A Small Peptide of SPARC Reverses Chemoresistance Figure 9. A model: SPARC-mediated apoptosis. A schematic of SPARC and Bcl2 interacting with caspase 8 to influence apoptosis. In this study, we demonstrate that in an environment low in SPARC, Bcl2 interacts with caspase 8 to inhibit apoptosis. However, in the presence of SPARC or its N- terminus, these proteins interact with caspase 8 to inhibit its interaction with Bcl2, leading to an augmentation of the apoptotic cascade. doi:10.1371/journal.pone.0026390.g009 RNA Interference. Caspase 8 gene expression knockdown using 40 nM caspase 8 siRNA (Stealth RNAi, Invitrogen) was assessed as previously described, using similar conditions that resulted in a 14-fold reduction in gene expression [8]. Transfection with siRNA was performed for 48-hrs before additional treatments or assays were performed. The conditions for Bid siRNA transfection were tested using 20–40 nM siRNA (Stealth RNAi. Invitrogen) and the degree of knock-down determined 48 and 72 hrs later (Figure S2A). The most effective knock-down was obtained following transfection with 40 nM of Bid siRNA for either 48 or 72 hrs. All subsequent experiments were performed using 40 nM siRNA for 48 hrs. For Bcl2, conditions were optimized using 40–80 nM siRNA (Stealth RNAi, Invitrogen) and 4.5 uL of HiperFect-Transfection Reagent (Qiagen) for 48– 72 hrs. At 72 hr following transfection with 40 and 80 nM of siRNA, a 40% and 60% reduction in Bcl2 expression was achieved, respectively (Figure S2B). In subsequent experiments, 80 nM Bcl2 siRNA for 72 hrs were used. As control, scramble oligonucleotide sequences were used. For RT-PCR, RNA was isolated with Trizol (Invitrogen), and 1 mg of total RNA was used to generate cDNA (Superscript III, Invitrogen). Specific primers used for PCR include: Bcl2 (forward)59-atctgggccacaagtgaagt-39, (reverse)59-cttctccccagcctccag-39; Bid (forward)59-aaaaccacatgg- cacagaga-39, (reverse)59-agagggaaccactttgctga-39; and under the following PCR conditions: 94uC62 min; 35 cycles of 94uC630 s, 58uC620 s, 72uC630 s; 72uC630 s, PCR products were separated on a 1.5% agarose gel electrophoresis, and levels of expression quantified using ImageJ. ELISA for quantification of SPARC and related peptides Stable cell lines (MIP/ZEO, MIP/SP, MIP/SP-N, MIP/SP-F and MIP/SP-C) or transiently transfected cells were grown to 90% confluence and proteins from whole cell lysates were isolated as previously described [4]. Microwells were coated with anti- SPARC antibody (1:5000) in coating buffer (0.1 M sodium carbonate, pH 9.5) overnight at 4uC, washed 36 with PBS-T (0.05% Tween-20), and blocked with 2%BSA in PBS for 1-hour at RT. 5 mg of total protein was added to each well in triplicate, incubated at 4uC for 4-hrs, and washed 36 with PBS-T. Wells were then incubated with either anti-His or anti-myc (1:5000, Sigma; all expression constructs express His6/myc-tagged fusion proteins) antibodies for 2-hours at RT, washed 56 with PBS-T, and incubated with appropriate secondary-HRP antibodies (1:10 000) for 1-hour at RT. Wells were then washed 56 with PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e26390