br Cell culture and compounds br LNCaP CRL and PC
2.2. Cell culture and compounds
LNCaP (CRL-1740) and PC-3 (CRL-1435) MG132 were purchased from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 and Ham’s F-12K medium, respectively. DU145 cells were a kind gift of Dr. Robert Bristow (University Health Network) and were maintained in alpha-Modified Eagle’s Medium (a-MEM). VCaP cells were a kind gift of Dr. Mathieu Lupien (University Health Network) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM). All cell lines were supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 mg/mL streptomycin. Cell lines were routinely confirmed to be mycoplasma-free using the MycoAlert My-coplasma Detection Kit (Lonza), and their authenticity was verified by short tandem repeat (STR) profiling. Fluvastatin (US Biological) was dissolved in ethanol, dipyridamole (Sigma) was dissolved in DMSO, mevalonate (Sigma) was dissolved in water, 25-hydroxycholesterol (Sigma) was dissolved in ethanol and doxycycline hyclate (Sigma) was dissolved in water.
2.3. Cell viability assays
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed as previously described . Briefly, PCa cells were seeded at 2,000e15,000 cells/well in 96-well plates overnight, then treated in triplicate with 0e400 mM fluvastatin for 72 h.
2.4. RNA interference
Two independent short hairpin RNAs (shRNAs) against SREBF2 were designed using The RNAi Consortium (TRC) Genetic Perturbation Platform (portals.broadinstitute.org/gpp/public) and cloned into the doxycycline-inducible pLKO shRNA lentiviral system (shRNA se-quences in Supplementary Table 1). HEK-293Tv cells were co-transfected with the shRNA constructs, pMD2.G and psPAX2 via cal-cium phosphate transfection. Viral supernatants were harvested 48 h post-transfection. LNCaP cells were transduced with the lentiviral supernatants in the presence of 8 mg/mL polybrene, after which they were selected in 1 mg/mL puromycin.
2.5. Cell death assays Cells were seeded at 0.5e1 106 cells/plate and treated the next day as indicated. After 72 h, cells were fixed in 70% ethanol for >24 h, stained with propidium iodide and analyzed by flow cytometry for DNA fragmentation (% pre-G1 population) as a measure of cell death, as previously described .
2.6. Xenograft experiments
All animal experiments were carried out in accordance with the regulations of the Canadian Council on Animal Care. 7e9 week-old male NOD/SCID (non-obese diabetic/severe combined immunode-ficiency) mice were injected with 5 million LNCaP cells subcuta-neously in the flank in a 1:1 mixture with Matrigel (Corning). When tumor volumes reached 200 mm3, mice were randomized and treated with one of the following: 50 mg/kg/day fluvastatin (resuspended in phosphate-buffered saline (PBS), administered orally), 120 mg/kg/day dipyridamole (5 mg/mL dipyridamole in 50 mg/mL polyethylene glycol 600 and 2 mg/mL tartaric acid, administered intraperitoneally), the combination of fluvastatin and dipyridamole or vehicle controls. For the patient-derived xenograft (PDX) experiment, 5 5 mm pieces of LTL-484  were surgically implanted subcutaneously in the flank of NOD/SCID mice. When tumor volumes reached 200 mm3, mice were randomized and treated with vehicle controls or fluvastatin and dipyridamole, as described above.
2.7. Fluvastatin quantification
Fluvastatin concentrations were quantified by high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/ MS) with atorvastatin used as the internal standard. Flash-frozen mouse prostate and liver tissues (up to 250 mg) were homoge-nized in liquid nitrogen and resuspended in 500 mL of water. Serum and tissue samples were subjected to methyl tert-butyl ether extraction, after which the supernatants were separated, dried at room temperature and reconstituted in 250 mL of meth-anol:water (1:1). 10 mL of sample was injected into a Shimadzu CBM-20A system coupled with a triple quadrupole mass spec-trometer (API 3200, Applied Biosystems/MDS SCIEX). Chromato-graphic separation was achieved using a Phenomenex HyperClone BDS C18 column (50 2.0 mm, 5 mm). The binary mobile phase consisted of 5 mM ammonium acetate in water (mobile phase A) and 5 mM ammonium acetate in acetonitrile (mobile phase B), and was delivered at a flow rate of 0.5 mL/min. The following gradient schedule was used: 10%e100% B (0.0e1.0 min), 100% B (1.0e 3.0 min), 100%e10% B (3.0e3.2 min) and 10% B (3.2e6.0 min). Data collection, peak integration and processing were performed using Analyst version 1.4.2 software (Applied Biosystems/MDS SCIEX).
MOLECULAR METABOLISM 25 (2019) 119e1302019 University Health Network. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).