PBS-T and 100 mL of TMD chromagen substrate was added to each well for 15–30-minutes. Absorbance was read at 650 nM. Results of the ELISA are provided in Figure S3. Apoptosis M30 Antibody staining. Cells were seeded and treated 48 hours later with 5 mM 5-FU for an additional 48 hours, harvested and fixed onto glass slides by Cytospin, and stained with M30 antibodies (M30 CytoDEATH monoclonal antibody, ALEXIS Biochemicals, Switzerland) as per manufacturer’s protocol. Caspase- 3/7 assay. 20 mg of total protein isolated from cell lysates were used in Caspase-Glo 3/7 Assay (Promega) as previously described [35]. The relative luminescence units (RLU) were measured using Viktor2 1320 Multilabel-platform (Perkin Elmer). Immunoblot analysis 48 hours after plating, cells were incubated with 5 mM 5-FU, and collected at 0–12 hours for immunoblotting as previously described [4,36]. Immunodetection was performed using antibod- ies against caspase 8 (1:1000, Santa Cruz Biotechnology, Inc) or cleaved-caspase 8, BID, and caspase 3 (all 1:1000, Cell Signaling Technologies) followed by incubation with the appropriate secondary antibody. All immunoblots were also probed with antibodies to b-actin (0.32 mg/mL, Abcam) as loading control. Proteins were detected with SuperSignal West Dura (Pierce). Subcellular Fractionation, Co-Immunoprecipitation Total protein was isolated from cells grown to ,80% confluence, then separated into nuclear, cytosolic, and membrane fractions as previously described [8]. 250 mg of individual cellular fractions were incubated with antibodies against His6 or myc (1:100), caspase 8 (N-terminus, 1:100, Abcam), Bcl2 (1:100, Cell Signaling Technologies) or a non-specific anti-mouse-IgG anti- body as control (Cell Signaling Technologies), in PBS overnight (4uC). Protein:Antibody mixture was then incubated with 30 mL of Protein A:Protein G beads (1:1,Sigma) for 4-hours (4uC), washed with PBS, and eluted with 26 SDS-loading buffer, and used for immunoblotting against caspase 8, His6, or Bcl2 [8]. Animal Studies Tumor xenograft animal models were used to assess how overexpression of the various fragments of SPARC could influence tumor progression and response to chemotherapy in-vivo. Nude mice were implanted with 16106 (MIP/ZEO, MIP/SP, MIP/SP- N, MIP/SP-F, or MIP/SP-C) cells into the flanks of each animal. Treatment with 5-FU using a 3-week cycle regimen (6 cycles), commenced once the average tumor size reached 75–100 mm3 [4]. Control animals received saline. All studies were approved by the Animal Care Committee at the University of British Columbia, Canada (protocol A06-1507). Doubling time was averaged from all tumor measurements and calculated as: t1/2 = (t2-t1)*ln(2)/ln(volume2/volume1). In order to minimize differ- ences in tumor engraftment as a potential reason for influencing the variability in cell doubling time, all xenografts were implanted with the same number of cells and only mice with tumors ,75– 100 mm3 in size by 2 weeks following implantation were used in the in-vivo studies. Immunohistochemistry. For caspase 8 (1:100), Bcl2 (1:100), and SPARC (1:100, Hematological technologies) expression, OCT-embedded tissues were sectioned and processed according to established protocols [4,36]; counterstained with TO-PRO-3 A Small Peptide of SPARC Reverses Chemoresistance iodide (Invitrogen). Confocal Zeiss-Nikon microscope was used for image capture and Olympus BX61 microscope for light microscopy. CD31 staining. Tumors embedded in OCT-media were sectioned and fixed in acetone for 10-minutes at 4uC, washed in 16PBS, blocked in 2% NCS for 20-minutes at RT, and incubated in CD31 antibody (1:100, DAKO) overnight at 4uC. Sections were counterstained with DAPI (Molecular probes). Zeiss-Axioplan2 fluorescence microscope was used for imaging. The number of CD31-positive cells were counted and averaged from three different fields (n = 3 independent tumors). Routine histology was also performed on paraffin-embedded tissues (6 mM thick) [8] for hematoxyline and eosin (H&E) or cleaved-caspase 3 staining (1:25 dilution). Leica MicroSystems Bond-Max was used to stain tissue slides with cleaved-caspase 3 based on the manufacturer’s protocols with the following modifications: Leica’s ER1 Solution (citrate based buffer) was used for antigen retrieval (10-min6100uC), followed by a 10-min peroxide block at RT. Cleaved-caspase 3 antibody was incubated overnight at 4uC, then stained with hematoxylin. H&E images were captured with a Zeiss Axioplan 2 microscope, while images of caspase 3 stained slides were analyzed with a Leica DM600B microsope and Surveyor (version 5.5.5.12). Supporting Information Figure S1 Histology of tumor xenografts. Tumor xeno- grafts of MIP/ZEO, MIP/SP, MIP/SP-N, MIP/SP-F, and MIP/ SP-C subcutaneously implanted cells were paraffin-embedded and processed and stained with Hematoxylin and Eosin (H&E), and cleaved caspase 3. (TIF) Figure S2 siRNA optimization for Bid and Bcl2; and interaction between Bcl2 and caspase 8 in various resistant cells. A) Bid siRNA optimization: MIP101 cells were transfected with 20–40 nM Bid siRNA (or scramble control). RNA was isolated from cells harvested after 48–72 hrs of transfection and Bid gene expression determined by RT-PCR. Optimal Bid gene expression knock-down was achieved after transfection of 40 nM of siRNA for either 48–72 hrs; B) Bcl2 siRNA optimiza- tion: MIP/5FU cells were transfected with 40–80 nM Bcl2 siRNA (or scramble control) for 72 hrs. A 40% and 60% reduction in Bcl2 gene expression was noted following transfection with 40 nM and 80 nM respectively. The optimal condition used in subsequent experiments included the transfection of cells with 80 nM of Bcl2 siRNA for 72 hrs; C) The interaction between Bcl2-caspase 8 occurs at the N-terminus of caspase 8 as cells incubated with antibodies blocking caspase 8 (N-term, 1.5–3 mg) prevented this Bcl2-caspase 8 interaction. (TIF) Figure S3 Levels of SPARC and related peptides in cells used in this study. Cell lysates from stable transfectants (in-vitro and in-vivo) or transiently transfected cells were isolated 120 hours post-transfection and levels of SPARC and SPARC-related peptide levels were assayed by ELISA. Results represent mean 6 s.e. (n = 3 independent studies). Student’s t-test, * statistical difference compared to control, where p,0.05. (TIF) Table S1 (DOC) Table S2 (DOC) List of abbreviations. Site-directed mutagenesis primers. PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e26390