Figure 6.

Effects of endogenous Olig1 and HHM on the growth of U373MG glioma cells in vitro and in vivo. (A) Knockdown of Olig1 and HHM using microRNA. U373MG cells were infected with lentivirus-encoding control microRNA, Olig1 microRNA, or HHM microRNA. Expression of Olig1 and HHM proteins were determined by immunoblot. Expression of α-tubulin was determined as a loading control. NC, negative-control microRNA. (B) In vitro proliferation assay of U373MG glioma cells in which Olig1 or HHM was knocked down. Results were expressed as the change from control (untreated cells). Error bars represent s.e. (C) TGF-β-induced expression of PDGF-B in U373MG glioma cells in which Olig1 or HHM was knocked down. mRNAs were prepared from cells treated with TGF-β (1 ng/ml) for 3 h. Expression of PDGF-B, Olig1, or HHM was determined by quantitative real-time PCR analyses. Values were normalized to the amount of GAPDH mRNA. Error bars represent s.d. (D) Association of Olig1 and Smad2/3 with the PDGF-B promoter region. ChIP analysis was performed using U373MG cells infected with lentivirus-encoding control microRNA, Olig1 microRNA, or HHM microRNA. Cells were treated with TGF-β (2.5 ng/ml) for 1 h and harvested. Eluted DNAs were subjected to quantitative real-time PCR analysis. Values were normalized to the amount of the first intron of hypoxanthine phosphoribosyltransferase 1. Error bars represent s.d. Primer sequences are listed on the Supplementary Table S2. (E) In vivo growth of U373MG cells in which Olig1 or HHM was knocked down by infection with lentivirus-encoding control microRNA, Olig1 microRNA, or HHM microRNA. Values are plotted as the mean tumour volume±s.e. of nine tumours per condition. Panels on the right side show the results of histological examination of the samples. Tumour tissues dissected at 3 weeks after transplantation appeared encapsulated and exhibited no significant variation histologically. Tissue sections were stained with haematoxylin–eosin or Azan. Scale bars: 60 μm.