Colony-forming cell assay and effects of <i>MSI1</i>-KD.

<p>(A,B,C and D) <i>MSI1</i> shRNA decreased the number of colony-forming cells. (A) Representative images of the secondary colonies formed by the control shRNA-treated and <i>MSI1-</i>KD U251MG cells. (Bar = 500 µm). The number of spheres was reduced in U251 cells exposed to 10 µM DAPT. (B) On Day 30, secondary spheres larger than 100 µm in diameter were counted. U251MG glioblastoma cells transduced with <i>MSI1</i> shRNA and cells treated with 10 µM DAPT (γ-secretase inhibitor) generated significantly fewer spheroid cell colonies than cells transduced with control shRNA. Error bars represent SEM, *P<0.01(C) Representative images of the secondary colonies obtained in cells transduced with control shRNA and in <i>MSI1-</i>KD PTEN-intact Daoy cells exposed to 10 µM LY294002 (PI₃ kinase inhibitor) and DAPT. (D) On Day 30, secondary spheres larger than 100 µm in diameter were counted. PTEN-intact Daoy cells transduced with <i>MSI1</i> shRNA and cells exposed to LY294002 and DAPT generated significantly fewer spheroid colonies than cells transduced with control shRNA. Error bars represent SEM, *P<0.01(E and G). Representative images of the secondary colonies formed by cells expressing low endogenous MSI1. GM1600 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033431#pone-0033431-g002" target="_blank">Fig. 2E</a>) and KNS42 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033431#pone-0033431-g002" target="_blank">Fig. 2G</a>) transduced with control shRNA and <i>MSI1</i> shRNA (F and H). On day 30, secondary spheres larger than 100 µm in diameter were counted. There was no significant difference between control shRNA and <i>MSI1</i> shRNA in GM1600 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033431#pone-0033431-g002" target="_blank">Fig. 2F</a>) or KNS42 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033431#pone-0033431-g002" target="_blank">Fig. 2H</a>). (I, J, and K) <i>MSI1</i>-KD inhibits colony formation. Proliferation was assessed in glioblastoma and medulloblastoma cell lines (cell density of 2×10³ cells/well) were assessed for cell proliferation by cell counting. The total cell count in the <i>MSI1</i>-KD groups on day 15 was reduced by 83%, and 55% in the (I) U251MG and (J) Daoy cells, respectively, as compared to the control groups.