All strains used in this study are listed in Tab.S1. Y. lipolytica and E. coli strains were maintained as described in [25 (link),26 ]. E. coli JM109 strain, used for sub-cloning, and its derivatives were grown at 37 °C with 250 rpm shaking in LB medium ((g L − 1): yeast extract (BTL, Lodz, Poland), 5; bactopeptone (BTL), 10; NaCl (POCh, Gliwice, Poland), 5), supplemented with kanamycin (Sigma Aldrich, Merck KGaA, St. Louis, USA, 40 (μg mL−1)) and agar (Biomaxima, Lublin, Poland; 15 (g L − 1)), when required. Y. lipolytica Po1f strain (ura‐ leu‐, ATCC MYA2613) was used as a parental strain for co-transformations. Y. lipolytica Po1h strain (ura-, leu+) transformed with a URA3 marker cassette was used as a negative control strain for assessment of background fluorescence. Yeast strains were routinely maintained in a minimal yeast nitrogen base medium (YNB; (g L − 1): YNB (Sigma-Aldrich, Merck KGaA), 1.7; (NH4)2SO4 (PoCh), 5; glucose (PoCh), 20), or in a rich yeast extract-peptone-dextrose medium (YPD; (g L − 1): yeast extract, 10; bactopeptone, 20; glucose, 20), solidified with agar (15 (g L − 1)) when required, at 30 °C and with 250 rpm shaking, for liquid cultures.
Bacto peptone
Manufactured by Merck Group
Sourced in Germany, United States, Poland
Bacto-peptone is a complex nutrient mixture used as a component in culture media for the cultivation of microorganisms. It provides organic nitrogen and growth-promoting substances to support microbial growth and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Bacto yeast extract, by Merck Group (6 mentions)
Yeast extract, by Merck Group (6 mentions)
Agar, by Merck Group (4 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Glucose, by Merck Group (3 mentions)
Bacto agar, by Merck Group (2 mentions)
Agar, by Biomaxima (2 mentions)
Kanamycin, by Merck Group (2 mentions)
D glucose, by Merck Group (2 mentions)
Beef extract, by Merck Group (2 mentions)
D glucose, by Chempur (2 mentions)
4 nitroquinoline n oxide, by Merck Group (2 mentions)
Dextrose, by Merck Group (2 mentions)
Agar agar, by Merck Group (2 mentions)
α d glucose, by Merck Group (2 mentions)
Sodium chloride (nacl), by Chempur (2 mentions)
Adenine hemisulfate, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Shields and sang m3 insect medium, by Merck Group (1 mentions)
Shields and sang m3 medium, by Merck Group (1 mentions)
22 protocols using bacto peptone
1
Yeast and Bacterial Strain Maintenance
2
Autophagy Induction in Budding Yeast
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All experiments were performed at 30°C. Budding yeast (S. cerevisiae) were cultured in YPA (1% Bacto-yeast extract (Y), 2% Bacto-peptone (P), 0.025% adenine hemi-sulfate (A; Sigma)) supplemented with 2% glucose (D; Sigma) or in csm (complete supplement mixture; Sunrise Science Products) to maintain selection for centromeric plasmids. For one experiment, we used S. pombe strain MKSP3186, which was cultured overnight in YE5S (yeast extract (YE), 250 mg/L adenine, histidine, leucine, uracil, and lysine hydrochloride).
To induce autophagy by nitrogen starvation, cells in log phase were washed and resuspended in synthetic defined medium lacking nitrogen (sd-N) (0.17% Difco Yeast Nitrogen Base without amino acids and ammonium sulfate, and 2% D). Samples were collected at time points indicated in the figures and were prepared for extraction of RNA, fluorescence microscopy, or immunoblotting as described below.
To induce autophagy by inhibiting Tor1 kinase by treatment with rapamycin, rapamycin (in DMSO; Sigma-Aldrich) was added to cells in log phase to a final concentration of 250 ng/mL. Samples collected at time points indicated in the figures were prepared for extraction of RNA for RT-qPCR.
