Tissue homogenizer

All experiments involving animals were conducted in compliance with, and with the approval of, the Animal Care Committee of the University of British Columbia (A18-0077). Male NOD/SCID mice at 6 to 8 weeks of age were subcutaneously injected with LNCaP cells (1 × 10⁷ cells/site) using Matrigel (Becton Dickinson). Mice were castrated once tumors reached ~100 mm³ and randomized into treatment groups to receive ralaniten (233 mg/kg), EPI-7170 (56.6 mg/kg) or vehicle control (5% DMSO/1% CMC/0.1% Tween 80) once daily by oral gavage. Treatment began one week following castration. Mouse body weight and tumor volume (defined as volume = length × width × height × 0.5236) were regularly recorded, and tumors were excised 24 h after the last treatment. To analyze tumor gene expression, tumors were flash frozen, and ~100 mg was added to 1 mL TRIzol reagent (Invitrogen) and homogenized using a tissue homogenizer (MP Biomedicals). RNA was extracted and reverse-transcribed as detailed below.

DNA was extracted from frozen necropsy tissues using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA), and eluted in 200μl AE buffer. All samples were adjusted to 20ng/μl using 1X TE buffer and FIV-C provirus was quantitated by qPCR using previously described reaction components, cycling parameters, and FIV-C primers and probes [52 (link), 53 (link)]. Proviral copy number within these tissues was quantitated using a standard curve with 1:10 serial dilutions of FIV-C gag plasmid into 1X TE buffer, ranging from 10⁵ copies to 10¹ copies per reaction. Resulting proviral copy numbers were normalized to copies per 10⁶ cells based on the total amount of DNA present in the reaction (100ng) as previously described [49 (link)].
RNA was extracted from frozen necropsy tissues using an RNeasy Mini Kit (Qiagen, Valencia, CA) and tissue homogenizer (MP Biomedicals, Solon, OH). Superscript II (Invitrogen), random primers (Invitrogen), and RNase Out (Invitrogen) were used to synthesize cDNA by reverse transcription. Real-time qPCR quantification of viral RNA was then performed on an iQ5 thermocycler (Bio-Rad, Hercules, CA) using previously described reaction components, cycling parameters, and FIV-C primers and probes [52 (link), 53 (link)]. Viral RNA was normalized to GAPDH expression as previously described [46 (link)] using the ΔΔCT method [59 (link)].

Half of the full thickness wound was mechanically disrupted with a tissue homogenizer in the presence of protease inhibitors, as previously described^{19 (link)}. Briefly, a 12 mm biopsy punch of a wound was cut in half with a razor blade. Half of the wound was frozen, and then wounds from the study were thawed, and mechanically ground in a tissue homogenizer (MP Biomedicals, Santa Ana, California) utilizing lysing matrix D (MP Biomedicals) in the presence of an EDTA-free protease inhibitor cocktail (Thermo). The amount of VEGF, PDGF-BB, and HB-EGF (WT and -PLGF-2_123–144 variants) present in the tissue lysate were assessed by ELISA (DuoSet, R&D Systems), per the manufacturer’s instructions.