Htb 35

Human osteosarcoma cells (U2OS, ATCC® HTB-96™), human cervix cancer cells (SiHa, ATCC® HTB-35™), human uterus cancer cells (MES-SA, ATCC® CRL-1976™), human cervix cancer cells (HeLa, ATCC® CCL-2™) mouse embryonic fibroblasts (MEFs) with/without S51A mutation of eIF2α, and Dcp1-YFP expressing U2OS cells were grown in Dulbecco’s Modified Eagle Medium with 4.5 g/l D-glucose (DMEM, Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and Penicillin-Streptomycin cocktail (Sigma-Aldrich). HAP1 cells: (a) parental (PAR), (b) eIF2α (S51A), (c) ΔHRI, (d) ΔGCN2, (e) ΔPKR, (f) ΔPERK (Horizon Discovery, UK) grown in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco) supplemented as described for DMEM. Kinase-negative HAP1 cells were verified by sequencing (Fig. S6).

Human osteosarcoma derived cell line U-2 OS, HPV16-positive carcinoma derived cell lines SiHa (ATCC® HTB-35™), SCC090 (ATCC® CRL-3239™) and U-2 OS stable cell lines were cultured in DMEM medium (Lonza, Verviers, Belgium). U-2 OS stably transfected clones were routinely cultured in the presence of 625 μg/mL G418 to maintain selection. HPV16-positive carcinoma derived cell line Ca Ski (ATCC® CRL-1550™) was cultured in RPMI-1640 medium (Lonza, Verviers, Belgium). All media were supplemented with 10% Fetal Calf Serum (Eurobio, Courtaboeuf, France). Undifferentiated W12 20861, 20862 and 20863 clones were cultured in monolayer on Mitomycin C treated mouse 3T3 cells as previously described^{58 (link)}. Cell lines were incubated at 37 °C in a humidified atmosphere with 5% of CO₂.

Cell lines SiHa (ATCC® HTB35™), C33A (ATCC HTB31™), and HaCaT (CLS 300493) were grown in DMEM (Dulbecco's modified Eagle medium) with Ham F10 (F10 Ham nutrient mixture). The HSC-3 cell line (JCRB: JCR 0623) was grown in DMEM with Ham F12 (F12 Ham nutrient mixture), hydrocortisone (0.4 μg/L), and ascorbic acid (50 mg/L). All media were supplemented with fetal bovine serum (10% v/v), penicillin (100 U/mL), streptomycin (100 μg/mL), amphotericin B (0.25 mg/L), kanamycin (0.1 g/L), and HEPES (5.96 g/L). Cultures were grown in a sterile atmosphere at 37°C under 5% CO₂.
Emodin, physcion, ethanolic extract, and hexane fraction from Rhamnus sphaerosperma var. pubescens stems were used to study cell death mechanisms. The working concentrations were established by a sulforhodamine B assay (data not shown). Chrysophanol did not induce cytotoxicity for all cell lines studied.

HPV16 positive cervical carcinoma cell lines CaSki (approximately 400 copies per cell of the HPV16 genome, ATCC CRL1550; American Type Culture Collection (ATCC), Manassas, VA, USA) and SiHa (1–2 copies of HPV16 per cell, ATCC HTB35; ATCC), previously characterised as having high and low CpG methylation levels respectively in the HPV16 URR [24 (link)], were used as methylation controls and during standardization of PCRs and pyrosequencing assays. DNA was extracted from cell lines on the MagNAPure 96 (Roche Diagnostics GmbH, Penzberg, Germany) using the DNA and Viral Nucleic Acid Small Volume Kit (Pathogen Universal 200 protocol).

Cervical cancer-derived cell lines SiHa (HPV16; ATCC^® HTB-35™, ATCC, Manassas, VA, USA), HeLa (HPV18; ATCC^® CCL-2™), and C33A (HPV-negative; ATCC^® CRM-HTB-31™) were cultured in MEM (Invitrogen, Thermo Fisher, Carlsbad, CA, USA) and HEK293T (ATCC^® CRL-3216™) and HaCaT cells were cultured in DMEM (Invitrogen), both supplemented with 10% FCS (Cultilab, Campinas, SP, Brazil) and maintained at 37 °C and 5% CO₂. Primary Human Keratinocytes (PHK) (Lonza Walkersville, Inc., Walkersville, MD, USA) were grown in serum-free medium (Invitrogen) supplemented with recombinant epidermal growth factor (5 ng/mL) and bovine pituitary extract (50 mg/mL). PHK were transduced with recombinant retroviruses carrying the control vector (pLXSN) or vectors encoding HPV16 E6 and/or E7 or HPV16 E6 mutant, E6–8S9A10T, which cannot degrade p53 [50 (link),51 (link),52 (link)]. After 24 h, cells were selected with 300 µg/mL of G418 for 2 days, when 100% of mocked infected controls were dead. Surviving cells were expanded and used to seed monolayers cultures.

The HPV16-positive cervical cancer cell line SiHa (ATCC^® HTB-35^™) was used as positive control and for determining the analytical sensitivity and PCR efficiency. SiHa cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator at 37 °C with 5% CO₂.