Mice were maintained in the Laboratory Animal Centre of the University of Helsinki. The National Animal Experiment Board in Finland approved animal experiments used in this study. We used the VEC-tTA/Tet-OS-Ang2 mouse line, which expresses mouse Ang2 under an inducible endothelial cell promoter19 (link). The driver VEC-tTA and responder transgenic mouse lines were bred together to obtain double-transgenic VEC-tTA/Tet-OS-Ang2 offspring. To overcome the embryonic lethality due to endothelial Ang2 overexpression in double-transgenic embryos, Ang2 expression was repressed during the entire pregnancy. Tetracycline (Sigma-Aldrich) at 2 mg ml−1 in 5% sucrose was added to the drinking water of pregnant females, starting at the time of mating and until birth, when Ang2 expression was induced by discontinuation of Tetracycline administration. Single-transgenic or WT littermates were used as controls for double-transgenic mice, both designated as WT. None of the control mice displayed any obvious phenotype.
Tetracycline
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Tetracycline is a type of antibiotic used for laboratory testing and research. It is a broad-spectrum antimicrobial agent effective against a variety of bacteria. Tetracycline is commonly used in microbiological studies and antimicrobial susceptibility testing.
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Lab products found in correlation
Chloramphenicol, by Merck Group (161 mentions)
Ampicillin, by Merck Group (143 mentions)
Erythromycin, by Merck Group (114 mentions)
Kanamycin, by Merck Group (110 mentions)
Ciprofloxacin, by Merck Group (103 mentions)
Gentamicin, by Merck Group (86 mentions)
Streptomycin, by Merck Group (80 mentions)
Vancomycin, by Merck Group (45 mentions)
Calcein, by Merck Group (34 mentions)
Rifampicin, by Merck Group (31 mentions)
Doxycycline, by Merck Group (29 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (27 mentions)
Penicillin g, by Merck Group (26 mentions)
Clindamycin, by Merck Group (26 mentions)
Amoxicillin, by Merck Group (26 mentions)
Streptomycin, by Thermo Fisher Scientific (25 mentions)
Trimethoprim, by Merck Group (23 mentions)
Penicillin, by Thermo Fisher Scientific (23 mentions)
Oxytetracycline, by Merck Group (22 mentions)
Norfloxacin, by Merck Group (22 mentions)
845 protocols using tetracycline
1
Inducible Overexpression of Angiopoietin-2 in Mice
2
Meningococcal Strain Cultivation and Genetic Manipulation
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Meningococcal strains were cultured under aerobic conditions with 5% CO2 at 37 °C on GC agar (GCA) or GC broth (GCB) (Oxoid) supplemented with 0.4% glucose, 0.01% glutamine, 0.2 mg of cocarboxylase per litre, and 5 mg of Fe(NO3)3 per litre. The wild-type strains and constructed mutants used in this study are shown in Supplementary Table S5 . Antibiotic selection for meningococcal mutants was performed on GCA containing 100 μg/ml of kanamycin (sulfate salt), 60 μg/ml of spectinomycin, 5 μg/ml of tetracycline or 2 μg/ml of erythromycin (Sigma). Escherichia coli DH5α was used as a host for all DNA manipulations and Rosetta™ (DE3) as a host for all protein expression. E. coli strains were routinely grown on Luria-Bertani broth (LBB) and agar (LBA, Oxoid) which, where appropriate, was supplemented with antibiotics at the following concentrations: ampicillin at 100 μg/ml, spectinomycin at 50 μg/ml, kanamycin at 50 μg/ml, erythromycin at 300 μg/ml, tetracycline at 12.5 μg/ml and chloramphenicol at 30 μg/ml (Sigma).
