Fig 1: Analysis of Cx40 expression (ventricular conduction system (VCS) marker protein) in 2-D and 3-D cultures of E11.5 ventricular cells. (A) Representative merged confocal image of 3-D cultures stained with Cx40 antibodies (green, arrows) and Hoechst nuclear dye, (B,C) The same field was imaged under brightfield (B) or Hoechst fluorescence alone (C). A–C: Arrows indicate the relative location of Cx40 expressing cells, Bar = 50 µm, (D) Western blot analysis of Cx40 in membrane lysates extracted from three independent samples each from E11.5 ventricular cells cultured in 2-D (N = 3) and 3-D (N = 3) conditions for 8 days, Lower panel represents pan-cadherin (Cadh) expression levels which was used to normalize the relative levels of Cx40, (E) Signal intensities for samples shown in panel D were quantified using the acquisition software from Odyssey DLx (LI-COR) and Cx40 expression levels relative to the pan-cadherin (Cadh) were shown. N = 3 independent extractions for both 2-D and 3-D cultures. Each bar represents mean ± SEM. Open circles represent individual data points for each group. * p < 0.05 for 2-D Vs. 3-D, Student’s unpaired t-test.