Gdc0980

Antibodies for p110α, p-85, AKT, p-AKT ^Thr308, p-AKT ^ser473, S6, p-S6 ^Ser235/236, cleaved PARP, total PARP, cyclin D1, p-MET (1234/1235) and anti-MAPK antibodies (ERK and p-ERK) were from Cell Signaling (Danvers, MA). Antibodies against total Met, p-MET (pY1349 and pY1003) and Alexa Fluor Phalloidin 594 were from Invitrogen (Grand Island, NY). PIP3 antibody was from MBL Co. Ltd (Japan). β-actin antibody was from Sigma (St. Louis, MO).
Recombinant human HGF was from R&D Systems (Minneapolis, MN). Wortmannin and LY294002 were from Cell Signaling. Crizotinib, GDC-0941, GDC-0980, ARQ 197 and NVP-BEZ235 were purchased from Selleck (Houston, TX). Stock solutions were prepared in DMSO and stored at −20°C till further use.

All cell lines were maintained in culture as previously described [3 (link)]. To evaluate response to dihydrotestosterone (DHT) cells were grown in charcoal-stripped (CS) media for two days prior to plating in 96-well tissue culture dishes. Media were then changed to fresh CS-media alone or increasing doses of DHT and viability determined by measuring fluorescent intensity after metabolic reduction of AlamarBlue five days later. For drug treatments, breast cancer cell lines and HMECs were seeded (3,000 to 10,000 cells) in quadruplicate wells in 96-well plates. After attachment, media was replaced with either fresh media (control) or media containing half-log serial dilutions of the following drugs: GDC-0941 (10 nM-3000 nM), GDC0980 (1 nM-300 nM), BKM120 (10 nM-3000 nM) and NVP-BEZ235 (1 nM-300 nM) purchased from Selleck Chemicals (Houston, TX) or in combination with bicalutamide (CDX) (Sigma, St. Louis, MO). Viability was determined by measuring fluorescent intensity after metabolic reduction of AlamarBlue in the presence/absence of drug incubation for 72 h. Viability assays were performed in triplicate and replicates were normalized to untreated wells. EC50 values were determined after double log-transformation of dose response curves as previously described [3 (link)].

PU-H71, PU-beads, control beads (CB), fluorescently labelled PU-H71 (PU-FITC), LSI-137 and the control derivative PU-FITC9 were synthesized using previously described protocols^{18 (link),48 (link)–50 (link)}. Signalling inhibitors used for in vitro experiments, such as, MEK/ERK pathway inhibitors, trametinib (GSK1120212) and pimasertib (AS-703026); ERK inhibitor, SCH779284; PI3K/mTOR pathway inhibitors, rapamycin, PP242 (Torkinib) and GDC0980 (Apitolisib); JAK/STAT inhibitor, AZD1480; AKT/PI3K inhibitors, LY294002, Idealisib (CAL-101, GS-1101); NFκB inhibitor, JSH-23; phospholipase-C inhibitor, U-73122 along with its inactive analogue, U-73343; and protein kinase A inhibitors; 6-22 amide and H-89, were purchased from Selleckchem. trametinib used for animal studies was purchased from LC laboratories. Debio 0932 (CUDC-305) was purchased from ChemieTek.