Figure 2.

nSMase2 is upregulated transcriptionally via a novel TSS. (a) MCF7 cells were seeded in 60 mm dishes and treated with vehicle or Doxorubicin for 24 h. After that, 10 nM of actinomycin D was added for 1, 3 and 6 h. Cells were lysed, RNA was isolated and transformed to cDNA and quantitative real-time PCR (qRT-PCR) was performed with nSMase2 primers. (b) MCF7 cells were seeded in 60 mm dishes and transfected with LacZ and pGL3 basic containing the first 1600 bp upstream of exon for 24 h, after which cells were treated with doxorubicin. Cells were then collected and both luciferase and β-galactosidase assays were performed as described under ‘Materials and Methods', **P<0.01. (c) Representation of exonic sequences of nSMase2. Cells were treated with vehicle and doxorubicin for 24 h, after which cells were collected, RNA was isolated and transformed to cDNA. qRT-PCR was performed using exonic intronic primers, exon1–intron2 (E1–I2), exon3–intron3 (E3–I3) and exon9–intron8 (E9–I8), **P<0.01