Ergosterol peroxide (EP) exerts anti-tumor activity in vivo. Six-week-old female BALB/c athymic nude mice were subcutaneously injected in the flank area with 2 × 106 cells of U266 in 100 μl of matrigel mixed PBS. Five days after inoculation, mice (n = 5/group) were each given every 2-3 day intraperitonial (i.p.) injection of EP at 100 mg/kg in 2% tween-80 or bortezomib at 0.25 mg/kg in PBS. Control mice were administered the solvent vehicle. Tumor volumes were measured every other day with caliper. (A) Body weight was measured every other day after administration of EP or bortezomib. (B) Tumor volume was calculated according to the formula V = 0.25a
b, where a is the smallest superficial diameter and b is the largest superficial diameter. (C) Mice were killed on day 20 after cell inoculation, and tumors were immediately removed, fixed, embedded and sectioned at 4 μm for immunostaining of biomarkers. Representative photomicrographs of apoptosis detected by immunostaining of CD34, phospho-STAT3 and TUNEL at ×400, 200 and 100 of magnification, respectively. The sections were detected as DAB substrate staining (brown) and counterstained with Mayer's hematoxylin solution (right). Graphs show the CD34 index (angiogenesis), TUNEL index (apoptosis) and, pSTAT3 index in tumor sections. **p < 0.01 and ***p < 0.001 compared with vehicle-treated control mice (left).