Pa5 106697

Ten paired normal and tumor tissues were collected from breast and lung adenocarcinoma patients who underwent surgical excision at either Southwest Hospital or Xinqiao Hospital of the Army Medical University, China (Supplementary Table S1). The permission for using human tissue samples in this research has been approved by the Ethics Committees of Southwest Hospital and Xinqiao Hospital, respectively. Tissue samples were processed and analyzed according to the methods described in our previous publications 17 (link), 18 (link), ensuring consistency and comparability experimental procedures. The primary antibody used for detecting NUSAP1 was purchased from Invitrogen (PA5-106697, Invitrogen, CA, USA).

All mice experiments were conducted in accordance with the protocols approved by the Ethical Committee for Animal Experimentation of the Army Medical University. SPF grade female nude mice (5 weeks old with a mean body weight of 20 g) were purchased from Vital River Laboratory Animal Technology (Beijing, China). To evaluate the effect of NUSAP1 on the cell proliferation ability in vivo, 1 × 10⁷ control or NUSAP1-knockdown A549 and MCF-7 cells were subcutaneously inoculated in the underarm region of the right foreleg of each mouse (5 mice for per group). The tumor diameter and weight of tumor-bearing mice were measured at 3, 6, 9, 12, 16, 20, and 22 days respectively after inoculated. Tumor volume was calculated using the formula: volume = (long diameter) × (short diameter)²/2. After 20 days, the mice were euthanized, and the tumors were isolated, weighted, photographed. The tumors were then lysed and total protein was extracted for western blotting analysis to assess the levels of NUSAP1 and KI67 proteins in each tumor. Antibodies used for western blotting was rabbit anti-NUSAP1 (PA5-106697, Invitrogen) at a 1:1000 dilution, rabbit anti-KI67 (MA5-14520, Invitrogen) at a 1:1000 dilution, and mouse anti-β-Actin (3700S, CST, MA, USA) at a 1:20000 dilution.