Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Ferroptosis‐associated zinc finger protein 36 (Zfp36) expression is reduced under cardiac hypertrophy. (A) Schematic of study design, and workflow of Nanogrid single‐cell selection and sequencing platform. (B) t‐Distributed stochastic neighbour embedding (t‐SNE) projection of cardiac tissue cells analysed by single‐cell RNA sequencing (RNA‐seq). Cells are coloured by distinct cell populations as indicated. (C) Top two to three distinct genes for each cell population, identified using an unsupervised approach. (D) Gene Set Enrichment Analysis (GSEA) showing that the genes related to lipid oxidation was upregulated in transverse aortic constriction (TAC) mice. (E and G) Western blot shown the protein expression level Gpx4 ( n = 6). (F and H) Representative images of transmission electron microscope. (I) BODIPY581/591‐C11 probe staining for lipid peroxidation in cardiomyocytes after treating with Ang II. (J) Representative images of transmission electron microscope and cell area stained by α‐actinin (red) in cardiomyocytes. Nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI) (blue). Scale bars: 50 µm. Right row was the averaged data of cell area stained by α‐actinin ( n = 9‒12). (K) Western blot results shown the protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 4). (L) Gene Ontology (GO) analysis using significantly changed genes of RNA‐seq. (M) Venn diagram of ferroptosis suppressor, RNA‐binding protein and significantly changed genes shown in cardiac hypertrophy. (N‒O) Western blot results showed the expression level of Zfp36 ( n = 5‒6). Statistical analysis was performed with Student's t ‐test or one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Expressing, Selection, Sequencing, RNA Sequencing, Western Blot, Transmission Assay, Microscopy, Staining, RNA Binding Assay, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Zinc finger protein 36 (Zfp36)‐attenuated ferroptosis in cardiac hypertrophy. (A) Detection of malondialdehyde (MDA) for lipid peroxidation level in Sham and transverse aortic constriction (TAC) mice hearts ( n = 5). (B) Western blot results shown the protein expression level of Gpx4 in mice hearts ( n = 5). (C) Representative images of transmission electron microscope in mice hearts. (D) Representative images of echocardiography recording, heart cross‐morphology, heart sections stained with haematoxylin and eosin (HE) and wheat germ agglutinin (WGA) in mice. (E) Left ventricular posterior wall thickness dimensions during diastole in indicated mice ( n = 6‒7). (F and G) Heart weight‐to‐body weight ratio and heart weight‐to‐tibia length ratio in mice ( n = 8‒10). (H) Quantification of myocyte area from WGA staining heart sections ( n = 5). (I and N) Western blot results shown the protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 6). (J) DCFH‐DA probe determined the levels of reactive oxygen species (ROS) in cardiomyocytes ( n = 6). (K) Detection of MDA for lipid peroxidation level ( n = 5‒6). (L) Western blot analysed the expression levels of Gpx4 ( n = 6). (M) Representative photographs of cardiomyocytes identified with α‐actinin antibody and the averaged data of cell area ( n = 9‒15). Statistical analysis was performed with Student's t ‐test or one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Western Blot, Expressing, Transmission Assay, Microscopy, Staining, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Knockdown zinc finger protein 36 (Zfp36) aggravate cardiac hypertrophy and ferroptosis. (A and J) Detection of malondialdehyde (MDA) for lipid peroxidation levels ( n = 5‒6). (B and K) The expression level of Gpx4 protein ( n = 6). (C) Representative images of echocardiography recording, heart cross‐morphology, heart sections stained with haematoxylin and eosin (HE) and wheat germ agglutinin (WGA) in mice. (D) Left ventricular wall thickness of mice ( n = 5‒7). (E and F) Ratio of heart weight‐to‐body weight and heart weight‐to‐tibia length in mice ( n = 6‒7). (G) Quantification of myocyte area from WGA staining heart sections ( n = 5). (H and M) Western blot analysed the protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 6). (I) DCFH‐DA probe determined the levels of reactive oxygen species (ROS) in cardiomyocytes ( n = 6). (L) Representative photographs of cardiomyocytes identified with α‐actinin antibody and the averaged data of cell area ( n = 11‒13). Statistical analysis was performed with one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Knockdown, Expressing, Staining, Western Blot, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Ythdc2 as a direct target of zinc finger protein 36 (Zfp36) is upregulated in cardiac hypertrophy. (A) Gene Ontology (GO) analysis used the potential targets genes of Zfp36 and also exhibited high expression levels in RNA sequencing (RNA‐seq) data. (B‒E) qRT‐PCR analysed the mRNA expression levels of the potential targets of Zfp36 which were enriched in m 6 A regulating enzymes pathway ( n = 3‒5). (F) Zfp36 protein structure diagram and the predicted binding region with Ythdc2 and docking results of Zfp36 protein with Ythdc2 3′ untranslated region (3′UTR) molecule, green chain (Ythdc2 3′UTR), the blue chain (Zfp36), the rod‐like structure represents the interacting amino acids and nucleic acids. (G) RNA‐pull down analysed the binding relation between Zfp36 and Ythdc2 mRNA. (H) RNA immunoprecipitation (RIP)‐PCR analysed the binding relation between Zfp36 and Ythdc2 mRNA ( n = 