Trypsin edta

Licochalcone A (LicA; BP0855) was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). LicA was dissolved the DMSO solution and stock concentration was 100 mM. MTT, dimethyl sulfoxide (DMSO), 4,6-diamidino-2-phenylindole (DAPI) and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). PF-753228 (FAK inhibitor) and PP2 (Src inhibitor) were purchased from BioVision Inc. (Milpitas, CA, USA). Penicillin/Streptomycin (P/S) reagent, fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM)/F12 (DMEM/F12) Minimum Essential Media (MEM), RPMI1640 and 0.25% Trypsin-EDTA were purchased from Cytiva Life Sciences (Marlborough, MA, USA). Immobilon-E polyvinylidene difluoride (PVDF) membrane was obtained from Merck (Darmstadt, Germany). Lipofectamine 3000 Transfection Reagent, RNAiMAX Transfection Reagent and Acridine Orange (AO) dye were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Polymerase chain reaction (PCR) primers were obtained from Mission Biotech (Taipei, Taiwan). Anti-LC3 antibody was purchased from Novus Biologicals, Inc. (Littleton, CO, USA). Anti-Sp1 and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-phosphoryl-FAK (p-FAK), total-FAK (t-FAK), phosphoryl-Src (p-Src) and total-Src (t-Src) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

The 3T3-L1 preadipocytes were obtained from the American Type Culture Collection (ATCC, MD, USA), and Dulbecco’s modified Eagle medium (DMEM) containing 10% bovine calf serum (HyClone Laboratories Inc., Logan, UT, USA) and 1% antibiotics was used as the culture medium. The cells were cultured in an incubator under the conditions of 5% CO₂ at 37 °C. After the cells had been suspended using Trypsin-EDTA (HyClone Laboratories Inc.) every 48 h, the cells were seeded at 1 × 10⁶ cells/mL and subcultured.

HCT116 cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were grown in RPMI-1640 medium (Hyclone Laboratories Inc.)containing 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C in a 5% CO₂ incubator. The cells were sub-cultured by detachment with Trypsin-EDTA (Hyclone Laboratories Inc.)and re-seeded at 1 × 10⁶ cells/mL per 100 mm plate every 48 h.

Curcumin, TPGS, d-mannitol (98%), and paclitaxel were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Hank’s balanced salt solution were also purchased from Sigma-Aldrich. Acetone was obtained from Merck KGaA (Darmstadt, Germany). Acetonitrile, methanol, and pure water were obtained from Fisher Scientific Korea, Ltd. (Seoul, Korea) and were of high-performance liquid chromatography (HPLC) grade. All other chemicals used were of analytical grade.
Caco-2 cells were purchased from American Type Culture Collection (Rockville, MD, USA). LLC-PK1-P-gp cells were purchased from Corning (Corning, NY, USA) and MDA-MB-231 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Fetal bovine serum and Dulbecco’s Modified Eagle’s medium (DMEM), Medium 199, penicillin–streptomycin, and Trypsin–EDTA were purchased from Hyclone Laboratories (Logan, UT, USA).

Treated cells were detached from 6-well plates by Trypsin-EDTA (HyClone Laboratories) and subsequently stained with PI using a BD Cycletest^TM Plus DNA kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. The percentage of cells exhibiting low PI intensity (Sub-G1 phase) was analyzed using an Accuri^TM C6 Plus flow cytometer (BD Biosciences). 10,000 cells were recorded in each experiment.

Methoxypoly (ethylene glycol)-amine (mPEG-NH₂) with a molecular weight of 2000 g/mol was purchased from JenKem Technology (Plano, TX, USA). N-hydroxy succinimide (NHS), 4-dimethylaminopyridine (DMAP), pyrene, mercaptopropionic acid, ethylenediamine, dicyclohexylcarbodiimide (DCC), sodium dodecyl sulfate (SDS) and piperidine were acquired from Sigma-Aldrich Corp. (St. Louis, MO, USA). D-α-tocopherol, N,N-bis[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine (Fmoc-Lys(Fmoc)-OH) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were obtained from TCI (Portland, OR, USA). Doxorubicin hydrochloride (DOX∙HCl) was procured from Lancrix Chemicals (Shanghai, China). All solvents used in this experiment were reagent grade. MCF-7 and HEK-293 cells were bought from the Korean Cell Line Bank (Seoul, Korea). RPMI-1640, fetal bovine serum (FBS) and trypsin-EDTA (0.25%) were purchased from HyClone Laboratories (Logan, UT, USA). The cell viability assay (CCK-8) kit was acquired from Donginbiotech Co. (Seoul, Korea).

Pectinase, cellulase, β-galactosidase, dextranase, β-mannase, and standards for maltose (purity ≥98%), maltotriose (purity ≥98%), malttetraose (purity ≥97%), and maltpentaose (purity ≥7%) were products of Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). D-glucose standard (purity ≥98%) was obtained from Sichuan Weikeqi Biotechnology Co., Ltd. (Chengdu, China). Moreover, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) was purchased from Meryer (Shanghai, China) Biochemical Technology Co., Ltd. (Shanghai, China). Aβ_25–35 and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Gibco (Burlington, ON, Canada), Annexin V-APC/PI and Hoechst33342/PI dual staining kits were from Jiangsu KeyGEN BioTECH Co., Ltd. (Nanjing, China), Dulbecco’s modified Eagles medium (DMEM) cell culture medium was purchased from Procell Life Science &Technology Co., Ltd. (Wuhan, China). Trypsin-EDTA from Hyclone Laboratories (Logan, UT, USA). Detection kits for ROS, SOD, LDH and MDA were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRIzol reagent and the RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher (Waltham, MA, USA).