Afterwards, the membranes were washed and incubated with a secondary antibody against rabbit or mouse IgG conjugated to horseradish peroxidase (Cell Signaling,

MA, USA) for 1 h, followed by washing and transferring into ECL solution (Millipore, Darmstadt, Germany), and exposed to X-ray film. Treatment with p38 isoforms, p53 and FOXO3a small interfering RNAs (siRNAs) For the transfection procedure, cells were seeded in 6-well or 96-well culture plates in RPMI 1640 medium containing 10% FBS (no antibodies), grown to 60% confluence, and p38 MAPK isoforms Ro 61-8048 α, β, p53, FOXO3a and control siRNAs were transfected using the lipofectamine 2000 reagent according to the manufacturer’s instructions. Briefly, Lipofectamine 2000 was incubated with see more Opti-MEM medium (Invitrogen, CA, USA) for 5 min, mixed with siRNA (up to 70 nM), and incubated for 20 min at RT before the mixture was added to cells. After culturing for up to 30 h, the cells were washed and resuspended in fresh media in the presence or absence of BBR for an additional 24 h for all other experiments. Cell apoptosis assays Cell apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit (BestBio, Shanghai, China) according to instructions from the manufacturer.

Briefly, after treated with BBR for 24 h, Tideglusib the apoptotic cells were harvested by Trypsin (no EDTA) and washed with PBS, then resuspended the cells in 500 μL binding buffer, Org 27569 5 μL Annexin V-FITC regent and 10 μL PI regents and incubated for 5 min at RT in the dark, followed by detecting cell apoptosis by flow cytometry. In parallel experiment, Hoechst 33258 staining was used to further analyze cell apoptosis. Cells were cultured in 12-well culture plates and treated with berberine for 24 h. Afterwards, the cells were washed with PBS, and incubated with 500 μL 4% methanal for 10 min, followed by staining with Hoechst 33258 (Sigma, St. Louis, MO, USA) at RT for

10 min, then observed with filters for blue fluorescence under fluorescence microscopy. Electroporated transfection assays NSCLC cells (1 × 107 cells/mL) were washed and centrifuged at 1200 rpm for 5 min, followed by removing the medium and PBS. Afterwards, the cells in the tubes were added Bio-Rad Gene Pulser electroporation buffer. After resuspending the cells, the desired N1-GFP or FoxO3a-GFP plasmid DNA (10 μg/mL) were added and the electroporation plate were put in the MXcell plate chamber and closed the lid in Gene Pulser II Electroporation System (Bio-Rad, CA, USA). The electroporation conditions on the plates to deliver 150 V/5 ms square wave were adjusted until reaching the optimal one. Once the condition has been set and then press “Pulse” to electroporate the cells. After electroporation was completed, the cells were transferred to a tissue culture plate.