<i>In vitro</i> effects of TCRP1 interfering RNA on human OSCC.

<p><b>A.</b> Real-time PCR analysis of TCRP1 mRNA expression. Treatment of Tca/PYM cells with TCRP1 interfering plasmid (pAU-siTCRP1) led to a significant decrease in TCRP1 mRNA levels. No reduction in expression was observed for untreated cells or cells treated with scrambled interfering plasmid. <b>B.</b> TCRP1 expression in Tca/PYM, Tca/PYM-Con, and Tca/PYM-siTCRP1 cells were examined by Western blot. Decreased expression of TCRP1 was observed after transfection with TCRP1 interfering plasmid in Tca/PYM cells. No reduction in expression was observed for untreated cells or cells treated with scrambled interfering plasmid. β-actin was used as the loading control. <b>C.</b> Responses of Tca/PYM and Tca/PYM-siTCRP1 cells to PYM. Tca8113/PYM cells were more resistant to PYM. <b>D.</b> Responses of Tca/PYM and Tca/PYM-siTCRP1 cells to DDP. Tca8113/PYM-siTCRP1 cells were more sensitive to DDP.</p

Identification of genes potentially involved in TCRP1-mediated multidrug-resistance phenotype.

<p><b>A.</b> Differentially expressed genes in the Tca/PYM-Con (left) and Tca/PYM-siTCRP1 (right) cells were analyzed by Human Toxicology and Drug Resistance Microarray (OHS-401). This microarray included 263 key genes critical in drug metabolism and resistance. <b>B.</b> A heat map generated from the microarray shows gene expression as Tca/PYM-siTCRP1 over Tca/PYM-Con cells for the genes whose expressions had increased or decreased by more than 1.5-fold in TCRP1 knockdown cells. <b>C.</b> GO analysis of functional gene grouping of the differentially expressed genes involved in TCRP1-associated multidrug-resistance phenotype.</p

Genes with downregulated expression in Tca/PYM-siTCRP1 cells.

a<p>Function: 1. Apoptosis genes; 2. Cell cycle genes; 3. Cell growth, proliferation and differentiation genes; 4. Transporters; 5. Response to stress; 6. Chaperones/heat shock proteins; 7. Transcription factors and regulators; 8. Drug metabolizing enzymes.</p

TCRP1 interacts with candidate proteins.

<p><b>A.</b> Tca8113/PYM cell lysates were incubated with normal mouse IgG-conjugated agarose (control IgG) or anti-TCRP1 antibody-conjugated agarose (TCRP1). The immunoprecipitants and cell lysates (input) were electrophoresed and immunoblotted with the indicated antibodies or TCRP1. <b>B.</b> GST pull-down assay. GST alone or GST-tagged TCRP1 was incubated with Tca8113/PYM cell lysates. The precipitated proteins and the input proteins were detected by immunoblotting with antibodies to GST, Akt or MT1X.</p

MT1X knockdown affects cellular apoptosis and soft agar colony formation.

<p><b>A.</b> Cells were treated with DDP for 48 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051413#s4" target="_blank">Materials and Methods</a>. Apoptotic cells were determined by flow cytometry after PI and Annexin-V-FITC double staining (Tca/PYM-siComb = Tca/PYM-siTCRP1 + MT1X ASO3#). <b>B.</b> Quantitative analysis of apoptosis data. Data represents mean ± S.D. of three independent experiments. *<i>P</i><0.05 versus untreated control. <b>C.</b> TCRP1 knockdown affects anchorage-independent growth of Tca/PYM, Tca/PYM-Con, and Tca/PYM-siTCRP1 cells. No. of colonies (>50 cells) per 35 mm² dish relative to those of untreated control cells were calculated. The assay was done in biological triplicates (soft agar plating in duplicate for each biological replicate; n = 3). *<i>P</i><0.05 versus untreated control.</p

Statistically significant molecular and immunohistochemical parameters.

<p>Clinical samples from primary oral squamous cell carcinoma were classified into cisplatin-resistant and cisplatin-sensitive groups on the basis of MTS assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051413#s4" target="_blank">Materials and Methods</a>.</p>a<p>Mann–Whitney U test was used to compare the differences in TCRP1 and MT1X with varying degrees of IHC Scores (−, +, ++, +++) between the resistant and sensitive groups. <i>P</i>-values for statistical significance are indicated.</p>b<p>Spearman’s correlation coefficient was used to analyze whether the relationships between expression of TCRP1 and MT1X in primary tumors correlate with their drug resistance patterns. <i>P</i><0.05 was considered statistically significant.</p

TCRP1 regulates the PI3K/Akt/NFκB pathway and protects oral squamous cells from cisplatin induced apoptosis.

<p>TCRP1-over-expressing cells (Tca/TCRP1) were treated with 30 µM DDP in the presence or absence of specific inhibitors of PI3K (LY2294002 [50 µM]), NFκB (Bay11-7082 [20 µM]) or vehicle (DMSO) and cultured for an additional 24 h. <b>A.</b> The degree of apoptosis was determined by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051413#pone-0051413-g006" target="_blank">Figure 6</a>. Significantly higher percentages of apoptotic cells were found in the presence of both inhibitors (*<i>P</i><0.05, compared with control). <b>B.</b> Western blot analyses showing the effect of TCRP1 on apoptosis associated proteins in Tca/TCRP1 cells. Nuclear histone and β-actin were included as loading controls. All experiments were repeated at least three times, and representative results are shown.</p

Genes with upregulated expression in Tca/PYM-siTCRP1 cells.

a<p>Function: 1. Apoptosis genes; 2. Cell cycle genes; 3. Cell growth, proliferation and differentiation genes; 4. Transporters; 5. Response to stress; 6. Chaperones/heat shock proteins; 7. Transcription factors and regulators; 8. Drug metabolizing enzymes.</p

Reduction in MT1X activity sensitizes cells to DDP <i>in vitro</i>.

<p><b>A.</b> MT1X expression in Tca/PYM cells was examined by Western blot. Decrease in expression of MT1X was observed after transfection with MT1X interfering RNA in Tca/PYM cells. No reduction in expression was observed in cells that were treated with scrambled interfering RNA or in untreated cells. β-actin was used as the loading control. <b>B.</b> MT1X expression in Tca/PYM cells was examined by Real time-PCR. <b>C.</b> MT1X and TCRP1 expression in genetically engineered Tca8113 cells were determined by Western blot. <b>D.</b> Responses of different cells to DDP and PYM were determined by MTS. Each point represents the mean of data from three independent experiments.</p

Representation of the candidate genes involved in the metabolism of DDP based on our microarray results.

<p>Representation of the candidate genes involved in the metabolism of DDP based on our microarray results.</p