H 89 2hcl

High mobility group box-1 protein monoclonal antibody (mAb) was from R&D (Minneapolis, MN, USA). GSTP, cPKC, chromosomal region maintenance 1 (CRM1), and phospho-Serine mAb were from Abcam (Cambridge, United Kingdom). Polyclonal antibodies against β-actin, laminB, phospho-GSTP (Ser184), His-tag, and Flag-tag were from Bioworld Biotechnology (Minneapolis, MN, USA). Rabbit monoclonal antibodies against phospho-Threonine and mouse mAb against phospho-Tyrosine were obtained from Cell Signaling Technology (Beverly, MA, USA). Secondary antibodies coupled to IRDye800 flurophore were purchased from Rockland (Gilbertsville, PA, USA). LPS (from Escherichia coli 0111:B4), phorbol-12-myristate-13-acetate (PMA; TPA, Phorbol 12-myristate 13-acetate), and ATP (adenosine triphosphate) were from Sigma-Aldrich (St. Louis, MO, USA). 6-(7-nitro-2,1,3-Benzoxadiazol-4-ylthio)-hexanol (NBDHEX) was synthesized as previously reported and dissolved in DMSO. The broad-spectrum PKC inhibitor Gö6983, the PKA inhibitor H 89 2HCl, and the cPKC inhibitor GF109203X were purchased from Selleckchem.

Cells were plated in 96 well cell culture plates at a seeding density of 1000 cells/well (A549 cells) or 2000 cells/well (A427, NCI-H23, NCI-H358, NCI-H1975, and NCI-H1650 cells). Cells were incubated overnight at 37° C and 5% CO₂. They were then treated with DMSO, A-674563 (AKT-1 inhibitor), MK-2206 (pan-AKT inhibitor), PHA-848125 (CDK2 inhibitor), or H-89 2HCl (PKA inhibitor) from Selleck Chemicals (Houston, TX). Media and inhibitor were replaced every 24 hours and survival was measured after 72 hours of treatment. Cells were incubated with 100μL of fresh media and 10μL of WST-1 reagent (Roche Canada, Mississauga, ON) for 2–4 hours. Optical density was determined at 450nm using the EL800 Universal Microplate Reader (BioTek, Winooski, VT) and CalcuSyn software (Biosoft, Cambridge, UK) was used to determine the IC50 concentrations.

A safe and effective in vitro ovarian culture system has been established in our lab (10 (link), 11 (link)). Neonatal mice were sacrificed by cervical dislocation at the designated times. The ovaries were separated in cold phosphate-buffered saline (PBS) under the microscope. The isolated ovaries were incubated in 6-well culture dishes (NEST, China), and an insert (PICM0RG50, Millipore, USA) was placed in every well with 3 mL Dulbecco Modified Eagle Medium/Ham F12 nutrient mixture (DMEM/F12) (Gibco, Life Technologies, CA) supplemented with insulin-transferrin-sodium selenite (1:100, Sigma, USA) and penicillin-streptomycin solution at 37 °C, 5% CO₂ and saturated humidity. Ovaries were randomly assigned, and cultured for 1–7 days in basal medium alone or basal medium supplemented with either dbcAMP (10 μM, D0627, Sigma, USA), MDL-12,330 (5 μM, M182, Sigma, USA), Milrinone (10 μM, 78415-72-2, J&K, China) or H 89 2HCL (5 μM, S1582, Selleck, China), respectively.