Journal: Nature Communications
Article Title: Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells
Figure Lengend Snippet: ITE inhibits hypoxia- and tumour sphere-induced Oct4 elevation and leads to the differentiation of stem-like cancer cells. ( a ) U87 cells were seeded at a density of 2 × 10 5 per well of the six-well plate, cultured under normoxia (21% O 2 ) in DMEM for 24 h before being switched to the F10 medium and treated for 8 h with either 21% O 2 (normoxia) or 1% O 2 (hypoxia) in the presence of ITE (10 μM), Kyn (50 μM), Trp (1 μM) or aTrp. Cells were harvested and analysed using qRT–PCR for the mRNA levels of the POU5F1 . The data were expressed as mean±s.d. of triplicate measurements from one of three independent experiments. ( b,c ) Adherent parental U87 cells (U87 parental), U87 tumour sphere cells treated with vehicle (U87 sphere) or 10 μM ITE (U87 sphere+ITE) for varying duration were harvested and analysed using qRT–PCR for the mRNA levels of the indicated genes ( b ) and by immunoblotting (samples treated for 72 h) for the indicated proteins ( c ). ( d ) U87 cells grown in DMEM were transfected with a Scr siRNA or an AhR siRNA in combination with a plasmid containing EGFP only or EGFP + OCT4 . The transfected cells were simultaneously treated with DMSO (vehicle or −) or 10 μM ITE. Twenty-four hours after transfection, cells were cultured in a neural stem cell medium to induce the formation of tumour spheres. The pictures were taken 3 days after transfection. Scale bars, 100 μm. ( e ) The cultures described in d were quantified for the numbers of tumour spheres formed and analysed for the indicated proteins by immunoblotting. The data were expressed as mean±s.d. of three independent experiments. ( f ) Heat map generated from DNA microarray data for adherent parental U87 cells (U87 parental), U87 tumour sphere cells treated with vehicle (U87 sphere) or 10 μM ITE (U87 sphere+ITE) for 7 days. In a , b , e , The statistical significance of compared measurements was evaluated using the one-way ANOVA. * P
Article Snippet: An aliquot of the lysates (100 μl) was used as a control for the amount of input DNA after being purified using the PCR clean-up kit (Axygen AP-PCR-50).
Techniques: Cell Culture, Quantitative RT-PCR, Transfection, Plasmid Preparation, Generated, Microarray