Cycloheximide (chx)

3-Methylcholanthrene (3MC), indole-3-carbinol (I3C), indole-3-acetate (IAA), cycloheximide (CHX), and β-naphthoflavone (βNF) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Indirubin was kindly provided by Dr. Tomonari Matsuda (Kyoto University, Kyoto, Japan). MG-132 was purchased from Abcam (Tokyo, Japan). All other chemicals and solvents used were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ligands were dissolved in dimethyl sulfoxide (DMSO) and added to media. The final concentration of DMSO was adjusted to 0.1% (v/v) in culture media.

HUVECs were treated with CONT or CUL3 siRNA and then incubated for 72 h. After treatment with 25 μg/mL cycloheximide (Wako, Osaka, Japan), the cells were lysed at various time points (0, 2, 4, 6, 9, and 12 h). Western blot analyses for DAXX were performed as described.

Cycloheximide, nocodazole, sodium azide, chloramphenicol, rifampin, nalidixic acid, and a cocktail of protease inhibitors cocktail were purchased from Wako. Cytochalasin D was purchased from Focus Biomolecules (Plymouth Meeting, PA) and staurosporine from Cayman Chemical Company (Ann Arbor, MI). The solvents used and final concentrations were as follows: Cycloheximide, 100 μg/ml in dimethyl sulfoxide (DMSO); nocodazole, 10 μg/ml in DMSO; sodium azide, 50 mM in PBS; chloramphenicol, 5 μg/ml in ethanol; rifampin, 0.25 mg/ml in DMSO; nalidixic acid, 5 μg/ml in 1N NaOH; protease inhibitor cocktail containing aminoethyl benzylsulfonyl fluoride (AEBSF), 100 mM in aprotinin, 80 μM in DMSO; E-64, 1.5 mM in DMSO, leupeptin, 2 mM, bestatin, 5 mM, pepstatin, 1 mM; Cytochalasin D, 1 μg/ml in DMSO; and staurosporine, 1 μM in ethyl acetate. All chemicals and solvents were tested at a concentration used for possible adverse effects in Ca9-22 cells, as compared with cells without the inhibitor, by examining the morphology of the cells and cell viability with MTT assay (S2 Fig). Moreover, all chemicals and solvents were tested at the appropriate concentrations and found to produce no reduction in P. gulae growth (S3 Fig).

Carnosic acid, carnosol, and rosmarinic acid were supplied by Nagase Co LTD. (Kobe, Japan). Piciferic acid was purchased from Tokyo Chemical Industry (Tokyo, Japan). Dimethylsulfoxide, actinomycin D, cycloheximide, tunicamycin, and hemin were obtained from Wako Pure Chemicals (Osaka, Japan). The tert-butylhydroquione (tBHQ) was obtained from Kanto Chemical (Tokyo, Japan). ISRIB was obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against ATF4 (CREB2) (C-20), Nrf2 (H-300), lamin B (M-20), and HSP90α/β (AC88) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and primary antibodies against eIF2α (9722S) and phospho-Ser51 eIF2α (9721S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies against rabbit and goat immunoglobulin G (IgG) were obtained from Life Technologies (Carlsbad, CA, USA) and Santa Cruz Biotechnology, respectively. hemin was purchased from ICN Biomedicals (Irvine, CA, USA). hemin stock solution (100×) was made by dissolving hemin in 20 mM NaOH.

The purity of all chemicals was >98%. ArtC was isolated and purified from Brazilian propolis [16 (link), 17 ]. In brief, propolis originating from Baccharis dracunculifolia was collected in Minas Gerais, Brazil in 2008, and stored at 4°C until use. The propolis sample was extracted with methanol, and pure ArtC was isolated from the extract by repeated column chromatography. The purity of the compound was >99% as determined from its ¹H-NMR spectrum. Dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), triiodothyronine (T3), and cycloheximide were obtained from Wako Pure Chemical Industries (Osaka, Japan). Rabbit polyclonal β-actin antibody (#4967) and rabbit polyclonal cytochrome c oxidase subunit IV (CoxIV) antibody (#4844) were obtained from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal UCP1 antibody (ab10983) and rabbit polyclonal PRDM16 antibody (ab106410) were obtained from Abcam (Tokyo, Japan).

A spot dilution assay was conducted to estimate the relative resistance of each yeast strain to fluconazole or cycloheximide [37 (link), 38 (link)]. Three independently derived ρ⁰ cells from each yeast strain were aerobically grown to an OD₆₀₀ of 0.6–0.9 at 30 °C in YPD medium. Five microliters of 10-fold serial dilutions of the logarithmic phase cultures containing the same number of cells were spotted on YPD plates containing or not containing 20 μg/mL fluconazole (Nacalai Tesque) (or 0.5 μg/mL cycloheximide (Wako)) and incubated at 30 °C for 7 days. Representative plate images of three replicates were captured after culturing at 30 °C for 7 days.