Thiazolyl blue tetrazolium bromide mtt powder

SH003 was prepared as described in our previous study [26 (link)]. In brief, SH003 was provided from Hanpoong Pharm and Foods Company (Jeonju, Republic of Korea). Am, Ag, and TK were mixed at 1 : 1 : 1 ratio (w/w) and then extracted with 30% ethanol at 100°C for 3 h. Dried extracts were dissolved in 30% ethanol and stored at −80°C until use. DTX (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO and stored at −20°C.
Thiazolyl blue tetrazolium bromide (MTT) powder was purchased from Sigma-Aldrich (St. Louis, MO, USA). FITC-conjugated Annexin V apoptosis Detection Kit and 7-aminoactinomycin D (7-AAD) were purchased from BD Pharmingen™ (BD Biosciences, San Jose, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Anticleaved caspase-3, -PARP, -GAPDH, -p-EGFR (Tyr 1068), -p-EGFR (Tyr 1173), -EGFR, -p-AKT (Ser 473), -AKT, -p-C-Raf (Ser 338), -p-STAT3, and -STAT3 antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-p-ERK and ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Ki67 and CD31 antibodies were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase- (HRP-) conjugated secondary antibodies for mouse and rabbit were purchased from SeraCare Life Sciences (Milford, MA, USA).

The cryptolepine used in this study was donated by Professor Kwesi Mensah Boadu of the Faculty of Pharmacy and Pharmaceutical Sciences at the Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. The cryptolepine was prepared to a stock concentration of 861 μM in phosphate-buffered saline, filter sterilized, aliquoted into Eppendorf tubes, wrapped with aluminium foil and stored at − 20 °C until used. Recombinant human IL-6 protein (cat # ab9627), niclosamide (cat # ab120868), STAT3 (Tyr705) In-Cell ELISA Kit (cat # ab126427) and IL-23 Human ELISA Kit (cat # ab64708) were purchased from Abcam (Cambridge, USA). Cignal Finder Reporter Array Plate (cat # CCA-901 L) and Attractene Transfection Reagent (cat # 301005) were purchased from Qiagen (Germantown, USA). Dual Luciferase Reporter Assay System (cat# E1960) was purchased from Promega (Madison, USA) and Thiazolyl blue tetrazolium bromide (MTT) powder (cat # M5655-1G) was purchased from Sigma Aldrich (St. Louis, USA). All reagents and chemicals were prepared according to the manufacturers' instructions.

First, 0.05% Trypsin-EDTA (1X) (Gibco, Sigma-Aldrich Company Ltd., Poole, UK), phosphate-buffered saline (PBS) solution tablets (prepared by dissolving one tablet in 200 mL of deionised water), Dulbecco’s Modified Eagle Medium (Gibco DMEM 1X, Sigma-Aldrich Company Ltd., Poole, UK), containing 4.5 g/L of glucose and 0.11 g/L of sodium pyruvate stored at 4 °C, antibiotics prepared with 100 µg/mL of streptomycin sulphate, 0.25 µg/mL of amphotericin B, and 100 U/mL penicillin G sodium, Gibco foetal bovine serum (FBS), thiazolyl blue tetrazolium bromide (MTT) powder, DMSO, menadione, DPPH (1,1-diphenyl-2-picrylhydrazyl), methanol, fluorescein diacetate (FD), propidium iodide (PI), and ascorbic acid were all purchased from Sigma-Aldrich (Poole, UK). L-glutamine, Gibco trypan blue stain (0.4%), 96-well microplates, 24-well microplates, and fluorescence-activated cell sorting (FACS) tubes were purchased from Sarstedt (Sarstedt Ltd., Nümbrecht, Germany). A ROS-Glo™ H2O2 Assay Kit was purchased from Promega (Promega UK Ltd., Southampton, UK). The antioxidant scavenging activity was measured by the use of a microtiter plate reader (Infinite 200 Pro, Tecan Trading, Männedorf, Switzerland). All other materials were obtained from Thermo Fisher Scientific (Loughborough, UK) and used as received.