For the xenograft mouse model, 1 x 10⁶ OS 143B cells in 100 uL PBS were subcutaneously injected with 1 x 10⁶ OS cells. For the OS lung metastasis model and in vivo imaging, 1 x 10⁶ 143B cells in 10 uL PBS were injected orthotopically into the medullary cavity of the tibia of mice. For bioluminescent imaging, we used 143B cells stably expressing luciferase, and the luciferase substrate D-Luciferin (Selleck, China) was retro-orbitally injected before imaging on days 7 and 21.

All animal experimentation was approved by the Ethics Committee of The Eighth Affiliated Hospital of Sun Yat-sen University. Each group consisted of five female BALB/c nude 4-week-old mice, and the studies were run twice. 143B cells were treated with ZOL or DMEM containing 10% FBS for 2 days. Two sets of BALB/c nude mice were used in the study (ZOL group and NC group). The ZOL group and NC group were subcutaneously injected with ZOL-treated 143B cells or normal 143B cells, respectively, to form xenograft tumors. After 2 weeks, we subcutaneously injected 100 ul ZOL (100 ug/kg) into the experimental group and 100 ul saline into the NC group twice a week. Then, after 4 weeks, mice from both groups were humanely killed, the tumor diameter was measured, and the tumor tissue was stained with HE.

Male BALB/c mice, aged 4 weeks (14-16 g), were purchased from SPF (Beijing) Biotechnology Co., Ltd. K7M2 osteosarcoma cells were harvested and suspended in PBS at 4 . Mice were anesthetized with isoflurane. The left hindlimbs were shaved and cleaned with 75% ethanol. The knees of the mice were flexed beyond 90 and the cortex of the proximal tibial crest was penetrated using a 25 gauge needle by a rotating action. Once the tibial bone cortex was penetrated, the needles were further inserted 2 mm along the diaphysis, followed by injection of 10 uL of K7M2 osteosarcoma cells (5 x 10⁵ cells) into the tibia.

Thirty BALB/c nude mice (20~22 g, five weeks) were SiPeiFu Biotechnology Co., Ltd. (Beijing, China). Animal experiment was conducted in accordance with China Animal Welfare Legislation and was approved by the Ethics Committee of our hospital. U2OS cells (1 x 10⁶ cells) were subcutaneously injected into mice to establish osteosarcoma mice models. The mice were randomized into Lv-si-NC and Lv-si-FANCD2 groups (n = 6). After modeling, lentivirus-packaged si-FANCD2-1 and si-NC were locally injected into tumor tissues of mice in the Lv-si-FANCD2 and Lv-si-NC groups, respectively. They were euthanized by intraperitoneal injection of sodium pentobarbital (50 mg/kg) after 28 days, the tumor tissues were collected and weight was measured.

A total of 24 BALB/c-nude mice (4-5 weeks old) were purchased and MG63 cells were injected into the right tibial bone marrow cavity of mice in a volume of 1 x 10⁶/100 ul. When the tumor volume was visible, all animals were randomly divided into four groups (n = 6): the control (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline) group, the baicalin (200 mg/kg/day) group, the Fer-1 (0.8 mg/kg/day) group and Fer-1 + baicalin group. The baicalin and Fer-1 were intraperitoneally administered every day for two consecutive weeks and tumor sizes were measured every two days.

The OS model of nude mice was constructed using the CDTX model. After transfection, the h143B cells were prepared into a single-cell suspension and subcutaneously injected into the left proximal tibia of 36 (3 mice per group) 4-weeks-old nude mice (1 x 10⁷ cells per mouse).

The OS model of nude mice was constructed using the CDTX model. After transfection, the h143B cells were prepared into a single-cell suspension and subcutaneously injected into the left proximal tibia of 36 (3 mice per group) 4-weeks-old nude mice (1 x 10⁷ cells per mouse).

A total of 24 BALB/c-nude mice (4-5 weeks old) were purchased and MG63 cells were injected into the right tibial bone marrow cavity of mice in a volume of 1 x 10⁶/100 ul. When the tumor volume was visible, all animals were randomly divided into four groups (n = 6): the control (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline) group, the baicalin (200 mg/kg/day) group, the Fer-1 (0.8 mg/kg/day) group and Fer-1 + baicalin group. The baicalin and Fer-1 were intraperitoneally administered every day for two consecutive weeks and tumor sizes were measured every two days.

The OS model of nude mice was constructed using the CDTX model. After transfection, the h143B cells were prepared into a single-cell suspension and subcutaneously injected into the left proximal tibia of 36 (3 mice per group) 4-weeks-old nude mice (1 x 10⁷ cells per mouse).

The OS model of nude mice was constructed using the CDTX model. After transfection, the h143B cells were prepared into a single-cell suspension and subcutaneously injected into the left proximal tibia of 36 (3 mice per group) 4-weeks-old nude mice (1 x 10⁷ cells per mouse).

NU/NU mice (the Fourth Military Medical University, Shaanxi, China) were injected with 143B cells (100 uL, 5 x 10⁷ cells/mL, i.h.). Seven days after the injection, the mice were divided into 6 different groups (n= 3) and intraperitoneally injected with different drugs twice a week. Then, on day 28, the mice were sacrificed, and the tumours in the different groups were weighed. Body weight and tumour size were measured every 3 days from day 7 to day 28. The tumour tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 um thick sections for haematoxylin-eosin (H&E) and immunofluorescence staining.

NU/NU mice (the Fourth Military Medical University, Shaanxi, China) were injected with 143B cells (100 uL, 5 x 10⁷ cells/mL, i.h.). Seven days after the injection, the mice were divided into 6 different groups (n= 3) and intraperitoneally injected with different drugs twice a week. Then, on day 28, the mice were sacrificed, and the tumours in the different groups were weighed. Body weight and tumour size were measured every 3 days from day 7 to day 28. The tumour tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 um thick sections for haematoxylin-eosin (H&E) and immunofluorescence staining.