Xenograft Model for Doxorubicin Response

All animal experiments were approved by the Animal Ethics Committee of Bilkent University. The studies were carried out in compliance with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations. 6–8-week-old female athymic nu/nu mice were subcutaneously injected with 2 × 10⁶ 231.Luc2.GFP cells prepared in 100 μL PBS:Matrigel (1:1) solution into the left and right mammary fat pads (MFPs). After tumors became palpable, their growth was measured regularly twice a week using a caliper, and tumor volume was calculated as (length × width²)/2. Development of doxorubicin-sensitive/resistance xenografts has previously been described [15 (link)]. To test the effects of miR-326 overexpression or shITGA5 on doxorubicin response, mice with palpable tumors were treated with doxorubicin (2.5 mg/kg) after induction of miR-326 or shITGA5 expressions with doxycycline (100 μg/mL). To perform the tumor bioluminescence imaging, mice were anesthetized with isoflurane, 150 mg/kg D-luciferin (Sigma Aldrich, St Louis, MO, USA) was injected into intraperitoneal (i.p.) cavity, and the images were acquired using Lumina III In vivo Imaging System (Perkin Elmer, Waltham, MA, USA). All mice were sacrificed when the tumors of control group reached 1500 mm³, and the tumors were collected and weighed.