Ammonium tetrathiomolybdate

Methyl-2-pyrrolidone (NMP, 99.5 wt%), polyvinylidene fluoride (PVDF, 99.5 wt%), elemental sulfur (99.5 wt%), lithium sulfide (Li₂S, 99.98 wt%), 1,3-dioxolane (DOL, 99.8 wt%), 1,2-dimethoxyethane (DME, 99.5 wt%), tetraglyme (99 wt%), lithium bis (trifluoromethanesulfonyl) imide (LiTFSI, 99.95 wt%), lithium nitrate (LiNO₃, 99.99 wt%), cobalt chloride hexahydrate (CoCl₂·6H₂O), potassium permanganate (KMnO₄, 99.5 wt%), graphite powder, ammonium tetrathiomolybdate ((NH₄)₂MoS₄, 99.95 wt%), reduced graphene oxide (rGO, chemically reduced) were purchased from Sigma-Aldrich. Graphene oxide (GO, >99 wt%) was purchased from Aladdin reagent. Hydrochloric acid (HCl, 37 wt%), sodium nitrate (NaNO₃, 99 wt%), hydrogen peroxide (H₂O₂, 30 wt%) were purchased from Chuandong Reagent. Super P carbon (99.5 wt%) from Timcal were used as received. The carbon paper (HCP120, thickness ∼0.21 mm) was purchased from Shanghai HESEN Co., Ltd.

Cells were treated with 50 μM CuCl₂ (Acros Organics, Morris Plains, NJ) or 10 μM ammonium tetrathiomolybdate (TTM; Sigma-Aldrich, St. Louis, MO) by supplementing normal growth medium and inoculums, beginning at 24 hours prior to subsequent treatments, i.e. infection. TTM is an efficient intracellular copper chelator [31 (link), 32 (link)]. Intracellular copper concentrations in complete lysates of untreated, 10 μM TTM, and 50 μM CuCl₂ treatment of A549 cells were assessed by inductively coupled plasma mass spectrometry (ICP-MS) elemental analysis (courtesy of M. Ralle, Oregon Health & Science University). Cytotoxicity of CuCl₂ and TTM on cell viability was assayed by chemiluminescent ATP quantitation; CellTiter-Glo (Promega, Madison, WI). No decrease in luminescence was observed below concentrations of CuCl₂ or TTM at least 5 fold higher than used for this study. Additionally, the possible effect of these treatments on virion viability was assayed. Copper ions have previously been seen to inactivate H9N2 virions [24 (link)]. To determine if such inactivation was occurring in our conditions, inoculums were prepared as for infections and incubated in the presence of CuCl₂ or TTM but without cells. No effect on titer was observed at the concentrations used for this study.

CMA was isolated from the culture broth of Streptomyces sp. as described elsewhere13 (link)14 (link). Dp44mT, deferoxamine mesylate salt, ammonium tetrathiomolybdate (TM), and N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) were purchased from Sigma-Aldrich. Antimycin A was obtained from the RIKEN Natural Products Depository (NPDepo)40 (link).

Antimony(III) sulfide (99.995%. Sigma Aldrich), antimony(III) selenide (99.99%, Sigma Aldrich), tin powder (99.8%, 325 mesh, Acros Organics), selenium powder (99.999%, 200 mesh, Alfa Aesar), sulfur (99.998%, Sigma Aldrich), copper powder (99%, Alfa Aesar), ammonium sulfide solution (40–48 wt% in H₂O, Sigma Aldrich), chloroplatinic acid solution (8 wt% in H₂O, Sigma Aldrich), ammonium tetrathiomolybdate (99.97%, Sigma Aldrich), 2-[2-(5-methylfuran-2-yl)vinyl]-4,6-bis(trichloromethyl)-1,3,5-triazine (MFVT, TCI), N-(trifluoromethylsulfonyloxy)-1,8-naphthalimide (IM-NIT, WIMAS Corp.), ethylenediamine (≥99.5%, Sigma Aldrich), 1,2-ethanedithiol (≥98.0%, Sigma Aldrich), anhydrous acetonitrile (99.8%, Sigma Aldrich), anhydrous toluene (99.8%, Sigma Aldrich), N-methyl formamide (99%, Sigma Aldrich), anhydrous dimethyl sulfoxide (≥99.9%, Sigma Aldrich), ethyl alcohol (99.5%, Samchun), and methyl alcohol (99.5%, Samchun) were used in the experiments. All chemicals and reagents were used as received without further purification.