Balb c athymic nu nu mice

3- to 5-week old female balb/c athymic (nu/nu) mice were purchased from Charles River Laboratories in Beijing. All mice were housed in a biosafety level 2 lab and maintained in accordance with institutional guidelines at Binzhou Medical University. All animal experiments were performed with the approval of Binzhou Medical University Committee on Animal Care. Mice were subcutaneously injected with 5 × 10⁶ A549 cells and randomly divided into different groups (5 mice per group) when the diameter of established tumors reached approximately 5 mm. Xenografted mice received vehicle (200 μl saline), DDP (1.5 mg/kg), SBE (50 mg/kg), or their combination intraperitoneally three times a week. Body weight and tumor volume was measured every second day. Tumor volume was calculated as 0.5 × a² × b (a and b represent tumors short and long diameter respectively). Mice were euthanized and executed after 5 weeks’ treatment and tumors were measured. Tumor tissues were collected from xenografts at the end of treatment and analyzed by immunohistochemistry staining.

Animal experiments were approved by the Animal Welfare Committee of Capital Medical University (AEEI-2014-101). Balb/c athymic (nu+/nu+) mice, 6 weeks of age, were purchased from Charles River Laboratories (Beijing, China). Tumors were produced by inoculating the mice with HCT-116 or HCT-116^Sphk2 cells (1 × 10⁷ per mouse) subcutaneously into the armpit of a mouse. Three weeks later, the rapidly proliferating HCT-116 or HCT-116^Sphk2 tumor tissue was cut into 1.5 mm thick pieces and inoculated subcutaneously into the armpit of each mouse with a puncture needle. When tumor volumes reached approximately 100 mm³, mice were randomly divided into a vehicle control group (n = 6, 0.2 ml olive oil by gavage) and ATRA treatment groups (n = 6, 20 and 30 mg/kg ATRA in 0.2 ml olive oil). All administrations were performed five times per week for three consecutive weeks. Mice were observed daily for any clinical symptoms. Tumor volume and body weight were measured every 3 days. Volume was calculated using the formula, 1/2 × L × W², where length (L) and width (W) were determined in mm [36 (link)]. Tumor growth was defined as a ratio to the tumor weight of vehicle controls. Specimens of HCT-116 or HCT-116^Sphk2 xenografts were removed for further analysis.

Equal numbers of HepG2 cells stably expressing either control or lincRNA-p21 knockdown, U6 or lincRNA-p21 overexpression vectors (5 × 10⁶) in 100 μl of a 1:1 mixture of culture medium and growth factor–reduced Matrigel were implanted subcutaneously into the forelegs of 4- to 5-week-old male BALB/c athymic nu/nu mice (Vital River). When the tumors reached approximately 7–10 mm in diameter, they were prepared to form a brei and then injected subcutaneously into nude mice. Tumor growth was monitored by tumor weight at the end of study (Four weeks for in vivo tumor growth). The study was approved by the Animal Research Ethics Committee of Second Military Medical University. The methods were carried out in accordance with the approved guidelines.

Four to five weeks of male BALB/c athymic (NU/NU) mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal research procedures were performed according to the protocols of the Animal Care and Use Ethics Committee of Shenzhen University Health Science Center and all animals were treated in strict accordance with protocols approved by the Institutional Animal Use Committee of the Health Science Center, Shenzhen University. The mice were injected with 5 ×10⁶ A549-empty vector, A549- TRPV1-FLAG, A549 and A549-DDP cells. The mice were randomly divided into two groups after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. The xenografted mice were then intraperitoneally administrated with 0.9% NaCl or DDP (3 mg/kg per day) for three times a week. The tumor volume was measured before every injection. Tumor volume was estimated as V = (L × W²)/2 (V, volume; L, length; W, width).

Equal numbers of MGC803 cells stably expressing luciferase and either control or Sirt7 knockdown or Sirt7 plus miR-34a knockdown vectors (5 × 10⁶) in 100 μl of a 1:1 mixture of culture medium and growth factor–reduced Matrigel were implanted subcutaneously into the forelegs of 4- to 5-week-old male BALB/c athymic nu/nu mice (Vital River). When the tumors reached approximately 7 to 10 mm in diameter, they were prepared to form a brei and then injected subcutaneously into nude mice. Tumor growth was monitored using calipers and visualized with a bioluminescence-based IVIS system (Caliper LifeSciences). The study was approved by the Animal Research Ethics Committee of Ningbo University. The methods were carried out in accordance with the approved guidelines.

Four-week-old BALB/c athymic nu/nu mice (Vital River) were injected in the right flank with AD-ARHGDIA-infected and in the left flank with AD-GFP-infected U87 MG cells (1 × 10⁶ cells in 100 μl physiological saline). The tumors were measured weekly or every 5 days thereafter. Tumor volume (V) was estimated using a caliper by measuring the length (L) and width (W), where V = (L × W²)/2. The data were analyzed by a two-tailed Student's t test. Intracranial orthotopic xenografts were established by implanting 5 ×10⁵ Luc-U87 MG stable cells. The plvx-U87 MG and ARHGDIA-U87 MG stable transfectants were constructed by Shanghai Chempartner. Five- to six-week-old BALB/c athymic nu/nu mice were anesthetized, and the implantation of U87 MG cells into the third ventricle was performed stereotactically (2 mm lateral and 0.5 mm anterior to the bregma; depth 1.5 mm from the dura). Tumor size was quantified by bioluminescence imaging.

The protocols of the Institutional Animal Care and Use Committee (IACUC) of the Affiliated Hospital of Qingdao University were used to guide the design of all animal studies, which received approval from the Affiliated Hospital of Qingdao University IACUC committee (AHQU‐MAL20190356). For these experiments, BALB/c athymic nu/nu mice (Vital River, 5 weeks old) received an injection in the left liver lobe of Huh7 cells (5 × 10⁶) stably expressing luciferase that had been transfected with siMock or siHULC constructs. Cells were injected in a 100 μl volume containing a 1:1 mixture of culture medium and growth factor‐reduced Matrigel. Orthotopic HCC xenograft tumor growth was measured every week after injection by injecting anesthetized mice with D‐Luciferin (100 μg/g), with a NightOWL II LB 983 System (Berthold, Bad Wildbad, Germany) then being used for murine imaging. The IndiGO2 software was used for luminescence calculations. At 4 weeks following tumor injection, nude mice were euthanized via carbon dioxide inhalation followed by cervical dislocation and the xenograft tumors were collected for histological and immunohistochemical staining.