Complete dulbecco s modified eagle medium dmem

17β-estradiol, dihydroethidium (DHE), and 2′,7′-dichloro dihydro fluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Antibodies (rabbit anti-human) against ERα, Akt, p-Akt, Foxo3a, p-Foxo3a, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, United States). The 17β-estradiol was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution at a concentration of 50 mg/ml, and was stored at −20°C. Complete Dulbecco’s modified Eagle medium (DMEM; Gibco, Gaithersburg, MD, United States) was added to dilute the 17β-estradiol to the appropriate concentrations prior to use.

African green monkey (Vero) cells were maintained in complete Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% Fetal Calf Serum (FBS) (ThermoFisher). VC2 was constructed as described previously (30 (link)). Briefly, the VC2 recombinant virus was constructed utilizing the two-step Double-Red Recombination protocol using the HSV-1(F) viral genome cloned as a bacterial artificial chromosome (BAC). The virus was cultivated in Vero cells. HSV-1((McKrae) was a gift by the late Dr. James Hill (Louisiana State University Health Sciences, Center, New Orleans, LA).

The chondrosarcoma cell line HCS-2/8 was purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China), OUMS-27 was purchased from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Tokyo, Japan), and SW1353 and JJ012 were purchased from American Type Cell Culture (ATCC, Manassas, VA, USA). All these cell lines were authenticated by Short Tandem Repeat assay in our laboratory once in a quarter, and they were tested for mycoplasma by PCR monthly. Mycoplasma-free cells were used in the in vitro and in vivo experiments.
HCS-2/8 and OUMS-27 cells were cultured in complete Dulbecco’s modified Eagle medium (DMEM) supplemented with 5 mM glucose and L-glutamine (Gibco, Grand Island, NY, USA), SW1353 cells were maintained in L-15 medium (Gibco, Grand Island, NY, USA), whereas JJ012 cells were grown in RPMI-1640 medium (Gibco, Grand Island, NY, USA). All these mediums were supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 μg/ml streptomycin, and 100 U/ml penicillin (Gibco, Grand Island, NY, USA). Cell culture was performed at 37 °C in a humidified incubator containing 5% CO₂.