Insulin

Melanoma stem cells and non-stem cells were previously sorted in our laboratory from cell lines MDA-MB-435 [18 (link)] and A375 [19 (link)], respectively. Aldehyde dehydrogenase 1 (ALDH1), a marker of cancer stem cells, was used to sort melanoma stem cells with fluorescence-activated cell sorting (FACS) using an ALDEFLUOR™ kit (Cyagen Biosciences Inc., USA) [18 (link), 19 (link)]. Briefly, MDA-MB-435 or A375 cells were suspended in ALDEFLUOR assay buffer containing ALDH1 fluorescent substrate BODIPY-aminoacetate (BAAA, 1 μM), followed by incubation for 40 min at 37 °C. As a negative control, an aliquot of cells was treated with 50 mM of ALDH1 inhibitor diethylaminobenzaldehyde (DEAB). The self-renewal capability and tumorigenicity of ALDH1-positive and ALDH1-negative cells were examined using tumorsphere forming assays and in vivo experiments to confirm the ALDH1-positive cells are melanoma stem cells [18 (link), 19 (link)]. Melanoma stem cells and non-stem cells were cultured in DMEM/F-12 medium (Invitrogen, USA) supplemented with 20 ng/mL epidermal growth factor (EGF) (Beyotime Biotechnology, China), 10 ng/mL basic fibroblast growth factor (bFGF) (Beyotime Biotechnology), 5 mg/mL of insulin (Beyotime Biotechnology), and 2% of B-27 (Sigma, USA) at 37 °C in a humidified atmosphere with 5% CO₂.

Breast cancer cell lines MCF7, T47D, HCC1954, MDA-MB-453 and SK-BR-3 were purchased from ATCC and SUM149 was purchased from Asterland. MCF7, T47D, HCC1954 and SK-BR-3 were cultured in RPMI medium 1640 (Gibco, USA) with 10% FBS (Gibco, USA). MDA-MB-453 was cultured in Leibovitz’s L-15 medium (Sigma, USA) supplemented with 10% FBS. SUM149 was cultured in Ham’s F-12 medium (Invitrogen, USA) supplemented with 5% FBS, 5 μg/mL of insulin (Beyotime, China) and 1 ug/mL of hydrocortisone (Sigma, USA). MCF7, T47D, HCC1954, SUM149 and SK-BR-3 cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 and MDA-MB-453 was cultured at 37 °C with 100% humidified atmosphere. The cell lines were profiled routinely by short tandem repeat analysis.

A549 cells were obtained from Stem Cell Bank, Chinese Academy of Sciences. A549 cells were cultured in RPMI-1640 medium (Bio-Channel, China), which was supplemented with 10% fetal bovine serum (FBS, BIOAGRIO SCIENCE, China). HCE-T cells were obtained from Wuhan Punosai Life Technology Co., LTD. HCE-T cells were cultured in the DMEM/F12 medium (Bio-Channel, China), which was supplemented with 10% FBS, 5 μg/mL Insulin (Beyotime, China), and 10 ng/mL human epidermal growth factor (EGF, BIOAGRIO SCIENCE, China). The cell culture was maintained in an incubator (Thermo Scientific, MA, USA) at a constant temperature of 37 ℃ and 5% CO₂. The supplemented RPMI-1640 and DMEM/F12 medium were further cited as complete cell medium (CCM).
A549 and HCE-T cells were passaged when 80–90% confluent. For all the exposure experiments, 50,000 cells/insert were cultured in 12-well culture plate inserts. These inserts have a surface area of 1.12 cm² and are equipped with a 0.4 μm pore polyester (PET) membrane (LABSELECT, China). For each well, 0.5 mL of CCM was added to the upper side and 1.5 mL to the basal side for each well. The cells were incubated for 24 h under immersion conditions, and the medium in the upper side was then removed after forming a tight contact cell layer. The cells were incubated for12 h at the ALI until the exposure experiment was conducted.

Normal human mammary cells MCF10A and human breast cancer cell lines, MCF7, T47D and BT-474 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MCF10A were cultured in special medium (Procell, China). MCF7 and T47D cells were maintained in DMEM high glucose medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Gibco, USA). BT-474 cell were maintained in RPMI-1640 (Gibco, USA) supplemented with 20% fetal bovine serum (Gibco, USA), 10 μg/mL Insulin (Beyotime, China) and 2mM L-glutamine (Procell, China). Cells were incubated at 37 °C in an atmosphere of 5% CO2.