mouse models  (Addgene inc)

Journal: Research

Article Title: CHAtRF Modulates Cardiac Hypertrophy via SRSF5-Dependent Regulation of Psmg4 Alternative Splicing

doi: 10.34133/research.1202

Figure Lengend Snippet: CHAtRF binds to splicing factor SRSF5 to regulate mRNA alternative splicing. (A) LC-MS/MS identification of SRSF5. (B) RNA immunoprecipitation using SRSF5 antibody followed by qPCR analysis showing CHAtRF enriched in SRSF5 fraction ( n = 6 independent experiments). (C) RNA pull-down assay was carried out using biotinylated CHAtRF (Bio-CHAtRF) or NC (Bio-NC) and WB analysis showing that CHAtRF binds with SRSF5 protein. (D) Data of RNA immunoprecipitation sequencing with anti-SRSF5 antibody in NC and CHAtRF knockdown cells. (E) Summary of differential splicing analysis performed using anta- or NC-transfected cardiomyocytes. (F) Numbers of predicted alternative splicing (AS) events in each category upon CHAtRF deletion. (G) Venn diagrams showing overlap of RIP-seq genes, RNA-seq genes, and AS genes in CHAtRF anta- or NC-transfected cardiomyocytes. (H) Alternative sites in Psmg4 gene bound directly by SRSF5 from RIP-seq and RNA-seq using IGV software. (I) RT-PCR analysis for AS event of Psmg4 gene in NC- and CHAtRF-overexpressed cardiomyocytes. The middle panels represent the schematic diagram of indicated AS exons. Right panels show the quantification of percent spliced-in (PSI) ( n = 7 independent experiments). Data are presented as mean ± SD. Data presented in (B) and (I) were analyzed by Student,s t test (2-tailed).

Article Snippet: The Srsf5 -cKO mouse model was created by Cyagen Biosciences Inc. (China) employing CRISPR/Cas9 genome editing.

Techniques: Alternative Splicing, Liquid Chromatography with Mass Spectroscopy, RNA Immunoprecipitation, Pull Down Assay, Sequencing, Knockdown, Transfection, RNA Sequencing, Software, Reverse Transcription Polymerase Chain Reaction

Journal: Research

Article Title: CHAtRF Modulates Cardiac Hypertrophy via SRSF5-Dependent Regulation of Psmg4 Alternative Splicing

doi: 10.34133/research.1202

Figure Lengend Snippet: CHAtRF blocks SRSF5 to mediate the AS of Psmg4. (A) RIP-qPCR analysis showing relative binding level of SRSF5 to Psmg4 mRNA in NC or CHAtRF-overexpressed cardiomyocytes ( n = 6 independent experiments). (B) WB analyses of the expression of Psmg4 protein in NC or CHAtRF-overexpressed cardiomyocytes. GAPDH was used as a loading control. (C) RIP-qPCR analysis showing relative binding level of SRSF5 to Psmg4 mRNA in NC- or anta-transfected cardiomyocytes ( n = 6 independent experiments). (D) RT-PCR analysis for AS events of Psmg4 gene in cardiomyocytes transfected with anta, si-NC, or si-Srsf5. (E) Quantification of PSI ( n = 8 independent experiments). (F) Cardiomyocytes were transfected with anta or NC for 24 h, and then cells were treated with AngII. RT-PCR analysis for AS events of Psmg4 gene. (G) Quantification of PSI ( n = 7 independent experiments). (H) WB analyses of the expression of Psmg4 protein in cardiomyocytes transfected with anta or NC for 24 h and then treated with AngII. GAPDH was used as a loading control ( n = 6 independent experiments). (I) Schematics for the full-length Psmg4 domains and the Psmg4 minigene-splicing reporter based on the Psmg4 genomic locus. Lengths of exons and introns are indicated. (J) 293T cells were transfected with Psmg4 mini plasmid and adenovirus harboring Srsf5. RT-PCR analysis of the splicing pattern of the Psmg4 mini reporter (left panel) and quantification of RT-PCR data (right panel) were shown ( n = 8 independent experiments). Data are presented as mean ± SD. Data presented in (A) and (B) were analyzed by Student,s t test (2-tailed). Data presented in (C) were analyzed by 2-way ANOVA with Tukey post hoc test. Data presented in (E), (G), (H), and (J) were analyzed by one-way ANOVA with Tukey post hoc test.

Article Snippet: The Srsf5 -cKO mouse model was created by Cyagen Biosciences Inc. (China) employing CRISPR/Cas9 genome editing.

