Luciferase activity in stable FLuc-αPIG-iPS cells is clone dependent and decreases after spontaneous differentiation.

<p><b>A.</b> Scheme of the pGL4.14-pUbC [<i>luc2/Hygro</i>] plasmid used to generate stable iPSC lines constitutively expressing FLuc. <b>B.</b> BL signal intensity in protein lysates (5 µg/well) of 14 hygromycin-resistant pUbC-FLuc iPSC clones measured in triplicates using the BrightGlo Kit in a microplate reader. Data are shown as relative luminescence units (RLU) per µg of protein (n = 3). <b>C.</b> The stability of FLuc activity in transgenic pUbC-FLuc iPSC clones C3 and C5 over prolonged <i>in vitro</i> expansion (n = 3). <b>D.</b> BL signal intensity in protein lysates of pUbC-FLuc EB collected at different days during spontaneous differentiation as determined in microplate reader (n = 6 for C3, n = 9 for C5). <b>E.</b> Relative levels of FLuc transcript expression during spontaneous differentiation of pUbC-FLuc iPSC as determined by RT-qPCR (n = 6 from two independent differentiations). <b>F.</b> BL signal intensity in two clones (C3 and C5) of undifferentiated pUbC-FLuc iPSC and puromycin purified pUbC-FLuc iPS-CM on day 16 of differentiation. Data are given as mean ± SD of 2 independent experiments for clone C3 and 3 for clone C5, each measured in technical triplicates. <b>G.</b> Relative FLuc-transcript expression in two clones of purified pUbC-FLuc iPS-CM relative to undifferentiated pUbC-FLuc iPSC as determined by RT-qPCR. Data are shown as mean ± SD of 2 independent experiments measured in technical triplicates. <b>H.</b> UbC promoter activity in native iPSC and purified native iPS-CM measured 24 hours after transient transfection with pGL4.14-pUbC [<i>luc2/Hygro</i>] plasmid and renilla luciferase-encoding control plasmid. Values shown are ratios of FLuc activity relative to co-transfected renilla lucifderase activity given as mean ± SD (n = 3), p>0.05. Statistical analyses were performed by two-tailed paired Student's t-test: *p<0.05; **p<0.01; ***p<0.001.