreated controls. Scale bars: 20 μm, n = 6. (G) Determination of oxygen consumption rate, ROS, and MDA levels in fresh tissue samples. Results were normalized to expression relative to untreated controls, n = 6. (H) Lipid deposition and paralipid mitochondria were observed in diabetic myocardial fibrosis mice under TEM. M represents paralipid mitochondria and L represents lipids. Scale bars: 1 μm, n = 6. (I) Heart sections from normal and diabetic mice were co-stained with mito tracker and BODIPY. Count the quantification of individual stains separately. Scale bars: 20 μm, n = 6. (J) Heart sections from normal and diabetic mice were co-stained with mitotracker, BODIPY, and Postn—a cardiac fibroblast marker. Count the quantification of individual stains separately. Scale bars: 50 μm, n = 6. All data are presented as mean ± SD; ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001.

Article Snippet: Anti-DECR1 rabbit polyclonal antibody , Sangon Biotech , Cat#:D161613;.

Techniques: Animal Model, Staining, Software, Expressing, Quantitative RT-PCR, Control, Western Blot, Immunohistochemistry, Marker

Journal: iScience

Article Title: WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation

doi: 10.1016/j.isci.2023.107931

Figure Lengend Snippet: HG/HF-induced enhanced mitochondrial lipid oxidation in CFs, dependent on Decr1 expression (A) Schematic diagram of cell culture. We used cardiac fibroblasts, 3T3, and cardiomyocytes to evaluate gene or protein expression and cell function under HG/HF conditions and after transfection with siRNA-Decr1. (B) The expression levels of Decr1, CPT1a, ATGL, HSL, collagen I, and PCNA in different cells under HG/HF were detected by RT-qPCR. Results were normalized to the β-actin levels in each sample and expressed relative to the untreated control. (C) The expression levels of Decr1, CPT1a, ATGL, HSL, collagen I, and PCNA in different cells under HG/HF were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (D) Cells in the normal control group and HG/HF group were co-stained with mitotracker and BODIPY. Calculation of mitochondrial bounding to lipid droplet rate. Scale bars: 25.375 μm, n = 6. (E) The EdU assay was performed to compare the proliferative capacity of cardiac fibroblasts, 3T3 cells, and cardiomyocytes under normal control conditions and HG/HF conditions. Scale bars: 25.375 μm, n = 6. (F) Transwell migration assays were performed to compare the migration ability of cardiac fibroblasts, 3T3 cells, and cardiomyocytes under normal control conditions and HG/HF conditions. Scale bars: 500 μm, n = 3. (G) Measurement of oxygen consumption rate, ROS, and MDA levels in cardiac fibroblasts and cardiomyocytes under normal control and HG/HF conditions. Results were normalized to expression relative to untreated controls, n = 6. (H) Detection of Decr1 protein level in cardiac fibroblasts after Decr1 knockout under HG/HF condition by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (I) Knockdown of different lipid-related enzymes under HG/HF conditions measured oxygen consumption rate, ROS, and MDA levels in cardiac fibroblasts. Results were normalized to expression relative to untreated controls, n = 6. (J) Cardiac fibroblasts were co-stained with mitotracker and BODIPY after Decr1 knockdown. Calculation of mitochondrial bounding to lipid droplet rate. Scale bars: 25.375 μm, n = 6. (K) The EdU assays were performed on the proliferative capacity of cardiac fibroblasts after the Decr1 knockdown. Scale bars: 20 μm. (mean ± SD; ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001,n = 6). (L) Transwell migration assays were performed to examine the migratory capacity of cardiac fibroblasts after the Decr1 knockdown. Scale bars: 500 μm, n = 3. All data are presented as mean ± SD; ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001.

Article Snippet: Anti-DECR1 rabbit polyclonal antibody , Sangon Biotech , Cat#:D161613;.

