KU 55933

All animal experiments were performed with the approval of Animal Care and Use Ethics Committee of Army Medical Center of PLA. (i) HeLa NC and HeLa shAPE1 cells (ii) HeLa WT cells (5 × 10⁶ in 100 ul medium with Matrigel) were injected subcutaneously in flank of 8 week-old male nude mice. Subcutaneous tumors were allowed to grow for 2 weeks before treatments. When palpable tumors were visible, mice of (i) were divided into four and (ii) were divided into seven groups (n = 7 in each group). Mice were received treatments for 9 days (Ku55933, 10 mg/kg, intraperitonially, daily; E3330, 40 mg/kg intraperitonially, daily; Inhibitor III, 20 mg/kg, intraperitonially, daily; IR, 5 Gy, every 2 days). Specifically, Mice were treated with (i) vehicle, Ku55933, IR, Ku55933 + IR (ii) vehicle, IR, E3330 + IR, Inhibitor III + IR, Ku55933 + IR, E3330 + Ku55933 + IR, and Inhibitor III + Ku55933 + IR. Unblinded tumor measurements were recorded once every 2 days and the volume as calculated by the formula: (width) 2 × length/3. The mice were euthanized in a gas canister with gradual fill carbon dioxide at the end of the treatment cycles and sacrificed. Weighed the tumor with electronic scale.

HeLa, SiHa WT cell lines were purchased form National Collection of Authentical Cell Cultures of China. HeLa C65S-APE1 and HeLa WT-APE1 (provided by Gianluca Tell Lab, Department of Medical and Biological Sciences, University of Udine, Udine, Italy). Cells were cultured in DMEM medium (Gibco, China), supplemented with 1% Penicillin/Streptomycin (Hyclone, US) and 10% fetal bovine serum (Gibco, US) and were monitored for mycoplasma contamination using the Mycoplasma PCR Detection Kit (Beyotime, catalog No.C0301S, Shanghai, China). All chemical reagents were supplied by Selleck (Shanghai, China) unless otherwise specified mentioned. E3330 (catalog No. S7445), Ku55933 (catalog No. S1092), MMS (catalog No. E0609), MG132 (catalog No. S2619), Doxycycline (catalog No. S5159). Cycloheximide (catalog No.C4859, Sigma, USA), Inhibitor III (Sigma, catalog No.262017, USA), TBHP (Sigma, catalog No.416665, USA). The 8MV X-rays at indicated doses were generated by (Elekta, synergy 2178).

All cell lines used in this paper were correctly authenticated. SCC011 cell line (RRID:CVCL_5986), previously described^{64 (link)}, has been reported having a “damaging” annotation which may lead to either loss-of-function or gain-of-function activities, in our experiments these cells behave as cells with a functional p53, at least for the D2 and p21 regulation, and are then used as a model of cells with a functional p53 protein. SCC011were cultured in RPMI 1640 Medium (HiMedia Leading BioSciences Company, Mumbai, Maharashtra, India, cod. AL028) supplemented with 10% Fetal Bovine Serum (HiMedia Leading BioSciences Company, Mumbai, Maharashtra, India, cod. RM10432), 1% L-Glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA, cod. 25030024) and 1% Penicillin/Streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA, cod. 15070063). p53 mutant SCC cell line (SCC13, RRID:CVCL_4029) and p53 mutant D2 TET-ON SCC cell line^{12 (link)}, derived from a skin SCC^{65 (link)}, were cultured in Keratinocyte-SFM (KSFM 1X, Gibco, Thermo Fisher Scientific, Waltham, MA, USA, cod. 17005059) medium [+] L-Glu (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with Bovine Pituitary Extract (BPE, 30.0 μg/mL, Gibco, Thermo Fisher Scientific, Waltham, MA, USA, cod. 11543530) and human recombinant Epidermal Growth Factor (EGF, 0.24 ng/mL, Gibco, Thermo Fisher Scientific, Waltham, MA, USA, cod. 11543530). HaCaT cells (RRID:CVCL_0038) were cultured in Dulbecco’s modified Eagle medium (DMEM, HiMedia Leading Bio Sciences Company, Mubai, India, cod. AL007) supplemented with 10% Fetal Bovine Serum, 1% L-Glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA, cod. 25030024) and 1% Penicillin/Streptomycin. All cell lines were mycoplasma free and were cultured at 37 °C in a humidified atmosphere with 5% CO₂. In all the experiments in which THs was applied to cells, we used a combination of T3 (30.0 nM/24 h, Sigma-Aldrich, St. Louis, Missouri, USA, cod. T6397) and T4 (30.0 nM/24 h, Sigma-Aldrich St. Louis, Missouri, USA, cod. T2501), indicated throughout the text as THs, thus resembling physiological exposure of cells to both the active hormone (T3) and its pro-hormone (T4). In experiments in which THs were removed from the serum, THs-deprivation was achieved by FBS Charcoal absorption. For the studies of DNA damage mechanisms, we used etoposide (50.0 µM/30 min, Sigma-Aldrich, St. Louis, MO, USA, cod. E1383) and KuDOS (KU-55933, 100.0 µM/1 h, Sigma-Aldrich, St. Louis, MO, USA, cod. SML1109). UV-A/UV-B radiation was performed by exposing cells to 60 mJ/cm² UV-A/UV-B light, using six Philips TL12/60W fluorescent lamps (Philips, Eindhoven, The Netherlands). UV-C radiation was performed by exposing cells to 100 µJ/cm² UV-C light, using UV Stratalinker 2400 (Agilent Genomics/Stratagene Stratalinker 1800 UV Crosslinker, Stock #53267-1).