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

Journal: Molecular Medicine Reports

Article Title: N-terminal truncated peroxisome proliferator-activated receptor-γ coactivator-1α alleviates phenylephrine-induced mitochondrial dysfunction and decreases lipid droplet accumulation in neonatal rat cardiomyocytes

doi: 10.3892/mmr.2018.9158

Figure Lengend Snippet: NT-PGC-1α overexpression reduces LD accumulation in NRCMs. Representative images of LD staining of NRCMs cultured in high lipid medium from (A) the BSA+vehicle group, (B) OA-BSA+vehicle group, (C) OA-BSA+AdV-NT-PGC-1α group and (D) OA-BSA+AdV-NT-PGC-1α+MK886 group. (E) NT-PGC-1α overexpression reduced LD accumulation in NRCMs. The number of LDs in each group was counted. **P<0.01, as indicated. BSA+vehicle, n=27; OA-BSA+vehicle, n=30; OA-BSA+AdV-NT-PGC-1α, n=50 and OA-BSA+AdV-NT-PGC-1α+MK886, n=31. AdV, adenovirus; NT-PGC-1α, N-terminal truncated peroxisome proliferator-activated receptor-γ coactivator-1α; LDs, lipid droplets; NRCMs, neonatal rat cardiomyocytes; OA, oleic acid; BSA, bovine serum albumin.

Article Snippet: Protein was extracted from cultured NRCMs using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitors (1:100; Sigma-Aldrich; Merck KGaA) and quantified with a bicinchoninic acid (BCA) protein assay.

Techniques: Over Expression, Staining, Cell Culture

Journal: Autophagy

Article Title: Autophagy modulates cell fate decisions during lineage commitment

doi: 10.1080/15548627.2021.2008691

Figure Lengend Snippet: Kinetics of autophagy during differentiation to mesoderm and neuroectoderm. (A) Autophagy kinetics during cardiac differentiation. AG27 human iPSCs were seeded and treated with CHIR on day 3 (8 µM). On day 6, cells were treated with IWP2 (5 µM). Cells started beating between day 8 and 17. Cell lysates were collected at different time points and processed for Western blotting using the indicated antibodies. The blots are representative of three independent experiments ( n = 3). ACTB was used as a loading control. (B) Quantification of SQSTM1 and LC3B-II levels (relative to ACTB) during mesoderm (cardiomyocyte) formation from AG27 cells. The relative expression levels were normalized to that at day 0 and are presented as the mean of three independent experiments ( n = 3); error bars represent SD. * P < 0.1, ** P < 0.01, *** P < 0.001. (C) Quantification of WDFY and p-ULK1 (Ser-555):total ULK1 levels (relative to ACTB) during mesoderm (cardiomyocyte) formation from AG27 cells. The relative expression levels were normalized to that at day 0 and are presented as the mean of three independent experiments ( n = 3); error bars represent SD. * P < 0.1, ** P < 0.01, *** P < 0.001. (D) AG27 hiPSCs with stable Dox-inducible expression of mCherry-EGFP-LC3B were seeded as single cells and differentiated to cardiomyocytes. The cells were fixed at different days after induction of cardiomyocytes differentiation until day 7 and processed for microscopy. Images are representative of three independent experiments. Scale bar: 10 µm (day 0–5), 100 µm (day 7). (E) The number of LC3B puncta formed during DE formation were quantified from a minimum of 100 cells from each time point (except day 7) as shown in ( D ) using ImageJ analysis. Error bars represent SD. *** P < 0.001. (F) Autophagy kinetics during neuroectoderm differentiation. AG27 cells were collected at the indicated time points during neuroectoderm differentiation and processed for Western blotting using the indicated antibodies ( n = 3). ACTB was used as a loading control. (G) Quantification of WDFY, p-ULK1:ULK1 and LC3B-II levels (relative to ACTB) during neuroectoderm formation (day 0-day 6) from AG27 cells. The relative expression levels were normalized to that at day 0 and are presented as the mean of three independent experiments ( n = 3); error bars represent SD. * P < 0.1, ** P < 0.01, *** P < 0.001. (H) AG27 hiPSCs with stable Dox-inducible expression of mCherry-EGFP-LC3B were seeded as colonies (EDTA splitting) and differentiated to neuroectoderm. The cells were fixed at different days after induction of neuroectoderm differentiation and processed for microscopy. Images are representative of three independent experiments. Scale bar: 10 µm. (I) The number of red puncta were quantified from a minimum of 100 cells from each time point as shown in ( H ) using ImageJ analysis. Error bars represent SD.

Article Snippet: CHIR99021 (for cardiomyocytes) , Tocris Bioscience , 4423.

