Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Platelet Internalization Mediates Ferroptosis in Myocardial Infarction

doi: 10.1161/atvbaha.122.318161

Figure Lengend Snippet: Figure 2. Platelets are internalized by cardiomyocytes. A, Distribution of platelets in myocardial tissue (n=4). Platelets were labeled with CD41 (green), cardiomyocytes were labeled with cTnI (red), and nuclei visualized with DAPI (blue). Scale bar, 500 μm. B, Neonatal mouse cardiomyocytes (CMs) internalize platelets (n=3). CMs were labeled by CM-Dil (red), platelets were labeled by CFSE (green). Scale bar, 100 µm. C, Three-dimensional reconstruction of confocal Z-stack images of CMs co-cultured with platelets to demonstrate uptake and perinuclear localization (n=3; view angle: 30, z-Scaling: 2).

Article Snippet: Primary antibodies included cTnI antibody (1:100, Proteintech, catalog 21652-1- AP) and CD41 antibody (1:200, Santa cruz, sc-365938).

Techniques: Labeling, Cell Culture

Journal: Scientific Reports

Article Title: Human platelet lysate combined with mesenchymal stem cells pretreated with platelet lysate improved cardiac function in rats with myocardial infarction

doi: 10.1038/s41598-024-79050-6

Figure Lengend Snippet: Immunofluorescent analysis indicates the expression of NKX2.5 and cTnI (green) in the hearts of rats with MI two and four weeks after PMSCs and HPL + PMSCs injection: ( A , B ): Sample micrographs of NKX2.5 and cTnI in an animal of each group. Quantification of the levels of NKX2.5 + and cTnI+ ( C and E ) and the number of Dil+/NKX2.5 + and Dil+/ cTnI+ ( D and F ) in the study groups. Stem cells stained with Dil dye (red) and nuclei with DAPI (blue). *** P < 0.001 vs. PMSCs 2 weeks; # < P < 0.05, ### P < 0.001 vs. HPL + PMSCs 2 weeks; &&& P < 0.01 vs. PMSCs; $$ P < 0.001, $$$ P < 0.001 vs. HPL + PMSCs. n = 3 in each group.

Article Snippet: Rabbit polyclonal primary antibody for NKX2 (No: orb540657), rabbit polyclonal antibody for cTnI (No: orb304638), and goat anti-rabbit IgG (H + L) antibody (FITC) (No orb688925) were purchased from Biorbyt, United Kingdom.

Techniques: Expressing, Injection, Staining

Journal: Nature Communications

Article Title: The nuclear receptor ERR cooperates with the cardiogenic factor GATA4 to orchestrate cardiomyocyte maturation

doi: 10.1038/s41467-022-29733-3

Figure Lengend Snippet: a Real-time quantitative polymerase chain reaction (RT-qPCR)-determined levels of the designated mRNAs during human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation. Levels of indicated genes are shown normalized to RPLP0 levels at each timepoint. Day 0 and 2, n = 9; Day 4, 6, 8, and 15, n = 8. * p < 0.05, *** p < 0.001, **** p < 0.0001 vs Day 0; one-way ANOVA followed by Dunnett’s multiple comparison test. b Representative immunoblots of estrogen-related receptor (ERR) α and γ protein levels during hiPSC-CM differentiation. α-Tubulin was used as a loading control. c Heatmap representing log 2 fold change [ERRα/γ knockout (KO)/wild-type control (WT)] of mRNA levels of genes involved in cardiac structural component, Ca 2+ handling function, and ion transport in two different KO cell lines (KO1, KO6). All changes were significant (Benjamini-Hochberg false discovery rate <0.05) except for SCN5A in KO1 ( p = 0.057). d Representative immunoblots of indicated protein levels in WT Control and ERRα/γ KO hiPSC-CMs. Total protein staining was shown as a loading control. e The mRNA levels of indicated genes determined by RT-qPCR in hiPSC-CMs following the infection of adenovirus expressing GFP or ERRγ (Ad-GFP or Ad-ERRγ). ** p < 0.01, *** p < 0.001, **** p < 0.0001, Ad-ERRγ vs Ad-GFP, two-tailed student’s t-test. PDK4 (Ad-GFP, n = 7 and Ad-ERRγ, n = 8); COX6A2 (Ad-GFP, n = 14 and Ad-ERRγ, n = 15); MDH1 and CKMT2 , (Ad-GFP, n = 7 and Ad-ERRγ, n = 8); TNNI3 (Ad-GFP, n = 14 and Ad-ERRγ, n = 15); TNNI1 (Ad-GFP, n = 10 and Ad-ERRγ, n = 11). f The mRNA levels of indicated hiPSC-CM maturation markers determined by RT-qPCR in WT and ERRα/γ KO hiPSC-CMs derived from KO1 and KO6 with ( n = 15) or without ( n = 9) maturation cocktail treatment for 7 days. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, WT vs ERRα/γ KO hiPSC-CMs, two-way ANOVA followed by Tukey’s multiple comparisons test. All graphs in a , e , and f represent the means ± SEM. n denotes independent biological replicates. g Representative immunoblot images to show ACSL1 and TNNI3 protein expression levels in WT and ERRα/γ KO hiPSC-CMs with or without maturation cocktail. Total protein staining was used as a loading control.

