Fig 1: The expression of Rasd1 in H9C2 cells was not affected by hypoxia. (A,B) qPCR analysis for Kmt2d (A) and Rasd1 (B) in hypoxia in H9C2-Ctl and H9C2-KO cells. (C) Representative images of western blots for KMT2D and RASD1 expression in normoxia/hypoxia conditions in the H9C2-Ctl and H9C2-KO cells. (D,E) Quantitative analysis of KMT2D (D) and RASD1 (E) expression in the H9C2-Ctl or the H9C2-KO cells. (n = 3 of each group; *p < 0.05, **p < 0.01, ***p < 0.001.)
Fig 2: Expression of Rasd1 in the heart was significantly reduced by ischemia and KMT2D deletion in mice. (A) qPCR measurements of Rasd1 following 24 h post-MI or sham surgery in the KMT2D-Ctl and the KMT2D-cKO mice. (B) Representative images of western blots for KMT2D and RASD1, showing expression in MI/sham-operated hearts in the KMT2D-Ctl and the KMT2D-cKO mice. (C) Quantitative analysis of KMT2D expression in the KMT2D-cKO mice and the KMT2D-Ctl mice. (D) Quantitative analysis of RASD1 expression in the KMT2D-Ctl and the KMT2D-cKO mice during MI and sham groups. (E) Schematic diagram for the GRE motif of Rasd1 gene. (F) ChIP-qPCR data quantifying enrichment of H3K4me1 at the GRE of Rasd1 enhancer in all groups. (n = 3 of each group; *p < 0.05, **p < 0.01, ***p < 0.001.)
Fig 3: The decreased expression of Rasd1 was caused by serum deficiency. (A,B) qPCR analysis for Kmt2d (A) and Rasd1 (B) in FBS or FBS-free conditions in H9C2 cells. (C) Representative images of western blots for KMT2D and RASD1 in FBS or FBS-free conditions in the H9C2-Ctl and the H9C2-KO cells. (D,E) Quantitative analysis of KMT2D (D) and RASD1 (E) expression in the H9C2-Ctl and the H9C2-KO cell. (n = 3 of each group; *p < 0.05, **p < 0.01, ***p < 0.001.)
Fig 4: Transcription of Rasd1 was weakened in the Monoclonal H9C2 cell line with deletion of KMT2D. (A) Schematic diagram showing targets of gRNAs on the 16th exon of Kmt2d gene in H9C2 cell. (B) Sanger sequencing of genomic DNA from CRISPR/Cas9-edited H9C2 monoclone. (C,D) qPCR analysis of Kmt2d (C) and Rasd1 (D) expression in the H9C2-Ctl and the H9C2-KO monoclone. (E) Representative images of western blots for KMT2D and RASD1, showing expression in the H9C2-Ctl and the H9C2-KO cells. (F) Quantitative analysis of KMT2D expression in the H9C2-KO and the H9C2-Ctl monoclone. (G) Quantitative analysis of RASD1 expression in the H9C2-Ctl cell and the H9C2-KO monoclone. (n = 3 of each group; *p < 0.05, **p < 0.01, ***p < 0.001.)
Fig 5: Schematic diagram summarizing the protective role of KMT2D during ischemia and the potential mediated mechanism. In normal condition, which is enriched with serum, KMT2D catalyzes H3K4 mono-methylation on the Rasd1 enhancer region and promotes Rasd1 transcription in cardiomyocyte. In ischemic condition, serum deficiency leads to interruption of H3K4me1 that modified by KMT2D on GRE of Rasd1 and then reduces Rasd1 transcription.
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