Figure 7. Hdac6 is necessary for ESC colony and EB formation.
(A) Colony formation assays. Indicated KD ESCs were plated at clonal density and grown for seven days, at which time they were stained with crystal violet for visualization. (B) Growth curve. ESCs were infected with shRNA expressing viruses as shown and cultured in normal ESC medium. Cells were counted at the times indicated. (C) EB formation assay. Left and upper right: Brightfield images of EBs formed by hanging-drop cultures of ESCs knocked down as indicated, then cultured in differentiation medium, as described in ‘Materials and methods’. Scale bars equal 400 μm. Lower right: box plot quantification of the range of EB sizes by diameter. The upper and lower limits of the box correspond to the 75th and 25th percentiles of each KD, respectively, and the dark line corresponds to the median of each box. At least 88 EBs were measured for each KD.