Xenograft Models

HCCLM3 and SMMC-7721 xenograft cell models were established in athymic nude mice aged 4–6 weeks, which were obtained from the Animal Experimental Center of Chongqing Medical University (Chongqing, China). Experiments were conducted in accordance with the guidelines on animal care and use established by the Chongqing Medical University Experimental Animal Management Committee. The animal study protocols were approved by the ethics committee of Chongqing Medical University.

A total of 5 × 10⁶ cells were implanted into the left armpit. When tumors grew to 150–200 mm³ (5–6 weeks), the mice were randomly assigned to experimental groups (n = 5 per group). G1, G15, AG, and U0126 were dissolved and diluted in absolute ethanol. The compounds (10 µl) were added to an aqueous vehicle (90 µl, 0.9% NaCl with 0.1% albumin and 0.1% Tween-20). Ethanol (10 µl) was added to the aqueous vehicle (90 µl) and used as the control group. Mice were subcutaneously injected daily with 0.1 ml of G1 (1 μM) alone, or G1 (1 μM) in combination with the GPER-specific antagonist G15 (1 μM), AG (10 μM), or U0126 (10 μM). Body weights were monitored daily. At the end of the 56 day experiment, tumors were removed and measured using a Vernier calliper, and tumor volumes were calculated using the following equation: Tumor voulume = 1/2 × length × (width)2. The expression of specific proteins was analysed by immunoblotting to determine the effects of treatment.