Expression of the GFP-tagged ABCG2 variants in HUES9 cells.

<p>(A) Confocal images for HUES9 cell clumps expressing the GFP-ABCG2 variants (green), HUES9 cells are cultured on mouse embryonic fibroblasts. Immunostaining for Oct4 (red), cell nuclei are stained with DAPI (blue). (B) Flow cytometry analysis of HUES9 cells expressing the GFP-ABCG2 variants. The cells were labeled by a monoclonal anti-ABCG2 antibody (5D3), and a phycoerythrin (PE) coupled anti-mouse IgG2b. The green fluorescence of GFP-ABCG2 is plotted against 5D3-PE red fluorescence. Control HUES9 cells, HUES9 cells expressing GFP-ABCG2-R482R, GFP-ABCG2-K86M, and GFP-ABCG2-R482G variants are shown. (C) Flow cytometry analysis of the pluripotency marker SSEA4 in parental HUES9 cells and the GFP-ABCG2-expressing variants. The cells were labeled by a monoclonal anti-ABCG2 antibody (5D3) visualized by a phycoerythrin (PE)-conjugated anti-mouse IgG2b, and by anti-SSEA4, conjugated to APC (green). HUES9 cells expressing GFP-ABCG2-R482R, GFP-ABCG2-K86M, and GFP-ABCG2-R482G variants are shown.</p

PhenGreen diacetate based assay for functional studies of multidrug resistance ABC transporters.

<p>The non-fluorescent, hydrophobic PGD rapidly enters the cells through the plasma membrane. In the cytoplasm, PGD is cleaved by nonspecific esterases to yield fluorescent Phengreen (*PG—for the structures of PGD and PG see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190629#pone.0190629.s008" target="_blank">S8 Fig</a></b> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190629#pone.0190629.ref019" target="_blank">19</a>]). The ABC transporters ABCG2, ABCB1, or ABCC1 efficiently extrude PGD (and potentially also PG) to the extracellular space.</p

Fluorescent PG accumulation in human PLB cells, examined by confocal microscopy.

<p><b>Effects of ABCG2 protein expression and the specific inhibition of ABCG2 function by Ko143.</b> Cellular fluorescence was observed by confocal microscopy. PG fluorescence (green) was examined after 30 minutes of the addition of 0.5μM PGD to the medium, either in the absence or presence of the ABCG2 inhibitor KO143 (2.5μM). The cells were pre-labeled with fluorescent anti-WGA (red) to indicate the plasma membranes.</p

PhenGreen diacetate toxicity assay.

<p><b>Panel A.</b> Effect of PhenGreen accumulation on cell growth in PLB cells and PLB-ABCG2 cell. Cell growth was measured after 0.5μM PGD treatment for 30 minutes at 37°C, followed by cell sorting. <b>Panels B and C.</b> Cytotoxic effects of PGD treatment in HEK and A431 cells. The cells were pre-treated with the indicated concentrations of PGD for 30 min at 37°C in the loading media, then washed and cultured in normal cell culturing media (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190629#sec002" target="_blank">Methods</a>) for 72 hours.</p

Fluorescent PG accumulation in human cells, effects of ABCB1 and ABCC1 expression—Flow cytometry studies.

<p><b>Panel A</b>. Control PLB cells and ABCB1-expressing PLB cells, <b>Panel B</b>. Control A431 cells and ABCB1-expressing A431 cells, <b>Panel C.</b> Control HL-60 cells and ABCC1-expressing HL-60 cells, <b>Panel D.</b> Control HEK cells and ABCC1-expressing HEK cells. <b>Panel E</b>. Control PLB cells and ABCB1-expressing PLB cells, Control A431 cells and ABCB1-expressing A431 cells. <b>Panel F</b>. Control HL-60 cells and ABCC1-expressing HL-60 cells, Control HEK cells and ABCC1-expressing HEK cells.</p

Flow cytometry detection of PG accumulation in human PLB cells—Recognition and separation of control PLB cells and PLB cells expressing the ABCG2 transporter.

<p>Control PLB cells and PLB cells expressing wild-type ABCG2 were mixed in various ratios (0.2–99.8%). PG accumulation was measured after the addition of 0.25μM PGD. Immunofluorescent detection of the ABCG2 protein on the cell surface of the same cells was measured by the ABCG2-specific 5D3 monoclonal antibody binding. The numbers on the graphs indicate the % values of the separated cells, measured based on the relative fluorescence values (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190629#sec002" target="_blank">Methods</a>).</p

Fluorescent PG accumulation in human cells, effect of ABCG2 expression—Flow cytometry studies.

<p><b>Panel A</b>. PGD-concentration dependent PG accumulation in control PLB cells (×) and in PLB-ABCG2 cells (●). The black squares (■: ABCG2) and triangles (▲: CTRL) demonstrate PG accumulation in the presence of 2.5μM KO143, a specific ABCG2 inhibitor. <b>Panel B.</b> PGD-concentration dependent PG accumulation in control A431 (×) and A431-ABCG2 (●) cells, measured in EDTA-DPBS medium for 30 minutes at 37°C. The black squares (■: ABCG2) and triangles (▲: CTRL) demonstrate PG accumulation in the presence of 2.5μM KO143 at 0.5μM PGD. ± SD values are indicated.</p

MDR activity factors based on PG and mitoxantrone (MX) accumulation in human PLB and A431 cells.

<p><b>Effects of the ABCG2 variants on dye extrusion capacity—flow cytometry studies. Panels A. and B.</b>: MDR activity factor (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190629#sec002" target="_blank">Methods</a>) calculated by PG accumulation in PLB cells (Panel A) and A431 cells (Panel B), expressing ABCG2 variants. Panels C. and D.: MDR activity factor calculated by MX accumulation in PLB cells (Panel C) and A431 cells (Panel D), expressing ABCG2 variants. +/- SD values are indicated.</p

Fluorescent PG accumulation in human PLB and HL-60 cells, examined by confocal microscopy.

<p><b>Effects of ABCB1 and ABCC1 protein expression and the specific inhibition of the transporter function by tariquidar (ABCB1) or by benzbromarone (ABCC1)</b>. Cellular fluorescence was observed by confocal microscopy. PG fluorescence (green) was examined after 30 minutes of the addition of 0.5μM PGD to the medium, either in the absence or presence of the transporter inhibitors (0.25μM tariquidar for ABCB1 or 50μM benzbromarone for ABCC1). The cells were pre-labeled with fluorescent anti-WGA (red) to indicate the plasma membranes.</p

Pedigrees of two families carrying different ABCG2 premature stop mutations – co-segregation of the heterozygous mutation with reduced erythrocyte ABCG2 expression levels.

<p>Blood samples obtained from the 14 family members of the two healthy volunteer probands, carrying the premature stop mutations (see Fig. 3 -indicated with arrowheads) were analyzed for ABCG2 expression and the respective mutations. The RBC-G2 factor values, reflecting ABCG2 expression in erythrocytes, are shown in parentheses. Family members not available for blood donation are labeled by N.A.</p