Cited2 regulates proliferation and survival in young and old mouse cardiac stem cells

Figures

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  Fig. 1. Age-dependent expression of Cited 2 in mice heart tissues and CSCs. a. RT-PCR for Cited2 mRNA expression in Young (2–3 months), Middle (4–6 months) and Old (20–22 months) mice heart tissues. b. The analysis of RT-PCR for Cited2 mRNA expression. c. DD-PCR for Cited2 mRNA expression in Young (2–3 months) and Old (20–22 months) CSCs. d-g. Immunoblotting of CITED2 protein levels in lentivector transduction groups and CSCs groups. OE represented Old CSCs transducted with lentiviral particles of Cited2-cDNA, KD represented Young CSCs transducted with lentiviral particles of Cited2-shRNA3, the control was transducted with empty lentivector. Data were from three independent experiments and presented as mean ± SD. Statistical analysis was implemented by GraphPad Prism 5, *p < 0.05, ** p < 0.01, and *** p < 0.001, Old-CSCs contrast to Young-CSCs

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  Fig. 2. The affect of Cited2 overexpression on differentiation in Old cardiac stem cells. a:DD-PCR of Cited2 mRNA expression for lentivector transduction. The control was transducted with empty lentivector. Overexpression represented Old CSCs transducted with lentiviral particles of Cited2-cDNA; Data were from three independent experiments and presented as mean ± SD. Statistical analysis was implemented by GraphPad Prism 5, *p < 0.05, ** p < 0.01, and *** p < 0.001. b: The differentiation efficiency of CSCs from Cited2 overexpression and negative control group. The percentage % of cells differentiated into cardiomyocytes was calculated cardiomyocytes marker cells among total GFP cells based on a mean of five independent experiments. Statistical analysis was implemented by GraphPad Prism 5, *p < 0.05, ** p < 0.01, and *** p < 0.001, contrast to control. c-e: Immunofluorescence staining (red) of cardiomyocytes-specific marker-cTnI; smooth muscle marker-α-SMA and enodthelial cell marker-VWF; transduction CSCs [17] staining with GFP; then merged images. Overexpression represented Old CSCs transfected with lentiviral particles of Cited2-cDNA; The control was transducted with empty…

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  Fig. 3. The affect of Cited2 knockdown on differentiation in Young cardiac stem cells. a: DD-PCR of Cited2 mRNA expression for lentivector transduction. The control was transducted with empty lentivector. Knockdown represented Young CSCs transducted with lentiviral particles of Cited2-shRNA1, Cited2-shRNA2 and Cited2-shRNA3; Data were from three independent experiments and presented as mean ± SD.Statistical analysis was implemented by GraphPad Prism 5, *p < 0.05, ** p < 0.01, and *** p < 0.001, contrast to control. b: The differentiation efficiency of CSCs from Cited2 knockdown and negative control group. The percentage % of cells differentiated into cardiomyocytes was calculated cardiomyocytes marker cells among total GFP cells based on a mean of five independent experiments. Statistical analysis was implemented by GraphPad Prism 5, *p < 0.05, ** p < 0.01, and *** p < 0.001, contrast to control. c-e: Immunofluorescence staining (red) of cardiomyocytes-specific marker-cTnI; smooth muscle marker-α-SMA and enodthelial cell marker-VWF; transfection CSCs staining with GFP; then merged images. Each experiment is repeated five times, and the picture represents a single result. Knockdown…

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  Fig. 4. Cited2 overexpression affects cell proliferation in Old CSCs. a The proliferation was tested by FCM with EdU assays. O-overexpression represented Old CSCs transducted with lentiviral particles of Cited2-cDNA. The control was transducted with empty lentivector. b Statistical analysis of proliferation rate was implemented by GraphPad Prism 5. c Cell cycle analysis was carried out by FCM, statistical analysis of apoptosis rate was implemented by GraphPad Prism 5, *p < 0.05, ** p < 0.01, and *** p < 0.001, contrast to control. Data are based on three independent experiments, and the picture represents a single result

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  Fig. 5. Cited2 knockdown affects cell proliferation in Young CSCs. a The proliferation was tested by FCM with EdU assays. Y-knockdown represented Young CSCs transducted with lentiviral particles of Cited2-shRNA3; The control was transducted with empty lentivector. b Statistical analysis of proliferation rate was implemented by GraphPad Prism 5. c Cell cycle analysis was carried out by FCM, statistical analysis of apoptosis rate was implemented by GraphPad Prism 5, *p < 0.05, ** p < 0.01, and *** p < 0.001, contrast to control. Data are based on three independent experiments, and the picture represents a single result. Knockdown represented Young CSCs transducted with lentiviral particles of Cited2-shRNA3. The negative control was transducted with empty lentivector

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  Fig. 6. Cited2 affects cell apoptosis with CSCs aging. The apoptosis was tested by FCM with Annexin V-PE apoptosis detection kit and the PE-positive cells percentage stand for the ratio of apoptosis cells to all numbered cells. a The apoptosis rate in Old CSCs transducted with lentiviral particles of Cited2-cDNA (left panel) and empty lentivector (right panel). b Statistical analysis of apoptosis rate was implemented by GraphPad Prism 5. c The apoptosis rate in Young CSCs transducted with lentiviral particles of Cited2-shRNA3 (left panel) and empty lentivector(right panel). d Statistical analysis of apoptosis rate was implemented by GraphPad Prism 5. *p < 0.05, ** p < 0.01, and *** p < 0.001, contrast to control. Data are based on three independent experiments and the picture represents a single result. Overexpression represented Old CSCs transducted with lentiviral particles of Cited2-cDNA. Knockdown represented Young CSCs transducted with lentiviral particles of Cited2-shRNA3.The negative control was transducted with empty lentivector