cardiomyocyte protein extracts  (Santa Cruz Biotechnology)

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Beclin1-mediated autophagy was upregulated with cardiac hypertrophy in vivo and in vitro . (A) The expression of LC3, beclin1 and p62 in hypertrophic heart were determined by western blotting at indicated time point after TAC (Student t test, n =5-6, * 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) Cardiomyocytes were challenged by ET-1 and the expression of LC3, beclin1 and p62 were examined by western blotting at indicated time point (one-way ANOVA, n =5-6, * P < 0.05 vs. 0h). (C) After infected with mRFP-GFP-LC3 adenoviral particles for 24h, the cells were challenged by ET-1. Fluorescent signals were captured with the confocal laser scanning microscopy at indicated time point and the number of autolysosomes and autophagosomes was determined by counting of red puncta or yellow puncta, respectively (one-way ANOVA, * P < 0.05 or ** P < 0.01 vs. 0h in yellow puncta. # P < 0.05, ## P < 0.01 vs. 0h in red puncta. Thirty randomly selected cells per experimental group were analyzed).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: In Vivo, In Vitro, Expressing, Western Blot, Control, Infection, Confocal Laser Scanning Microscopy

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Inhibition of Beclin1-mediated autophagy mitigated adverse cardiac remodeling and cardiac hypertrophy. (A) Ultrastructure analysis showing the autophagosomes (multimembrane vacuoles, arrow heads) and autolysosomes (electron dense structures, arrows) in the myocardium 8 weeks after TAC. (B) Representative cross-sectional images with H&E staining. (C) Representative cross-sectional images showing interstitial fibrosis with Masson staining. (D) Analysis results for cardiomyocyte cross-sectional area (one-way ANOVA, n =5, * P < 0.05 vs . sham, # P < 0.05 vs. TAC). (E) Analysis results for fibrosis (one-way ANOVA, n =5, * P < 0.05 vs. sham, # P < 0.05 vs. TAC). (F) mRNA level of β-MHC and BNP in hypertrophic heart were determined by real time RCR from mice in each experimental group (one-way ANOVA, n =5, * P < 0.05 vs. sham and # P < 0.05 vs. TAC, β-actin served as a loading control). (G) Neonatal cardiomyocytes pretreated with Beclin1 shRNA were subjected to ET-1 for 24h. 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 < 0.05 or # P < 0.05 vs. Control in yellow puncta or red puncta, respectively. $ P < 0.05 vs. ET in yellow puncta. △ P < 0.05 vs. ET in red puncta. Thirty randomly selected cells per experimental group were analyzed). (H) The cell size was assessed by Image J (one-way ANOVA, n =30, * P < 0.05 vs. control, # P < 0.05 vs. ET). (I) mRNA levels of β-MHC and BNP in cardiomyocytes were determined by real time RCR (one-way ANOVA, n =5, * P < 0.05 vs. control, # P < 0.05 vs. ET-1, β-actin served as a loading control).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: Inhibition, Staining, Control, shRNA

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: 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).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: In Vivo, In Vitro, Expressing, Western Blot, Control, shRNA, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Deficiency of DGKζ promoted cardiac dysfunction by augmenting Beclin1-mediated autophagy. (A) Representative M-mode echocardiograms, transmitral flow and tissue doppler echocardiograms. (B) The analyzed results of LVAW and LVPW obtained from mice in each experimental group ((one-way ANOVA, n =6, * P <0.05 vs. TAC, # P <0.05 vs. DGKζ shRNA+TAC). (C) The analyzed results of EF and FS obtained from mice in each experimental group ((one-way ANOVA, n =6, * P <0.05 vs. TAC, # P <0.05 vs. DGKζ shRNA+TAC). (D) The analyzed results of E/A and e'/a' obtained from mice in each experimental group ((one-way ANOVA, n =6, * P <0.05 vs. TAC, # P <0.05 vs. DGKζ shRNA+TAC). (E) Representative cross-sectional images with H&E staining. (F) Representative cross-sectional images with masson staining. (G) Analysis results for cardiomyocyte cross-sectional area (one-way ANOVA, n =5, * P < 0.05 vs. TAC, # P <0.05 vs. DGKζ shRNA+TAC). (G) Analysis results for fibrosis (one-way ANOVA, n =5, * P < 0.05 vs. TAC, # P < 0.05 vs. DGKζ shRNA+TAC).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: shRNA, Staining

