Siha cells

Cervical cancer cells (HeLa, ME-180, and SiHa cells) were purchased from ATCC (Manassas, USA), and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, Invitrogen, USA) and 1% penicillin/streptomycin (Gibco/Invitrogen) at 37°C in a 5% CO₂ atmosphere in an incubator. Normal cervical epithelial cells were obtained from ATCC, and maintained in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C.

The cell lines used in this study were derived from a variety of carcinoma types. HeLa cells (HPV18-positive cervical epithelial adenocarcinoma), FaDu cells (HPV-negative hypopharyngeal carcinoma), Detroit 562 cells (HPV-negative pharyngeal carcinoma), CaSki cells (HPV16-positive cervical squamous cell carcinoma), and SiHa cells (HPV16-positive cervical squamous cell carcinoma) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). MCF-7 cells (HPV-negative breast carcinoma) were kindly provided by Dr. Libor Macurek (Institute of Molecular Genetics, Czech Republic).
HeLa, CaSki, and SiHa cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Sigma‒Aldrich, St. Louis, MO, USA); FaDu and Detroit 562 cells in Eagle's minimum essential medium (EMEM; Sigma‒Aldrich); and MCF-7 cells in Dulbecco's modified Eagle's medium (DMEM; Sigma‒Aldrich). All cell culture media were supplemented with 10% fetal bovine serum (FBS; Sigma‒Aldrich), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Biosera, Kansas, MO, USA). The cells were cultured in an incubator at 37°C in 5% CO₂ and periodically checked for mycoplasma contamination. The cells were harvested at 80% confluency using a 0.05% trypsin-EDTA solution in phosphate-buffered saline (PBS) for further examination.

The cervical cancer SiHa cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in an incubator using an RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone) at 37°C with 5% CO₂. Stable HeLa cells were also maintained using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone) in an incubator at 37°C with 5% CO₂. The cells were transfected with siRNA using the Lipofectamine 2000 transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions.

HeLa cell lines were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. SiHa cells were obtained from the American Type Culture Collection (ATCC). The HPV16-type SiHa, HPV18-type HeLa, and non-HPV-type Yumoto cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (Life Technologies, CA, USA), supplemented with 10% fetal bovine serum. Cell lines were maintained in a humidified incubator containing 5% CO₂ at 37 °C. Cells were used for functional experiments within 3 months of passaging post-receipt. The SiHa, HeLa, and Yumoto cell lines were trypsinized and plated in culture dishes. At ~ 50% confluency, the cell lines were transfected with an annealed ADAR1 siRNA (siADAR1, sc-37657; Santa Cruz Biotechnology, TX, USA), control siRNA (siControl, sc-37007; Santa Cruz Biotechnology, TX, USA), or an empty vector (mock) for gene silencing (final concentration, 100 nmol/L) using an siRNA transfection reagent (sc-29528; Santa Cruz Biotechnology, TX, USA).

Human cervical cancer cells transformed with HPV16 (SiHa cells), and HPV18 (HeLa cells) were obtained from the American Type Culture Collection (ATCC). The cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10 % fetal bovine serum (FBS), penicillin/streptomycin (50 μg/ml), 2 mM L-glutamine, 250 ng/ml fungizone, and maintained at 37 °C in 5 % CO₂. The total RNA isolation was carried out with TriPure isolation reagent (Roche, Indianapolis, IN) for the end-point RT-PCR and real time RT-qPCR assays. The cellular protein isolation was performed and protein concentration was determined by the BCA protein kit (Pierce, Rockford, IL.) for the Western Blot assays. The cells were attached on a slide for epifluorescence microscopy assays or fixed in ethanol for the flow cytometry assays. In addition, the cells which were used in transfection assays were analyzed for the luciferase activity assays.