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Procedure

Animal study was carried out in compliance with the ARRIVE guidelines as follows. Animal studies were conducted following approved protocol (2018-0138) established by Yonsei University Health System’s Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. After receiving the approval, eighty 5-week-old male BALB/c athymic nude mice, weighing around 20 g, were obtained from OrientBio (Seongnam, Korea). All mice were housed for 1 week for a period of adjustment, and free feeding to food (PicoLab Rodent diet 20 5053) and purified water by reverse osmosis in individually ventilated cages were provided at a temperature of 21 ± 0.2 °C, humidity of 50 ± 10%, and a 12/12-h light/dark cycle. The maximum caging density was five mice and aspen chip after autoclave was used as bedding. To generate a xenograft tumor model, each of 1 × $107$ A549 or DU145 cancer cells were injected with Matrigel into the subcutaneous abdominal region of mice. When the tumors reached an average size of 100–120 mm3, 1 × 109 plaque forming unit (PFU) of each adenoviruses diluted in 50 μl PBS or PBS alone was intratumorally injected into the nude mice. Each mice group with similar average and standard deviation values of tumor size was separated (n = 7 per group). The adenoviruses injected were Ad-3484-NC, Ad-3484-shDaxx, Ad-3484-pTP, Ad-3484-pTP-shDaxx. Intratumoral injection was repeated every other day for a total of three injections. Five mice per group were used for the tumor size measurement, and two mice per group were used for the immunohistochemistry. Regression of tumor growth was assessed by measuring the length (L) and width (W) of tumors. Tumor volume was calculated using the following formula: Volume (mm3) = 0.52 × L × W2.

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