3
Tetracycline Effects on Cell Cultures
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Tetracycline (Sigma-Aldrich, St. Louis, United States) at concentrations of 0, 25, 50, 100, 250, 500 μg/mL, drug-containing culture medium were prepared by dissolving Tetracycline powder into DMEM cell culture medium for cell survival rate, colony-forming efficiency, cell migration test and proliferation ability test. Osteogenic induction solution and adipogenic induction solution containing the above concentrations of Tetracycline were prepared specifically. The composition of each kind of solutions is shown in Table 1 .
4
Antimicrobial Phytochemical Evaluation
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Phytochemicals (berberine chloride, 8-hydroxyquinoline, salicylic acid, tannic acid, and sanguinarine chloride) and their synthetic analogs [chloroxine (5,7-dichloroquinolin-8-ol), nitroxoline (5-nitroquinolin-8-ol), ferron (7-iodo-8-hydroxyquinoline-5-sulfonic acid), bismuth subsalicylate, and zinc pyrithione], as well as antibiotics (ceftriaxone sodium, ciprofloxacin, chloramphenicol, metronidazole, tetracycline, and vancomycin hydrochloride), used in this study were purchased from Sigma-Aldrich (Prague, Czech Republic). Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Prague, Czech Republic) was used to prepare the stock solutions of all test compounds, except those of metronidazole, salicylic acid, vancomycin, and zinc pyrithione, which were prepared using distilled water. Stock solutions of chloramphenicol, tannic acid, and tetracycline were prepared using 96% ethanol (Sigma-Aldrich, Prague, Czech Republic). The chemical structures of individual compounds tested are shown in Figure 3 .
5
Assessing Antibiotic Resistance in Bacteria
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In addition to being plated on selective agar, 100 μL from each dilution of EC and PBS was also plated onto nutrient agar (DifcoTM, BD), MacConkey containing tetracycline (16 μg/mL, Sigma-Aldrich (SA), Saint Louis, MO, USA), ceftiofur hydrochloride (8 μg/mL, SA), or ampicillin (32 μg/mL, SA), or m-Enterococcus containing tetracycline (16 μg/mL), ceftiofur hydrochloride (8 μg/mL), or ampicillin (concentration of 16 μg/mL). Plates were incubated and counted; isolates were characterized and saved as described above. Isolates were considered resistant if they grew on their respective media containing antibiotics. This design is shown in Figure 1 . The antibiotic concentrations utilized in the plates are the CLSI breakpoints for the bacteria–antibiotic combination being tested. This was done to assess bacterial growth and resistance of human implication following veterinary administration of florfenicol. The MIC interpretive CLSI breakpoints can be found in Table 1 .
6
Isolation and Characterization of Anthracnose Fruit Rot Fungus
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Seventeen fields were sampled for anthracnose fruit rot infection in the major production areas in Trinidad (see Tables 1 and 2 for sampling locations). Symptomatic fruit with diagnostic symptoms were collected and surface disinfested by rinsing in 70% ethanol for 1 min followed by another rinse in 0.6% sodium hypochlorite solution for 1 min. Samples were then washed three times in sterilized distilled water and dried on sterilized tissue paper in a laminar flow hood. A 4-mm3 block of tissue was cut from the edge of the lesion and placed in the center of STEA plates (2% water agar amended with 50 mg/l streptomycin and 50 mg/l tetracycline [Sigma-Aldrich Co., St. Louis, MO, USA] and 5% absolute ethanol). STEA plates were incubated for five days at 25°C in the dark. After incubation, a 4-mm3 block of agar taken from the advancing mycelial edge of a 5-day old STEA culture was removed and placed in the center of a potato dextrose agar plate (PDA; Oxoid Ltd., Basingstoke, UK) supplemented with 50 mg/l streptomycin and 50 mg/l tetracycline (Sigma-Aldrich Co.). Cultures were incubated for seven days at 25°C in the dark. Monoconidial cultures were subsequently obtained through serial dilution. Isolates were maintained on PDA slants at 4°C for temporary storage, and as conidial suspensions in 50% glycerol at −70°C for long-term storage.
