We used a gene appearance profiling approach in order to define the genes and networks regulated by the SIK2/TORC/CREB1 transcriptional pathway

We used a gene appearance profiling approach in order to define the genes and networks regulated by the SIK2/TORC/CREB1 transcriptional pathway. Treatment with a small molecule SIK2 inhibitor (ARN-3236), currently in preclinical development, also led to enhanced CREB1 activity in a dose- and time-dependent manner. Since CREB1 is usually a transcription factor and proto-oncogene, it was posited that the effects of SIK2 on cell proliferation and viability might be mediated by changes in gene expression. To test this, gene expression array profiling was performed and whilst SIK2 knockdown or over-expression of the kinase-dead mutant affected established CREB1 target genes; the overlap with transcripts regulated by forskolin (FSK), the adenylate cyclase/CREB1 pathway activator, was incomplete. Implications This study demonstrates that targeting SIK2 genetically or therapeutically will have pleiotropic effects on cell cycle progression and transcription factor activation, which should be accounted for when characterizing SIK2 inhibitors. cells (Agilent Technologies) and were purified using HiSpeed Plasmid Midi Kit (Qiagen) according to manufacturers recommendations. Cell counting and Cell viability Cells were seeded in triplicate at a density of 300,000 cells per well in a 6-well plate. At each time point, the supernatant was harvested to include lifeless or detached cells and live cells were harvested using 0.25 % Trypsin-EDTA (Invitrogen). Dead cells and live cells were then pooled together, pelleted, resuspended in 500 l 1 PBS and transferred to a vial for cell counting and estimation of cell viability using a Rabbit polyclonal to IQCA1 Beckman Coulter? Vi-Cell. IncuCyte growth assays Cells were seeded in four replicates at a density of 20,000 cells per well in a 48-well plate. Plates were placed in the IncuCyte? CMK and nine time-lapse images of each well were taken at 3 hour intervals for seven days. IncuCyte? 2010A software was used to assess changes in cell confluence as a surrogate for switch in cell number. MTS Cell proliferation assay Cells were seeded in four replicates at a density of 10,000 cells per well in a 96-well plate. At each time point, 20 l of CellTiter 96? AQueous Assay reagent (Promega) were added directly to each CMK well with minimal exposure to light. Plates were incubated for 1 h at 37C, 5 % CO2. Formazan absorption was measured at 490 nm using an Infinite M200 spectrophotometer (Tecan). The mean absorbance of wells was displayed as optical density to estimate proliferation status. Soft agar colony formation assay Cell were resuspended in DMEM (Cell Biolabs) supplemented with 6 % Fbs and made up of 0.4 % agar. They were then seeded in six replicates at a density of 1 1,000 cells per well in a 96-well plate containing a bottom layer of DMEM supplemented with 10 %10 % Fbs and made up of 0.6 % agar. Cell-agar suspension was overlayed with media containing 10 %10 % Fbs and cultured for seven days. After seven days, CMK the soft agar layer was solubilised, cells were lysed and quantity of colonies was decided using the CyQuant CMK GR dye and measure of fluorescence at 520 nm. To measure colony formation of cells after transient knock-down, cells were transfected with siRNA, trypsinised 24 h later and 10,000 cells were reseeded in soft agar as explained above. Cell cycle analysis For DNA content analysis, cells were seeded in triplicate at a density of 300,000 cells per well in a 6-well plate and were produced for 48 h or 72 h. At each time point, cells were trypsinized using 0.25 %25 % Trypsin-EDTA (Invitrogen), were washed in 1 PBS and were fixed with 1 % paraformaldehyde (Electron Microscopy Science) for 1 h at 4C. Cells were then washed in chilly 1 PBS (Gibco), resuspended in 80 % ice chilly methanol and stored at ?20C CMK until staining. Methanol-fixed cells were treated with 3 M DAPI (Sigma-Aldrich) overnight at 4C. Fluorescence activated cell sorting (FACS) analysis was carried out using a BD LSRII instrument (Becton&Dickinson, San Jose, CA) and data acquisition was performed using BD FACSDiva software (v.5.0.3.). The fluorescence emitted by DAPI was collected using a UV-450/50 bandpass filter. Data were analysed after doublet discrimination [23] using the FlowJo software (Tree Star, v.8.8.4.) and applying the curve-fitting algorithm contained in the software. Annexin V Apoptosis assay Cells were seeded in triplicate at a density of 300,000 cells per well in a 6-well plate. At each time point, the supernatant was harvested to include lifeless or detached cells and live cells were harvested using 0.25 %25 % Trypsin-EDTA (Invitrogen). Dead cells and live cells.