wells contained 1% DMSO. Following treatment, cells were incubated (37uC and 5% CO2) for 72 hours prior to adding CellTiter-Glo reagents and followed by a 10 min incubation at room temperature in the dark. Luminescence intensity (LI) was recorded using a Wallac 1420 plate reader (PerkinElmer, Norwalk, CT). The percentage of normalized viable cells was calculated using the following equation: % viability = 1006 (LI experiment - LI background)/(LI vehicle control - LI background). Molecular Modeling/Docking Protein Modeling and Docking The sequence of the human ABCB6 transporter as extracted from the UniProt database [23,24] (accession code: Q9NP58) was aligned using the T-Coffee package [25] with both the Val33- Thr626 and Leu684-Ala1271 parts of the sequence of the mouse ABCB1a transporter retrieved from the RCSB Protein Data Bank (PDB entry: 3G5U). The alignment was further manually refined in order to remove the insertions and deletions in the alpha helices (Fig. S3). The 3D model of the dimer human ABCB6 transporter was generated using the protein structure modeling package Modeller [26], and was prepared for docking using the standard protocol implemented in Fred_Receptor, a wizard like graphical utility that prepares an active site for docking with FRED (OpenEye Scientific Software Inc. Santa Fe, NM. www.eyesopen. com). Three dimensional (3D) conformations for the chosen ligands (tomatine, verteporfin, benzethonium, piperlongumine, and the endogenous substrate coproporphyrinogen III) were generated using OMEGA (OpenEye Scientific Software Inc. Santa Fe, NM. www.eyesopen.com) and the ligands were docked to the protein with FRED, using the default docking parameters, and a box big enough to include most part of the transmembrane region. The Chemgauss3 scoring function, which uses Gaussian smoothed potentials to measure the complementarity of ligand poses within the active site, was used to evaluate and compare how well the different ligands fit in the receptor binding site. Primary Hit Validation Selectivity/specificity and Functional Relevance Compounds identified to be non-toxic to the primary cells were screened for selectivity/specificity using four different assays. 1) Hemin agarose affinity chromatography. The assay was performed as described previously [17]. Briefly, mitochondrial lysate (20 mg) or purified ABCB6 protein (300 ng for immunoblots and 3 mg for silver staining and coomassie staining) with a flag tag were incubated at room temperature for 15 min in the presence of 167.5 nM hemin-agarose (Sigma, StLouis, MO). The reaction mixture was centrifuged at 4uC. The resulting pellet was washed thrice with 1 mL lysis buffer, resuspended in 20 mL of 2X SDS sample-loading buffer, and then centrifuged. The supernatant from the final spin was analyzed on a 5–20% gradient gel, transferred to a nylon membrane, and probed with a monoclonal antibody to the flag epitope to identify ABCB6-flag (Sigma, StLouis, MO). A schematic representation of the hemin-agarose affinity chromatography assay is presented in Fig. S4. Purification of ABCB6 protein with a flag tag used in hemin agarose affinity chromatography described above. A detailed description of the purification protocol is presented in a manuscript that is currently in preparation. Briefly, HEK293 cells expressing ABCB6-flag (transduced with ABCB6- flag lentiviral particles) were harvested and lysed in lysis buffer (0.2%Triton X 100 in 1X PBS containing 1 mM phenylmehane- sulfonylfluoride (PMSF), Pepstatin, Aprotinin and Leupeptin). Following centrifugation at 1,000 rpm for 10 min at 4uC the supernatant was incubated with 1 mL of anti-flag M2 affinity HTS Assay for Functional Modulators of ABCB6 beads (Sigma, StLouis, MO) overnight at 4uC. The antibody- ABCB6-flag complex formed following overnight incubation was washed twice with lysis buffer and centrifuged to remove the supernatant. The antibody-ABCB6-flag-bead complex (pellet) was resuspended in lysis buffer and transferred to handee spin cups (Peirce, Rockford, IL) followed by additional washing steps using lysis buffer and wash buffer (10 mM Tis, 0.2% Triton X-100). ABCB6-flag protein was finally eluted from the column using elution buffer containing Glycine and 0.2% Triton –X-100. The eluted protein was quantified and purity analyzed by SDS-PAGE following coomassie blue staining and silver staining. 2) Mitochondrial transport assay: Hemin competition. The assay was performed as described previously [17]. Briefly, mitochondrial preparations containing 20 mg of protein were incubated with an ATP regenerating mix along with 23 nM Fe55-hemin in either the presence or absence of the potential hit compound. The reaction was stopped at the indicated time and filtered through a cellulose filter. The filter was washed and analyzed by liquid scintillation counting. Fe55 hemin uptake was defined as the difference between substrate binding to mitochondria at 4uC and after incubation at 37uC in the presence or absence of ATP. 3) Mitochondrial transport assay: verteporfin and tomatine uptake studies. The assay was performed as described above with four modifications; a) to increase the power of the assay to distinguish between ABCB6 dependent and ABCB6 independent transport we performed transport using mitochondria isolated from cells expressing either a transport competent ABCB6 protein (ABCB6-wildtype; referred to as ABCB6) or a transport incompetent ABCB6 protein (ABCB6-mutant; referred to as ABCB6-MT) protein. The ABCB6-MT carries a single aminoacid change in the walker A domain of ABCB6, which is the nucleotide binding domain in ATP binding cassette transporters that is essential for transport function (1, 17); b) the amount of mitochondria used in these assays were 100 mg (mitochondrial protein) instead of the 20 mg used in the competition studies c) in these assays radioactive hemin was omitted and only verteporfin or tomatine hydrochloride were used as substrates, d) following uptake the reaction was stopped at the indicated time and instead of filtering through a cellulose filter, the mitochondria were pelleted at 17,0006 g for 10 min at 4uC. The pelleted mitochondria were washed three times with transport buffer. The final pellet was resuspended in 100 mL of acetonitrile and the amount of verteporfin or tomatine taken up into isolated mitochondria was analyzed using Ultra Performance Liquid Chromatography-time-of-flight mass spectrometry (UPLC- TOFMS) as previously described [27]. UPLC takes full advantage of chromatographic principles to run separations using columns packed with smaller particles for superior resolution and signal-to- noise ratio. Increased analyte concentration and reduced chro- matographic dispersion achieved by UPLC promote increased electrospray ionization efficiency giving improvements in response and in confirmation of identity. The concentration of verteporfin and tomatine hydrochloride following uptake into mitochondria was calculated from a standard curve generated using the respective compounds. 4) ATPase activity assay. ATPase activity was measured as described previously [17]. Briefly, vanadate-sensitive ATPase activity was measured in crude mitochondrial fraction isolated from K562-ABCB6-flag, and K562-ABCB6-Mutant-flag (K562- MT-flag) cells by colorimetric assay as described [17]. Prepara- tions of the isolated mitochondria containing 30 mg of protein were incubated with the indicated concentrations of verteporfin or tomatine hydrochloride in methanol; the reaction was started by PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40005