10.1371/journal.pone.0023810.g003 Basha L. Stankovich Basha L. Stankovich Esmeralda Aguayo Esmeralda Aguayo Fatima Barragan Fatima Barragan Aniket Sharma Aniket Sharma Maria G. Pallavicini Maria G. Pallavicini ESC with knockdown of junction molecules by shRNA maintain ability to differentiate to Flk-1 cells. Public Library of Science 2011 knockdown junction molecules shrna differentiate flk-1 2011-09-06 02:36:31 Figure https://plos.figshare.com/articles/figure/_ESC_with_knockdown_of_junction_molecules_by_shRNA_maintain_ability_to_differentiate_to_Flk_1_cells_/409391 <p>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.</p>