Phenylarsine oxide pao

Toxicity of drugs to mammalian cells was carried out in the human cell line HFF, using a previously described method [41 (link)], with slight modifications. Briefly, HFF cells were grown in a medium consisting of 500 mL Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma), 50 mL new-born calf serum (NBCS; Gibco, Cleveland, TN, USA), 5 mL penicillin/streptomycin (Gibco), and 5 mL L-Glutamax (200 mM, Gibco), at 37 °C/5% CO₂ up to ~80% confluence in vented flasks. For the assay, 100 µL of the cell suspension (3 × 10⁵ cells/mL) was added to each well of a 96-well plate. The plate was incubated at 37 °C/5% CO₂ for 24 h to allow for cell adhesion, after which 100 µL of a serial drug dilution was added (prepared in a separate sterile plate). Phenylarsine oxide (PAO; Sigma) was used as the positive control. The cells were then incubated for a further 30 h before the addition of 10 µL of 125 mg/L resazurin solution, and underwent a final incubation for 24 h. Fluorescence measurements and data analysis were performed, as described above. The selectivity index was calculated as EC₅₀ (HFF)/EC₅₀(427-WT).

Mito-Tracker Orange CM-H2TMRos (Molecular Probes, Cat No. M-7511) was supplied by Life Technologies. PI4KIIIα inhibitor, phenylarsine oxide (PAO, Sigma, Cat No. P-3075) and PI4KIIIβ inhibitor, PIK-93 (AdooQ BioScience, Cat No. A10731), were acquired from Sigma and AdooQ BioScience, respectively. Transfection reagents, polyethylenimine (PEI, Sigma Cat No. 408727) and GenJet in vitro DNA tranfection reagent for Huh-7 Cells (SignaGen, Cat No. SL100489-HUH) were purchased from Sigma and SignaGen, respectively. Hydrogen peroxide (Cat No. 1100066) was purchased from International Laboratory, USA). Apoptotic cells were detected by by TUNEL staining using In situ cell death detection kit, TMR red (Roche, Cat no. 12156792910).
For sample preparation for electron microscopy (EM), fixatives glutaldehyde, EM Grade (Electron Microscopy Sciences, Cat No. 16200) and osmium tetroxide (Electron Microscopy Sciences, Cat No. 19110); embedding medium, Embed-812 embedding kit (Electron Microscopy Sciences, Cat No. 14120); and stains, uranyl acetate (Electron Microscopy Sciences, Cat No. 22400) and lead citrate (Electron Microscopy Sciences, Cat No. 17800) were purchased from Electron Microscopy Sciences.

To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x10⁵ cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na₃VO₄, Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [22 (link)]; using Catalase, Sigma, Cat. no. C1345 and H₂O₂ Sigma, Cat. no. 31642) for 1 hour and lysed. Following concentrations of inhibitors were used: DMSO (Sigma-Aldrich, Cat. no. D2438, solvent control), 10 μM erlotinib (Tarceva, Molecula; Cat. no. 89983631), 0.5 μM afatinib (Selleckchem, Cat. no. S1011); 5 μM gefitinib (CellSignaling; 4765), 5 μM lapatinib (Selleckchem, Cat. no. S1028), 300 nM flavopiridol (Selleckchem, Cat. no. S1230), 5 μM arry380 (Gentaur Europe, Cat. no. A8366), 10 μM Nu7441 (Selleckchem, Cat. no. S2638) and 10 μM ruxolitinib (Selleckchem, Cat. no. S1378).

Primary as well as immortalized eIF2αP-proficient or deficient MEFs, IMR90, BJ-hTERT, HT1080, and A549 cells were generated and maintained as described previously [18 (link), 19 (link)]. ATF4 KO MEFs as well as HT1080 cells expressing ATF4 shATF4 were established as previously described [18 (link)]. LATS1 KO MEFs were previously reported [52 (link)]. The shRNA-mediated knockdown of LATS1 in HT1080 cells was performed with as previously described targeting vector [53 (link)]. Cells were cultured in Dulbecco modified Eagle medium (DMEM; Wisent) supplemented with 10% fetal bovine serum (FBS; Gibco), antibiotics (100 U/ml of penicillin-streptomycin; Gibco) and 2.5 μg/ml of puromycin (Sigma). H₂O₂ was purchased from Bioshop, Canada; thapsigargin, trolox, phenylarsine oxide (PAO) and propidium iodide were obtained from Sigma; MG132 was purchased from Enzo Life Sciences. In control treatments, an equivalent volume of the solvent of each drug was added in culture.

All reagents were of analytical grade. Milli-Q water (Millipore, Bedford, MA, USA) was used throughout the experiment. Trizma^® HCl and Trizma^® Base, phenazene methosulfate, decylubiquinone, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and phenylarsine oxide (PAO) were purchased from Sigma (St. Louis, MO, USA). Sodium arsenite (iAs^III), dimethylarsinic acid [(CH₃)₂AsO(OH)] (DMA^V) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Monomethylarsonic acid (MMA^V) was obtained from Tri Chemicals (Yamanashi, Japan). The arsenic standard solutions were stored in the dark at 4 °C. Diluted standard solutions for experiments were prepared daily prior to use.

ITC experiments were carried out at 25 ± 0.2 °C by a MicroCal PEAQ-ITC calorimeter. Arsenic binding flagellin variants in monomeric form were prepared in 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) ((Sigma Aldrich), 150 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma Aldrich) (pH 7.0). Before the measurement 10% v/v DMSO was added to the solution. As a titrant, phenylarsine oxide (PAO) (Sigma Aldrich) was first dissolved in pure dimethyl sulfoxide (DMSO) (Sigma Aldrich) to an appropriate concentration then diluted 10 times with 100 mM HEPES, 150 mM NaCl (pH 7.0) buffer containing 1 mM TCEP. A typical experiment consisted of a ~ 0.5 mM PAO solution titrated into a ~ 5 µM protein solution in the same buffer. Interaction between PAO and the buffer or the buffer and protein was checked by PAO-to-buffer and buffer-to-protein titrations, respectively. The ITC data were fitted to a one-binding-site model with the MicroCal PEAQ-ITC Analysis Software package provided by MicroCal, using non-linear least-squares algorithm.