Structures of cisplatin and carboplatin.

<p>Structures of cisplatin and carboplatin.</p

HSP90α expression and cell viability.

<p>(A) MTT assay on cells treated with cisplatin for 2 days showed that kidney proximal tubule cells (KPT11 and KPT2) had lower viability compared to kidney distal tubule KDT3 cells (**<i>P</i><0.01 between either one of proximal tubule cell lines and KDT3). (B) Western blotting confirmed that KPT2-HSP90α cell lines (HSP90α#1 and #2) express higher levels of HSP90α compared to parental KPT2 or KPT2 with empty pBabe vector control. (C) Cellular growth for 2 days was assessed by MTT assay, and KPT2-HSP90α cell lines showed faster cellular growth compared to KPT2-pBabe cell lines (**<i>P</i><0.01). (D) Cisplatin treatment for 2 days showed that KPT2-HSP90α cell lines had lower viability compared to KPT2-pBabe cell lines (**<i>P</i><0.01 between either one of KPT2-HSP90α and either one of KPT2-pBabe).</p

Synthesis of Pt complexes 3 and 5.

<p>Synthesis of Pt complexes 3 and 5.</p

Protein expression and cisplatin-binding of HSP90α and HSP90β.

<p>(A) HSP90 protein expression analysis. Total cell lysates from cell lines used for pull-down assay were analyzed by Western blotting, using antibodies for HSP90α and HSP90β. KPT11 cell sample showed very low expression of HSP90α. (B) Pt-agarose pull-down assay using 293T cells transfected with FLAG-HSP90α or -HSP90β. 2NH₃Pt-agarose pulled down the two HSP90 isoforms with similar amounts.</p

Cisplatin-binding protein pull-down assay results.

<p>(A) Pull-down assay samples using HEI-OC1 cells. Agarose without Pt conjugation (No Pt) was used as a control. 2NH₃Pt-agarose pulled down several proteins in significant amounts, while 2ClPt-agarose only pulled down fewer proteins in lower amounts. The total cell lysate (Lysate) sample before pull-down is to show relative expression levels of proteins with different molecular mass. (B) 2NH₃Pt-agarose pull-down assay results. Several protein bands with high intensity appeared in all cells tested, with some minor band differences in different lanes. Gel bands were excised from HEI-OC1 samples and analyzed by mass spectrometry. Protein names identified in gel regions a, b, c, d and e are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066220#pone-0066220-t001" target="_blank">Table 1</a>.</p

Cisplatin-binding proteins identified in excised gel bands.

<p>Protein and gene names are those used in the SwissProt database. Gel regions of the identified proteins are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066220#pone-0066220-g006" target="_blank">Fig. 6B</a>. “Protein MW” is the molecular weight of the protein sequence from the SwissProt database. “Total spectra” is the number of MS/MS spectra matched to the protein sequence. “Unique spectra” is the number of MS/MS spectra that are not shared between other protein sequences in the database. “Unique peptides” is the number of different amino acid sequences matched. “Sequence coverage” is the percentage of the total protein sequence matched to assigned MS/MS spectra. “Histidine content” is the percentage of the histidine residues in the total amino acids of each protein. Myosin-9 is the same as myosin IIA. *Endoplasmin is the same as GRP94. **Transitional endoplasmic reticulum ATPase is the same as VCP. The seven identified isoforms of tubulin are not included in the table.</p

¹⁹⁵PtNMR spectra of complexes 3 and 5 in D₂O.

<p>Pt complex <b>5</b> shows an upfield chemical shift as compared to that of <b>3</b>. Similar chemical shifts are observed for the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066220#pone.0066220-Campbell1" target="_blank">[15]</a>N-labeled (top) and non-labeled (middle) complex <b>5</b>.</p

Heterologous expression of SGLT2 in KDT3 cells increased cellular uptake of GTTR.

<p>(A–C) KDT3-SGLT2 cells with positive SGLT2 immunofluorescence displayed robust GTTR uptake (B, C). (D–F) Empty vector control clones (KPT2-pBabe) showed negligible SGLT2 immunofluorescence (D) and weak, uniform levels of GTTR fluorescence (E, F) compared to (B, C). (H, I) GTTR fluorescence in KDT3-SGLT2 cells in the presence of phlorizin (100 µg/ml) was visibly less intense than in KDT3-SGLT2 cells without phlorizin treatment (B, C). (K, L) GTTR fluorescence in phlorizin-treated KDT3-pBabe cells showed weak levels of GTTR fluorescence as untreated in KDT3-pBabe cells (E, F). Scale bar = 20 µm. (M) Fluorescence intensities of GTTR in KDT3-SGLT2 or KDT3-pBabe cells in the presence or absence of phlorizin (100 µg/ml; **<i>p</i><0.01).</p

SGLT2 immunofluorescence in the kidney and cochlea.

<p>Two different SGLT2 antibodies were used, a rabbit polyclonal IgG to synthetic peptide derived from residues 250–350 of human SGLT2 and a goat polyclonal IgG against a murine peptide sequence within the N-terminal extracellular domain of SGLT2. (A, C) In wild-type mice, SGLT2 was immunolocalized at the apical membranes (arrows) of proximal tubules (p), but not in adjacent glomerular (not shown) or distal tubule (d) regions. (B, D) In <i>Sglt2^−/−</i> mice, no immunoexpression for renal SGLT2 was observed with either antibody. (E, F) No labeling above background was observed in cochlear marginal cells of wild-type or <i>Sglt2^−/−</i> mice with rabbit antisera for SGLT2. (G, H) Goat antisera for SGLT2 produced labeling patterns in cochlear marginal cells of both wild-type mice and <i>Sglt2^−/−</i> mice, suggestive if substantial non-specificity in this cell type. (I, K) In the intra-strial layer of wild-type mice, predominantly composed of both marginal and intermediate cells, both antisera exhibited a punctate labeling pattern not observed in <i>Sglt2^−/−</i> mice (J, L). Scale bar = 20 µm. (M) Immunoblotting with the goat antibody for SGLT2 revealed SGLT2 protein expression in wild-type and <i>Sglt2^+/−</i> mice, but not <i>Sglt2^−/−</i> mice. The ratio of SGLT2 to actin expression in kidney tissues of wild-type and <i>Sglt2^+/−</i> mice were significantly higher than that in <i>Sglt2^−/−</i> mice. There was no statistical difference in SGLT2 protein expression between wild-type and <i>Sglt2^+/−</i> mice. (N) Genotyping demonstrated the absence of wild-type SGLT2 alleles in <i>Sglt2^−/−</i> mice.</p

Uptake of the fluorescent glucose analog 2-NBDG is mediated by SGLT2 in KPT2 cells.

<p>KPT2 cells (A) had robust SGLT2 immunolabeling compared to KDT3 cells (B). Increasing doses of (C–F) D-glucose (molar ratios of 1∶0, 1∶1, 1∶50 or 1∶1000 [2-NBDG/D-glucose]), or (G–J) phlorizin (molar ratios of 1∶0, 1∶1, 1∶10 or 1∶50 [2-NBDG/phlorizin]) dose-dependently decreased 2-NBDG fluorescence in KPT2. Scale bar = 20 µm. (K, L). The fluorescence intensity of 2-NBDG in KPT2 cells was significantly decreased with increasing doses of D-glucose (K) or phlorizin (L; **<i>p</i><0.01).</p