Techniques: Expressing, Cell Culture, Cell Function Assay, Transfection, Quantitative RT-PCR, Control, Western Blot, Staining, EdU Assay, Migration, Knock-Out, Knockdown

Journal: iScience

Article Title: WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation

doi: 10.1016/j.isci.2023.107931

Figure Lengend Snippet: Mitochondrial lipid oxidation and CFs proliferation and migration decrease with AR overexpression and increase with AR knockdown (A) Schematic diagram of cell culture. We used cardiac fibroblasts, 3T3, and cardiomyocytes to evaluate gene or protein expression and cell function under HG/HF conditions and after transfection with pcDNA3.1-AR or siRNA-AR. (B) Sequence motif identified from top 1,000 AR peaks and transcription factor AR-specific site on the Decr1 promoter. (C) ChIP assay of AR binding to three different sites on Decr1. ChIP-qPCR quantification of AR binding at the Decr1 promoter site. Each group of Input was used as baseline control, n = 3. (D) The expression levels of AR in different cells under HG/HF conditions were detected by RT-qPCR. Results were normalized to the β-actin levels in each sample and expressed relative to the untreated control, n = 6. (E) The expression levels of AR in different cells under HG/HF conditions were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (F) The expression levels of AR in diabetic cardiac fibrosis mice were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (G) The predicted site 2 was selected as the site for subsequent experiments and CHIP analysis of AR on Decr1 under HG/HF conditions was performed. ChIP-qPCR quantification of AR binding to site 2 at the Decr1 promoter. Each set of inputs served as baseline control, n = 3. (H) Predicted ARE-site 2 on wild-type Decr1 and mutated sites on the DNA promoter of mutant Decr1. ChIP-qPCR quantification of AR binding to the Decr1 promoter in wild-type and mutant-type. Each set of inputs served as baseline control, n = 3. (I) Cardiac fibroblasts were stably transfected with plasmids containing the gene indicated by mutant Decr1 and subjected to DNA pull-down, followed by western blot analysis with anti-AR antibody, n = 3. (J) The expression levels of AR, Decr1, CPT1a, ATGL, HSL, collagen I, and PCNA in cardiac fibroblasts after AR overexpression under HG/HF conditions were detected by western blotting analysis. β-actin was used as an internal protein loading control. Representative samples for each protein are shown n = 6. (K) Transwell migration assays were performed to compare the migration ability of cardiac fibroblasts and 3T3 cells after AR overexpression. Scale bars: 500 μm, n = 3. (L) The EdU assays were performed on the proliferative capacity of cardiac fibroblasts and 3T3 after AR overexpression. Scale bars: 20 μm, n = 6. (M) Cardiac fibroblasts were co-stained with mitotracker and BODIPY after AR overexpression. Calculation of mitochondrial bounding to lipid droplet rate. Scale bars: 25.375 μm, n = 6. (N) The expression levels of AR, Decr1, collagen I, and PCNA in cardiac fibroblasts and 3T3 after AR knockdown under HG/HF conditions were detected by western blotting analysis. β-actin was used as an internal protein loading control. Representative samples for each protein are shown n = 6. (O) The EdU assays were performed on the proliferative capacity of cardiac fibroblasts and 3T3 after AR knockdown. Scale bars: 20 μm, n = 6. (P) Scratch-healing assays were performed to measure the migratory capacity of cardiac fibroblasts and 3T3 after AR knockdown. Scale bars: 500 μm, n = 6. (Q) Cardiac fibroblasts were co-stained with mitotracker and BODIPY after AR knockdown. Calculation of mitochondrial bounding to lipid droplet rate. Scale bars: 25.375 μm, n = 6. All data are presented as mean ± SD; ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001.

Article Snippet: Anti-DECR1 rabbit polyclonal antibody , Sangon Biotech , Cat#:D161613;.

Techniques: Migration, Over Expression, Knockdown, Cell Culture, Expressing, Cell Function Assay, Transfection, Sequencing, Binding Assay, ChIP-qPCR, Control, Quantitative RT-PCR, Western Blot, Mutagenesis, Stable Transfection, Staining

