Bisindolylmaleimide 1

All reagents were purchased from Sigma-Aldrich unless otherwise specified. Bisindolylmaleimide I was purchased from Cayman Chemical. [³H]glucosamine was purchased from PerkinElmer. All fluorescent reagents, including WGA-Alexa 647, CFSE, and CellMask Deep Red, and secondary antibodies were from Thermo. Caspase 3, caspase 9, and PARP were from Cell Signaling. Enzyme-linked immunosorbent assay (ELISA) DuoSet kits for IL-1β, IL-1α, and TNF-α as well as the anti-Vamp8 antibody were from R&D. VAMP8-pHluorin was a kind gift from Rytis Prekeris (University of Colorado), and pmRuby2-MUC2CK was synthesized by Genscript as previously described (15 (link), 20 (link), 22 (link)). Mouse 41-plex cytokine arrays were constructed by Eve Technologies on the basis of tissue lysates normalized to 1 mg/ml.

Recombinant human tumor necrosis-α (TNF-α) was from Peprotech (Rocky Hill, NJ). U0126, SB-203580, and bisindolylmaleimide I were from Cayman Chemical (Ann Arbor, MI). BAY 11–7082 was from Focus Biomolecules (Plymouth Meeting, PA). Goat anti-rabbit IgG-HRP was from GE Healthcare Japan Corporation (Tokyo, Japan). Antibodies for phospho-NF-κB p65 (Ser536) (93H1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14C10) were from Cell Signaling Technology (Danvers, MA). Fatty acid-free bovine serum albumin (BSA) was from EMD chemicals (San Diego, CA). Dexamethasone (DEX), Dulbecco’s Modified Eagle Medium (DMEM), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

PC9 cells were from Kyushu University, Japan. EVSA-T and MKN-45 cells were from DSMZ. All other cell lines were from American Type Culture Collection. All cell lines were banked at the Genentech cell line core facility that routinely performs single nucleotide polymorphism (SNP) and short tandem repeats (STR) analysis to confirm cell line identity and detect possible mycoplasma contamination. PC9 cells were cultured in RPMI medium (Gibco) containing 4.5 g l⁻¹ of D-glucose and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 U ml⁻¹ of penicillin and streptomycin. All other cell lines were maintained in RPMI medium (Gibco) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 U ml⁻¹ of penicillin and streptomycin. Lapatinib was from LC Laboratories, cinnamycin was from Sigma, AACOCF3 was from Santa Cruz Biotechnology, paclitaxel was from US Biologicals, carboplatin was from Selleckchem, AZ628 was from Tocris Bioscience, Bisindolylmaleimide I, XI and XI were from Cayman Chemicals, PKC-20-28 was from EMD Millipore, phorbol-12-myristate13-acetate was from VWR Scientific, and erlotinib and GDC-0980 were synthesized at Genentech.

Tetrodotoxin, phentolamine, iberiotoxin, NS-1619, Y-27632, apamin, NS-309, and ryanodine were purchased from Abcam (Cambridge, MA, USA). Atropine, propranolol, suramin, nisoldipine, and SKF-96365 were purchased from Sigma-Aldrich (St. Louis, MO USA). Bisindolylmaleimide 1 and H₂O₂ were obtained from Cayman Chemical (Ann Arbor, MI, USA) and Fisher Scientific (Pittsburg, PA, USA), respectively.
NS-1619, nisoldipine, NS 309, and SKF-96365 were dissolved in dimethylsulphoxide (DMSO). ryanodine was dissolved in 100% ethanol. All other drugs were prepared with water. Final ethanol and DMSO concentrations in the bath solution did not exceed 0.1% and were shown not to affect spontaneous contractions or bladder function in previous reports [35 (link)–37 (link)].