Figure 5: Endothelial marker expression in early and late cultured cardiac endothelial cells. (a) q-RT-PCR of endothelial cell genes revealed a decreased expression of CD31, CD34, eNOS, VE-Cad, and vWF in late cultured EC (passage 6, black bar) compared to early cultured EC (passage 1, white bar). In contrast, expression of sFlt-1 was increased. (b) the table shows average Ct values of each endothelial genes expressed by sorted cardiac endothelial cells from atrium in passage 1 and 6. The experiment was run from three different samples in each passage represented as Sample 1, 2, and 3, respectively. Each sample was performed in triplicate per each target gene. The RNA from mouse whole heart was used as positive control. (c) the table represents efficiency ( ) of primers that we used for q-RT-PCR. We calculated the percentage of primer efficiency by using the formula; where is the primer efficiency of the q-RT-PCR reaction and slope refers to the slope of the plot of Ct value versus the log of the input RNA amount. Our primer list demonstrates a slope between −4.19 and −3.42 which corresponds to an estimated efficiency between 73.4% and 95.9%, respectively [63]. (d) western blot for phosphorylated eNOS showed active eNOS in both early (passage 1 samples 1, 2, and 3) and late passaged EC (passage 6 samples 4, 5, and 6).