Western blot analysis, qRTPCR, and immunohistochemistry

Western blot analysis was conducted as previously described³ with NuPAGE 4 to 12% bis-tris gel and visualized with ECL Prime Western Blotting Detection Reagent (Amersham) with CytoSMART Live Imaging System (Lonza). For all lysis buffers, fresh protease inhibitor (Roche) was added immediately. All antibodies were purchased from Cell Signaling except anti-DR5 antibody (Abcam ab1675).

RNA was isolated using RNeasy kit (Qiagen) or Quick-RNA Miniprep kit (Zymo Research) according to manufacturers’ instructions. RNA was quantitated using a Nanodrop spectrophotometer. cDNA was synthesized using a SuperScript II RT kit while real-time PCR was performed using a Quantitect SYBR Green PCR mix. Relative amounts of target mRNA were quantitated using the 2^ΔΔCt method using GAPDH as internal control. At least three technical replicates per biological replicate were analyzed.

After tissue fixation, the tumor samples were embedded in paraffin and 8 μm sections were cut and mounted on slides. The sections were then processed and analyzed using immunohistochemistry with TRAIL, Ki67, and caspase-3 antibodies similar to the method described previously.³