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3 protocols using pitavastatin

1

Apoptosis pathway inhibitors protocol

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ABT-263, A-1331852 and A-1210477 were from AbbVie (North Chicago, IL, USA),
ABT-199, epigallocatechin gallate (EGCG), CB-839, simvastatin, rapamycin and
torin-1 from Selleck Chemicals (Houston, TX, USA),
gamma-L-glutamyl-p-nitroanilide (GPNA) from Insight
Biotechnology (Wembley, Middlesex, UK), azaserine from Cambridge Bioscience
(Cambridge, UK), aminooxyacetate (AOA), sodium palmitate, dimethyl
α-ketoglutarate, oxaloacetate and citrate from Sigma-Aldrich
(Gillingham, UK), L-glutamine from Life Technologies (Paisley, UK) and
GSK2194069, SB204990, atorvastatin, pitavastatin and bafilomycin A1 from Tocris
(Abingdon, UK). Antibodies against PARP, BCL-2, MCL-1, BAX, BAK and GAPDH were
from Santa Cruz Biotechnology (Santa Cruz, CA, USA), caspase-3, caspase-9,
BCL-XL, BCL-w, BIM, PUMA, BAD, IDH2, ACL, ACO2, ATG5 and ATG7
from Cell Signaling Technology (MA, USA), BID from Prof. J. Borst (The
Netherlands Cancer Institute, Amsterdam, the Netherlands), NOXA from Millipore
(Watford, UK) and SLC1A5, GLS, GFAT, GLUD1, IDH3, FASN and HMGR from Abcam
(Cambridge, UK).
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2

Pitavastatin Treatment Protocol

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All chemicals and cell culture reagents were obtained from commercial sources. Pitavastatin was obtained from Tocris and dissolved in dimethyl sulfoxide (DMSO), and the latter was used as vehicle control for cellular studies where noted.
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3

Evaluating Combination Treatments on REH Cell Viability

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REH cells were treated for 2 days with an ATP citrate lyase inhibitor, BMS-303141 (10 μM) (Tocris, Minneapolis, MN, USA), an HMG-CoA reductase inhibitor, pitavastatin (1 μM) (Tocris, Minneapolis, MN, USA), a chemotherapy agent, cytarabine (Ara-C, 0.1 μM) (Sigma Aldrich, St. Louis, MO, USA) or the combinations, as indicated. Following treatment, the media cells (M), suspended cells (S), and phase dim cells (PD) were recovered, and the viability and total cell count were analyzed using the trypan blue exclusion method.
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