Techniques: Western Blot, Control, Expressing, Microscopy

Journal: Autophagy

Article Title: Autophagy modulates cell fate decisions during lineage commitment

doi: 10.1080/15548627.2021.2008691

Figure Lengend Snippet: Cells, antibodies and other reagents used in the study are indicated below.

Article Snippet: CHIR99021 (for cardiomyocytes) , Tocris Bioscience , 4423.

Techniques: Recombinant, Membrane, Derivative Assay, Knock-Out, CRISPR, Bicinchoninic Acid Protein Assay, Protease Inhibitor, Extraction, Reverse Transcription, SYBR Green Assay, Gene Expression, Virus, Plasmid Preparation, Software

Journal: Autophagy

Article Title: Autophagy modulates cell fate decisions during lineage commitment

doi: 10.1080/15548627.2021.2008691

Figure Lengend Snippet: Autophagy modulation affects DE differentiation through SOX2 degradation. (A) AG27 iPSCs were treated with CHIR to induce DE formation in the absence or presence of the MTOR inhibitor rapamycin. After 48 h, SOX17 levels were assessed by RTqPCR. The graph is representative of three independent experiments ( n = 3). Error bars represent SD. *** P < 0.001. (B) AG27 iPSCs were treated as in ( A ) and processed for Western blotting using a SOX17 antibody ( n = 3). ACTB was used as a loading control. (C) AG27 cells were treated with CHIR to induce DE differentiation in the absence or presence of the ULK1 inhibitor MRT68921. Cell lysates were collected at different timepoints of DE differentiation and analyzed by Western blotting using antibodies against the pluripotency factors POU5F1, SOX2 and NANOG ( n = 3). ACTB was used as a loading control. (D) Quantification of SOX2 levels (relative to ACTB) in lysates from cells treated as in (C) at 0, 12 and 48 h during DE differentiation. The relative expression levels were normalized to that at day 0 and are presented as the mean of three independent experiments ( n = 3); error bars represent SD. * P < 0.1, ** P < 0.01, *** P < 0.001. (E) Cell lysates from AG27 cells treated or not with autophagy inhibitor, BafA1 or autophagy inducer, rapamycin and were collected at different timepoints (0, 12 and 48 h) of DE differentiation, followed by immunoblotting with the indicated antibodies ( n = 3). ACTB was used as loading control. (F) Quantification of SQSTM1, SOX2 and SOX17 levels (relative to ACTB) in cell lysates from BafA1 and rapamycin treated cells during DE differentiation (0, 12 and 48 h). The relative expression levels were normalized to that at day 0 and are presented as the mean of three independent experiments ( n = 3); error bars represent SEM. * P < 0.1, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (G) Cell lysates from ATG7 ± cells directed toward DE differentiation at different time points (0, 12 and 48 h) were analyzed by Western blotting using antibodies against FOXA2 and SOX2. ACTB was used as loading control. Blot is representative of three independent experiments. (H) Quantification of SOX2, FOXA2 and SOX17 levels (relative to ACTB) in cell lysates from ATG7 ± cells during DE differentiation (0, 12 and 48 h). The relative expression levels were normalized to that at day 0 and are presented as the mean of three independent experiments ( n = 3); error bars represent SD. * P < 0.1, ** P < 0.01, *** P < 0.001. (I) AG27 with stable Dox-inducible expression of mCherry-EGFP-tagged SOX2 were treated with CHIR or CHIR+MRT to induce DE formation. MRT was removed after 8 h of treatment. The cells were fixed at 24 h (left panels) or 48 h (right panels) post treatment and processed for microscopy. Red puncta represent SOX2 in autolysosomes. Images are representative of three independent experiments. Scale bar: 10 µm. (J) Quantification of the number of red-only puncta formed (autophagic flux) during DE differentiation from images as shown in (J). A minimum of 20 images at each time point ( n = 20) were used for quantification with ImageJ analysis. Error bars represent SD. ** P < 0.01. (K) AG27 were treated with CHIR or CHIR+MRT to induce DE formation. MRT was removed after 8 h of treatment. Cells were fixed at 24 (upper left two panels) or 48 h (upper right two panels) post differentiation, followed by immunofluorescence analysis against endogenous SOX2 and LAMP1 (lysosome marker). Nuclei were counterstained with DAPI. The lower panel represents zoomed images on the indicated region. Images are representative of three independent experiments. Scale bar: 10 µm. (L) AG27 hiPSCs were treated with CHIR to induce DE formation and fixed 24 h post differentiation, followed by immunofluorescence analysis against endogenous SOX2 and SQSTM1. Nuclei were counterstained with Hoechst. Arrowheads show colocalization. Image is representative of three independent experiments. Scale bar: 10 µm.

Article Snippet: CHIR99021 (for cardiomyocytes) , Tocris Bioscience , 4423.

Techniques: Western Blot, Control, Expressing, Microscopy, Immunofluorescence, Marker