Article Snippet: The following antibodies were used: ERRα; SMYD1; VDAC; ACSL3; TNNI3 (Abcam catalog and clone #s: ab76228; EPR46Y, ab181372; EPR13574(B)-30, ab15895, ab151959, and ab47003 respectively); His tag; α-tubulin; ACSL1 (Cell Signaling Technology catalog and clone #s: 12698S; D3I1O, 3873; DM1A, and 9189; D2H5 respectively); HA tag and MYL2 (Proteintech catalog #s 51064-2-AP and 10906-1-AP); α-Actinin, FLAG M2, TNNI1, ACTB, and PGC-1α (Sigma-Aldrich catalog and clone #s: A7732-100UL; EA-53, F1804-50UG; M2, AV42117-100UL, A5316; AC-74, and ST1202; 4C1.3); GATA4 and MYBPC3 (SANTA CRUZ BIOTECHNOLOGY catalog and clone #s, sc-25310; G-4, and sc-137180; E-7).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Comparison, Western Blot, Control, Knock-Out, Staining, Infection, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: The nuclear receptor ERR cooperates with the cardiogenic factor GATA4 to orchestrate cardiomyocyte maturation

doi: 10.1038/s41467-022-29733-3

Figure Lengend Snippet: a Venn diagram indicates the overlapped peaks from published GATA4 chromatin immunoprecipitation sequencing (ChIP-seq; GSE85631) and ERRγ ChIP-seq (GSE113784) datasets generated from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). GATA4 and ERRγ-shared peaks (ERRγ + GATA4) are presented by orange-colored region. p values were calculated using Fisher’s exact test. b Pie chart represents the overlapped sites from published GATA4 ChIP-seq, cardiac super-enhancers (SEs; GSE85631), and ERRγ ChIP-seq (GSE113784) datasets in hiPSC-CMs. The 45 SEs where ERRγ and GATA4 peaks are colocalized within 200 base pairs are highlighted with yellow. c Top 20 transcription factors that significantly overlapped with ERRγ + GATA4 and ERRγ-GATA4 are plotted. ESRRA was ranked in the top 30 in the ERRγ + GATA4 analysis. Y axis represents the similarity (GIGGLE score) between published datasets and each peak set. d Bars indicate enrichment score for Gene Ontology (GO) Biological Process and GO Cellular Component terms significantly enriched in ERRγ + GATA4 targets. e Representative genomic browser tracks of TNNI3 , MYBPC3 , and MYH6-7 regions. f Bar graphs represent mRNA expression levels of a subset of ERRγ + GATA4 and ERRγ targets in Negative Control (NC) ( n = 9) and siGATA4#1 ( n = 7) or siGATA4#2 ( n = 7) transfected hiPSC-CMs. * p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparisons test. All bars represent the means ± SEM. n denotes independent biological replicates.

Article Snippet: The following antibodies were used: ERRα; SMYD1; VDAC; ACSL3; TNNI3 (Abcam catalog and clone #s: ab76228; EPR46Y, ab181372; EPR13574(B)-30, ab15895, ab151959, and ab47003 respectively); His tag; α-tubulin; ACSL1 (Cell Signaling Technology catalog and clone #s: 12698S; D3I1O, 3873; DM1A, and 9189; D2H5 respectively); HA tag and MYL2 (Proteintech catalog #s 51064-2-AP and 10906-1-AP); α-Actinin, FLAG M2, TNNI1, ACTB, and PGC-1α (Sigma-Aldrich catalog and clone #s: A7732-100UL; EA-53, F1804-50UG; M2, AV42117-100UL, A5316; AC-74, and ST1202; 4C1.3); GATA4 and MYBPC3 (SANTA CRUZ BIOTECHNOLOGY catalog and clone #s, sc-25310; G-4, and sc-137180; E-7).

