Establishment of tumor xenografts in mice

Six-week-old male SCID (severe combined immune deficiency) mice (nu/nu) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China) and maintained under specific pathogen-free conditions at Nanjing University. AGS cells were infected with a control lentivirus or a miR-93 overexpression lentivirus, or transfected with a PDCD4 overexpression plasmid, or co-transfected with a miR-93 overexpression lentivirus and a PDCD4 overexpression plasmid. After infection and transfection, AGS cells were injected subcutaneously into xenograft mice (2 × 10⁶ cells per mouse, 5 mice per group). Mice were sacrificed after 60 days. The tumor xenografts were removed, and the weight of the tumors was measured. Parts of the tissues were used for protein and total RNA extraction, and the remains were fixed in 4% paraformaldehyde for 24 h and then processed for Hematoxylin and eosin (H&E) staining or immunohistochemical staining for PDCD4 and Ki-67.