Palbociclib pd 0332991 hcl

Human breast cancer cell lines were maintained in appropriate culture media (e.g., RPMI 1640, DMEM, L-15) supplemented with 10 to 15% heat-inactivated fetal bovine serum (FBS), 2 mmol/L glutamine, and 1% penicillin G-streptomycin-fungizone solution (PSF, Irvine Scientific) as previously described [22 (link)]. Cells were routinely assessed for mycoplasma contamination using a multiplex PCR method, and STR profiling by the GenePrint 10 System (Promega) was used for cell line authentication. Ribociclib (LEE011-succinate), alpelisib (BYL719), and everolimus (RAD001) were provided by Novartis. Palbociclib (PD-0332991-HCL) and abemaciclib (LY2835219-mesylate salt) were purchased from Selleck Chemicals (Houston, TX). The palbociclib-resistant EFM19 (EFM19-PR) breast cancer cell line was established through long-term culture in the presence of increasing concentrations (10–1000 nmol/L) of palbociclib. For molecular analysis, PR cells were removed from the drug for 7 days prior to experiments.

Palbociclib (PD0332991) HCl (catalog no. S1116) was purchased from Selleckchem (www.selleckchem.com). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (CellTiter 96^® Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI, USA) was utilized to assess the effect of palbociclib on cell viability. Cells were seeded in culture media (3 × 10⁴ cells/well) in triplicate in a 96-well plate and incubated overnight. Palbociclib was added at eleven different concentrations (0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 µM) at 37 °C. After 48 h incubation, the cells were washed with fresh media and were grown in palbociclib-free media for an additional 24 h, until MTT assays were done as specified by the manufacturer’s instructions. IC₅₀, a concentration at which cell viability was down to half, was calculated using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).
For apoptosis assays, 10⁶ cells were initially plated on 6-well plates and incubated overnight. Cells were treated with 5 µM of palbociclib, incubated for 24 h, and subjected to apoptosis assay with flow cytometry by using Annexin V-FITC and 7-AAD (catalog no. 640922; BioLegend, San Diego, CA, USA). All experiments were independently repeated in quadruplicate.

CDK4 knockdown was performed by CDK4 specific siRNA transfection in synovial sarcoma cells. The human nonspecific siRNA and CDK4 siRNAs (#SASI_Hs01_00122488, NM_000075.2, 5′-CUCUUAUCUACAUAAGGAU-3′ and #SASI_Hs01_00122490, NM_000075.2, 5′-CACUUACACCCGUGGUUGU-3′) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The nonspecific siRNA oligonucleotides were used as negative controls. Increasing concentrations (0, 10, 30, and 60 nM) of CDK4 siRNAs or nonspecific siRNA (60 nM) were transfected into cells using Lipofectamine^® RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h for western blotting or 5 days for MTT assay, transfected cells were subjected to subsequent analysis.
CDK4 inhibition in synovial sarcoma cells was carried out by palbociclib treatment. Palbociclib (PD-0332991) HCl (#S1116, Selleck Chemicals, Houston, TX, USA) is a highly selective inhibitor of CDK4/6 activity. SYO-1 and Fuji cells were grown on 96-well plates for cell proliferation assays, 6-well plates for wound healing assays, or 12-well plates for western blotting analysis, and incubated with various concentrations of palbociclib, followed by subsequent experiments.