Cal 62

Breast cancer cell line
MDA-MB-231, anaplastic thyroid cancer cell line CAL-62 and human keratinocytes
HaCaT were purchased from ATCC and were cultured in RPMI 1640 medium
(R0883 Sigma Aldrich-MERCK) (MDA-MB-231 and CAL-62) or DMEM medium
(11965092, Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich-MERCK,
F9665), 2 mM glutamine (G7513; Sigma-Aldrich-MERCK), 100 units/mL
penicillin and 0.1 mg/mL streptomycin (P0781; Sigma-Aldrich-MERCK).
Adherent cells were detached by Trypsin–EDTA solution (TA049;
Sigma-Aldrich-MERCK). Cells were maintained at 37 °C in humidified
incubator with 5% CO₂. Cells were treated with cobalt(III)
chloride hexahydrate (CoCl₂) (Merck; C8661) dissolved in
distilled, sterile water for 24 h to induce hypoxia (200 μM).
Hypoxic and normoxic cells were also treated with 10/50 μM of
the indicated compounds (dissolved in DMSO) for 48 h, maintaining
the CoCl₂ in the culture medium for hypoxic conditions.
SIRT3 inhibitor 3-TYP was dissolved in DMSO and added to a final concentration
of 1 μM for 24 and/or 48 h.

Human ATC cell lines CAL‐62 and C‐643 were purchased from the China Center for Type Culture Collection. CAL‐62 and C‐643 cells were cultured in DMEM and RPMI‐1640 medium supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C in 5% CO₂, respectively. Apatinib mesylate (YN968D1), VX‐765 and Z‐DEVD‐FMK were purchased from TargetMol (Target Molecule Corp, USA). These agents were first dissolved in DMSO and then diluted with DMEM or RPMI‐1640 medium with a final DMSO concentration of less than 0.4% for in vitro studies. Melittin was purchased from Sigma‐Aldrich and was first dissolved in PBS and then diluted with DMEM or RPMI‐1640. N‐Acetyl‐L‐cysteine (NAC) was purchased from Beyotime (China). All cells were cultured in well plate for at least 24 h before each treatment.

Human thyroid cancer cell lines CAL-62, KHM-5M, BHT-101, B-CPAP, and normal thyroid cell lines Nthy-ori-3-1 were purchased from the China Center for Type Culture Collection (CCTCC, China). The CAL-62 and BHT-101 cells were cultured in DMEM medium supplemented with 10% and 20% fetal bovine serum (Gibco, USA) at 37°C in 5% CO2, respectively. KHM-5M, B-CPAP and Nthy-ori-3-1 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C in 5% CO2. A total 15 of DTC patients who underwent radical thyroidectomy in Ruijin Hospital of Shanghai Jiaotong University Medical College and were confirmed as DTC by postoperative histopathological examination were included in this study. All samples were obtained with the patients’ informed consent, and the samples were histologically confirmed by at least 2 pathologists independently in a double-blinded fashion.

CAL-62 and BHT-101 cell lines were purchased from FuHeng Cell Center (Shanghai, China) and cultured in DMEM medium (KeyGen BioTECH, Jiangsu, China) supplemented with 10% fetal bovine serum (FBS) (Gibco, NY, USA) for CAL-62 and 20% FBS for BHT-101. A total of 4–6 × 10⁴ cells/mL cells were plated into culture dishes (Corning, NY, USA) and incubated in atmosphere of 5% CO₂ at 37 °C for 12–24 h before the experiments were performed. For immunofluorescent labeling, several cell-bearing coverslips were prepared under similar conditions by using coverslip preparation dishes (JET Bio-Filtration, Guangzhou, China) for multiple experiments.