KSR1 AS-ODN suppresses KSR1 expression in PANC-1 pancreatic cancer xenografts. After 5 days of treatment with 40 mg/kg/d KSR1 AS-ODN or PBS vehicle control via subcutaneous Alzet osmotic minipump, tumors were harvested, weighed, pared of connective tissue, and minced into small pieces. 0.2−0.5 g tumor tissue was Dounce homogenized in 4 vol NP-40 lysis buffer, centrifuged at 10,000 xg for 10 min and protein concentration of tumor lysate was determined using the Bio-Rad protein assay. 250 μg tumor lysate were mixed with Laemmli SDS-loading buffer and resolved by 7% SDS-PAGE. Separated proteins were transferred to a Hybond-ECL nitrocellulose membrane. KSR1 was detected by autoradiography using a mouse monoclonal anti-KSR1 antibody and HRP-conjugated sheep anti-mouse secondary antibody. The lane marked (+) contains 5 μg of a COS-7 cell lysate obtained from cells overexpressing Myc-tagged human KSR1. The housekeeping gene GAPDH was used as loading control. KSR1 and GAPDH bands were quantified by densitometry using Imagej 1.37v software. Percent KSR1 expression in AS-ODN-treated tumors was calculated as a percentage of densitometry arbitrary units in each drug-treated tumor compared to the mean densitometric value of PBS-treated control tumors.

Detection of KSR1 AS-ODN in plasma. After 5 days of treatment with 20, 30 or 40 mg/kg/d KSR1 AS-ODN or PBS vehicle control via subcutaneous Alzet osmotic minipump, mice were anesthetized and 0.7−1.0 ml of blood was extracted from left ventricle in EDTA anticoagulant. Blood samples were centrifuged for 15 min at 800xg at room temperature. Subsequently plasma was centrifuged for 5 min at 15,000xg to remove remaining cells and platelets. 5−20 μl plasma in 100 μl lysate buffer was subjected to ELISA to quantify AS-ODN level as described in Figure 2. Data (mean ± SEM) are collated from 6 independent experiments.

Establishment of an ultrasensitive NCHL-ELISA for quantitation of KSR1 AS-ODN levels. (A) Schematic principle of the ELISA. Template was added to plasma or tumor lysate for hybridization followed by binding to a streptavidin-coated plate. Subsequently, signal probe with a digoxigenin group was ligated to KSR1 AS-ODN and detected by anti-digoxigenin antibody conjugated to alkaline phosphatase. Note that the template sequences complementary to KSR1 AS-ODN and signal probe are in the same color as their cognate binding partners, respectively. (B) Representative standard curve. A serial dilution of KSR1 AS-ODN from 0.05−20 nM was used as standard. To construct the standard curve, 100 μl template probe solution was added to different concentrations of standard at 37°C for 1h in a polypropylene 96-well plate. After hybridization, 150 μl of solution was transferred to a NeutrAvidin coated 96-well plate and incubated at 37°C for 30 min to allow binding of biotin to streptavidin-coated wells. After washing, ligation was performed by adding 150 μl ligation probe solution followed by 2 h incubation at room temperature. The plate was then washed with washing buffer and then deionized water. Subsequently, 150 μl of the 1:2000 dilution anti-digoxigenin-AP was added. After 30 min, AttoPhos was added for 30 min, and fluorescence intensity was determined using a Cytofluor microtiter plate reader (excitation 450/50 and emission 580/50). Each calibration point was run in duplicate (open and filled circles). Sample fluorescence arbitrary units were quantified against the standard curve, and R² calculated in R 2.5.0 (http://cran.r-project.org/).

Correlation of KSR1 expression in PANC-1 xenografts with tumor AS-ODN levels. Tumor-bearing mice were treated with 10−75 mg/kg/d KSR1 AS-ODN or PBS via subcutaneous Alzet osmotic minipump. After 5 days of treatment, plasma and tumors were co-harvested, and KSR1 AS-ODN levels in both were quantified using our ultrasensitive ELISA. KSR1 expression in tumor tissue was measured by western blot as described in Figure 1. Pearson and Spearman correlation coefficients and corresponding p values were computed to illustrate strength of association between plasma or tumor drug levels and tumor KSR1 protein expression. (A) KSR1 downregulation in tumor tissues correlates highly with KSR1 AS-ODN drug uptake in tumor tissues. (B) KSR1 AS-ODN plasma levels do not correlate with PANC-1 tumor xenograft drug levels.