Figure 5. N2a and CHLA-02-ATRT cultures transfected with MLH1 siRNA continue to form higher numbers of MCTS following post-transfection passage.

Negative control and MLH1 siRNA transfected cultures were grown for 4 days post-transfection and analysed for MCTS formation (passage number 0). Control and MLH1 transfected cultures were then scraped from the plate surface, treated with non-enzymatic cell-dissociation, strained over a 70 µm filter to produce a single cell suspension and re-seeded as separate populations in new 60 mm dishes at a density of 1×10³ cells per dish. MCTS number was analysed 7 days post passage. This procedure was repeated for a total of 3 passages. N2a MLH1 siRNA transfected cultures were seen to retain an increased MCTS forming ability even after 3 passages. CHLA-02-ATRT MLH1 transfected cultures showed an increased propensity to form higher numbers of MCTS for one post-transfection passage only. Each bar represents the mean +/− s.e.m. for at least three independent experiments. Significant differences (P<0.05) are indicated by the asterisks (*) and were calculated using the Student's t-test.