the mechanism of action is not limited nor is specificity ensured. Because ABCB6 activity is dependent on ATP hydrolysis, compounds that affect ATP synthesis and accumulation could also appear as false positives. However, as with the differentially localized ABCB6, these issues can be easily resolved by secondary validation assays and specificity studies described above. Further analysis of the compounds identified here as well as those resulting from future screening of the small Molecular Libraries Small Molecule Repository (MLPCN) is expected to yield significant inhibitors and activators of ABCB6 and potential new ABCB6 substrates. Availability of these inhibitors, activators and substrates in multiple classes will also be useful in understanding the biochemical, physiological, and clinical impli- cations of ABCB6 in development, tumorigenesis and therapy related drug resistance. Supporting Information Figure S1 Human erythroleukemia cells (K562) ex- pressing ABCB6 have stable expression of ABCB6. The data show comparable ABCB6 expression in ABCB6 overexpress- ing cells that have undergone either 2 or 60 passages, indicating that ABCB6 expression in overexpressing cells is stable for upto 60 passages. Porin is used as the mitochondrial loading control. Figure representative of 3 independent experiments. (TIF) ABCB6 high-throughput screening assay flow- Figure S2 chart. (TIF) Figure S3 The sequence of the human ABCB6 trans- porter (UniProt accesion code: Q9NP58) aligned with both the Val33-Thr626 and Leu684-Ala1271 parts of the sequence of the mouse ABCB1a (PDB code: 3G5U). (TIF) Figure S4 Schematic representation of ABCB6 hemin- agarose affinity chromatography. (TIF) Figure S5 Flow cytometric HTS of Prestwick Chemical Library (PCL). The figure depicts data from a representative PCL plate demonstrating decrease in FL2 fluorescence (repre- senting PPIX fluorescence) mediated by (a) benzethonium chloride, (b) piperlongumine, and (c) tomatine hydrochloride (tomatine). Figure (c) also demonstrates three potential activators of porphyrin biosynthesis (doxorubicin, ellipticine, and hesperidin). The Y-axis displays the amount of fluorescence (FL2 log channel representing PPIX fluorescence). The X-axis displays each well. In the assays time bins were automatically drawn around the clusters by using IDLQuery software programs, each cluster corresponds to one well. The red square represents the PPIX fluorescence in untreated ABCB6 overexpressing cells while the white square represents the PPIX fluorescence in vector control cells. The arrow highlights the decrease in PPIX fluorescence of ABCB6 References 1. Dean M, Rzhetsky A, Allikmets R (2001) The human ATP-binding cassette (ABC) transporter superfamily. Genome Res 11: 1156–1166. 2. Rees DC, Johnson E, Lewinson O (2009) ABC transporters: the power to change. Nat Rev Mol Cell Biol 10: 218–227. 3. Zutz A, Gompf S, Schagger H, Tampe R (2009) Mitochondrial ABC proteins in health and disease. Biochim Biophys Acta 1787: 681–690. 4. Fletcher JI, Haber M, Henderson MJ, Norris MD (2010) ABC transporters in cancer: more than just drug efflux pumps. Nat Rev Cancer 10: 147–156. HTS Assay for Functional Modulators of ABCB6 expressing cells in the presence of potential inhibitors while the arrowhead highlights the increase in PPIX fluorescence in the presence of potential activators. (TIF) Figure S6 Chemical structures of the identified lead compounds, benzethonium chloride (BCL), verteporfin (VRP) tomatine hydrochloride (THC) and piperlongu- mine (PLG). (TIF) Figure S7 SDS-PAGE analysis of purified ABCB6 and selectivity and validation of HTS identified compounds by hemin-agarose affinity chromatography using puri- fied ABCB6. (a) Purified ABCB6 sample was analyzed by SDS- PAAGE. The figure shows silver staining of SDS gel (lane legends are 1, protein marker; 2, purified ABCB6-flag 3 mg protein). b) verteporfin and (c) tomatine hydrochloride potently disrupt the interaction between purified ABCB6 protein and hemin-agarose. 3 mg of purified ABCB6-flag protein was incubated in the presence or absence of increasing concentration of the indicated compound and hemin-agarose and the resulting complex was analyzed on a SDS-PAGE gel. The figure shows coomassie staining of SDS gel. Results representative of two independent experiments. (TIF) Figure S8 Selectivity and validation of HTS identified compounds by hemin-agarose affinity chromatography using purified ABCB6. a) verteporfin and (b) tomatine hydrochloride potently disrupt the interaction between purified ABCB6 protein and hemin-agarose. Three hundred nanograms of purified ABCB6-flag protein was incubated in the presence or absence of increasing concentration of the indicated compound and hemin-agarose and the resulting complex was immunoblotted using a monoclonal antibody to the flag-tag. Results show the entire immunoblot. Results are representative of three indepen- dent experiments. (TIF) Figure S9 Viability of K562 cells exposed to DMSO. DMSO at a concentration of 1% does not affect K562 cell survival following 24 hr exposure. (TIF) Acknowledgments The authors would like to thank Kristine Gouvea for critical review of the manuscript. Author Contributions Conceived and designed the experiments: PK GPT LAS MMTK. Performed the experiments: KP MMTK DP SC SY MG XM HC PK. Analyzed the data: AW MMTK KP PK XM. Contributed reagents/ materials/analysis tools: TO JS LAS PK. Wrote the paper: KP MMTK AW JS MKH CB GPT LAS PK. 5. Furuya KN, Bradley G, Sun D, Schuetz EG, Schuetz JD (1997) Identification of a new P-glycoprotein-like ATP-binding cassette transporter gene that is overexpressed during hepatocarcinogenesis. Cancer Res 57: 3708–3716. 6. Polireddy K, Chavan H, Abdulkarim BA, Krishnamurthy P (2011) Functional significance of the ATP-binding cassette transporter B6 in hepatocellular carcinoma. Mol Oncol 5: 410–425. 7. Borel F, Han R, Visser A, Petry H, van Deventer SJ, et al. (2011) ATP-Binding Cassette Transporter Genes Up-Regulation in Untreated Hepatocellular Carcinoma is Mediated by Cellular microRNAs. Hepatology. PLoS ONE | www.plosone.org 13 July 2012 | Volume 7 | Issue 7 | e40005