Cell cultures, antibodies, and reagents
MeWo, SkMel2, SkMel28 cells (American Type Culture Collection; ATCC; Rockville, MD), A375, VMM5A, VMM39, SLM2, DM122, DM331 (kind gift from Dr. Craig Slingluff, University of Virginia (12
)) and SLM2 (kind gift from Dr. Angela Zarling) were propagated in RPMI Medium 1640 (Invitrogen, Grand Island, NY) supplemented with 5% or 10% fetal bovine serum (FBS; Gemini Bio-Products, West Sacramento, CA). All cultures were maintained in a humidified chamber at 37°C with 5% CO2
. An OncoMap analysis was performed at the Broad Institute to identify the mutational status of over 30 known oncogenes and tumor suppressor genes (13
). The cell lines were authenticated by comparing the tumor mutation profile determined by OncoMap to published reports.
Antibodies were obtained from the following sources: anti-phosphoERK (Sigma-Aldrich, St. Louis, MO), anti-tubulin (Calbiochem, Gibbstown, NJ), anti-ERK (B3B9) from the UVa hybridoma facility, anti-cPLA2 (Cell Signaling Technology, Beverly, MA), and anti-phospho-cPLA2 (Santa Cruz Biotechnology, Santa Cruz, CA).
The following small molecule inhibitors were obtained from EMD Chemicals (Gibbstown, NJ): 5-Aza-2-Deoxycytidine, AACOCF3, AG490, AKT Inhibitor IX, AMPK Inhibitor, Anacardic Acid, Celecoxib, Cyclopamine-KAAD, D4476, Diclofenac Na, DMAT, DNA Dependent Protein Kinase Inhibitor, Geldanamycin, GM6001, H-89, Indirubin-3′-Monoxime, IP3 Kinase Inhibitor, Jak I Inhibitor, K-252c, ML-7, NDGA, Okadaic Acid, Olomoucine, PD173074, S3I-201, SANT-1, SB203580, SC-514, Sphingosine Kinase Inhibitor, STO-609, SU6656, TGFβ Receptor II Inhibitor, Trichostatin A, TX-1918, U0126, Withaferin A, Wortmannin, and WP1066. Bortezomib, Dasitinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Lestaurtinib, Nilotinib, Rapamycin, Sorafenib, Sunitinib, Temsirolimus, and Vandetanib were acquired from LC Laboratories (Woburn, MA). 5-AIQ-hydrochloride, Bevacizumab, D609 Pro-drug, GF109203X, GW441756, Picropodophyllotoxin (PPP) and SP600125 were obtained from Sigma-Alrich (St. Louis, MO). Debromohymeniadlisine (DBH) was purchased from Enzo Life Sciences (Farmingdale, NY). OSU-03012 was obtained from Cayman Chemical (Ann Arbor, MI). Y27632 dihydrochloride was acquired from Tocris Bioscience (Ellisville, MO). PD325901 was a gift from Pfizer (New York, NY). Slo-101 was a gift from Dr. Deborah Lannigan (University of Virginia). Compounds were diluted in vehicle as specified by the manufacturer. Interferon (IFN) alpha and was a gift from Dr. Craig Slingluff (University of Virginia) and SAHA was a gift from Dr. David Jones (University of Virginia).
Synthetic Lethal Pathway Screen
Cell lines were grown in their normal growth media to 80% confluence and then washed with 1x PBS, trypsinized, collected, counted (via hemacytometer), and re-suspended in phenol-red free RPMI 1640 + 5% FBS at concentrations that would result in 100% confluence of the vehicle-treated control wells after 3 days of growth. Plating of the cells was carried out using the BioMek NX (Beckman Coulter, Indianapolis, IN) workstation. 90 μL of cell suspension was added per well in 96-well format. Small molecular inhibitors were diluted to 10x concentration and plated by hand into master drug plates. The BioMek NX workstation was used to add 10 μL of drug from the master plates to each well. The cells were then incubated for 3 days at 37°C and 5% CO2. Following this incubation, the BioMek NX workstation was used to add 10 μL of alamarBlue (Invitrogen, Grand Island, NY) to each well. The plates were incubated for 4 hours and fluorescence was measured at 560 nm excitation/590 nm emission on a Synergy 2 plate reader (BioTek Instruments, Winooski, VT). Mean results and standard error were calculated for triplicate samples.
alamarBlue: Four hours after being plated in 96-well plates, cells were treated with inhibitors or vehicle control in phenol red-free RPMI Medium 1640 (Invitrogen, Grand Island, NY) without fetal bovine serum and incubated for 3 days at 37°C. alamarBlue (Invitrogen, Grand Island, NY) was added to wells and incubated for 4 hours at 37°C. Fluorescence was measured as described above.
