Patf2 t71

1,3-Diethylbenzimidazoliumiodide (MC1), chloro-(1,3-diethylbenzimidazol- 2-ylidene)gold(I) (MC2), [di-(1,3-diethylbenzylimidazol-2-ylidene)]gold(I) iodide (MC3), [triphenylphosphine-1,3-diethylbenzylimidazol-2-ylidene)]gold(I) iodide (MC4) were synthesized as described
[9 (link), 10 (link)]. Structures and purities were ascertained by ¹³C- and ¹H-NMR, and mass spectroscopy and elemental analyses. N-acetyl-L-cysteine (NAC), glutathione (Glu), gemcitabine (Gem) and p38 inhibitor (p38i, SB203580) were purchased by Sigma-Aldrich (Germany). p38 (cat: #9212), pp38 (T180/Y182, cat: #9215), PARP (cat: #9542), pp53 (S15, cat: #9284) and pATF2 (T71, cat: #5112) were obtained from cell signaling (NEB, Germany). ASK1 (cat: sc-7931) and Trx (cat: sc-20146) were from Santa Cruz (Germany).

The cisplatin modified DNA (ab103261) and γH2A.X (ab26350) antibodies were from Abcam (Cambridge, USA). The phospho-STAT3 S727 (#9134), phospho-Akt S473 (#9271), phospho-NF-κB S536 (#3033), pERK T202/Y204 (#9101), pATF2 T71 (#5112), (P70S6K (#2708), phospho-P70S6K T389 (#9205), p21 (#2947), cleaved caspase 3 (#9661) and cleaved caspase 7 (#9491) antibodies were from Cell Signaling (MA, United States). The γH2A.X (S139) antibody (AB26350) was from Abcam (MA, USA). The p53 antibody (sc-126) was from Santa Cruz Biotechnology (TX, USA). The p63 antibody (NB100-691) was from Novus Biologicals (CO, USA). The actin monoclonal antibody (AC-15) was from Sigma-Aldrich (MO, United States). The SignalSilence p70/85 S6 Kinase siRNA was from Cell Signaling (MA, United States). The plasmids for FUCCI live cell imaging, mVenus-hGeminin(1/110) and mCherry-hCdt1(30/120), were a kind gift from Dr Atsushi Miyawaki (Riken, Japan). Dactolisib (NZP-BEZ235), MK2206, S3I-201, UO126 and SC75741 were all from Selleck Chem (MA, USA).

FaDu and Detroit 562 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured per ATCC recommendations. HPV(−) cell line UMSCC 1 and HPV(+) cell lines UMSCC 47 and UMSCC 104 were kindly provided by the laboratory of Dr. Thomas Carey, University of Michigan. Cells were maintained at 37°C in a humidified, 5% CO₂ atmosphere and cultured in Dulbecco's Modified Eagle's medium (DMEM), supplemented with 10% FBS, 1% penicillin and streptomycin, and MEM non-essential amino acids (Gibco™, Life Sciences Solutions Group, Thermo Fisher Scientific, Carlsbad, CA). Rigosertib (ON 01910.Na) and ON 01911.Na (an inactive control analog) (Figure 1) were provided by Onconova Therapeutics, Inc. (Newtown, PA). These compounds were dissolved in DMSO. Cisplatin was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies to cleaved PARP, cleaved Caspase-3, cleaved Caspase-9, p-mTOR, p-Akt (Ser 473), Akt, p-MSK-1 (T581), p-Erk1/2, Erk1/2, JNK, p-JNK, p-ATF-2 (T71), ATF-2, p-Jun (S63) and COX IV were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to Mcl-1, GAPDH, CDK2, CDK4, Cyt C, and Oct1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).