Fig. 3

Knock out of PAX6 alters morphology of colonies and increases colony-formation ability. Five hundred cells (WT, Control or KO) per well in a 6-well plate was seeded and left for (a) 16 days or (b) 12 days to study colony forming abilities. c Five hundred cells of the Dox inducible EGFP-PAX6 pooled rescue cell lines and the pooled PAX6 KO cells were seeded per well in 6-well plates and left for 12 days. Colonies were micro-photographed. d Colonies were stained with 0.005% crystal violet before counting. Arrows indicate WT U251 N colonies containing less than 50 cells, and PAX6 KO colonies containing over 50 cells. e Five hundred cells were seeded in three wells in 6-well plates and left to form colonies for 12 days for each of the PAX6 KO cell lines (2.10 KO, 2A.3 KO and 2A.28 KO), and their Rescued counterparts (2.10 R, 2A.3 R and 2A.28 R) as well as the WT cell line U251 N. Pooled versions containing equal amounts of each of the three individual cell lines within each group were also included (3xKO, 3xR and 3xR + Dox). The term “+DOX” indicate Rescue cells treated with Dox 18–24 h before FACS sorting. The day after FACS sorting they were seeded for the colony formation assay. Rescue cells without Dox treatment were also included. Colony-forming abilities were measured by counting colonies consisting of over 50 cells. The figure is representative of three experiments done in triplicate. For each experiment the colonies were counted by two persons.