Matrigel basement membrane matrix

Female nude mice (5–6 weeks old) were purchased from Vital River Laboratories (VRL, Beijing, China) and fed a standard animal diet and water. The animal studies were approved by the University Institutional Animal Care and Use Committee. MDA-MB-231 cells were suspended in a 1:1 ratio in DMEM medium with a Matrigel basement membrane matrix (Sigma, E1270). Cells (4 × 10⁷) were inoculated in the right legs of mice. After tumor inoculation, the mice were randomly divided into four treatment groups (16 mice per group; six mice were used for body weight and tumor volume measurement, the others for survival analysis). The mice were treated with either vehicle, CQ (40 mg/kg) or IH (20 mg/kg), or a combination of CQ/IH by intraperitoneal injection once every 2 days. The body weight and tumor volume (mm³) were measured. The mice were euthanized 30 days after medication, the tumors were excised and were either formalin-fixed or flash-frozen at − 20 °C. H&E, TUNEL, and immunohistochemical analyses were performed as previously described [22 (link)].

5 × 10⁴ HUVECs were plated and co‐cultured with the same amount of transfected ovarian cancer cells (SKOV3 and CAOV3) on the plate that was precoated with Matrigel Basement Membrane Matrix (Sigma‐Aldrich). After 6 h's incubation, cells were fixed with 4% PFA for 10–15 min at room temperature followed by PBS wash. The tube formation ability of HUVECs was measured with a light microscopy.

Nude mice (5 weeks old) were purchased from Vital River Laboratories (VRL, Beijing, China) and fed a standard animal diet and water. Animal studies were approved by the University Institutional Animal Care and Use Committee. The lower back of each mouse was subcutaneously inoculated with 2 × 10⁶ U937 cells in serum-free RPMI-1640 medium with a Matrigel basement membrane matrix (Sigma, E1270). The mice were randomized into three groups (n = 12 for each group). Five days after tumor inoculation, the mice were treated with MC-3129 (10 mg/kg, 50 mg/kg intraperitoneally for 30 days, respectively) or an equal volume of vehicle. The tumor volumes and body weights were monitored every 5 days after treatment. The tumor volumes were determined by measuring tumor length (l) and width (w), and subsequently, the tumor volume was calculated by using V = lw²/2. The mice were killed after 30 days of exposure, and tumor tissues from the representative mice were fixed in paraformaldehyde, embedded in paraffin, sectioned and processed for hematoxylin and eosin (H&E) staining. The TUNEL assay was performed according to the manufacturer’s instructions by using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) to detect apoptosis in the tumor tissues. Immunohistochemistry was performed as previously described^{34 (link)}.

Female nude mice (5–6 weeks old) were purchased from Vital River Laboratories (VRL, Beijing, China) and fed a standard animal diet and water. The animal studies were approved by the University Institutional Animal Care and Use Committee. MDA-MB-231 cells were suspended in a 1:1 ratio in DMEM medium with a Matrigel basement membrane matrix (Sigma, E1270). Cells (4 × 10⁷) were inoculated in the right legs of mice. After tumor inoculation, the mice were randomly assigned into 3 treatment groups (16 mice per group, 6 mice were used for body weight and tumor volume measurement, the others were used for survival analysis). The mice were treated with Arnidiol (40 mg/kg, 80 mg/kg) or an equal volume of vehicle by intraperitoneal injection. The body weight and tumor diameters were measured every 5 days. The mice were euthanized 30 d after medication. The tumors were excised and were either formalin-fixed or flash-frozen at − 20 °C. H&E, TUNEL, and immunohistochemical analyses were performed as previously described [30 (link)].