Journal: iScience

Article Title: WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation

doi: 10.1016/j.isci.2023.107931

Figure Lengend Snippet: WTAP-dependent m6A upregulation suppresses AR expression (A) M6A-sites prediction and the sites’ confidence score for AR. M6A sites prediction by bioinformatic analysis using SRAMP software ( http://www.cuilab.cn/sramp ). (B) The expression levels of METTL3, METTL14, and WTAP in different cells under HG/HF conditions were detected by RT-qPCR. Results were normalized to the β-actin levels in each sample and expressed relative to the untreated control, n = 6. (C) The expression levels of METTL3, METTL14, and WTAP in different cells under HG/HF conditions were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (D) The expression levels of METTL3, METTL14, and WTAP in diabetic cardiac fibrosis mice were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (E) Global m6A levels of RNA extracted from db/db mice heart tissue were measured via m6A dot blot assays. The gray value of m6A dot blot and methylene blue were evaluated by ImageJ software and quantitative analysis of 400 ng gray value was performed, n = 3. (F) Global m6A levels in RNA extracted from cardiac fibroblasts and 3T3 cells cultured under HG/HF were measured by m6A dot blot assay. The gray value of m6A dot blot and methylene blue were evaluated by ImageJ software and quantitative analysis of 400 ng gray value was performed, n = 3. (G) The expression levels of AR in cardiac fibroblasts after METTL3/METTL14/WTAP knockdown were detected by RT-qPCR. Results were normalized to the β-actin levels in each sample and expressed relative to the untreated control, n = 6. (H) Global m6A levels in RNA extracted from cardiac fibroblasts cultured under HG/HF after WTAP knockdown were measured by m6A dot blot assay. The gray value of m6A dot blot and methylene blue were evaluated by ImageJ software and quantitative analysis of 400 ng gray value was performed, n = 3. (I) The stability of AR transcripts in ActD-treated cardiac fibroblasts after transfection with WTAP siRNA was detected by RT-qPCR, n = 3. (J) MeRIP-qPCR analysis of m6A levels at three different sites of mRNAs from cardiac fibroblasts in siRNA negative control and siRNA-WTAP, n = 6. (K) The predicted ARE-site 2 was selected for subsequent experiments and CHIP analysis of AR on Decr1 under HG/HF conditions after WTAP knockdown was performed. ChIP-qPCR quantification of AR binding to the Decr1 promoter. Each set of inputs served as baseline control, n = 3. (L) Selection of m6A-site C for point mutation and relative quantification of AR by WTAP-RIP. Quantitative qPCR was analyzed by t-test. Each set of inputs served as baseline control, n = 6. (M) RNA pull-down was used to detect the binding pull-down ability of wild-type AR to WTAP or mutant AR to WTAP, followed by western blot analysis with anti-AR antibody. (N) The EdU assays were performed on the proliferative capacity of cardiac fibroblasts and 3T3 after WTAP knockdown. Scale bars: 20 μm, n = 6. (O) Transwell migration assays were performed to compare the migration ability of cardiac fibroblasts and 3T3 cells after WTAP knockdown. Scale bars: 500 μm, n = 3. (P) Cardiac fibroblasts were co-stained with mitotracker and BODIPY after WTAP knockdown. Calculation of mitochondrial bounding to lipid droplet rate. Scale bars: 25.375 μm, n = 6. All data are presented as mean ± SD; ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001.

Article Snippet: Anti-DECR1 rabbit polyclonal antibody , Sangon Biotech , Cat#:D161613;.

Techniques: Expressing, Software, Quantitative RT-PCR, Control, Western Blot, Dot Blot, Cell Culture, Knockdown, Transfection, Negative Control, ChIP-qPCR, Binding Assay, Selection, Mutagenesis, Quantitative Proteomics, Migration, Staining