To induce autophagy by nitrogen starvation, cells in log phase were washed and resuspended in synthetic defined medium lacking nitrogen (sd-N) (0.17% Difco Yeast Nitrogen Base without amino acids and ammonium sulfate, and 2% D). Samples were collected at time points indicated in the figures and were prepared for extraction of RNA, fluorescence microscopy, or immunoblotting as described below.
To induce autophagy by inhibiting Tor1 kinase by treatment with rapamycin, rapamycin (in DMSO; Sigma-Aldrich) was added to cells in log phase to a final concentration of 250 ng/mL. Samples collected at time points indicated in the figures were prepared for extraction of RNA for RT-qPCR.
3
Cultivation of Common Microbial Strains
4
Drosophila Cell Culture Protocols
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Cell culture was done according to the protocols established by the DGRC (Drosophila Genome Resource Center). S2-R+ cells were grown in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin. Kc167 cells were grown in Shields and Sang M3 insect medium (Sigma) supplemented with 0.25% Bacto Peptone, 0.1% Yeast Extract, and 10% FBS. BG3-c2 cells were grown in Shields and Sang M3 medium (Sigma) supplemented with 10% FBS and 20 µg/ml insulin (Sigma). 20-hydroxyecdysone (20-HE) stimulation was done by adding 20-HE at the final concentration of 5 µM and incubating cells for the indicated times. The presence of mycoplasma in the cell lines used in this study (S2-R+, Kc167 and BG3-c2) was tested by Universal Mycoplasma Detection Kit (ATCC, #30-1012K) and no mycoplasma contamination was detected.
5
Caenorhabditis elegans Larval Growth Assay
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Bristol N2 (wild type) Caenorhabditis elegans obtained from the Caenorhabditis Genetic Center were used in the larval development and growth assay. The transgenic C. elegans strain PE255 [ ; rol-6(su1006)] used in the ATP assay (Lagido et al. 2009 (link)) was also obtained from the Caenorhabditis Genetic Center; this strain expresses luciferase to measure in vivo ATP levels and GFP (green fluorescent protein) as a marker of live animals. All nematodes were maintained at 20°C on K-agar plates seeded with OP50 Escherichia coli (Caenorhabditis Genetic Center) as a food source (Williams and Dusenbery 1988 (link)), or as age-synchronized cultures of first stage larvae (L1s) by hatching embryos in the absence of food, causing L1 development to arrest, as previously described (Boyd et al. 2009 (link)). The K-agar plates are made at NIEHS from 2% bacto-agar, 0.25% bacto-peptone, sodium chloride, potassium chloride, and cholesterol (Sigma-Aldrich); complete K-medium consists of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and cholesterol was also made at NIEHS.
6
Cryptococcus neoformans Transformation
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C. neoformans var. grubii type strain H99 was stored in 15% glycerol at -80°C until use, at which point it was maintained on YPD at 4°C for a maximum of two weeks; this strain was used for all transformations. C. neoformans was cultured in liquid (2% bacto-peptone, 1% yeast extract, and 2% glucose (Sigma, USA)) or solid (2% agar added) YPD media at 30°C. Biolistic transformations were performed on solid YPD media supplemented with 1 M sorbitol (Sigma, USA), and transformants were selected on solid YPD medium containing 100 μg/mL G418 (Sigma, USA). Adenine auxotrophy was detected on YNB media (0.45% yeast nitrogen base w/o amino acids and ammonium sulfate, 2% glucose, 0.5% ammonium sulfate, 2% agar (Sigma, USA)), and melanin production on L-DOPA media (Sigma, USA) [28 (link)].
