ESC with knockdown of junction molecules by shRNA maintain ability to differentiate to Flk-1 cells.
A) shRNA constructs efficiently silence endogenous mRNA in mESC. Quantitative RT-PCR was performed on GFP expressing cells generated with lentiviral constructs containing shRNA specific to E-Cadherin, Cx43, ZO1 and ZO2 to analyze transcript expression level. Samples were normalized to GAPDH and expressed relative to transcript levels in untransfected D3-ESC (dashed line). Error bars represent standard deviation of n = 3 samples. B) Flk-1 expression does not significantly differ in knockdown ESC lines. ESC lines stably expressing shRNA sequences specific to E-Cad, Cx43, ZO-1 and ZO-2 and ESC lines with increased knockdown levels of E-Cad and ZO-1 were differentiated in EB for 5 days and Flk-1 expression was analyzed flow cytometrically using anti-Flk-1 conjugated to PE. Non-viable cells were excluded based on fluorescence of DAPI. Paired t-tests with D3-ESC were performed with n = 7 (Kd-E-cad, Kd-Cx43, Kd-ZO-1) or n = 6 (Kd-ZO-2). * p<0.05; *** p<0.0005. Error bars represent standard error of the mean.