Am7021

Mice were euthanized by CO₂ inhalation followed by decapitation. The gut was flushed of fecal contents, bisected along the longitudinal axis, and divided into samples for immunohistochemistry, biochemistry, and molecular biology. For immunohistochemistry, gut was pinned flat in Sylgard-coated Petri dishes with the lumen facing up, and tissue was fixed for 2–18 h in 4% formaldehyde/sucrose and then washed 3 times in PBS. For gut whole mounts, intestinal segments were dissected and trimmed into 1.5-cm cylinders and then fixed and processed using a modification of the method of Li et al. (81 (link)). For longitudinal muscle of the myenteric plexus, the villi and fatty gut tissues were gently scraped away under a dissecting microscope as described previously (40 (link)). Tissue for protein extraction was rapidly frozen on dry ice and then stored at −80 °C until use. Tissues for RNA extraction were preserved in RNALater solution (AM7021, Ambion, Thermo Fisher) as per the manufacturer's instructions. Before RNA extraction, tissues were frozen on liquid nitrogen and then crushed, with miRNA extraction performed immediately as described below. To measure gut length in 15-month-old littermates (n = 6), whole gut was cut from the base of the stomach to the anus. The entire gut was then carefully extended along a ruler, and the length in cm was documented.

Female NOD/SCID/γ mice were used for sunitinib resistance modelling. Animal procedures were done according to the institutional animal care guidelines. 5-week-old mice were subcutaneously injected with 786-O human RCC cell line (ATCC CRL-1932), 1.5 × 10⁶ cells in both flanks. Tumor size was measured twice per week using calipers, and tumor volume was calculated as width² × lenght × 0.5. After tumor formation (∼1 week), animals received oral gavage of sunitinib (purchased from Selleckchem, USA) as a citrate-buffered (pH 3.5) solution daily (7 days a week) at the dosage of 40 mg/kg or vehicle. Treatment response was assessed accourding to the RECIST criteria. Mice were sacrificed at the following timepoints: vehicle control (N = 4), sunitinib-sensitive (3 weeks of treatment, with response and before signs of resistance) (N = 5), and the resistant group (10 weeks of treatment, with resistance after initial resposne) (N = 7). Upon autopsy, xenograft tumors were stored in RNA later solution (Thermofisher Scientific, AM7021) until further use. Kidneys, livers and lungs were also collected for formalin fixation and immunhistochemical analysis.

At day 21 of age, spleens from sacrificed birds were collected and placed in tubes containing an RNA stabilization solution (AM7021, ThermoFisher Scientific) before being frozen. Three spleen samples for every six treatments (control, BACI, CP1, CP2, COH150, and COH300) were defrosted at room temperature for RNA extraction. Total RNA was prepared using the RNeasy® Mini Kit (Cat. No./ID: 74104 Qiagen) according to the manufacturer's instructions. Briefly, 10–15 mg of spleen samples were cut with sterile forceps and surgical blades and transferred into 600 μl RLT buffer with 1% beta-mercaptoethanol (Fisher Scientific). Cells were homogenized using a Pro 200 homogenizer (Pro Scientific). The homogenates were centrifuged for 3 min at 13,000 rpm, and the supernatants were transferred to microcentrifuge tubes followed by the addition of 70% ethanol. RNA was eluted using RNeasy Mini column (Cat. No./ID: 74104 Qiagen). The RNA quality was checked on an agarose gel, and the quantity and purity were measured with a Nanodrop spectrophotometer (260 and 260/280 nm respectively). The absence of genomic contamination was confirmed by running RNA samples with the GAPDH housekeeping gene using real-time PCR.