Five-week-old male BALB/c nude mice (nu/nu) were maintained in a pathogen-free environment and allowed free access to water and food. These mice were randomly assigned to 4 groups (5 mice each group) and received subcutaneous inoculation of RBM5-AS1-depleted or control DAOY cells (2 × 106 cells/mouse) with or without radiation exposure. For radiation treatment, mice were given a single dose of 10 Gy on day 3 after cell injection. Tumor volume was calculated weekly for 4 weeks. After the last measurement, mice were sacrificed and tumors were removed. Tumor sections were deparaffinized with xylene and rehydrated by alcohol gradient. Endogenous peroxidase activity was blocked using a 0.3% hydrogen peroxide solution. The sections were subjected to immunostaining with anti-Ki-67 antibody (ab15580, Abcam; 1:100 dilution). For apoptosis analysis, TUNEL staining was performed on tumor sections with the TUNEL Staining kit (Beyotime, Haimen, China). The percentage of Ki-67- and TUNEL-positive cells was calculated in 5 random microscopic fields. All studies involving animals were approved by the Animal Care and Use Committee of Shanghai Jiaotong University School of Medicine (Shanghai, China; approval number: 2018-0269).

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