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: Journal of Molecular Histology

Article Title: CLDN11 deficiency upregulates FOXM1 to facilitate breast tumor progression through hedgehog signaling pathway

doi: 10.1007/s10735-024-10267-5

Figure Lengend Snippet: CLDN11 deficiency upregulated FOXM1 to stimulate breast cancer development. A , The top 30 upregulated genes in CLDN11 low patients compared with CLDN11 high patients with breast cancer ( n = 817 in total). B , Kaplan-Meier overall survival curve was shown according to high and low expression of FOXM1 in breast cancer patients derived from TCGA data ( n = 1044). C , mRNA expression of FOXM1 in normal tissues ( n = 292) and tumor tissues from TCGA derived patients ( n = 1092). D , Immunohistochemical staining of FOXM1 in tumor tissues from breast cancer patients with lymph node metastasis ( n = 14) or not ( n = 12). E , Western blotting of CLDN11 and FOXM1 in MCF-7/MDA-MB-231 cells treated with scramble or CLDN11 siRNAs. F and G , MCF-7/MDA-MB-231 cells were treated with scramble or CLDN11 siRNAs. Then cells were treated with PBS or RCM-1 (80 nM, 48 h). Cell proliferation ( F ) and migration ( G ) were analyzed. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s, no significant difference

Article Snippet: The FOXM1 inhibitor RCM-1 was sourced from Selleck (USA), while the Hedgehog signaling pathway inhibitor Hedgehog IN-3 was obtained from MedChemExpress (USA).

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining, Western Blot, Migration

Journal: Journal of Molecular Histology

Article Title: CLDN11 deficiency upregulates FOXM1 to facilitate breast tumor progression through hedgehog signaling pathway

doi: 10.1007/s10735-024-10267-5

Figure Lengend Snippet: CLDN11 deficiency upregulated FOXM1 through hedgehog signaling pathway. A , The differentially expressed genes were compared between CLDN11 low and high patients with breast cancer ( n = 817 in total) from TCGA database. Kyoto encyclopedia of genes and genomes enrichment was then performed. B , mRNA expression of PT1, PT2 and SMO in MCF-7/MDA-MB-231 cells treated with scramble or CLDN11 siRNAs. C , Western blotting of CLDN11, Gli1 and Gli2 in MCF-7/MDA-MB-231 cells treated with scramble or CLDN11 siRNAs. D , Western blotting of CLDN11, Gli, Gli2 and FOXM1 in MCF-7/MDA-MB-231 cells treated with scramble or CLDN11 siRNAs (combined with 5 nM hedgehog-IN3 or not). E and F , MCF-7/MDA-MB-231 cells were treated with scramble or CLDN11 siRNAs. Then cells were treated with PBS or hedgehog-IN3 (5 nM, 48 h). Cell proliferation ( E ) and migration ( F ) were analyzed. *, p < 0.05; **, p < 0.01; n.s, no significant difference

Article Snippet: The FOXM1 inhibitor RCM-1 was sourced from Selleck (USA), while the Hedgehog signaling pathway inhibitor Hedgehog IN-3 was obtained from MedChemExpress (USA).

Techniques: Expressing, Western Blot, Migration

Journal: Journal of Molecular Histology

Article Title: CLDN11 deficiency upregulates FOXM1 to facilitate breast tumor progression through hedgehog signaling pathway

doi: 10.1007/s10735-024-10267-5

Figure Lengend Snippet: CLDN11 modulated breast tumor growth in vivo. A , The quantification of tumor volume in subcutaneous MCF-7 (scramble or CLDN11 siRNAs pre-treatment) bearing mice ( n = 6 per group). B ~ D , Tumor tissues were isolated on day 25 in ( A ). The Ki-67 expression was analyzed by immunohistochemical staining ( B ). The expression of PT1, PT2, and SMO was analyzed by qPCR ( C ). The expression of CLDN11, Gli1, Gli2 and FOXM1 was analyzed by western blotting ( D ). E , Subcutaneous MCF-7 (scramble or CLDN11 siRNAs pre-treatment) bearing mice were treated with PBS or hedgehog-IN3. Tumor volume was quantified ( n = 6 per group). *, p < 0.05; **, p < 0.01; n.s, no significant difference

Article Snippet: The FOXM1 inhibitor RCM-1 was sourced from Selleck (USA), while the Hedgehog signaling pathway inhibitor Hedgehog IN-3 was obtained from MedChemExpress (USA).

Techniques: In Vivo, Isolation, Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: Biochemical Journal

Article Title: Calpain-2 specifically cleaves Junctophilin-2 at the same site as Calpain-1 but with less efficacy

doi: 10.1042/BCJ20210629

Figure Lengend Snippet: Representative Western blots and quantification of in vitro β2-Spectrin ( A , B ), cardiac troponin T (cTnT) ( C , D ), and cardiac troponin I (cTnI) ( E , F ) cleavage assays in response to CAPN1 and CAPN2 expression and different Ca 2+ concentrations. Assays were performed as indicated and as described in . n = 3 for each group. Data are mean ± SEM. One-way ANOVA followed by the Tukey multiple-comparisons test. * P < 0.05.

Article Snippet: The pCMV6-XL5-CAPN1 and pCMV6-entry-CAPN2 plasmids expressing human CAPN1 and CAPN2 were provided by Dr. Tianqing Peng (Western University, Canada). cDNA of human cTnT (Origene, RC201218), human cTnI (Origene, RC214740) were fused with C-terminal Flag tag and human β2-Spectrin (Addgene, #31070) were fused with C-terminal HA tag.

Techniques: Western Blot, In Vitro, Expressing

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