Mtt assay

To analyze PTHrP secretion from ER+ tumor cells and its E₂ dependency, ER+ MCF-7 cells or ER+ tumor cells isolated from MCF-7 BMET were plated in 24-well plates at a density of 1.3 × 10⁵ cells/well in E₂-depleted media [phenol red-free DMEM (Invitrogen), 10% charcoal-stripped FBS (Valley Biomedical, Winchester, VA), 1% penicillin/streptomycin (Thermo Fisher), and 200 mM L-Glutamine (Sigma Aldrich, St. Louis, MO)] for 4 days, during which time cell number did not change for any cell line (data not shown), prior to treatment with E₂ (10⁻¹¹-10⁻⁶ M, as indicated; Sigma Aldrich), an ERα specific agonist propyl pyrazole triol (PPT; 10⁻⁸ M; Tocris, Minneapolis, MN), an ERα specific antagonist methyl-piperidinopyrazole hydrate (MPP; 10⁻⁶ M, Tocris), or vehicle control for 48 or 52 h, as indicated. Conditioned media, stored at −80 °C after addition of protease inhibitors (Sigma Aldrich), were assayed for secreted PTHrP using a commercial immunoradiometric assay (Beckman Coulter, Brea, CA). A lack of treatment effect on cell number during the 48 or 52-h incubation was verified using a commercial MTT assay (ATCC).

Proliferating 3T3-L1, H460, and HeLa cells were plated in a 96-well plate, with a seeding density of 1 × 10⁴ cells/cm². Quiescent 3T3-L1 cells were plated with seeding density of 1 × 10⁵ cells/cm². Cells were treated with vehicle control (DMSO) or 500 μM SSO dissolved in DMSO and incubated for 48 h. The proliferation/viability tests were carried out by using an MTT assay (ATCC) and performed according to the manufacturer’s instructions (see Supplemental Experimental Procedures).

Cell uptake was monitored by using MDA-MB-231 cells, a generous gift from Dr. Schiemann, Case Western Reserve University, and sulfo-Cy5 fluorescently labeled TMV and flow cytometry methods were as previously described.¹¹ Data were recorded with a BD LSRII flow cytometer and analyzed using FlowJo 8.63 software. The intracellular distribution of phenanthriplatin and PhenPt-TMV was determined following a 24 h incubation with A2780 cells. Cell components were separated by using a commercially available kit (Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit) and atomic absorption spectroscopy as previously described. Efficacy was analyzed by using the MTT assay (ATCC) and a panel of human cancer cell lines: A2780 (ovarian cancer), A2780/CP70 (ovarian cancer resistant to cisplatin), OV81.2 (ovarian cancer, primary patient cells; cells were a generous gift from Dr. DiFeo, Case Western Reserve University), 8988T (pancreatic 375 cancer) cells were a generous gift from Dr. Ghoroghchian, MIT. LNCAP (prostate cancer), MCF-7 (breast cancer), MDA-MB231 (breast cancer) cells were obtained from ATCC unless indicated otherwise. The assay was performed as per manufacturer’s recommendation; a BioTek Synergy HT multidetection microplate reader was used for read-out.

GBM6, GBM10, GBM12 and GBM39 xenograft lines were plated in 96 well plates in DMEM containing 10%FBS and 1X, penicillin/streptomycin at a density of 10⁴ cells per well. Twenty-four hours after plating the cells were exposed to different concentrations of alisertib. Cell proliferation was measured by MTT assay (ATCC, Manassas, VA) at days 1, 2, 3, 7, and 10 post exposure to alisertib and the IC50 was calculated for each xenograft line, as previously described [19 (link)].

HPASMC proliferation was determined with a quantitative colorimetric assay employing dimethylthiazol (MTT assay; ATCC) as described earlier [19 (link)]. Briefly, cells transfected with non-targeting control siRNA (si-Con) or with siRNA against PPARγ (si-PPARγ) were treated with or without PEG-catalase (1000 u/ml) during the last 24 hours of the 72 hour recovery period. The cells were then incubated with the MTT reagent for 4 hours. The mitochondrial reductase present in living cells reduces MTT to purple formazan, which is detected by spectrophotometry. Samples were then analyzed using an ELISA plate reader (λ = 570 nm), and values from treated cells were normalized to values from corresponding control cells. To assess proliferation by cell counting, HPASMCs were counted using a hemocytometer and cell viability was determined by Trypan blue exclusion assay as described previously [24 (link)]. We previously reported that MTT assays and cell counting methods produced similar results in hypoxia-exposed pulmonary vascular wall cells [24 (link)].