Sp600125

Twenty-three 6-week-old athymic (NCR-nu/nu) male nude mice were purchased from the Ohio State University Comprehensive Cancer Center (OSUCCC) Target Validation Shared Resource (TVSR). Mice were subcutaneously transplanted with Ace-1-Dkk-1^YFP-LUC cells (n = 12) or Ace-1-Vector^YFP-LUC (n = 11) cells in equal number (1–2 × 10⁷ cells in 0.1 mL PBS). Treatment with SP600125 (Santa Cruz Biotechnology) or vehicle (2% DMSO, 2% polyethylene glycol 600 and 2% Tween 80 in PBS) was initiated when the tumor size reached 100–250 mm³ (treatment started on day 8 after injection). Mice bearing Ace-1-Dkk-1^YFP-LUC or Ace-1-Vector^YFP-LUC tumors were treated with intraperitoneal (IP) injection of an equal volume of either SP600125 or vehicle (5 mg/kg) every other day for 4 weeks [27 (link)]. Tumor size and body weight were measured every 2–3 days.

Measurement of sphere formation and clonogenicity [30 (link), 56 (link)] was performed by plating single cells in type I collagen coated (1 μg/cm², BD Biosciences) 6-well or 96-well cell culture dishes, respectively. After a 24 hours settling time, cells were treated with AIIB2 (10 μg/ml), SP600125 (2.5–200 μM, Santa Cruz Biotechnology), LBH589 (0.625–80 nM, Novartis AG, [39 (link)]) and combinations thereof for 24 hours and then washed. Cells were irradiated 1 h after addition of inhibitors with 2, 4 or 6 Gy single X-ray doses. Non-specific IgG isotype antibodies (10 μg/ml) or DMSO (equal amount as inhibitors, < 0.1 % v/v) (AppliChem) were used as control. After a cell line-dependent growth period, cell colonies were fixed with 80% EtOH, stained with Coomassie blue (Merck Millipore) and colonies containing > 50 cells were counted. After a 6 d growth period, spheres containing > 25 cells were microscopically counted [56 (link)].

Cells were lysed by NP-40 with protease inhibitors and phosphatase inhibitors; the proteins were separated via SDS-PAGE electrophoresis, transferred onto nitrocellulose membranes, and then incubated with primary and secondary antibodies, respectively. Primary antibodies against JNK1/2/3 (YT2441) (1 : 1000) and phospho-JNK1/2/3 (phospho Thr183/Y185) (1 : 1000) were obtained from ImmunoWay Biotechnology (Newark, DE, USA). Primary antibodies against ERK (ab196883, 1 : 1000), p-ERK (ab50011, 1 : 5000), p38 (ab27986, 1 : 1000), and p-p38 (ab45381, 1 : 5000) were purchased from Abcam (Abcam, Cambridge, MA, United States). The JNK inhibitor SP600125 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The immunoreactive bands were detected with an enhanced chemiluminescence detection system from Pierce (Thermo Fisher Scientific Inc., Rockford, IL, USA). Protein band was analyzed with Quantity One software (Bio-Rad Laboratories, Life Science Research, CA, USA). Protein levels were normalized to that of the internal control β-actin purchased from Abcam (Cambrige, MA, USA) (ab8226, 1 : 1000).

Human bronchial epithelial BEAS-2B cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, San Diego, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, NY, USA). ATF3 KO BEAS-2B cells were custom developed by using a CRISPR/Cas9 gene-editing method by Cyagen Biosciences (Suzhou, China). Sodium meta-arsenite (NaAsO₂) was purchased from Sigma (St Louis, MO, USA). The inhibitors, SB203580, SP600125, U0126 and LY294002, were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against ATF3 (33593), DR5 (3696), JNK (9252), p-JNK (4668), p38 (8690), p-p38 (9215), Bcl-xL (2764), caspase 8 (9746), cleaved caspase 8 (8592), caspase 3 (14220), cleaved caspase 3 (9664), caspase 9 (9502), cleaved caspase 9 (9509), α-tubulin (2125), AKT (C67E7), p-AKT (4060T), ERK (4695), p-ERK (91015) and β-actin (4970) were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Nicotine, N-acetyl-L-cysteine (NAC), and 2,2,6,6-Tetramethyl-1-piperidinyloxy, free radical, 2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO), methyllycaconitine citrate (MLA), mecamylamine hydrochloride (MEC), VAS2870 (VAS), were purchased from Sigma-Aldrich (St. Louis, MO). SP600125 and SB203580 were purchased from Santa Cruz Biotechnology (Dallas, TX), and PD98059 was from Cell Signaling Technology (Danvers, MA).