3). (I) Co‐localisation of Zfp36 and Ythdc2 mRNA in cardiomyocyte. Zfp36 identified with Zfp36 antibody (green), Ythdc2 mRNA identified by its probes (red) and nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI) (blue). (J) Luciferase reporters of Ythdc2 3′UTR and ACTB 3′UTR in Hek293T cells transfected with increasing Zfp36 ( n = 3). (K) qRT‐PCR analysed the mRNA expression levels of Ythdc2 treated with or not Act D in over‐expression or knockdown Zfp36 ( n = 3‒6). (L and M) Over‐expression or knockdown Zfp36 CM was transfected with Ythdc2 3′UTR or ACTB 3′UTR luciferase plasmids, then subjected to analysis the expression level of luciferase mRNA at indicated time points treated with or not Act D ( n = 4). (N) RIP‐PCR analysed the binding relation between Zfp36 and Ythdc2 mRNA ( n = 3). (O and P) Real‐time PCR and western blotting analysed the expression levels of Ythdc2 ( n = 6). (Q) DCFH‐DA probe staining for the reactive oxygen species (ROS) levels of cardiomyocytes ( n = 6). (R) Detection of malondialdehyde (MDA) for lipid peroxidation level ( n = 5). (S) Western blot results shown the protein expression level Gpx4 ( n = 6). (T) The representative photographs of cardiomyocytes identified with α‐actinin and averaged data of cell area ( n = 11‒12). (U) The protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 6). Statistical analysis was performed with Student's t ‐test or one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Binding Assay, RNA Immunoprecipitation, Staining, Luciferase, Transfection, Over Expression, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Knockdown Ythdc2‐attenuated ferroptosis in hypertrophy induced by transverse aortic constriction (TAC). (A and K) Detection of malondialdehyde (MDA) for lipid peroxidation level ( n = 4‒5). (B and L) Western blot results show the protein expression level of Gpx4 ( n = 6). (C) Representative images of transmission electron microscope in mice. (D) Representative images of echocardiography recording, heart cross‐morphology, heart sections stained with haematoxylin and eosin (HE) and wheat germ agglutinin (WGA) in mice. (E) Left ventricular posterior wall thickness dimensions during diastole in indicated mice ( n = 7‒9). (F and G) Heart weight‐to‐body weight ratio and heart weight‐to‐tibia length ratio ( n = 6‒8). (H) Quantification of myocyte area from WGA staining heart sections ( n = 5). (I and N) Western blot results shown the protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 6). (J) DCFH‐DA probe staining for the reactive oxygen species (ROS) levels of cardiomyocytes ( n = 6). (M) Representative photographs and averaged data of cell area stained by α‐actinin antibody the averaged data of cell area ( n = 10‒14). Statistical analysis was performed with one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Knockdown, Western Blot, Expressing, Transmission Assay, Microscopy, Staining, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Ythdc2 exacerbates ferroptosis and cardiac hypertrophy. (A and J) Detection of malondialdehyde (MDA) for lipid peroxidation level ( n = 5‒6). (B and K) Western blot results shown the protein expression level Gpx4 ( n = 5). (C) Representative images of echocardiography recording, heart cross‐morphology, heart sections stained with haematoxylin and eosin (HE) and wheat germ agglutinin (WGA) in mice. (D) Left ventricular posterior wall thickness dimensions during diastole in indicated mice ( n = 6‒7). (E and F) Heart weight‐to‐body weight ratio and heart weight‐to‐tibia length ratio ( n = 5‒7). (G) Quantification of myocyte area from WGA staining heart sections ( n = 5). (H and M) Western blot results show the protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 5). (I) DCFH‐DA probe staining for the reactive oxygen species (ROS) levels of cardiomyocytes ( n = 6). (L) The representative photographs and averaged data of cell area stained by α‐actinin antibody (red), nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI) (blue) ( n = 13‒16). Statistical analysis was performed with one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Western Blot, Expressing, Staining, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Regulated of Ythdc2 expression by zinc finger protein 36 (Zfp36) mediates the ferroptosis and cardiac hypertrophy phenotypes. (A and I) Detection of malondialdehyde (MDA) for lipid peroxidation level in mice hearts ( n = 5‒7). (B and J) Western blot results shown the protein expression level Gpx4 ( n = 5‒6). (C and K) Representative images of heart cross‐morphology, heart sections stained with haematoxylin and eosin (HE) and wheat germ agglutinin (WGA) in mice. (D and L) Left ventricular (LV) posterior wall thickness dimensions in indicated mice ( n = 5‒7). (E and M) Heart weight‐to‐body weight ratio ( n = 6‒7). (F and N) Heart weight‐to‐tibia length ratio ( n = 5‒7). (G and O) Quantification of myocyte area from WGA stained ( n = 5). (H and P) Protein expression level of beta‐myosin heavy chain (β‐MHC) ( n = 5‒6). Statistical analysis was performed with one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Skim milk was blocked the nitrocellulose membranes for 2 h. Primary antibodies Zfp36, Ythdc2, SLC7A11, β‐MHC (Cat. no. 12737‐1‐AP, 27779‐1‐AP, 26864‐1‐AP, 22280‐1‐AP, Proteintech) and Gpx4 (Cat. no. A1933, ABclonal) were incubated overnight at 4°C. β‐Actin (Cat. no. 66009‐1‐Ig, Proteintech) was used as the internal control antibody.

Techniques: Expressing, Western Blot, Staining, Standard Deviation