Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: (A) Comprehensive flowchart of adult cardiomyocyte isolation, transfection with siCocktail or siControl, and RNAseq analysis. (B) Bar graph representing the significant knockdown of Rb1 and Meis2 after siRNA transfection. (C) Venn diagram represents the comparative analysis approach to analyze the differentially expressed genes among the groups. (D) Heatmap demonstrates differentially expressed genes after knocking down Rb1 and Meis2 at early and late time points versus control. (E) Sankey and dot plot represents the pathways associated with the up-regulated genes from both early and late-responding groups. (F) Venn diagram showing the 25 genes, commonly up-regulated with ≥2-fold change in siCocktail transfected group (at 48h and D7 after transfection). This analysis further revealed that all 25 genes were downregulated in the control groups at both time points (48h vs D7 post-transfection). (G) Heat map showing the expression of all 25 genes (identified in sub-panel ‘F’) among different study groups. (H) The bar graph shows the expression of MCM2 at early (48h) and late (D7) time points after knocking down Rb1 and Meis2 . (I) The immuno-blot images show reduced expression of MCM2 in adult hearts versus neonatal hearts, and the (J) corresponding bar graph shows the quantification of Western blot. N=6 mice per group. Data represented as mean±SE. * = p-value ≤0.05. Statistical analysis: T -test was used to compare the two groups. P value ≤ 0.05 was considered statistically significant. siCocktail = siRb1+ siMeis2.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Isolation, Transfection, Knockdown, Control, Expressing, Western Blot

Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: (A) Comprehensive flowchart representing the hypothesis that inhibition of Rb1 improves cardioprotection through MCM2 . (B) Schematics showing in vivo experimental design, illustrating LAD ligation, intramyocardial injection of MCM2 overexpressing plasmid, or placebo. Animals were observed for 21 days before euthanization and sample collection for molecular and histochemical analysis. Trans- thoracic echocardiography was performed on day 3 and day 21, post-surgery. (C) Dot plot representation of heart-to-body weight ratio among the study groups. (D) Representative echocardiography images showing the cardiac function in different study groups. Dot plots representing (E) ejection fraction, and (F) cardiac output among the study groups. (G) Representative Masson’s trichrome-stained images show cardiac remodeling among the groups. (H) Dot plot representing the quantitative analysis of Masson’s trichrome stained heart sections in all study groups. N=6 mice per group. Data represented as mean±SE. Scale bars represent the magnification of the corresponding image. Statistical analysis: One-way ANOVA was used to compare multiple groups, and post-hoc analysis (Bonferroni test) was performed to correct the p values from multiple comparisons. P value ≤ 0.05 was considered statistically significant. * = p- value ≤0.05. # = statically nonsignificant.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Inhibition, In Vivo, Ligation, Injection, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: (A) Representative immunohistology images showing vascular density in different study groups. Vascular density was analyzed through αSMA (green), and vWF (red) staining. (B) The dot plot shows the quantification of measured vascular density per field in different study groups. (C) Schematic representation of hypothesis, showing that the paracrine factors from MCM2 overexpressing adult CMs communicate with endothelial cells and improve angiogenesis. (D) Illustration of tube formation assay, showing ACM transfection and conditioned medium (CM) isolation, which was used to treat HUVECs and the subsequent tube formation assay. (E) Representative bright-field images illustrating endothelial tube formation potential in MCM2-CoMed treated groups versus control. (F-H) Dot plots represent the quantification of various parameters of tube formation. Quantification of endothelial tube formation was performed by Image J. Data represented as mean±SE. N = ≥ 10 images per group. Statistical significance was calculated through a T -test to compare the data between the groups. However, ANOVA was used to compare the data among the groups, and post hoc analysis (Bonferroni test) was performed to correct the p values from multiple comparisons. P value ≤ 0.05 was considered statistically significant. * = p-value ≤0.05. # = statically nonsignificant. Scale bars represent the magnification of the corresponding image. In vivo analysis: N =6 animals per group. In vitro: more than 10 images per group were analyzed for vascular density analysis.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Staining, Tube Formation Assay, Transfection, Isolation, Control, In Vivo, In Vitro

Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: (A) Schematic illustration of protein-protein interaction analysis using TurboID technology. (B) Silver staining image of a 2D SDS-PAGE showing the enrichment of proteins, interacting with MCM2. (C) Venn diagram revealing 243 proteins, interacting with MCM2. (D) Sankey and dot plot shows the biological pathways associated with the proteins that physically interact with MCM2. The size of the bubble corresponds to the number of genes in that component. Whereas, the color of the bubble corresponds to the significance value. (E) The chord plot demonstrates the MCM2 interacting proteins, which also occur in extracellular space and may contribute to paracrine signaling.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Silver Staining, SDS Page

Journal: bioRxiv

Article Title: MCM2 mediates post-MI cardioprotection by promoting the pro-angiogenic cardiosome signaling

doi: 10.1101/2024.12.12.628232

Figure Lengend Snippet: Schematics illustration of MCM2 overexpression in adult CM using AAV9 viral vector, collection of exosomes from MCM2 overexpressing CM, and proteomic analysis. The secretory proteins were characterized through Mass spectrometry. (B) Venn diagram revealed 23 unique proteins, which are secreted from the MCM2 overexpressing CM versus control. (C) The heatmap shows the expression pattern of the MCM2-induced 23 secretory proteins in previous RNAseq data after knocking down Rb1 and Meis2 in adult CM. The expression patterns of all 23 proteins were similar in the RNA seq analysis at early and late time points. (D) In silico analysis shows interaction between the 31 MCM2 interacting proteins with 13 proteins, which are secreted from MCM2 overexpressing CM. (E) In silico analysis using FunRich3.1.3 shows that many of the 23 proteins, which are specifically secreted from MCM2 overexpressing CMs are being regulated by hypoxia and stress-associated transcription factors.

Article Snippet: First, the AAV9-TNT- MCM2 vector was created by amplifying MCM2 cDNA from mEmerald-MCM2- N-22 (Addgene ID: 54164) using forward primer: TAAGCAggatccaccaATGCAAGCGGGCCCGGCC , and reverse primer: TGCTTAaagcttGAATTCTCAGAACTGCTGCAG .

Techniques: Over Expression, Plasmid Preparation, Mass Spectrometry, Control, Expressing, RNA Sequencing Assay, In Silico