Techniques: ChIP-sequencing, Generated, Derivative Assay, Expressing, Negative Control, Transfection

Journal: Nature Communications

Article Title: The nuclear receptor ERR cooperates with the cardiogenic factor GATA4 to orchestrate cardiomyocyte maturation

doi: 10.1038/s41467-022-29733-3

Figure Lengend Snippet: a mRNA expression level of TNNI3 in wild-type (WT) control ( n = 6) and ERRα/γ knockout (KO) line 1 or 6 ( n = 3) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) following the overexpression of PGC-1α and/or GATA4 (Ad-PGC-1α and/or Ad-GATA4). ** p < 0.01, **** p < 0.0001, two-way ANOVA followed by Tukey’s multiple comparisons test. b Representative immunoblot images to show hemagglutinin (HA)-tagged GATA4, His-tagged PGC-1α, TNNI3, and TNNI1. c Levels of HA-tagged GATA4 occupation on the indicated targets in WT ( n = 5 or n = 4 for TNNI3 and POU5F1 ) and ERRα/γ KO hiPSC-CMs ( n = 5). * p < 0.05 vs WT, two-tailed student’s t -test. MYH6 -7 denotes the enhancer site of MYH6-7 cluster. d Bar graphs represent relative light units (RLU) derived from TNNI3 promoter-luciferase reporter ( TNNI3 -luc) with overexpression of ERRγ, GATA4, and PGC-1α in H9c2 myoblast. of TNNI3 -luc. n = 12, **** p < 0.0001, one-way ANOVA followed by Tukey multiple comparisons test. e Bar graphs represent the RLU derived from TNNI3 -luc or COX6A2 luciferase reporter ( COX6A2 -luc) in H9c2 myoblasts transduced with CRISPR/Cas9 and non-targeting control (NTC) guide RNA (gRNA) or gRNA targeting ERRα and γ (ERRα/γ KD). n = 12 for NTC in COX6A2 -luc or n = 9 for others. *** p < 0.001, **** p < 0.0001, two-way ANOVA followed by Tukey’s multiple comparisons test. f Schematic of TNNI3 promoter with putative ERR response elements (ERRE) and GATA binding sites. g WT ( n = 25), ERRE-deleted ( n = 12) or GATA binding sites-deleted ( n = 16) - TNNI3 -luc were assessed with overexpression of ERRγ and/or GATA4 in H9c2 myoblasts. * p < 0.05, **** p < 0.0001, two-way ANOVA followed by Tukey’s multiple comparisons test. h Bar graphs represents RLU derived from TNNI3 -luc with overexpression of ERRγ ( n = 15), GATA4 WT ( n = 15), C-teriminal zinc finger deleted GATA4 (C-zincΔ, n = 12), N-terminal zinc finger deleted GATA4 (N-zincΔ, n = 6), the combination of ERRγ and WT (n = 15) or each GATA4 mutant (C-zincΔ, n = 12; N-zincΔ, n = 6) in H9c2 myoblasts. * p < 0.05, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test. i Bar graphs represents RLU derived from control reporter (empty-luc; n = 8 for ERRγ + PGC-1α or n = 9 for others) and MYH6-7 enhancer-luc ( n = 9) with overexpressed indicated factors in AC16 cells. **** p < 0.0001, two-way ANOVA followed by Tukey’s multiple comparisons test. All bars represent the means ± SEM. n denotes independent biological replicates.

Article Snippet: The following antibodies were used: ERRα; SMYD1; VDAC; ACSL3; TNNI3 (Abcam catalog and clone #s: ab76228; EPR46Y, ab181372; EPR13574(B)-30, ab15895, ab151959, and ab47003 respectively); His tag; α-tubulin; ACSL1 (Cell Signaling Technology catalog and clone #s: 12698S; D3I1O, 3873; DM1A, and 9189; D2H5 respectively); HA tag and MYL2 (Proteintech catalog #s 51064-2-AP and 10906-1-AP); α-Actinin, FLAG M2, TNNI1, ACTB, and PGC-1α (Sigma-Aldrich catalog and clone #s: A7732-100UL; EA-53, F1804-50UG; M2, AV42117-100UL, A5316; AC-74, and ST1202; 4C1.3); GATA4 and MYBPC3 (SANTA CRUZ BIOTECHNOLOGY catalog and clone #s, sc-25310; G-4, and sc-137180; E-7).