Journal: iScience

Article Title: WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation

doi: 10.1016/j.isci.2023.107931

Figure Lengend Snippet: WTAP enhances AR methylation and subsequent binding of YTHDF2 to AR suppresses its expression (A) A protein-protein interaction network revealed that WTAP could interact with five common m6A readers, namely, YTHDF1-3 and YTHDC1-2. (B) The expression levels of YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 in different cells under HG/HF conditions were detected by RT-qPCR. Results were normalized to the β-actin levels in each sample and expressed relative to the untreated control, n = 6. (C) The expression levels of YTHDF1 and YTHDF2 in different cells under HG/HF conditions were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (D) The expression levels of YTHDF1 and YTHDF2 in diabetic cardiac fibrosis mice were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (E) Bioinformatics analysis by SRAMP software revealed that the most common m6A motif in AR is the consistent RRACH. (where R = G or A, H = A, C, or U). (F) MeRIP-qPCR analysis of m6A levels at three different sites of mRNAs from cardiac fibroblasts in siRNA negative control and siRNA-YTHDF2, n = 6. (G) The expression levels of YTHDF2 and AR in different cells under HG/HF conditions after YTHDF2 knockdown were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (H) The stability of AR transcripts in ActD-treated cardiac fibroblasts after transfection with YTHDF2 siRNA was detected by RT-qPCR, n = 3. (I) Gene ontology enrichment analysis revealed that YTHDF2 was involved in mRNA processing, mRNA binding, and metabolism. (J) WTAP interacts with YTHDF2 by reciprocal coIP assays in cardiac fibroblasts. Input groups were used as an internal protein loading control, n = 3. (K) The expression levels of AR in cardiac fibroblasts under HG/HF conditions after WTAP knockdown or WTAP+YTHDF2 knockdown were detected by RT-qPCR. Results were normalized to the β-actin levels in each sample and expressed relative to the untreated control, n = 6. (L) CHIP analysis of AR on Decr1 under HG/HF environment after YTHDF2 knockdown was performed. ChIP-qPCR quantification of AR binding to the Decr1 promoter. Each set of inputs served as baseline control, n = 3. (M) Selection of m6A site C for point mutation and relative quantification of AR by YTHDF2-RIP. Quantitative PCR signals were analyzed by t-test. Each set of inputs served as baseline control, n = 6. (N) RNA pull-down was used to detect the binding pull-down ability of wild-type AR to YTHDF2 or mutant AR to YYTHDF2, followed by western blot analysis with anti-AR antibody. (O) The EdU assays were performed on the proliferative capacity of cardiac fibroblasts and 3T3 after YTHDF2 knockdown. Scale bars: 20 μm, n = 6. (P) Cardiac fibroblasts were co-stained with mitotracker and BODIPY after YTHDF2 knockdown. Calculation of mitochondrial bounding to lipid droplet rate. Scale bars: 25.375 μm, n = 6. (Q) Determination of oxygen consumption rate, ROS, and MDA levels in cardiac fibroblasts under HG/HF conditions after YTHDF2 knockdown. Results were normalized to expression relative to untreated controls, n = 6. All data are presented as mean ± SD; ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001.

Article Snippet: Anti-DECR1 rabbit polyclonal antibody , Sangon Biotech , Cat#:D161613;.

Techniques: Methylation, Binding Assay, Expressing, Quantitative RT-PCR, Control, Western Blot, Software, Negative Control, Knockdown, Transfection, ChIP-qPCR, Selection, Mutagenesis, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Staining

Journal: iScience

Article Title: WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation

doi: 10.1016/j.isci.2023.107931

Figure Lengend Snippet: Knockdown of WTAP/YTHDF2 ameliorated Db/db mice diabetic cardiac fibrosis by increasing AR expression (A) An animal model of lentivirus targeting cardiac fibroblasts injected from the tail vein in db/db mice. (B) The expression levels of AR, Decr1, Postn, and collagen I in the heart tissue of LV3-WTAP-injected or LV3-YTHDF2-injected mice were detected by RT-qPCR. Results were normalized to the β-actin levels in each sample and expressed relative to the untreated control, n = 6. (C) The expression levels of AR, Decr1, Postn, and collagen I in the heart tissue of LV3-WTAP-injected or LV3-YTHDF2-injected mice were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (D) The expression levels of Decr1, collagen I, and Postn in the heart tissue of LV3-WTAP-injected or LV3-YTHDF2-injected mice were detected by immunohistochemistry. Results were normalized to expression relative to untreated controls. Scale bars: 20 μm, n = 6. (E) Sirius Red and Masson trichrome staining of heart sections from LV3-WTAP, LV3-YTHDF2, and LV3-Vector-injected mice were performed. Semi-quantitative analysis of Masson trichrome and Sirius red staining was performed by using ImageJ software. Scale bars: 20 μm, n = 6. (F) Global m6A levels in RNA extracted from LV3-WTAP/LV3-Vector lentivirus-injected hearts were measured by m6A dot blot assay. The gray value of m6A dot blot and methylene blue were evaluated by ImageJ software and quantitative analysis of 400 ng gray value was performed, n = 3. (G) Representative M-mode recordings of echocardiography from LV3-Vector and LV3-WTAP/LV3-YTHDF2. Quantitative analysis of ejection fraction (EF), fractional shortening (FS), E/e’, or E/A in each group of lentivirus-injected mice was determined by echocardiography, n = 6. (H) Heart sections from LV3-Vector and LV3-WTAP/YTHDF2 mice were co-stained with mitotracker and BODIPY. Count the quantification of individual stains separately. Scale bars: 20 μm, n = 6. (I and J) AR expression in cardiac sections from LV3-Vector or LV3-WTAP/YTHDF2 mice was detected by tissue immunofluorescence. Scale bars: 20 μm, n = 6. All data are presented as mean ± SD; ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001.

