Four of the Arginine Biosynthetic Genes in the Yeast S

Supporting Information

Reagents. THIO and verapamil were purchased from Sigma-Aldrich (St. Louis, MO, USA).Vinblastine (VIB) was purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Antibodies. Antibodies against pChk2, c-IAP1, Bcl-XL, pp70S6K, PCNA, p53, p21 and cleaved poly ADP ribose polymerase (C-PARP) were purchased from CellSignaling Technology (Danvers, MA, USA). Antibodies against c-IAP2, pRb, Cdk4, Survivin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).Antibody against pH2AX was obtained from Abcam (Cambridge, UK).

Cell culture.Human oral squamous carcinoma cell lines,KB and its multidrug-resistant subline,KBV20C, were obtained from Dr. Yong Kee Kim, and they were previously described (14-17). All cell lines were cultured inRPMI 1640 containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomycin (WelGENE, Daegu, South Korea).

Cellular viabilityassay. Cell proliferation was measured by a colorimetric assay using the EZ-Cy Tox cell viability assay kit (Daeillab, Korea) according to the manufacturer’s instructions. Briefly, Cells grown on wells of 96-well plates were incubated with 10 l of EZ-Cy Tox solution for 1-2 h at 37°C. Then, absorbance at 450 nm was determined immediately using a multi-well spectrometer (Bio-Tek Instruments, Winooski, VT, USA). Cell viability assays wereperformedin quadruplicates, but some of the results presented were obtained from experiments performedin triplicates. We performed two independent experiments.

Western blot analysis. All cellular proteins were extracted using a previously described trichloroacetic acid (TCA) method (18). Briefly,Cells grown in 60-mm diameter dishes were washed three times with PBS. Next, 20% TCA were added to each plate. The cells were then dislodge by scraping and transferred to Eppendorf tubes. Proteins were pelleted by centrifugation for 5 min at 3000 rpm and resuspended in 1 M Tris-HCL (pH 8.0) buffer. The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis as described previously (18).

Rho and Calcein-AM uptake tests. The tests were used for determination of ability for inhibition of P-gp using a previously described method (14,15). Briefly, cells grown in

6-well plates were treated with indicated drugs and incubated for 24 h at 37°C. Cells were then incubated with and 1 g/mL Rho or 0.1 g/mL Calcein-AM for 1 h 30 min at 37°C. The medium were removed and the cells were washedwith PBS. The stained cells were analyzed using a FACSCalibur flow cytometry system (BD Bioscience, Franklin Lakes, NJ, USA). We performed two independent experiments.

Hoechst staining. Hoechst staining was used to identify nuclear disruption, an indicator of apoptosis (14, 16). The staining was performed as previously described (14, 16).Briefly, the cells plated in 6-well plates were treated with the indicated drugs and incubated for 24 or 48 h at 37°C. Cells were then incubated with 9.4 M Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min in the dark at 37°Cbefore image acquisition. The medium was removed and the cells were washed twice with phosphate-buffered saline (PBS). Stained cells were subsequently examined using an inverted fluorescence microscope.We performed two independent experiments.

Fluorescence-activated cell sorting (FACS) analysis.FACS analysis was performed as previously described (18).Cells were grown in 60-mm diameter dishes and treated with the indicated drugs for the prescribed times. The cells were then dislodged by trypsin and pelleted by centrifugation. The pelleted cells were washed thoroughly with PBS, suspended in 75% ethanol for at least 1 h at 4°C, washed with PBS, and resuspended in a cold propidium iodide (PI) staining solution (100 g/mL RNase A and 50 g/mL PI in PBS) for 40 min at 37°C. The stained cells were analyzed for relative DNA content using a FACSCalibur flow cytometry system (BD Bioscience, Franklin Lakes, NJ, USA). We performed two independent experiments.

Statistical analysis. Data are presented as the mean  standard deviation (S.D.). Statistical analysis was conducted using Student's t-test and a one-way analysis of variance (ANOVA) followed by a multiple-comparison test. Results were considered to be statistically significant compared to the control (*) whenP <0.05.