Techniques: Expressing, Control, Knock-Out, Derivative Assay, Over Expression, Western Blot, Two Tailed Test, Luciferase, Transduction, CRISPR, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: The nuclear receptor ERR cooperates with the cardiogenic factor GATA4 to orchestrate cardiomyocyte maturation

doi: 10.1038/s41467-022-29733-3

Figure Lengend Snippet: a Bar graphs represent the relative light unit (RLU) from pG5luc construct. ERRγ-Gal4, GATA4/PGC-1α/ERRγ-Gal4, GATA4/ERRγ-Gal4, and PGC-1α/ERRγ-Gal4, n = 18; Gal4, n = 17; PGC-1α/Gal4, n = 16; GATA4/Gal4, n = 15; GATA4/PGC-1α/Gal4, PGC-1α m/ERRγ-Gal4, PGC-1α m/ERRγ-Gal4, n = 9; PGC-1α m/Gal4, n = 6, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test. b Bar graphs represent the mRNA levels of indicated genes in wild-type control (WT, n = 8) and PGC-1α knockdown (KD, n = 8) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs WT, two-tailed Student’s t-test. c Levels of ERRγ occupation on the indicated targets in WT (n = 6 for MYH6 -7 and NPPA-NPPB , n = 8 for POU5F1 , n = 5 for others), PGC-1α KD ( n = 8 for TNNI3 , TNNC1 , FABP3 , and CPT1B , n = 6 for MYBPC3 and POU5F1 , n = 7 for MYH6 -7, NPPA-NPPB , and ATP5B ), and ERRα/γ knockout (KO) hiPSC-CMs ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 vs WT, one-way ANOVA followed by Dunnett’s multiple comparisons test. Stem cell enhancer region on POU5F1 was used as a negative control. MYH6 -7 and NPPA-NPPB denote the distal enhancer sites upstream of each gene cluster. d Representative immunoblot images to show the interaction between FLAG-tagged ERRγ and endogenous GATA4 in FLAG-ERRγ overexpressed hiPSC-CMs. e Schematic to indicate reported naturally occurring GATA4 mutations. NLS denotes nuclear localization signal. ERRγ-Gal4 experiment was performed with PGC-1α and GATA4 natural mutants. Bar graphs indicate RLU from pG5luc construct in AD-293 cells. ** p < 0.01, *** p < 0.001, vs ERRγ, PGC-1α, and WT GATA4 transfected group, one-way ANOVA followed by Tukey multiple comparisons test. n = 6. f TNNI3 -luc reporter experiment with overexpression of ERRγ and GATA4 WT or GATA4 G296S in H9c2 myoblast. Bar graphs represent RLU of TNNI3 -luc. n = 9, **** p < 0.0001, one-way ANOVA followed by Tukey multiple comparisons test. g Venn diagram showing significant overlap (using Fisher’s exact test) between RNA-seq datasets generated in G296S GATA4 hiPSC-CMs (GSE85631) and ERRα/γ KO hiPSC-CMs (GSE165963). Bar graphs represent enrichment score for significantly enriched Gene Ontology (GO) Biological Process and GO Cellular Component with the commonly downregulated genes. All bars in a , b , c , e , and f represent the means ± SEM. n denotes independent biological replicates.

Article Snippet: The following antibodies were used: ERRα; SMYD1; VDAC; ACSL3; TNNI3 (Abcam catalog and clone #s: ab76228; EPR46Y, ab181372; EPR13574(B)-30, ab15895, ab151959, and ab47003 respectively); His tag; α-tubulin; ACSL1 (Cell Signaling Technology catalog and clone #s: 12698S; D3I1O, 3873; DM1A, and 9189; D2H5 respectively); HA tag and MYL2 (Proteintech catalog #s 51064-2-AP and 10906-1-AP); α-Actinin, FLAG M2, TNNI1, ACTB, and PGC-1α (Sigma-Aldrich catalog and clone #s: A7732-100UL; EA-53, F1804-50UG; M2, AV42117-100UL, A5316; AC-74, and ST1202; 4C1.3); GATA4 and MYBPC3 (SANTA CRUZ BIOTECHNOLOGY catalog and clone #s, sc-25310; G-4, and sc-137180; E-7).

Techniques: Construct, Control, Knockdown, Derivative Assay, Two Tailed Test, Knock-Out, Negative Control, Western Blot, Transfection, Over Expression, RNA Sequencing, Generated