Cell lines and reagents. Human endometrial carcinoma cell line KLE and human cervical cancer cell line HeLa were purchased from ATCC. KLE cells were maintained in DMEM-F12 medium without HEPES supplemented with 10% FBS and 50 mg/mL gentamycin; HeLa cells were maintained in DMEM-F12 medium supplemented with 2% BGS and 50 mg/mL gentamycin. XIAP plasmid constructs were a kind gift from Dr. Robert G. Korneluk (University of Ottawa Eye Institute, Ottawa, Ont, Canada). All antibodies were from Cell Signaling Technology (Beverly, MA, USA) except for mouse monoclonal anti-actin antibody (Sigma, St-Louis, MO, USA), goat anti-rabbit, HRP-conjugated antibody (Bio-Rad Laboratories, Mississauga, ON, Canada), and anti-TGF-β antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Recombinant TGF-βs were purchased from Calbiochem (San Diego, CA, USA). LY294002 and PD98059 were purchased from Cell Signaling Technology. SB431542 was purchased from Sigma.
Immunofluorescence-based detection of TGF-β1 and TGF-β2 in clinical samples
. Preparation and image analysis was performed as previously described [6
]. Specificity of anti-TGF-βantibodies had previously been confirmed by checkerboard peptide blocking experiments [26
]. Briefly, the working dilution of each antibody (TGF-β1 (sc-146) and TGF-β2 (sc-90) from Santa Cruz Biotechnology) was incubated with a 10-fold excess of blocking peptide (Santa Cruz Biotechnology) overnight at 4°C before staining. In all cases, staining was abolished by homologous peptide but unaffected by pre-incubation with peptides corresponding to other isoforms [26
Cell treatments. Cells were seeded in 6 well-plates at the required density to reach approximately 60% confluency after 24 h (0.2 × 106 KLE cells and 0.5 × 106 HeLa cells per well). The following day, medium was changed and replaced with fresh media containing the appropriate treatment.
Western blots. Equal amounts of total cell lysates or subcellular fractions (as determined using Bio-Rad DC protein assay) were separated onto 8-15% polyacrylamide gels and then transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% milk in PBS-0.05% Tween-20 for 1 h at RT, probed with primary antibody (PTEN #9559; XIAP #2042; P-ERK #4377; P-Akt (Ser-473) #7291; Akt #9272; Smad3 #9513; Smad4 #9515; TGF-βRI #3712; all antibodies from Cell Signaling) overnight at 4°C, washed in PBS-0.05% Tween-20 and incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Bio-Rad). Detection was performed using SuperSignal West FemtoTM substrate (Pierce, Arlington Heights, IL, USA), as described by the manufacturer.
RNA extraction and RT-PCR analysis. Total RNA was isolated from cells using Trizol Reagent (Invitrogen, Burlington, ON, Canada) according to manufacturer's instructions. First strand cDNA was synthesized from 0.4 μg RNA using MMLV reverse transcriptase (Invitrogen). Primers for PCR amplification of XIAP were 5'-gagaagatgacttttaacagttttga-3' (sense) and 5'-ttttttgcttgaaagtaatgactgtgt-3' (antisense). Primers for amplification of PTEN were 5'-accaggaccagaggaaact-3' (sense) and 5'-gctagcctctggatttgacg-3' (antisense). Primers for amplification of Smad4 were 5'-gttgatggatacgtggaccc-3' (sense) and 5'-acctttgcctatgtgcaacc-3' (antisense). Primers for amplification of GAPDH were 5'-gtcagtggtggacctgacct-3' (sense) and 5'-tgagcttgacaaagtggtcg-3' (antisense). PCR reactions were conducted in a MJ Research Thermal cycler (model PTC-100), using the following parameters: 30 sec. at 94°C, 30 sec. at 58°C, and 1 min. at 72°C, for 35 cycles except for GAPDH (25 cycles). The reaction mixture was size-separated on an agarose gel and visualized using SYBR-SafeTM (Invitrogen) staining upon ultraviolet transillumination.
Transfection with siRNAs. Cells were seeded in 6-well plates at a required density to reach approximately 60% confluency in 24 h (0.2 × 106 KLE cells and 0.5 × 106 HeLA cells per well), and allowed to adhere overnight. The day of experiment, TGF-βRI (5'-ggacccuucauuagaucgctt-3' and 5'-gcgaucuaaugaagggucctc-3'), Smad4 (5'-ggucuuugauuugcgucagtt-3' and 5'-cugacgcaaaucaaagacctt-3') or control (5'-acucuaucugcacgcugacuu-3' and 5'-aagucagcgugcagauagagu-3') siRNAs were mixed with Mirus Trans-it TKO transfection reagent (Fisher Scientific, Ottawa, ONT, Canada) following supplier's instructions and added to the cells (100 nM working concentration). After 8 h-transfection, medium was replaced and plates were incubated for 16 additional hours (total: 24 h) or 40 additional hours (total: 48 h), as indicated in Figure legends, at 37°C before cells were collected.
Transfection with shRNAs. Cells were seeded in 6-well plates at the required density to reach approximately 60% confluency after 24 h. The day of transfection, XIAP shRNAs shRNA (5'-GCCACGCAGTCTACAAATTCT-3') or control (scrambled) shRNA (all shRNA inserted into pGeneClip (SABiosciences, Frederick, MD, USA)) were added to cells using a ratio of 3.6 μL Fugene:1.2 μg DNA/well. After 8 h-transfection, medium was replaced and plates were incubated for 40 additional hours (total: 48 h) at 37°C before cells were collected.
Data were subjected to one-way ANOVA (PRISM software version 3.03; GraphPad, San Diego, CA). Differences between experimental groups were determined by the Tukey's test. Statistical significance was accepted when p < 0.05 and indicated as asterisk above individual graph bars.