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. 2024 Jan 1;21(3):439–453. doi: 10.7150/ijms.88134

Figure 4.

Figure 4

DGKζ was decreased in response to the prohypertrophic stimuli in vivo and in vitro, and DGKζ deficiency linked to Beclin1-mediated autophagy during myocardial hypertrophy (A) Cardiac hypertrophy was induced by TAC in mice and the expression of DGKζ in the heart was determined by western blotting at indicated time point after surgery (Student t test, n=5, *P< 0.05 or **P< 0.01 vs. sham. The expression of proteins in TAC mice was normalized to that of the sham mice in each time point, and β-actin served as a loading control). (B) The expression of DGKζ was determined by western blotting in ET-1-treated cardiomyocytes for indicated time point (one-way ANOVA, n=5, *P<0.05 or **P<0.01 vs. 0h). (C) The level of DGKζ was successfully downregulated in cardiomyocytes by infecting lentiviral DGKζ shRNA. Lentiviral plasmid containing no targeted sequences or scramble sequences served as vector control and non-silencing control (one-way ANOVA, n=5, *P<0.05 vs. control). (D) The level of DAG was measured by ELISA kits in heart tissue 12 weeks after TAC and in cardiomyocytes after ET-1 treatment for 24h (one-way ANOVA, n=5). (E) Cardiomyocytes were challenged by ET-1 and the expression of LC3, beclin1 and p62 were examined by western blotting (one-way ANOVA, n=5-6, *P< 0.05 vs. control, #P< 0.05 vs. ET). (F) Autolysosomes and autophagosomes were determined by use of a tandem mRFP-GFP-LC3 adenovirus in cardiomyocytes. The red puncta indicated autolysosomes and the yellow puncta indicated autophagosomes, respectively (one-way ANOVA, **P< 0.01 vs. control in yellow puncta. #P< 0.05 or ##P< 0.01 vs. control in red puncta. $P< 0.05 vs. DGKζ shRNA+ET in yellow puncta. △P< 0.05 vs. DGKζ shRNA+ET in red puncta. Thirty randomly selected cells per experimental group were analyzed). (G) The cell size was assessed by Image J (one-way ANOVA, n=30, **P<0.01 vs. control, ##P<0.01 vs. ET). (H) mRNA level of β-MHC and BNP in cardiomyocytes were determined by real time RCR (one-way ANOVA, n=3, *P< 0.05 vs. control, #P< 0.05 vs. ET, $P< 0.05 vs. DGKζ shRNA+ET, β-actin served as a loading control).