Journal: PLoS ONE

Article Title: Vorinostat Induces Apoptosis and Differentiation in Myeloid Malignancies: Genetic and Molecular Mechanisms

doi: 10.1371/journal.pone.0053766

Figure Lengend Snippet: A, K562 cells were transfected with pGL3-basic vector or a series of IER3 reporters as indicated plus β-galactosidase vector, and then treated with vorinostat (2 µM) or vehicle (Control) for 24 h and reporter activity measured. Results are shown as average fold luciferase/β-galactosidase induction versus control cells transfected with pGL3 ± S.D. from one representative of at least three independent assays done in triplicate using each reporter construct at least from two different clones. B, Scheme of wild type and mutated −124/+32 reporter constructs showing the −91 to −43 nucleotide sequence of IER3 promoter containing the putative TF binding sites present in the −91/−61 region (lane 1) and the mutated nucleotides for the different TF binding sites present in this region (lanes 2 to 6). The mutated nucleotides present in the mutant luciferase reporter plasmids for the different TF binding sites are boldfaced and underlined. +1, denotes transcription start site. C, K562 and HL60 cells were transfected with pGL3-basic vector or with wild type and mutated −124/+32 IER3 reporter constructs for the indicated TF plus β-galactosidase vector and the rest of the procedure was done as in (A).The results are average fold luciferase/β-galactosidase induction versus control cells transfected with pGL3 ± S.D. from one representative assay done in triplicate using mutated plasmids for the same putative TF binding site from different clones, of at least three independent assays performed in both K562 and HL60 cells. Data were analyzed using the ANOVA and the Tukey-Kramer multiple comparison test. *p<0.05.

Article Snippet: K562 and HL60 cells (10 5 cells/ml well of 24-well plate) were transiently co-transfected with 400 ng indicated wild-type and mutated reporter constructs or with pGL3-basic control vector (Promega) plus 25 ng β-galactosidase reporter control expression vector (Promega) using 4 µl lipofectamine 2000 (Life Technologies) as per manufactureŕs protocol.

Techniques: Transfection, Plasmid Preparation, Activity Assay, Luciferase, Construct, Clone Assay, Sequencing, Binding Assay, Mutagenesis