Female balb c nude mice

Female BALB/c nude mice were obtained from Oriental Yeast Corporation (Tokyo, JAPAN). Five-week-old mice were used for ALKBH4 shRNA stable cell-xenograft experiments. Animals were kept under 12 h light–dark cycles at 22–24 °C. A549 cells, which had been stably transfected with ALKBH4 shRNA (A549-shALKBH4) and control shRNA (A549-shControl), were both adjusted to a concentration of 0.6 × 10⁷ cells in 100 μL of serum-free DMEM. The cell suspensions, together with 100 μL of Matrigel Matrix High Concentration (Corning, New York, USA) were then injected subcutaneously into the right flanks of BALB/c nude mice (A549-shALKBH4, n = 8; A549-shControl, n = 8). One xenografted mouse, which was inoculated with A549-shALKBH4, died during the experiment. The tumour volume was calculated as follows: (tumour length × tumour width²)/2. All procedures were performed under a protocol approved by the Animal Experimentation Committee at Osaka University. All methods were carried out in accordance with relevant ARRIVE guidelines and regulations. We confirmed that all methods were carried out in accordance with relevant guidelines and regulations. Developed tumours were resected 52 days after xenografts.