Penicillin streptomycin

The five ovarian mucinous adenocarcinoma cell lines used in this study (MCAS, RMUG‐S, MN‐1, OMC‐1, OMC‐3) were obtained from the following sources: MN‐1 from Scienstuff Co. Ltd (Nara, Japan) 20, OMC‐1 from Dr. Tsuyoshi Saito (School of Medicine, Sapporo Medical University, Sapporo, Japan) 21, MCAS and RMUG‐S from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, and OMC‐3 from RIKEN Bio Resource Center Cell Bank (Institute of Physical and Chemical Research, Tsukuba, Japan). The DLD‐1 colorectal cancer cell line used as a positive control was obtained from the American Type Culture Collection. MCAS cells were grown in Eagle's minimal essential medium (Wako, Osaka, Japan) with 20% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA). MN‐1 cells were grown in Dulbecco's minimal essential medium (Wako) with 10% FBS and penicillin/streptomycin. RMUG‐S and OMC‐3 cells were grown in Ham's F12 medium (Wako) with 10% FBS and penicillin/streptomycin. OMC‐1 cells were grown in Minimum essential medium α medium (Wako) with 10% FBS and penicillin/streptomycin. All cell lines were maintained at 37°C in 5% CO₂.

Rat C6 glioma cells47 (link) were cultured in Dulbecco’s modified Eagle’s medium (High Glucose) (D-MEM, 044–29765, Wako, Tokyo, Japan) supplemented with 10% foetal bovine serum (172012; Sigma-Aldrich) and 1% penicillin/streptomycin (168–23191; Wako) and maintained in 5% CO₂ at 37 °C. The cells were plated at a density of 1.0 × 10⁵ cells per well on a coverslip sheet (Cell Desk LF, MS-92132; Sumitomo Bakelite, Tokyo, Japan) for 2 days. For glucose deprivation, the cells were incubated in D-MEM (no glucose) (042–32255, Wako) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin for 3 days. The cells were immunostained for NSE with essentially a similar protocol as used for cerebellar slices. The NSE immunofluorescence images were captured using a confocal microscope and the same settings from 10 randomly selected regions from 2 independent cultures (5 regions from one culture) in each experimental group. The fluorescence intensity was measured using ImageJ. The background intensity was subtracted from the fluorescence intensity. The averaged fluorescence intensity of control cells was taken as 100%, and the relative values were calculated.

The estrogen receptor (ER)-positive human breast cancer line MCF-7 (RCB1904) and the ER-negative human breast cancer cell line MDA-MB-453 (RCB1192) were purchased from the RIKEN cell bank. MCF-7 cells were cultured at 37 °C with 5% CO₂ in E-MEM with L-glutamine and phenol red (Wako Pure Chemical Industries, Ltd., Osaka, Japan) containing 10% fetal bovine serum (GE Healthcare Life Sciences, Inc., Logan, UT, USA), 1% penicillin/streptomycin (Wako Pure Chemical Industries, Ltd.), 1.0 mM sodium pyruvate solution (Wako Pure Chemical Industries, Ltd.), and 1% MEM non-essential amino acid solution (Thermo Fisher Scientific, Inc., Waltham, MA, USA). MDA-MB-453 cells were cultured at 37 °C without CO₂ in Leibovitz′s L-15 Medium with L-glutamine, phenol red, and sodium pyruvate (Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (GE Healthcare Life Sciences, Inc.) and 1% penicillin/streptomycin (Wako Pure Chemical Industries, Ltd.).
The hyperthermia treatment was based on our previous study [11 (link)]. The MCF-7 cells were incubated at 42 °C with 5% CO₂ for 1 h using a standard incubator, and the MDA-MB-453 cells were incubated in the same way, but without CO₂.