br HeLa cells are HPV positive Homo
HeLa cells are HPV 18 positive Homo sapiens adenocarcinomas grown in Dulbecco's Modified Eagle Medium (ThermoFisher Scientific, catalog number: 12100046), supplemented with 10% fetal bovine serum (VWR, catalog number: 89510-194) and Pen-strep (Calsson Labs, catalog number: PSL02-6X100ML). Human foreskin keratinocytes or HFKs were derived from neonatal human foreskins and were grown in EpiLife medium supplemented with calcium chloride (60 μM), human keratinocyte growth supplement (Cascade Biologics, Portland, OR), and penicillin-streptomycin. Multiple cell lines were derived from several donors for this work. The cells were incubated at 37 °C in a 5% CO2 environment. Cells were washed with 0.1% EDTA (Invitrogen catalog #15-576-028) and raised from plate with 0.05% Trypsin (Sigma-Aldrich catalog # T4049-500ML).
acetic 1 , 2Distearoyl-sn-glycero3PC (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 20 mM NaF, 1% Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 mM Na3VO4, 1% Triton X-100, 10 mg/mL Aprotinin and 10 mg/mL Leupeptin) for 30 min. The lysates were cen-trifuged at 12,000 rpm for 10 min and the supernatant was collected. Equal amount of protein lysates were electrophoresed on a 12% SDS polyacrylamide gel and transferred onto nitrocellulose filter (NC) membrane. The membrane was washed 3 times for 10 min in TBST (Tris-Buﬀered Saline and Tween-20), then after blocking with 2.5% skim milk suspended in TBST for 1 h, membranes were individually incubated respectively with the appropriate primary antibodies Tubulin, P53, Bax, HPV E7 (1:1000) overnight followed by secondary antibodies (1:1000) for 1 h at room temperature. Protein bands were detected using ECL assay kit (Invitrogen, Australia) and exposed using a Kodak medical X-ray processor (Kodak, USA). Anti-human p53 and β-tubulin monoclonal antibodies were purchased from Sigma (Sydney, Australia). Anti-HPV 18E7 polyclonal antibody was purchased from Santa Cruz Biotechnology. Anti-human Bax polyclonal antibody was from Cell Signaling Technology.
2.4. Resistant colony generation and isolation
Colony isolation was performed using sterile cloning disks (SP Scienceware catalog # F37847-0001). Cells were grown at low con-fluence in a 10 cm plate and treated 4 times every 2 weeks for a 2-month period. Cells were observed from single cells, with their growth monitored microscopically. They were washed with 0.1% EDTA, before colonies were isolated by placing a sterile cloning disks soaked in trypsin on them. Individual disks were then put in a well of a 24-well plate containing growth media, allowing cells to detach from the disk. Cells were then expanded, and acquisition of resistance was assessed.
2.5. Colony formation assays
Colony formation assays were performed by seeding low con-centrations of cells on six well plates. They were then treated with UV 48 h after seeding and allowed to grow until colonies appeared. Twenty-four hours after the first colonies with 15 or more cells were seen, colonies were stained with a crystal violet solution (0.2% crystal violet, 5% ascetic acid, and 2.5% 2-proponol) and hand counted.
2.6. shRNA transfections
Cell transduction was basically according to the method described previously (Gu et al., 2006). Briefly, HeLa cells were maintained in complete DMEM medium (Gibco-Invitrogen) and were plated in T75 flasks (4 × 105/flask) overnight culture. LV-shRNAs was diluted in 2.0 mL cultural medium containing polybrene (8 μg/mL) and added to the cells for culture for 1 h at 37 °C. After this, 8 mL of fresh polybrene medium was added to the cells and incubation continued for 24 h. Polybrene medium was then replaced with fresh DMEM culture medium, and the cells were further cultured for assays.
To prepare cells for UV irradiation the media was aspirated, and the cells were washed with 1× PBS (Bio Basic catalog # PD8117). A thin layer of PBS was added to the wells or plate and then cells were irra-diated in a UV Stratalinker 2400 (Stratagene Catalog # 400075-03) at specified doses. Media was then added, and the cells were incubated at 37 °C.
Flow cytometry analysis was carried out with the FACSCalibur™ (Becton Dickinson). The acquisition and analysis of data was done with the program Cell Quest Pro.
2.9. Apoptosis assay
The Dead Cell Apoptosis Kit (ThermoFisher Scientific catalog #V13242) and the Countess™ II FL Automated Cell Counter (ThermoFisher Scientific catalog #AMQAF1000) were used to measure apoptosis. Cells were seeded at 50,000 cells/well in a 6-well plate and incubated at 37 °C overnight. Cells were then UV irradiated at specified intervals and incubated at 37 °C for 48 h. Cells were then washed with 0.1% EDTA and incubated with Trypsin for 3 min at 37 °C. DMEM was then added to cells and they were incubated at 37 °C for 30 min. Cells were then pelleted by centrifuging at 188 ×g for 5 min. After aspiration of DMEM, they were resuspended in ~1 × 106 cells/mL in 1× annexin binding buﬀer and incubated with propidium iodide for 15 min at RT. Cells then were loaded onto slides and imaged using appropriate fluorescent filters on Countess™ II FL Automated Cell Counter.