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: AKT/mTOR dependent signaling is involved in the enhancement of cardiac autophagy by DGKζ deficiency Cardiomyocytes infected with lentivirus expressing DGKζ shRNA were subjected to ET-1 for 24h. The total protein expression and phosphorylated level of AKT, mTOR, AMPK, ULK1 and Beclin1 were examined by western blotting, respectively. (A) Representative blots depicting total and phosphorylated proteins. (B) The analyzed results of p-AKT/AKT level, p-mTOR/mTOR level, p-AMPK/AMPK level, p-ULK1/ULK1 level and p-Beclin1 level (one-way ANOVA, n =5, *P< 0.05 vs. control. # P< 0.05 or ## P< 0.01 vs. ET).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, shRNA, Western Blot, Control

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Deficiency of DGKζ impaired the interaction of mTOR with TFEB and favored the nuclear translocation of TFEB. (A) Representative blot and the analyzed results of the nuclear TFEB level and cytoplasm TFEB levels in heart tissues after TAC (Student t test, n =7-8, * 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) Representative blot and the analyzed results of the nuclear TFEB level and cytoplasm TFEB levels in ET-1-treated cardiomyocytes (one-way ANOVA, n =6, * P <0.05 vs. 0h). (C) Immunofluorescent staining showing the translocation of TFEB from cytoplasm to nucleus induced by ET-1. (D) The representative image of immunoprecipitation assay showing that mTOR interacted with TFEB in cardiomyocytes. Input was used as a positive control and IgG was used as a negative control. (E) Representative blot and the analyzed results of the nuclear TFEB level and cytoplasm TFEB levels in cardiomyocytes after DGKζ knockdown (one-way ANOVA, n =5-6, * P <0.05 vs. control, # P< 0.05 vs. ET). (F) Immunofluorescent staining showing the translocation of TFEB from cytoplasm to nucleus induced by ET-1 after DGKζ knockdown.

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: Translocation Assay, Expressing, Control, Staining, Immunoprecipitation, Positive Control, Negative Control, Knockdown

Journal: International Journal of Medical Sciences

Article Title: Deficiency of diacylglycerol Kinase ζ promotes Beclin1-mediated autophagy via the mTOR/TFEB signaling pathway: Relevance to maladaptive cardiac hypertrophy

doi: 10.7150/ijms.88134

Figure Lengend Snippet: Genetic knockdown of TFEB abrogates Beclin1-mediated autophagy and ameliorate ET-1-induced myocardial hypertrophy. (A) Representative blot and the analyzed results of the expression of TFEB and Beclin1 in ET-1-treated cardiomyocytes (one-way ANOVA, n =5 , *P< 0.05 vs. scramble+ET, # P <0.05 vs. (scramble+DGKζ) shRNA+ET). (B) mRNA levels of Beclin1 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). (C) Schematic representation of conserved binding sequences identified in the promoter regions of Becn1 using JASPAR. (D) Occupancy of Becn1 promoter regions -1420 to -1290, 540 to -340, and -880 to -670 by TFEB, as determined by ChIP assay (one-way ANOVA, n =3, * P < 0.05 vs. control, # P <0.05 vs. ET, β-actin served as a loading control). (E) Luciferase activity in cardiomyocytes transfected with the Becn1 luciferase reporter constructs (one-way ANOVA, n =3, * P <0.05 vs. control, # P <0.05 vs. ET, & P <0.05 vs. DGKζ shRNA+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 (Student t test, * P < 0.05 vs. ET in yellow puncta. ## P < 0.01 vs. 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).

Article Snippet: Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, shRNA, Control, Binding Assay, Luciferase, Activity Assay, Transfection, Construct