Dimethyl sulfoxide (dmso)

A total of 20 male BALB/c nude mice with body weights of 10–14 g, were purchased from Hunan SJA Laboratory Animal Limited Company and maintained in a specific pathogen-free environment. Animal experiments were approved by the Guangdong Medical University Institutional Animal Care and Use Committee. After 2 weeks, mice were injected with DLD-1 cells (1 × 10⁷ cell in a volume of 0.2 mL) into the subcutaneous tissue of the right auxiliary region. The volume of the tumour was measured every other day by caliper and calculated by the following formula: (length × width × width)/2. When tumours reached a volume of 62.5 mm³, mice were divided randomly into the following four groups and given different drugs by intraperitoneal injection for a total of 13 days: control, DMSO (1% DMSO, every two days), deoxyshikonin (20 mg/kg in 1% DMSO, every two days), irinotecan (66.7 mg/kg, every four days, Qilu Pharmaceutical Co. Ltd, Hainan, China). On day 13, all mice were sacrificed. The tumours were segregated, measured and stored in liquid nitrogen for later use.

PB was purchased from Merck KgaA (Sigma-Aldrich). Fetal bovine serum (FBS) and 5-fluorouracil were purchased from Gemini Co. and Xudong Haipu pharmaceutical Co., respectively. Cisplatin was bought from Qilu pharmaceutical Co. DMEM, DMSO, CCK-8 kit, Giemsa, Matrigel, Hoechst 33342, phosphate buffer saline (PBS), bovine serum albumin (BSA), RIPA buffer, protease inhibitor cocktail, BCA protein assay kit, and PVDF were all purchased from Solarbio Science and Technology Co., Ltd (Beijing, China). EDU kits were purchased from Guangzhou RiboBio Co., Ltd. Cell cycle kits were bought from Lianke Biological Technology Co. AnnexinV-FITC/propidium iodide apoptosis detection kits were purchased from Beibo Co. Primary antibodies against Bcl-2, Bcl-xL, Bax, Bad, PI3K, AKT, phosphorylated (p)-AKT, mTOR, p-mTOR, p70S6K, and p-p70S6K were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against Ki67 and β-actin was purchased from Proteintech (Wuhan, China). Rabbit and mouse secondary antibodies (horseradish peroxidase-conjugated antibodies) were purchased from Kangwei Co.