Letrozole

Nine drugs were purchased from Santa Cruz Biotechnology, Inc.: Meticrane, tolazamide, β-sitosterol, memantine hydrochloride (herein referred to as memantine), valproic acid, letrozole, todralazine, thiostrepton, and levocabastine. Niridazole was not available. For in vitro screening, all compounds were dissolved in 100% dimethyl sulfoxide (DMSO) to a stock concentration of 250 mM. All stock solutions were subsequently diluted in complete DMEM to nine working solutions ranging from 0.2 to 1312.3 μM. This dose spectrum covered well below and above the reported dose levels for all drugs described in cMap. PLX4720 (ChemieTek) was solubilized and diluted in a similar manner to nine working solutions ranging from 0.01 to 1562.5 μM. Prior to in vitro testing, we pre-warmed (37 °C) and sonicated all working solutions.

Reagents used were as follows: letrozole (Sigma, L6545), 17β-estradiol (E2) (Sigma, 491187), celecoxib (Sigma, PZ0008), PGE2 (Sigma, P5640), tunicamycin (Sigma, T7765), salubrinal (Calbiochem, 324895), rapamycin (Sigma, R0395), arachidonic acid (Sigma, A9673), MTT (Sigma, M2128), Z-VAD-fmk (Sigma, V116), bafilomycin A1 (Sigma, B1793), 3-MA (Sigma, 08592), Hoechst 33342 (Sigma, B2261), acridine orange (Sigma, A6014), DMSO (Sigma, D2650). letrozole, E2, celecoxib, PGE2, tunicamycin, and salubrinal were dissolved in DMSO, while MTT, Hoechst 33342, and acridine orange were dissolved in phosphate-buffered saline (PBS).
Antibodies were obtained from the following sources: antibodies against Beclin 1, COX-2, EP-4, β-actin, Bcl-2, BAX, phospho-4EBP1 and phospho-Akt (S473) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); Phospho-p70-S6K(T389), eIF2α, phospho-eIF2α, Raptor, phospho-mTOR(S2448), caspase 3, and phospho-S6(S235/236) were from Cell Signaling Inc (Beverly, MA); LC3, ATG5, protein disulfide isomerase (PDI) were from Abcam (Cambridge, MA); horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

The azole-based drugs ketoconazole, posaconazole,
voriconazole, ravuconazole, clotrimazole, miconazole, letrozole, and
anastrozole were purchased from Santa Cruz Biotechnology (Dallas,
TX), and fluconazole was from ICN Biomedicals. VNI and VFV were synthesized
by the Chemical Synthesis Core Facility (Vanderbilt Institute of Chemical
Biology).^{70 (link)} The synthesis of their analogue
LFV was described previously.^{68 (link)} The purity
of the compounds was >95%. The pyridine derivatives UDO and UDD
were
from DNDi;^{46 (link)} the tetrazole-based compounds
VT1161^{47 (link)} and VT1598^{48 (link)} were from Mycovia Pharmaceuticals (Durham, NC). Hydroxypropyl-β-cyclodextrin
(HPCD) was purchased from Cyclodextrin Technology Development (Gainesville,
FL). Q- and CM-Sepharose resins were from GE Healthcare, and Ni²⁺-nitrilotriacetate (NTA) agarose was from Qiagen. The synthesis
of the CYP51 reaction product (3β,5α)-4,4-dimethyl-cholesta-8,14,24-trien-3-ol
(dihydro-FF-MAS) was reported elsewhere.^{71 (link)} All compounds were >95% pure by high-performance liquid chromatography
(HPLC).