Figure_2.tif (2.87 MB)

Luciferase activity and CD4 coreceptor expression in stable pCAG-CD4/FLuc iPSC clones decrease during differentiation in vitro.

figure

posted on 2014-09-16, 03:12 authored by Vera Lepperhof, Olga Polchynski, Klaus Kruttwig, Chantal Brüggemann, Klaus Neef, Florian Drey, Yunjie Zheng, Justus P. Ackermann, Yeong-Hoon Choi, Thomas F. Wunderlich, Mathias Hoehn, Jürgen Hescheler, Tomo Šarić

A. Schematic representation of the plasmid used to generate transgenic cell line expressing FLuc and extracellular domain of CD4 receptor under the control of CAG-promoter. B. FLuc-activity in 17 pCAG-CD4/FLuc αPIG-iPSC clones after hygromycin selection. BL was measured in iPSC lysates in a luminescence plate reader (n = 3). Values are given as mean relative luminescent units (RLU) ± SD per µg protein. C. FLuc-activity of the two pCAG-CD4/FLuc αPIG-iPSC clones was measured in cell lysates before and after spontaneous in vitro differentiation and selection of cardiomyocytes (d16 CM). Values are given as mean RLU ± SD per µg protein (n = 3). *p<0.05; ***p<0.001. D. Flow cytometric analysis of CD4 expression on the surface of pCAG-CD4/FLuc iPSC clone C4 in pluripotent state, day 8 EB, day 16 EB and puromycin selected CM at day 16 of differentiation. E. Quantification of CD4 expression levels in pCAG-CD4/FLuc iPSC clones A1 and C4 at different stages of differentiation. Values are given as mean fluorescent intensity (MFI) ± SD of triplicate measurements. *p<0.05; **p<0.01, ***p<0.001, compared to MFI of CD4 expression in iPSC.