P53 do 1

Total cell lysates were prepared in 1% CHAPS buffer [5mM MgCl2, 140 mMNaCl, 1mM EDTA, 1mM EGTA, 1% CHAPS, 20mM Tris-HCl (pH 7.5), and protease inhibitors (cOmplete ULTRA, Roche)]. AlgiMatrix dissolving buffer (ThermoFisher Scientific) was used to harvest spheroids before lysis in 1% CHAPS buffer. Proteins (600–1000 μg) were immunoprecipitated with BCL-2 (#4223, Cell Signaling), BCL-XL (#2762, Cell Signaling), MCL-1 (S-19, Santa Cruz) antibodies at 4°C for 16h and coimmunoprecipitates were captured by Dynabeads Protein G at 4°C for 2 h. Beads were recovered using DynaMag spin magnet and washed twice in 1% CHAPS buffer. Total cell lysates and immunoprecipitates were separated on NuPage 10% Bis-Tris gels. After SDS-PAGE, proteins were transferred onto PVDF membranes (Millipore) and then blocked with 5% dried milk in PBS-Tween20. Membranes were incubated with primary and secondary antibodies (GE Healthcare) in a buffer containing 10% milk diluent-blocking concentrate (KPL), detected with Luminata Crescendo Western HRP substrate (Millipore). Blots were imaged with LAS4000 image analyzer (Fujifilm) on chemiluminescence mode. The following antibodies were used for immunoblotting: BCL-2 (#2872, Cell Signaling), BCL-XL (#2762, Cell Signaling), Actin (#8457, Cell Signaling), BIK (#4592, Cell Signaling), MCL-1 (S-19, Santa Cruz), p53 (DO-1, Santa Cruz).

Proteins were extracted using a buffer composed of: 10 mM PIPES; 300 mM NaCl; 1 mM EDTA; 300 mM sucrose; 1 mM MgCl₂; 0.5% TritonX-100; cOmplete™, EDTA-free Protease Inhibitor Cocktail 1x (Roche, Mannheim, Germany); DTT 1 mM; NaF 1 mM; Na₃VO₄ 0.2 mM. Protein quantification was done using Protein Assay Dye Reagent Concentrate (Bio-Rad, Munich, Germany). The Western blotting analysis was performed using 40 µg protein and incubated with the following antibodies: HPV16 E6 (2 µg/mL, #849 Arbor Vita Corporation); HPV16 E7 NM2 (kindly provided by Prof. Martin Müller, DKFZ); p53 (DO-1, Santa Cruz-126); pRb (C-15, Santa Cruz-50); β-Actin clone C4 (Millipore 691001). Intensities of bands were quantified by ImageJ.

Western blot analyses were performed as described previously and repeated twice.^{14 (link),32 (link)} Primary antibodies used in this study include rabbit polyclonal antibodies to human NS (Ab138, Tsai Laboratory, Houston, TX, USA), p-cdc2 (Y15, Cell Signaling, Danvers, MA, USA), MDM2 (SMP-14, Santa Cruz, Santa Cruz, CA, USA), p53 (DO-1, Santa Cruz), p-histone H3 (S10, Upstate, Lake Placid, NY, USA), and α-tubulin (Sigma). Secondary antibodies were conjugated to peroxidase (Jackson ImmunoResearch, West Grove, PA, USA).

Cell proteins were prepared by lysing cells for 30 min at 4⁰ C in a lysis buffer composed of 150 mM NaCl, 50 mM Tris (pH 8.0), 5 mM EDTA, 1% (v/v) Nonidet p-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 μg/ml aprotinin and 25 μg/ml leupeptin. Equal amounts of protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose filter. After blocking with buffer containing 5% non-fat milk, 20 mM Tris-HCl (pH 7.5), and 500 mM NaCl for 1 h at room temperature, the filter was incubated with specific antibodies for 1 h at room temperature; washed and then incubated with HRP-labeled secondary antibody; and developed using a chemiluminescent detection system (ECL, Amersham Life Science, Buckinghamshire, England). The specific antibodies used included MYCN (sc-53993, Santa Cruz), MDM2 (SMP14, Sigma; 2A10, Invitrogen), p53 (DO-1, Santa Cruz), HuD (AB5971, Sigma), ARID3B (A06737, Boster) and HMGA2 (PA5-25276, Invitrogen). All antibodies were used according to the manufacturers’ instruction.