Crystal Violet: Cells were allowed to adhere to 12-well plates overnight before being treated with inhibitors or vehicle control in phenol red-free RPMI Medium 1640 (Invitrogen, Grand Island, NY) without fetal bovine serum for 7 days at 37°C. Plates were placed on ice and media was aspirated off cells. Cells were rinsed twice with cold 1x PBS (Invitrogen, Grand Island, NY) and fixed with cold 100% methanol for 10 minutes. Monolayers were stained with 0.5% crystal violet in 25% methanol:water for 10 minutes at room temperature and rinsed with distilled water until excess crystal violet solution has been removed. Plates were inverted to allow cells to air-dry overnight. Micrographs were taken with a Nikon SMZ1000 microscope equipped with a Nikon Coolpix 4300 digital camera. Digital images were processed with Adobe Photoshop CS2 version 9.0.2.
Immunoblot and Immunoprecipitation analysis
Cells were allowed to adhere to plates overnight before being treated with inhibitors or vehicle control in phenol red-free RPMI Medium 1640 (Invitrogen, Grand Island, NY) without fetal bovine serum for 1 hour at 37°C. Cells were rinsed with cold 1x PBS and lysed in Triton lysis buffer [10% Triton X-100, 5% 1M Tris (pH 7.5), 2.5% 4M NaCl, 0.5% 0.5M NaF, 0.01% 0.5M EDTA, 80% water] plus the following protease and phosphatase inhibitors: 1 μg/ml pepstatin, 1 μg/ml leupeptin, 0.4 TIU/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 200 μM orthovanadate, 50 mM β-glycerophosphate, and 0.4 μM Microcystin. Lysates were centrifuged at 13000 rpm at 4°C for 10 minutes and supernatants were collected. Protein concentrations were determined using BCA protein assay (Thermo Scientific, Waltham, MA). Protein extracts were prepared with Triton lysis buffer and LSB/beta-mercaptoethanol, boiled, loaded into acrylamide gels, and then run at 75V for 30 minutes then at 125V for 1 hour. Gels were transferred onto nitrocellulose membranes at 90V for 60 minutes at 4°C. Membranes were stained with ponceau blue and then were blocked with 5% bovine serum albumin (BSA) in PBS-T for one hour. Blots were incubated overnight at 4°C in primary antibodies, washed with PBS-T, incubated in a secondary antibody (LI-COR, Lincoln, NE) for 1 hour at room temperature, and imaged using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). Quantification was done using the Odyssey software and mean results and standard error were calculated for triplicate samples.
For immunoprecipitation analysis, protein extracts were incubated with antibody at 4°C overnight. Agarose beads (Roche) were washed with Triton lysis buffer with protease inhibitors and were added to the sample-antibody solution and incubated for 4 hours at 4°C. The beads were then washed with Triton lysis buffer with protease and phosphatase inhibitors, re-suspended in LSB/beta-mercaptoethanol, and loaded into acrylamide gels. Gels were run, transferred and immunoblotted as describe above with the following exception: membranes were washed with TBS-T.
SLM2, VMM39, and DM331 cells were plated and incubated overnight before being treated, in duplicate, with inhibitors or vehicle control in phenol red-free RPMI Medium 1640 without fetal bovine serum for 8 hours at 37°C. Cells were placed on ice and rinsed with cold 1x PBS. Cells were collected and RNA was isolated using the Qiashredder (Qiagen, Valencia, CA) and RNeasy Mini Kit (Qiagen, Valencia, CA). The gene array was performed using Illumina 3′IVT human HT-12 BeadChip arrays by Gene Analysis (Durham, NC). Principal component analysis and clustering QC analyses of the results indicated that two of the inhibitor-treated DM331 samples exhibited irregular quantities unseen in the majority of samples, leading us to conservatively exclude all DM331 sample results from further analyses. Differential expression analyses of quantile-normalized, log2-transformed expression values were performed using moderated ANOVA hypothesis testing (14
), as provided by the R/BioConductor (15
) limma package (16
), with Benjamini-Hochberg corrections for multiple hypothesis testing to control false discovery rate. All microarray data have been deposited with the NCBI Gene Expression Omnibus (GEO) under accession GSE39192.