Tumor xenograft experiments

All experiments involving animals were approved by the Ulsan National Institute of Science and Technology (UNIST, UNISTIACUC-16-27). Cancer cells (7 × 10⁶ 22Rv1) were suspended in 200 μL of PBS and then injected subcutaneously into both flanks of 6-week-old BALB/c nu/nu male mice (Charles River Laboratories). Tumors were allowed to grow to an average volume of approximately 100 mm³. Next, animals were grouped randomly (two tumors/mouse, five mice/group). Subsequently, vehicle (DMSO), DMAG, and gamitrinib were dissolved in 20% Cremophor EL (Sigma) in PBS. DMAG and/or gamitrinib (10 mg/kg) were administered intraperitoneally twice a week. Tumors were measured using calipers, and tumor volumes were calculated using the following formula: V = 1/2 × (width)² × length. At the end of the experiment, animals were euthanized, and organs (brain, heart, kidney, liver, lung, spleen, stomach, and tumor) were collected for histology and western blot analysis. In addition, harvested organ specimens were fixed in 10% formalin and embedded in paraffin for histological analyses. Briefly, Section (5 μm thick) were placed on high-adhesive slides, stained with H&E, and scanned at ×20 magnification using the Dot slide system (Olympus). For western blot analyses, tissue samples were homogenized in RIPA buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40, and 0.25% N-deoxycholate, plus protease inhibitor and phosphatase inhibitor cocktails (Calbiochem).