HTS Assay for Functional Modulators of ABCB6 (b) verteporfin, (d) tomatine hydrochloride and (f) benzethonium chloride for 14C-labeled hemin in the presence of ATP. Results representative of 3 independent experiments. ‘*’ Significantly different from untreated controls. P,0.05. doi:10.1371/journal.pone.0040005.g007 forms of ABCB6. In addition our results suggest that the two isoforms could have different substrate specificities as only the ,80 kDa form of ABCB6 appears to interact with hemin but not the ,50 kDa form. Although it is not clear at this time which of the two isoforms localize to the mitochondria, based on our earlier studies where we have shown that the ,80 kDa form of ABCB6 interacts with heme and localizes to the mitochondria we hypothesize that the ,80 kDa form of the ABCB6 isoform localizes to the mitochondria. As optimized, the HTS assay is amenable to high-throughput screening applications for identification of ABCB6 modulators and in combination with the secondary assays can identify potential mitochondrial ABCB6 substrates and inhibitors. The assay is also appropriate to establish the relative potencies of active com- pounds. Potential pitfalls include possibility of false positives due to cytotoxic compounds or pore-forming proteins, which could allow ABCB6 function-independent leakage of PPIX, leading to a decrease in the overall cellular PPIX levels. Finally, although the assay is configured to only detect compounds that block ABCB6, Figure 8. Verteporfin and tomatine hydrochloride are ABCB6 transport substrates. (a) verteporfin and (b) tomatine hydrochloride are transported by transport competent ABCB6 protein (ABCB6) in the presence of ATP which is significantly higher than transport by transport incompetent ABCB6 protein (ABCB6-MT) in the presence of ATP in mitochondria isolated from ABCB6 or ABCB6-MToverexpressing cells. Results are representative of three independent experiments. ‘*’ significantly different from ABCB6-MT and AMP treated ABCB6 expressing mitochondria; P,0.05. ‘**’ significantly different from ABCB6-MT and AMP treated ABCB6 expressing mitochondria; P,0.01. (c) and (d) vanadate sensitive ATPase activity (fold change relative to basal activity) was stimulated by (c) verteporfin and (d) tomatine hydrochloride in mitochondria from cells expressing transport competent ABCB6 protein (ABCB6) relative to mitochondria isolated from transport incompetent ABCB6 protein (ABCB6-MT). Values are means +/2 SEM. ‘*’ significantly different from ABCB6-MT cells at each time point; P,0.05. doi:10.1371/journal.pone.0040005.g008 PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e40005