Hela cells

Synthesized CerPCho (d18:1/18:1 CerPCho, d18:1/24:0 CerPCho, d18:1/(D₃₁)-16:0 CerPCho and d18:1/(D₉)-18:1 CerPCho) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). All chemicals used in mobile phases were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). HeLa cells (Riken Cell Bank, Riken Bioresource Center, Ibaraki, Japan) were cultured in minimum essential medium (Sigma-Aldrich, Inc., St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 2 mM l-glutamine (Thermo Fisher Scientific Inc., Waltham, MA, USA), 100 U mL⁻¹ penicillin and 100 µg mL⁻¹ streptomycin (Sigma-Aldrich, Inc.). After washing cells with phosphate-buffered saline three times, cell layers were scraped from the dishes and homogenized in methanol (Wako Pure Chemical Industries, Ltd.) using a vortex mixer and sonication bath. Homogenate protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.).

HeLa cells were purchased from RIKEN BioResource Center and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a 5% CO₂ incubator. One day before transfection, cells were dissociated and transferred onto a custom-made glass bottom dish with a 35-mm cell culture dish (430165, NUNK) and a coverglass (No.1S grade, Matsunami Glass)). 4.0 μg of plasmid DNA was mixed with calcium phosphate buffer (25 mM HEPES-NaOH, pH 7.1, 140 mM NaCl, 0.7 mM Na₂HPO₄ and 125 mM CaCl₂) and then we added the mixture to the cell. After 10–12 hours, the cells were washed with DMEM containing 10% FBS and cultured for 20–40 hours.

HeLa cells were sourced from RIKEN BioResource Center Cell Bank (Tsukuba, Japan), and Dulbecco’s Modified Eagle’s Medium (DMEM) (Nacalai Tesque, Inc., Kyoto, Japan) was used for culture supplemented with 10% heat-inactivated fetal bovine serum (Biowest, Nuaillé, France) and a mixture of antibiotic–antimycotic solution (Nacalai Tesque, Inc., Kyoto, Japan) at 37 °C under a 5% CO₂ atmosphere. The cells were passaged every 2 or 3 days before cell seeding. Trypan Blue exclusion assays were used to measure cell viability and density [28 (link)]. The cells were seeded in a 12-well plate at a density of 6.0 × 10⁴ cells/well for the quantitative reverse transcription PCR and in a 24-well plate at a density of 3.3 × 10⁴ cells/well for the luciferase reporter assay. About 48 h after seeding, the cells were treated with zinc for 6 h. ZnSO₄ was used for zinc treatment. Chelex^®-100 chelating resin was purchased from Bio-Rad (Hercules, CA, USA). The cells were seeded and cultured in DMEM for the zinc-deficient culture and supplemented with 10% Chelex^®-100-treated FBS (zinc-deficient medium) prepared according to the manufacturer’s protocols.

Cells of the human embryonic kidney line 293 (HEK293) were obtained from the American Type Culture Collection, Manassas VA (cat. CRL1573) and HeLa cells were obtained from the RIKEN BRC Cell Bank (cat. RCB0007, Tsukuba, Japan). Cells were grown in Eagle’s Minimum Essential Medium (cat. 30–2003, American Type Culture Collection, Manassas VA, USA) or Dulbecco’s Modified Eagle’s Medium (cat. D5796, Sigma-Aldrich) supplemented with 10% fetal bovine serum (cat. 10270-106, Thermo-Fisher, Grand Island NY; or cat. SH30070, HyClone GE Life Sciences, Logan UT).

HeLa cells were obtained from the RIKEN BRC Cell Bank for use as the cytokeratin‐positive control in the immunostaining experiments; SCC‐9 cells were obtained from ATCC for use as the p53 and SCCA1/2‐positive control. Both lines were maintained in the growth medium at 37 °C with 5% CO₂.