Techniques: Binding Assay, Expressing, Control, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

Journal: Research

Article Title: CHAtRF Modulates Cardiac Hypertrophy via SRSF5-Dependent Regulation of Psmg4 Alternative Splicing

doi: 10.34133/research.1202

Figure Lengend Snippet: Psmg4 and SRSF5 function as downstream molecules of CHAtRF in cardiac hypertrophy. (A to C) Mice were treated with AngII and transfected with CHAtRF anta or its NC, while infected with adenovirus harboring shPsmg4 or shCTRL. (A) Top row: Representative images of gross morphology of hearts. Scale bar, 2 mm. Bottom row: Representative images of left ventricular muscle sections stained with WGA. Scale bar, 25 μm. (B) HW/BW ratio ( n = 6 to 8 mice per group). (C) Analysis of the cardiomyocyte sizes in histological sections stained with WGA ( n = 7 to 8 mice per group). (D and E) Cardiomyocytes were treated with AngII and transfected with CHAtRF anta or its NC, while infected with adenovirus harboring shPsmg4 or shCTRL. (D) Cardiomyocytes were stained by phalloidin, and quantitative analysis of the cell surface area was assessed ( n = 6 independent experiments). (E) qPCR results showing BNP mRNA level ( n = 6 independent experiments). (F) Schematic diagram of tamoxifen (TAM)-induced generation in SRSF5-cKO mice. NC mice were SRSF5 flox/flox mice, and SRSF5-cKO mice were crosses between α-MHCMerCreMer mice and SRSF5 fl/fl mice. (G) WB showed SRSF5 expression in SRSF5 fl/fl mice and SRSF5-cKO mice ( n = 8 mice per group). (H) Representative images of gross morphology of SRSF5-cKO mice and SRSF5 fl/fl mouse hearts. Scale bar, 2 mm. (I) Analysis of HW/BW ratio in the SRSF5-cKO and SRSF5 fl/fl mice ( n = 9 mice per group). (J to M) SRSF5-cKO and SRSF5 fl/fl mice were injected with AngII and transfected with CHAtRF anta or its NC. (J) HW/BW ratio ( n = 6 to 8 mice per group). (K) qPCR results showing BNP mRNA level ( n = 6 mice per group). (L) RT-PCR analysis of the splicing pattern of the Psmg4 gene (left panel) and quantification of RT-PCR data (right panel) ( n = 6 independent experiments). Data are presented as mean ± SD. Data presented in (B) to (E) were analyzed by one-way ANOVA with Tukey post hoc test. Data presented in (G) to (I) were analyzed by Student,s t test (2-tailed). Data presented in (J), (K), and (M) were analyzed by 2-way ANOVA with Tukey post hoc test.

Article Snippet: The Srsf5 -cKO mouse model was created by Cyagen Biosciences Inc. (China) employing CRISPR/Cas9 genome editing.

Techniques: Transfection, Infection, Staining, Expressing, Injection, Reverse Transcription Polymerase Chain Reaction

Journal: Research

Article Title: CHAtRF Modulates Cardiac Hypertrophy via SRSF5-Dependent Regulation of Psmg4 Alternative Splicing

doi: 10.34133/research.1202

Figure Lengend Snippet: The effects of CHAtRF knockdown in established cardiac hypertrophy. (A) Schematic diagram of the CHAtRF anta injection and the experimental procedure. (B) qPCR results showing CHAtRF levels ( n = 6 mice per group). (C) Top row: Analysis of cardiac morphology. Scale bar, 2 mm. Bottom row: Representative images of coronal sections of heart stained with hematoxylin and eosin. Scale bar, 2 mm. (D) HW/BW ratio ( n = 8 to 9 mice per group). (E) Analysis of cardiomyocytes size ( n = 6 to 8 mice per group). (F) qPCR results showing BNP mRNA levels ( n = 6 mice per group). (G) Representative images of Masson,s trichrome-stained and semiquantitative analysis of histological sections of left ventricle. Scale bar, 20 μm ( n = 6 to 8 mice per group). (H) Cardiac function measured by left ventricle ejection fraction (EF) using echocardiography ( n = 6 to 8 mice per group). (I) Schematic diagram of CHAtRF function in hypertrophic signaling. CHAtRF participates in the regulation of cardiac hypertrophy by targeting the SRSF5/Psmg4 pathway. In our model, overexpression of CHAtRF prevents the binding of SRSF5 to Psmg4 pre-mRNA, resulting in AS of Psmg4 (exon 2 skipping) and a shift in Psmg4 full-length or short isoform expression, which subsequently promotes cardiac hypertrophy. Data are presented as mean ± SD. Data presented in (B) and (D) to (H) were analyzed by one-way ANOVA with Tukey post hoc test.

Article Snippet: The Srsf5 -cKO mouse model was created by Cyagen Biosciences Inc. (China) employing CRISPR/Cas9 genome editing.

Techniques: Knockdown, Injection, Staining, Over Expression, Binding Assay, Expressing