β-arrestin2 Affects Cardiac Progenitor Cell Survival through Cell Mobility and Tube Formation in Severe Hypoxia

Figures

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  Figure 1. Basic morphologic difference of 2 types of CPC and higher apoptotic rate of CPC-KO than CPC-WT in the regular culture condition. (A) Morphologic distinction of CPC-WT and CPC-KO in the routine culture. (B) c-kit positivity (red) was observed in more than 95% of cell in CPC-WT and CPC-KO in passage 3. Blue was nucleus of CPCs stained with DAPI. (C, D) Protein expression of β-arr2 was strong in CPC-WT, but was not observed in CPC-KO; brain tissue of SD-rat was used as positive control. (E-H) CPC-KO was more apoptotic and necrotic compared to CPC-WT in in the routine culture. CPC = cardiac progenitor cell; CPC-KO = CPC obtained from β-arr2 knock-out mouse; CPC-WT = CPC obtained from wild-type mouse; DAPI = 4′,6-diamidino-2-phenylindole; PI = propodium iodide; SD = Sprague Dawley; β-arr2 = β-arrestin2. *p<0.05, †p<0.01.

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  Figure 2. CPC-WT showed better expression profiles of proteins related with mobility especially in serum starvation compared to CPC-KO. (A-C) t-ERK expression was similar in CPC-WT and CPC-KO in all culture conditions. p-ERK/t-ERK expression increased similarly both in CPC-WT and CPC-KO in serum-free culture for 24 and 48 hours. (D-F) Expression of t-Akt was higher in CPC-WT in control condition and p-Akt/t-Akt was significantly higher in CPC-WT at all culture condition compared to CPC-KO. (G-I) β-catenin expression was sharply decreased in CPC-KO at 48 hours serum starvation culture. GSK-3β expression was significantly higher in CPC-WT than in CPC-KO in all culture conditions. Con = control; CPC-KO = cardiac progenitor cell obtained from β-arr2 knock-out mouse; CPC-WT = cardiac progenitor cell obtained from wild-type mouse; GSK-3β = glycogen synthase kinase-3β; p-Akt = phosphorylated-protein kinase-B; p-ERK = phosphorylated-extracellular signal-regulated kinase; star = serum starvation; t-Akt = total-protein kinase-B; t-ERK = total-extracellular signal-regulated kinase; β-arr2 = β-arrestin2. *p<0.05.

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  Figure 3. CPC-KO showed worse mobility compared to CPC-WT in normoxic and hypoxic cultures. (A) Normoxic and (B) hypoxic culture. (C, D) Percentage of area scratched wound closure was higher in CPC-WT than in CPC-KO. CPC-KO = cardiac progenitor cell obtained from β-arr2 knock-out mouse; CPC-WT = cardiac progenitor cell obtained from wild-type mouse; β-arr2 = β-arrestin2. *p<0.05.

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  Figure 4. CPC-WT showed better expression profiles of proteins related cell mobility and tube formation in normoxic and hypoxic cultures compared to CPC-KO. Proteins of Con-WT and Con-KO were obtained from the routine culture condition. And other cells were pre-treated with serum-free medium for 24 hours. And the experiment was carried out under each condition. (A-C) p-ERK/t-ERK expression level was higher in CPC-KO than CPC-WT in all culture conditions except the O2+/serum+ culture. (D-F) p-Akt/t-Akt was higher in CPC-WT than in CPC-KO in all cultures. (G-I) CPC-WT showed higher expression of β-catenin in control and O2+/serum+ culture. CPC-KO showed higher expression of β-catenin in O2−/serum− culture. GSK-3β was significantly higher in CPC-WT than in CPC-KO in all cultures. Con-KO = β-arrestin2 knock-out cardiac progenitor cell in the routine culture condition; Con-WT = wild-type cardiac progenitor cell in the routine culture condition; CPC-WT = cardiac progenitor cell obtained from wild-type mouse; CPC-KO = cardiac progenitor cell obtained from β-arr2 knock-out mouse; GSK-3β = glycogen synthase kinase-3β; p-Akt = phosphorylated-protein kinase-B; p-ERK = phosphorylated-extracellular…

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  Figure 5. CPC-WT showed better tube formation ability compared to CPC-KO in normoxic and hypoxic cultures. (A-C) Constituted tube length and total tube number was higher in CPC-WT than in CPC-KO under normoxic condition after pretreatment of serum-starvation. (D-G) Constituted tube length and total tube number was higher in CPC-WT than in CPC-KO under normoxic and hypoxic culture without pretreatment of serum starvation. CPC-KO = cardiac progenitor cell obtained from β-arr2 knock-out mouse; CPC-WT = cardiac progenitor cell obtained from wild-type mouse. *p<0.05, †p<0.01.