Figure 3.

Two days after plating each EB into a well of a 24-well plate (nine days after removal of LIF), DT-3 and DT-3 + PKCβ inhibitor, or DT-3 + PKCζ inhibitor were added to different EBs. Carrier alone and untreated EBs were used as controls. EBs were then analyzed for beating two days later (Day 11 post LIF removal). As an added control, all four authors routinely performed blind counts of EBs to decrease subjectiveness. Discrepancies between authors were rare, but when they occurred, the morphology observed by the majority of authors was used as the data point. (a) Inhibiting PKG alone or in combination with PKCbeta or zeta resulted in significantly more EBs with at least one beating area when compared to controls. Significance is shown in Table 1 at each time point. The combination of DT-3 + PKCbeta or zeta inhibitors revealed in a trend towards more EBs that had at least one beating area when compared to DT-3 alone. Significance (P < .05) was attained on Day 14. (b) Inhibiting PKG alone or in combination with PKCbeta or zeta resulted in significantly more beating areas within EBs when compared to controls. Significance is shown in Table 2 at each time point. (c) A typical Day 14 EB outgrowth that has been subjected to DT-3 and PKCbeta specific inhibitor. NB = nonbeating area, B = beating area. Arrows depict the directional flow of contraction of the larger beating area. Scale bar = 100 μmeter. The number of EBs analyzed for both (a) and (b) is depicted below their specific treatment (e.g., n = 211 EBs for DT-3 treatment).