Palbociclib

Compounds for in vitro assays were obtained from Developmental Therapeutics Program at NCI (palbociclib, gedatolisib, afatinib, neratinib, buparlisib, alpelisib,), BEZ235 and trametinib from ChemieTek (Indianapolis, IN, USA), rapamycin from LC Laboratories (Woburn, MA, USA) and were dissolved in 100% DMSO. Primary mammary cancer cells were plated at 2,500 cells per well in opaque white 96-well plates. Single compound treatment was conducted at a range of concentrations between 0.1 and 50,000 nM in 0.5% DMSO. Compounds were added once to wells, 24 h after plating cells, and drug-treated wells were run in triplicate. At 72 h after drug exposure, cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) as per the manufacturer’s instruction. DMSO-treated wells were considered as 100% viability for each treatment plate.

The following antibodies were used: Anti-JUNB(ab128878), anti-JUND(ab134067), anti-FOSB(ab184938) and anti-FOSL2(ab124830) were purchased from Abcam (Cambridge, UK); anti-FOS(sc-8047), anti-FOSL1(#sc-183), anti-p-JNK(sc-6254) and anti-β-actin(AC-15) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-JUN(#9165), anti-JNK(#9252), anti–p-P38 MAPK(#9211), anti-P38(#9212), anti-p-ERK1/2(#9106), anti-ERK1/2(#9102), anti-ubiquitin(#3936) and anti-p-Rb(#9308) were purchased from Cell Signalling Technology (Danvers, MA, USA); anti-p-JUN (Ser73) (#06–659) was purchased from EMD Millipore (Temecula, CA, USA).
Palbociclib was synthesized by ChemieTek (Indianapolis, IN, USA). SP600125 and MG132 were purchased from Sigma-Aldrich (St Louis, MO, USA). All compounds were dissolved in dimethyl sulfoxide (DMSO). TNFα was purchased from Genzyme (Cambridge, MA, USA) and IL-1β was purchased from PeproTech (Rocky Hill, NJ, USA).