Article Snippet: Anti-DECR1 rabbit polyclonal antibody , Sangon Biotech , Cat#:D161613;.

Techniques: Knockdown, Expressing, Animal Model, Injection, Quantitative RT-PCR, Control, Western Blot, Immunohistochemistry, Staining, Plasmid Preparation, Software, Dot Blot, Immunofluorescence

Journal: iScience

Article Title: WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation

doi: 10.1016/j.isci.2023.107931

Figure Lengend Snippet: Downregulated AR expression correlates with upregulated WTAP, YTHDF2, and Decr1 expression in human DCM patients (A) Flow chart of obtaining human myocardial tissue samples. (B) Global m6A levels in RNA extracted from Control healthy and DCM patients' myocardial tissue were measured by m6A dot blot assay. The gray value of m6A dot blot and methylene blue were evaluated by ImageJ software and quantitative analysis of 400 ng gray value was performed, n = 6. (C) The expression levels of WTAP, YTHDF2, AR, Decr1, Postn, collagen I, and α-SMA in the heart tissue of Control healthy and DCM patients were detected by western blotting analysis. β-actin was used as an internal protein loading control. A representative sample is shown for each protein, n = 6. (D) The expression levels of WTAP, YTHDF2, AR, Decr1, Postn, collagen I, and α-SMA in the heart tissue of Control healthy and DCM patients were detected by RT-qPCR. Results were normalized to the β-actin levels in each sample and expressed relative to the untreated control, n = 6. (E) H&E, Sirius Red, and Masson trichrome staining of heart sections from Control healthy and DCM patients were performed. Semi-quantitative analysis of H&E, Masson trichrome, and Sirius red staining was performed by using ImageJ software. Scale bars: 20 μm, n = 6. (F) The expression levels of WTAP, YTHDF2, Decr1, and Postn in the control healthy group and DCM patients group were detected by immunohistochemistry. Results were normalized to expression relative to untreated controls. Scale bars: 20 μm, n = 6. (G) Lipid deposition and paralipid mitochondria were observed in the control healthy group and DCM patients group under TEM. M represents paralipid mitochondria and L represents lipids. Scale bars: 1 μm, n = 6. (H) Heart sections from the control healthy group and DCM patients group were co-stained with mitotracker, BODIPY, and Postn—a cardiac fibroblast marker. Count the quantification of individual stains separately. Scale bars: 50 μm, n = 6. (I) WTAP/YTHDF2 expression in cardiac sections from the control healthy group and DCM patients group was detected by tissue immunofluorescence. Scale bars: 20 μm, n = 6. All data are presented as mean ± SD; ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001.

Article Snippet: Anti-DECR1 rabbit polyclonal antibody , Sangon Biotech , Cat#:D161613;.

Techniques: Expressing, Control, Dot Blot, Software, Western Blot, Quantitative RT-PCR, Staining, Immunohistochemistry, Marker, Immunofluorescence

Journal: iScience

Article Title: WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation

doi: 10.1016/j.isci.2023.107931

Figure Lengend Snippet:

Article Snippet: Anti-DECR1 rabbit polyclonal antibody , Sangon Biotech , Cat#:D161613;.

Techniques: Recombinant, Modification, Staining, ROS Assay, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, EdU Assay, Next-Generation Sequencing, Sequencing, Software