Transfected cells were also used for MTT and TUNEL assays. Statistical analysis Statistical significances were analyzed by ANOVA and paired Student t test with

Statistics Package for Social Science (SPSS) software (Version 14). Qualitative data were expressed as means ± S.D, and p < 0.05 was considered statistically significant difference. Results Paclitaxel induced cytotoxicity and apoptosis in FLCN-deficient renal cancer cells To determine whether paclitaxel treatment leads to apoptosis in FLCN-deficient renal cancer cells, cell lines with (ACHN-sc and UOK257-2) and without (ACHN-5968 and UOK257) FLCN expression were treated with paclitaxel. The cell viability was analyzed by MTT assay after treatment. As shown in Figure 1A, suppression of cell growth by paclitaxel on FLCN-deficient UOK257 and ACHN-5968 cells was C646 more significant than that on matched UOK257-2 and ACHN-sc cells,

indicating more severe paclitaxel-induced cytotoxicity to FLCN-deficient cells. We further analyzed apoptosis in these cell line pairs by using in situ colorimetric TUNEL assay. As shown in Figure 1B, paclitaxel could induce apoptosis in all treated cells with or without FLCN expression. However, a much greater number of apoptotic cells were detected in UOK257 and ACHN 5968 lines compared to UOK257-2 and ACHN-sc lines. The differences were also dose-dependent and reached maximum at 100 nM of paclitaxel. After paclitaxel treatment, cell nuclear morphological changes were observed using DAPI staining assay (Figure 1C). Cyclopamine Paclitaxel caused more apoptosis with

destroyed DNA in UOK257 and ACHN 5968 cells (indicated as the strong blue fluorescence). Furthermore, after the treatment of paclitaxel, the 35 kDa protein caspase-3 was cleaved into 17 kDa fragments in cells with or without FLCN expression (Figure 1D). The levels of cleaved caspase-3 were obviously higher in UOK257 and ACHN 5968 cells upon the treatment IMP dehydrogenase with 100 nM paclitaxel, indicating more apoptosis was induced in cells without FLCN expression. These results supported the conclusion that paclitaxel induces more apoptosis in FLCN-deficient renal cancer cells. Figure 1 Paclitaxel induced cytotoxicity and apoptosis in FLCN-deficient renal cancer cells. A. Cells were treated with 100 nM paclitaxel or a control vehicle, cell viability was measured by MTT assay. Compared with UOK257-2 and ACHN-sc cells, FLCN-deficient UOK257 and ACHN-5968 cells were more sensitive to paclitaxel-mediated cytotoxicity. (*: p < 0.05. UOK257 with Paclitaxel vs UOK257-2 with Paclitaxel; ACHN-sc with Paclitaxel vs ACHN 5968 with Paclitaxel; n = 15) B. Cells were treated with 50, 80, and 100 nM paclitaxel for 24 hours. TUNEL assay was used for apoptosis analysis. FLCN-deficient cells (UOK257 and AHN-5968) showed more cell death compared to FLCN-expressing counterparts. (*: p < 0.05. UOK257 vs UOK257-2; ACHN-sc vs ACHN 5968; n = 15). C.