Q vd oph

The Approved Oncology Drugs Set VIII screening panel was obtained from the NCI-Chemotherapeutic Agents Repository, through Fisher Bioservices. Marizomib was obtained from Adipogen. N-acetyl cysteine (NaC) and Q-VD-OPh were obtained from Sigma–Aldrich. Carfilzomib was obtained from Selleckchem. Chloroquine diphosphate was obtained from Sigma–Aldrich.

Single cell suspensions of splenocytes were labeled with CellTrace Violet stain according to the manufacturer’s instructions (Life Technologies and concentrated to 4 × 10⁵–5 × 10⁵ cells in 96 flat-well plates in 200 μl T cell media (RPMI with 10% FCS, 10 mM HEPES, 50 mM 2-mercaptoethanol, 2 mM L-Gln, 1% non-essential amino acids (MEM), 100 units/ml penicillin, 100 mg/ml streptomycin and 1 mM sodium pyruvate) or B-cell media (same as T-cell media but DMEM instead of RPMI). All conditions contained 1 μM 4-OH TAM to induce the HMGCR deletion in vitro. For T-cell stimulation, the conditions contained 1 μg/ml αCD3, and 6 ng/ml αCD28 (BioXCell); for B-cell stimulation 20 μg/ml LPS (Sigma) was added. In some conditions (±)-mevalonolactone (Sigma), a cell-permeable version of GGPP-geranylgeraniol (Sigma), Q-VD-OPh (Sigma) and NBD-6 Cholesterol (Avanti) was used. The cultured cells were analyzed on days 1–3 via FACS analysis.

Dissociated DRG neurons were obtained after enzymatic digestion of brachial DRG from E11.5 embryos using trypsin (0.05% Trypsin–EDTA; Gibco). Cells were plated in 24-well plates pre-coated with poly-d-lysine (0.01%; Sigma)/laminin (10 μg/ml; R&D Systems). Cells or whole DRG were cultured in N2 medium (DMEM-F12/glutamax medium with N2 supplement; Gibco) supplemented with pen/strep, gentamicin and with NT3 (Peprotech) when specified. For in vitro RUNX3 induction in whole chicken DRGs, NT3 (10 ng/ml, R&D systems), RA (500 ng/ml, Sigma) and the pan-caspase inhibitor Q-VD-OPh (10 μM, Sigma) were used. For the survival assay using mouse E11.5 DRG neurons, the majority of neurons die during the first 24 h in the absence of NTs^{66 (link)}, and the addition of NT3 promotes selectively the survival of PSNs^{67 (link)}. For transcripts (qPCR) measurements, whole DRGs were cultured in 2-ml open tubes in closed Petri dishes for the indicated time.

Harvested HeLa cells were lysed in lysis-buffer (10 mM HEPES, 1.5 mM MgCl₂, 300 mM sucrose, 10 mM KCl, 0.5% NP40, Roche Complete Protease Inhibitor Cocktail (Roche, Vienna, Austria), 5 mM DTT prepared freshly from a 1 M stock) on ice for at least one hour. Proteins were separated by SDS/PAGE, transferred to a nitrocellulose membrane and incubated with the relevant antibodies: cleaved Caspase-3 ([Asp175] Cell Signalling), Parp (#9542, Cell Signalling), Hsp 90 α/β (sc-13119, Santa Cruz Biotechnology). Treatments: NoA compound (6.7 μM) for 12/24/36/48 h and Staurosporine 1 μM for 4 h (♯S5921, Sigma), co-treatment: pan-caspase inhibitor Q-VD-OPh (10 μM, ♯SML0063, Sigma) was added one hour before.

CPTIO or QVD-OPH (Sigma) was used at a final concentration of 50 mM and 50 μM, respectively, in 0.5% dimethylsulphoxide in fish water. Control fish were incubated in 0.5% dimethylsulphoxide only. Fish were incubated immediately following infection and fresh inhibitor was added every 24 hr until experiment end point.

Three or more independent experiments were performed for all experiments. Animals and cell cultures for each group were randomized with the website www.randomization.com. Treatment, data collection, and data analyses were blinded by using different investigators or by masking sample labels.
For in vitro experiments, we tested different dosages of Hemin (0, 10, 50, or 100 μM) and RSL3 (0, 2, 4, 8, 16, or 32 μM) at 12 and 24 h respectively. We added 2 μM Ferrostatin-1 (Fer-1, S7243, Selleck), 100 μM Deferoxamine (DFO, Y0001937, Sigma), 100 μM Necrostatin-1 (Nec-1, N9037, Sigma), 1 mM 3-Methyladenine (3-MA, HY-19312, MCE, USA), or 10 μM Q-VD-OPh (SML0063, Sigma) at the same time with Hemin or RSL3 based on previously published work [21 (link), 48 (link)]. We treated cells with 20 μM t-BOOH (458139, Sigma, USA) for 12 h, with or without Fer-1 or 0.5 mM GSH (G4251, Sigma, USA) at the same time. Cells were collected for PI staining, TUNEL staining, immunofluorescence staining, flow cytometry, GPx4 activity assay, RT-qPCR, and RNA-seq.
For in vivo experiments, Fer-1 (2 mg/kg) or vehicle (0.1% DMSO (D1418, Sigma), 2.5% PEG300 (202371, Sigma) and 0.25% Tween80 (P1754, Sigma) in saline) was injected daily (i.p.) for the initial 7 days beginning 2 h after IVH. The assessment included behavior tests, neuroimaging, histology, and TEM.