mcm2 cdna  (Addgene inc)

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

Journal: PLoS ONE

Article Title: Whole Exome Sequencing Identified MCM2 as a Novel Causative Gene for Autosomal Dominant Nonsyndromic Deafness in a Chinese Family

doi: 10.1371/journal.pone.0133522

Figure Lengend Snippet: (a) Pedigree of Family #51 with hearing loss. This pedigree demonstrates nonsyndromic hearing loss of autosomal dominant inheritance. Open symbols, unaffected; solid symbols, affected. Squares, male; circles, female; slashed, deceased individual. Slanting arrow, the proband. Family members with the C>T symbol denote c.130C>T variant in MCM2 , those with the C/C symbol had no variant in MCM2 , and those without the symbols were not examined. Segregation of hearing loss with the c.130C>T variant in MCM2 is remarkable in this pedigree. (b) Results of pure tone audiometry for eight members of Family #51 with hearing loss. Threshold data were obtained from the worse ears of the eight affected family members. We can clearly deduce from this graph that hearing loss is variable and mild to severe in degree in these cases. (c) A heterozygous c.130C>T variant in exon 2 of the MCM2 gene was identified in the affected members in this pedigree. (d) Conservation analysis showed that Arg44 in human MCM2 is conserved across human, bovine, zebra finch, green anole, zebrafish, nile tilapia, acorn worm, sea urchin lineages.

Article Snippet: A mammalian expression plasmid containing an open reading frame of human MCM2 cDNA with a green fluorescent protein (GFP) tag at the 3′ end was obtained from Origene (Rockville, MD, USA).

Techniques: Variant Assay

Journal: PLoS ONE

Article Title: Whole Exome Sequencing Identified MCM2 as a Novel Causative Gene for Autosomal Dominant Nonsyndromic Deafness in a Chinese Family

doi: 10.1371/journal.pone.0133522

Figure Lengend Snippet: (a) Western blot of cochlear tissues from guinea pigs. The three lanes were total cochlear protein (Total), cochlear tissues without spiral ligament or stria vasculari (without sl. or sv.), and spiral ligament and stria vascularis parts (sl. and sv.), respectively. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the reference. (b-f) Whole mounts of the hearing sensory epithelial layer from mature rat were prepared for immunofluorescence using anti-MCM2 antibody. Serial sections of confocal microscopic scanning were taken from superficial to deeper layers with an interval of 1 μm/layer at the site of one line of inner hair cells (double arrows) and three lines of outer hair cells (1, 2, 3 in the panels). MCM2 was stained with Alexa Fluor 488 and nuclei with DAPI. The nuclei of the outer hair cells are not shown as they lie deeper than the level of section G. MCM2 was distributed in the cytoplasm and was highly expressed in the hair skin plate of the two kinds of hair cell. Scale = 10 μm. (g) Cochlear frozen sections from mature rat were prepared for immunofluorescence using anti-MCM2 antibody. MCM2 was stained with Alexa Fluor 488. Three outer hair cells on the left and one inner hair cell on the right were shown in the panel. Scale = 10 μm.

Article Snippet: A mammalian expression plasmid containing an open reading frame of human MCM2 cDNA with a green fluorescent protein (GFP) tag at the 3′ end was obtained from Origene (Rockville, MD, USA).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Whole Exome Sequencing Identified MCM2 as a Novel Causative Gene for Autosomal Dominant Nonsyndromic Deafness in a Chinese Family

doi: 10.1371/journal.pone.0133522

Figure Lengend Snippet: (a-b) HEK293 cells were treated with liposome transfection reagent only (lipo), empty vector transfection (empty vector), wild-type MCM2 plamid transfection (WT), and variant MCM2 plasmid transfection (M) respectively. After transfection for 48 h, apoptotic cell ratios were determined with annexin-V-PE-staining. Data represent the mean and SD of 3 experiments. The asterisks (**) indicate significant differences between the control and experimental groups or differences between the WT group and the M group (**p<0.001). (c-d) The western blot analysis of whole cell lysates from HEK293 cells showed a level of endogenic expression of MCM2 (as shown in lipo and empty vector groups). The upper bands in lane 3 and 4 (from the left) represent the transfected exogenous MCM2 with a GFP tag. The endogenous level of MCM2 is shown in the lower band. The ratio of cleaved caspase3 densitometry to exogenous MCM2 densitometry is compared. In the histogram, whole cell lysates from the variant MCM2 (M) transfected cells showed an upper regulated expression of cleaved caspase-3 compared with wild-type MCM2 (WT) transfected cells. Data represent the mean and SD of 3 experiments. The asterisks (*) indicate significant differences between the WT group and the M group (*p<0.01). (e) Cell cycle phases of HEK293 cells transiently transfected with wild-type or c.130C>T variant MCM2 cDNA plasmid for 48 h. Cell cycle phases were measured by flow cytometry using a cell cycle assay kit. In cells transfected with wild-type MCM2 cDNA plasmid, G1, S, and G2 phases were 27.08±4.99, 50.69±2.14, 22.21±6.04, respectively; N = 3. In cells transfected with the variant MCM2 cDNA plasmid, G1, S, and G2 phases were 28.61±1.29, 44.80±0.64, 26.57±0.80, respectively; N = 3.

Article Snippet: A mammalian expression plasmid containing an open reading frame of human MCM2 cDNA with a green fluorescent protein (GFP) tag at the 3′ end was obtained from Origene (Rockville, MD, USA).

Techniques: Transfection, Plasmid Preparation, Variant Assay, Staining, Control, Western Blot, Expressing, Flow Cytometry, Cell Cycle Assay