Waymouth s mb 752 1 medium

DMS114 cells (human small cell lung cancer, ATCC
CRL-2066) obtained from American Type Culture Collection (Manassas,
VA) were cultured in the Waymouth’s MB 752/1 medium from Gibco
(Waltham, MA). MCF7 cells (human adenocarcinoma, ATCC HTB-22) obtained
from American Type Culture Collection were cultured in DMEM high glucose
with stable glutamine and sodium pyruvate from Biowest (France). The
MCF7 cells stably expressing FGFR1 (MCF7-R1) were generated by transfection
of pcDNA3-FGFR1 using DOTAP (Roche Diagnostics). Clones were selected
with 1 mg/mL G-418 and chosen based on their receptor expression level
analyzed by immunofluorescence and immunoblotting. MCF7-R1 cells were
cultured in DMEM high glucose (Biowest) with 50 μg/mL gentamicin
sulfate (Thermo Fisher Scientific), stable glutamine, and sodium pyruvate
(Biowest).

The human cervical normal cell line Etc1/E6E7 and cancer cell lines (HeLa, C4‐1, SiHa, Ca Ski cells) were purchased from the American type culture collection (ATCC; Manassas, VA). Etc1/E6E7 cells were grown in Keratinocyte‐Serum Free medium (Gibco, Carlsbad, CA) with 0.1 ng/mL human recombinant EGF, 0.05 mg/mL bovine pituitary extract, and additional calcium chloride 44.1 mg/L (final concentration 0.4 mM). HeLa, C4‐1, SiHa, and Ca Ski cells were grown separately in Eagle's Minimum Essential Medium(EMEM, Gibco), Waymouth's MB 752/1 medium (Gibco), EMEM (Gibco), and RPMI‐1640 Medium (Gibco) complemented with 10% FBS (Life Technologies, Grand Island), and all cell lines were incubated at 37°C in a 5% CO2 incubator.

Rat pancreatic cancer cells (DSL-6A/C1) were transfected with luciferase (Luc)/red fluorescence protein (RFP) /lentivirus gene to create Luc/RFP-positive cells according to the protocol provided by the manufacturer (GeneCopoeia Inc., Rockville, MD). Luc/RFP-positive cells were sorted out using fluorescence-activated cell sorting technique (Aria II, Becton Dickinson, Franklin Lakes, NJ). Cells were then seeded in four-chamber cell culture slides (Nalge Nunc International, Rochester, NY) and maintained in Waymouth's MB 752/1 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Gibco). RF hyperthermia was performed as described in the literature [20 (link)]. Cells in different groups were treated by (a) Gemcitabine (5.0 μM) plus 30-min RFH at approximately 42^oC; (b) Gemcitabine alone; (c) 30-min RFH alone; and (d) no treatment to serve as a control. We used the 50-percentage inhibitory concentration (IC50) dose of gemcitabine for cell treatment, which was decided by CellTiter 96 Aqueous One Solution Cell Proliferation-assay (Promega Corporation, Madison, WI).

Paired ovaries of 28-day-old ICR female mice were excised and washed three times in Waymouth’s medium containing 3 mg/ml BSA. Ovaries (paired ovaries/well) were cultured on Millicell inserts (Millipore, Billerica, MA, USA) in 24-well plates (Corning, NY, USA) with 600 μl of Waymouth’s MB752/1 medium (Gibco) supplemented with 1 mM sodium pyruvate, 100 IU/ml penicillin, 100 μg/ml streptomycin, 3 mg/ml BSA, 10% FBS, and 0.6 IU/ml rFSH (Merck, Germany). After 24 h of culture, the ovaries were infected with Ad-LacZ and AdmiRa-mmu-miR-181a-off adenovirus (2.5 × 10¹⁰ pfu/ovary) for another 48 h before the medium was changed. The ovaries were then cultured with 2 μg/ml 3-NP for an additional 48 h at 37 °C in a humidified atmosphere of 5% CO₂. The cultured ovaries were collected for measurements of mRNA and protein expression.

Both ovaries of 4-week-old female ICR mice were removed and washed three times in Waymouth medium containing 3 mg/ml bovine serum albumin. Ovaries (paired ovaries/well) were cultured on Millicell inserts (Millipore) in 24-well plates (Corning) with 600 μl of Waymouth’s MB752/1 medium (Gibco) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 3 mg/ml bovine serum albumin, 10% fetal bovine serum, 1 mM sodium pyruvate, and 0.6 IU/ml rFSH (Merck). After 24 h of culture, the ovaries were infected with Ad-LacZ, Ad-FLAG-KLF12, and Ad-SPHK1 adenovirus (2.5 × 10¹⁰ plaque-forming unit/ovary) for another 48 h before the medium was changed. The ovaries were then cultured with 2 μg/ml 3-NP for an additional 48 h at 37 °C in a humidified atmosphere of 5% CO₂. The cultured ovaries were collected for measurements of mRNA and protein expression.