7
Proteomics Analysis of Platelet Activation
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Bactopeptone, bis-benzimide, glutaraldehyde, penicillin, propidium iodide, sodium citrate, streptomycin, trypsin, and yeast extract were acquired from Sigma Aldrich (Saint Luis, MO, USA); anti CD61-PerCP, anti CD62-PE, and PAC-1-FITC (Becton-Dickinson, Franklin Lakes, NJ, USA); Hoechst 33342 from Santa Cruz Biotechnology (Dallas, TX, USA); McCoy’s 5A medium and fetal bovine serum (FBS) from Biowest (Nuaillé, France) and XTT test from Biotium (Fremont, CA, USA). Bromophenol blue, dithiothreitol (DTT), glycerol, iodoacetamid, Immobiline Dry Strip gels, pH 4–7, Precast 12.5% polyacrylamide gel and buffer for 2-D DIGE, sodium dodecyl sulfate (SDS), Tris-HCl, UREA, (GE Healthcare, Little Chalfont, UK). Refraction-2D Labeling Kit and DyeAgnostics was obtained from LKB Biotech, Warsaw, Poland. All other reagents were obtained from POCH SA (Gliwice, Poland). Standard polystyrene flasks (T-75 flasks) and flat bottom cell culture microplates (12, 24, 48 and 96 wells) were from TPP Techno Plastic Products AG (Trasadingen, Switzerland). All other disposables were from VWR Int. (Gdansk, Poland).
8
Polyphenol Analytical Procedures via HPLC
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Reagents of analytical grade used for HPLC analyses included the standards trans-piceid, ɛ-viniferin, and trans-resveratrol (Sigma-Aldrich, St. Louis, MI, USA). Other used reagents were methyljasmonate (MeJA), cis-jasmonate (cJA), dihydrojasmonic acid (2HJA), cellulase from Trichoderma viride (TvC), sodium orthovanadate (NaVO) (Sigma-Aldrich, St. Louis, MI, USA), naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), sucrose, agar, yeast extract, bacto-peptone, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, sodium dodecylsulphate (SDS) (Sigma-Aldrich, St. Louis, MI, USA; Duchefa BIOCHEMIE B.V, Netherlands; Imuna, Slovakia, Merck, Germany). Water was purified by the Direct-Q® 3 UV Water Purification System (Merck Millipore, Darmstadt, Germany). Reference standard solutions of polyphenols were prepared in mixture methanol:water (1:1, v/v) and stored at 4 °C in dark.
9
Spore Suspension Preparation from Molds
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Molds were cultured on MEA at 25°C for 7-14 days. Spores were suspended in saline peptone (SPO) solution (per liter; 5 g NaCl (Merck), 0.3 g Na2HPO4⋅2H2O (Merck), 1 g bactopeptone (BD) with pH adjusted to 5.3 ± 0.1) containing 0.01% Tween 80 (Sigma-Aldrich, St. Louis, United States) followed by filtering through six layers of sterilized gauze to remove hyphal fragments. Spore concentrations were estimated by bright field microscopy (Olympus BX40, Japan) using a Neubauer counting chamber and adjusted to a final concentration of 104 spores/mL using SPO solution.
10
Antifungal Drug Resistance Profiling
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The isolates used are listed in Table S1 . The isolates were maintained on YPD agar (1% Bacto Yeast Extract [212750, Sigma], 2% Bacto Peptone [211677, Sigma], 2% Bacto Agar [214010, Sigma], 2% D-[+]-Glucose [G8270, Sigma]), and liquid cultures were grown in 5 mL YPD broth without agar at 30°C and 200 rpm shaking overnight. For serial dilutions, 0.5 mL of the overnight cultures were harvested at 13,000 rpm at room temperature for 1 min, washed twice in 0.5 mL of phosphate-buffered saline (PBS) buffer (BR0014G, Thermo Fisher), resuspended in 0.5 mL PBS, and diluted to A600 = 0.0625 (approximately 6.25 × 105 cells/mL) in PBS. Five-fold serial dilutions were made in PBS and transferred with a pinner to YPD agar plates containing miltefosine (M5571, Sigma-Aldrich) or fluconazole (F8929, Sigma-Aldrich) at the indicated concentrations. The pinned plates were incubated at 30°C for the indicated times and photographed using a Singer PhenoBooth.
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