7
Conditional Ang2 Expression in Mice for Vascular Studies
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The VE-cadherin-tTA (TET-Off) driver mice where generously provided by Dr. Laura Benjamin (Sun et al. 2005 (link)). The TetO-Ang2 responder has been previously described in detail (Holopainen et al. 2012b (link)) and was backcrossed to C57Bl/6J for at least seven generations in this study. The driver and responder transgenic mouse lines were crossed to obtain DTG VE-cadherin;TetO-Ang2 offspring. Ang2 expression in DTG embryos was repressed by 2 mg/mL tetracycline (Sigma-Aldrich) in 5% sucrose in the drinking water for the pregnant females, starting within 2 d post-coitum, until Ang2 expression was induced by discontinuing tetracycline administration. The tetracycline-containing water was changed every 2–3 d. Single transgenic or wild-type littermates were used as controls for DTG mice or embryos. Ang2−/− mice were bred as previously reported (Gale et al. 2002 (link); Fiedler et al. 2006 (link)). Eight-week-old male mice or P5 pups were analyzed for their LEC junctions. The Pdgfb-Cre-EGFP mice have been previously described (Claxton et al. 2008 (link)). PDGFB was visualized by staining for EGFP. The embryonic time points were determined according to vaginal plugs. The Committee for Animal Experiments of the District of Southern Finland approved all animal experiments.
8
Growth of M. synoviae Strains
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M. synoviae strains/transformants, listed in Table 1 , were grown to late exponential phase (~ pH 6.8) at 37°C, unless otherwise stated, in mycoplasma broth (MB) as described previously [26 ], except MB and mycoplasma agar (MA) contained 3 μg mL –1 tetracycline (Sigma, St. Louis, Missouri, USA) for the growth of tetracycline resistant M. synoviae strains transformed with pMAS-LoriC and pKS-VOTL plasmids. Escherichia coli α-Select (DH5 α derivative) competent cells (Bioline, Alexandria, New South Wales, Australia) were used as host for gene cloning and propagation of different plasmids following manufacturer’s instructions.
9
Preparation and Storage of RNAi Library Clones
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Plates from Ahringer’s RNAi library containing the clones of interest (purchased from the Medical Research Council (MRC) GeneService) were replicated to Luria-Bertani (LB) agar supplemented with ampicillin (100 μg/ml, Sigma-Aldrich) and tetracycline (15 μg/ml, Sigma-Aldrich) using a pin replicator (Boekel) and grown overnight at 37 °C. The day after, the selected clones were inoculated in 1.3 ml of LB supplemented with ampicillin (100 μg/ml, Sigma-Aldrich), tetracycline (15 μg/ml, Sigma-Aldrich) and 8% glycerol in deep well plates (VWR) and incubated overnight at 37 °C with shaking (180 rpm, New Brunswick™ Innova® 44/44R incubator shaker). The last column of the plates was left free for convenient control. The next day, 120 μl of the culture was transferred to microtitre plates using a Precision XS Microplate Sample Processor (Biotek, Winooski, VT, USA) and frozen at −80 °C.
10
Measurement of H2O2 Levels in Fibroblasts
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H2O2 levels were measured using ROS-Glo™ H2O2 Assay (Promega Corporation) according to manufacturer instruction. Briefly, fibroblasts were plated on 96-well white plate with a clear bottom and cultured with or without 10 ng/mL TGF-β2 for 5 days. As control of the assay, tetracycline-inducible NOX4 overexpression system NOX4 T-Rex™ cells were incubated with tetracycline (1 μg/mL, Sigma) for 24h to induce NOX4 expression [40 (link)]. Detection of H2O2 was performed by incubating cells with H2O2 Substrate Solution for 6h at 37 °C. Then, cells were lysed with Detection Solution for 20 min in RT, after which luminescence was measured using SpectraMax L 96w at READS unit, University of Geneva, Switzerland.
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