Ivis lumina optical imaging system

After the final RTKI treatments, fluorocoxib A was administered (1 mg/kg, s.c.) to all mice. Four hours after fluorocoxib A administration, the mice were sacrificed, tumor and other organs were dissected, and imaged ex vivo using the Xenogen IVIS Lumina optical imaging system with DsRed filters with excitation 500 to 550 nm, emission 575 to 650 nm, and bac kground 460 to 490 nm (Perkin Elmer, Waltham, MA, USA). The obtained total flux (p/s) and average radiant efficiency ([p/s/cm^²/sr] / [μW/cm^²]) of labeled regions of interest (ROI) of dissected tumor and other organs/tissues (heart, lung, kidney, liver, blood, spleen, pancreas, small intestine, muscle, and fat) were evaluated. Tumor-to-noise ratio (TNR) was calculated using the following equation: TNR = (tumor radiant efficiency values)/(blood radiant efficiency values). The TNR for each treatment groups (AB1010 and imatinib) were normalized to Group 1 (control). Each dissected tumor was divided into pieces; one piece of tumor was fixed in a 10% neutral buffered-formalin for histology and immunohistochemistry (IHC) analysis. Another piece of tumor was kept in RNAlater solution and stored at −80° C freezer until western blotting (WB) analyses were performed.

Cancer cells were collected and lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentration was measured by BCA assay. Samples in SDS sample buffer were heat-denatured at 95 °C for 5 min. The proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with primary antibodies including anti-MCM2 (sc-373702, 1:1000 dilution), anti-MCM3 (sc-390480, 1:1000 dilution), anti-MCM4 (sc-28317, 1:1000 dilution), anti-MCM6 (sc-393618, 1:1000 dilution), anti-AR (sc-7305, 1:1000 dilution), anti-SYP (sc-17750, 1:2000 dilution), anti-CD56 (sc-7326, 1:500 dilution), anti-cleaved PARP1 (sc-56196, 1:1000 dilution), anti-β-Actin (sc-47778, 1:2000 dilution), and anti-GAPDH (sc-47724, 1:2000 dilution) purchased from Santa Cruz Biotechnology. Secondary HRP conjugated antibody (PI31432, 1:6000 dilution) was purchased from Fisher Scientific. Western blot development and detection was performed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) and IVIS Lumina optical imaging system (PerkinElmer). All the raw images of Western blot were summarized in Supplementary Fig. 4.