For Western blot analyses, cells were lysed in RIPA buffer (50 mm Tris-HCl ph 7.2,

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1 Western blot assay For Western blot analyses, cells were lysed in RIPA buffer (50 mm Tris-HCl ph 7.2, 150 mm NaCl, 1% NP40, 0.1% SDS, 0.5% DOC, 1 mm PMSF, 25 mm MgCl 2, and supplemented with a phosphatase inhibitor cocktail). Protein concentration was determined by the BCA assay (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of protein were electrophoresed on SDS polyacrylamide gels. PageRuler Prestained Protein Ladder (Fermentas, USA) were used to determine molecular weight. The gels were then electroblotted onto PVDF membranes. After blocked with 5% milk, membranes were incubated with specific primary anti-human antibodies against SBP1 (1:1,000; MBL International), GPX1 (1:800; Abcam), HIF-1α (1:2000; Abcam), or glyceraldehyde-3-phosphate dehydrogenase (1:5,000; KangChen). Finally, the relevant protein was visualized by staining with the appropriate secondary horseradish-peroxidase-labeled antibody for 1 h at room temperature. The reactions were detected by enhanced chemiluminescence assay. qrt-pcr assay Total RNA was extracted from cell lines using Trizol Reagent (Invitrogen, USA). Total RNA (2 μg) was reverse transcribed using a PrimeScript RT reagent Kit (Takara, Japan). Reverse transcription-pcr was performed before quantitative realtime PCR. SBP1 mrna expressions was determined by real-time PCR using SYBR Premix Ex TaqTM II (Takara, Japan). PCR amplification cycles were programmed for 10 s at 95 ºC, followed by 40 cycles of 95 ºC for 5 s and 60 ºC for 30 s. Data were collected after each annealing step. GAPDH was used as an endogenous control to normalize for differences in the amount of total RNA in each sample. Relative expression of genes was calculated and expressed as 2 - ΔCt, as previously described 1.

2 The following primers were used: SBP1 forward primer 5 - GACGACCGCTTCCTCTACTTCA-3 and reverse primer 5 - GGGCTCTGGCTGGGACTTTA-3 ; GAPDH forward primer 5 -GGTATG ACAACGAATTTGGC-3 and reverse primer 5 - GAGCACAGGGTACTTTATTG- 3. Migration analysis Chemotactic cell migration was detected via Transwell assay using an 8 μm pore transwell plate (Corning) following the manufactures protocol. The migrated cells were stained with Giemsa. Spontaneous cell migration was assessed by wound healing assay. Cells were densely plated and allowed to grow to 100% confluence. Following serum starvation for 24 hours, cell monolayers were scratched by sterile p200 tips and the digital image of predetermined locations in each well were taken by microscope every 12 hours. Proliferation analysis To observe the effect of the SBP1 expression on cell proliferation in vitro, Cell Counting Kit-8 (Dojindo Molecular Technologies, Japan) was employed to draw cell growth curves. SMMC7721 cells were plated onto 96-well cell culture plates at a density of 5000 cells/well grown overnight and the cells were transfected with SBP1- sirna the next day. Cell proliferation was measured by absorbance of 450 nm at 0, 24, 48 and 72 hours after transfection. Apoptosis assay We used flow cytometry with annexin V/PI staining to detect apoptosis. Cells were harvested and resuspended in phosphate buffered saline (PBS) buffer at a

3 concentration of cell/ml. After centrifuged at 1000 g for 5 min, 195 μl FITCconjugated annexin V binding buffer and 5 μl of annexin V-FITC were added. Following gentle vortex, the mixture was incubated for 15 min at room temperature (20-25 C) in the dark. After centrifuged at 1000 g for 5 min, 190 μl FITC-conjugated annexin V binding buffer and 10 μl propidium iodide were added. Following gentle vortex, the sample was analyzed by flow cytometry (FACS Calibur, BD) within one hour period. The percentages of apoptotic and necrotic cell for each sample were calculated. Immunofluorescence assay Immunofluorescence analysis for SBP1 and GPX1 was performed, as described previously 1. Briefly, Cells were plated onto 35mm glass bottom dishes (MatTek, Ashland, MA) in RPMI 1640 supplemented with 10% FBS and incubated overnight at 37ºC in normoxia. The day after, cells were treated with various stimuli and cultured for indicated time. At the end of treatments, cells were washed with PBS, fixed in icecold methanol:acetone (1:1) at -20 ºC. Then, cells were blocked for 30 minutes in 1% bovine serum albumin (BSA). Primary antibodies (mouse monoclonal anti-human SBP1, diluted 1:160; rabbit polyclonal anti-human GPX1, diluted 1:200) were applied after blocking overnight in 4ºC, followed by three 5-minute washes in PBS, then the secondary antibodies (1:250) Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 555 goat anti-mouse (Invitrogen Life Technologies, Carlsbad, CA) were applied for 1 hour at room temperature also followed by three 5-minute washes in PBS. Finally, cells were stained with Hoechst (0.5μg/mL; Invitrogen-Molecular Probes) for one minute. Cover slips were mounted in 50% glycerol solution and stored in the dark at 4ºC. Negative controls were consisted of slides in which only the second antibody was added. Images were taken using confocal microscope (Leica, TCS SPE

4 Microscope). Measurement of GPX1 activity Cell extracts were prepared using cell lysis buffer for Western blot and immunoprecipitation (20 mmol/l Tris (ph 7.5), 150mmol/L NaCl, 1% Triton X-100, 5mmol/L sodium pyrophosphate, 5 mmol/l β-glycerophosphate,1 mmol/l EDTA, 0.5 mmol/l Na 3 VO 4, and 1 mg/ml leupeptin). Cell lysates were centrifuged at 12,000g for 10 min at 4 C. GPX1 activity (nmol NADPH/min/mL) was measured in the supernatant using a Cellular Glutathione Peroxidase Assay Kit (Cayman Chemical Company, USA) that measures the coupled oxidation of NADPH during glutathione reductase (GR) recycling of oxidized glutathione from GPX-mediated reduction of t- butyl peroxide. In the assay, excess GR, glutathione, and NADPH were added according to the manufacturer s instruction. The results were quantified spectrophotometrically and the GPX1 activity was then calculated from the results according to the manufacturer s instruction 2. Immunohistochemical Scoring. The immunostains were scored using a 4-point scale ( ) system, based on the number of positive cells and the intensity of staining; no staining was recorded as 0, weak staining in fewer than one-third of cells as +, moderate staining in one-third to two-thirds of cells as ++, and strong staining in more than two-thirds of cells as +++. Duplicate spots for each tumor showed a good level of homogeneity for the percentage of cells stained and intensity of staining. The higher score was taken as the final score in cases of a difference between duplicate tissue cores.

5 1. Li JC, Yang XR, Sun HX, Xu Y, Zhou J, Qiu SJ, Ke AW, Cui YH, Wang ZJ, Wang WM, Liu KD, Fan J. Up-regulation of Kruppel-like factor 8 promotes tumor invasion and indicates poor prognosis for hepatocellular carcinoma. Gastroenterology 2010;139: e Flohe L, Gunzler WA. Assays of glutathione peroxidase. Methods Enzymol 1984;105: