Cells were seeded into 25 cm2 flasks and grown for 3 h, then supplemented with 2 mM Thymidine (Sigma). Cells were released 16 h later by washing 2 times with prewarmed WT medium. 8 h later, cells were supplemented with 3 μg/ml aphidicolin (Sigma) and 2 mM 4sT (Carbosynth). Cells were released 16 h later by washing 2 times with PBS and addition of medium containing 2 mM 4sT (Carbosynth). For Sororin-AID experiments, 500 μM Indole-3-acetic acid (Sigma) was added 1 h prior to S-phase release. For S-phase release experiments, samples were taken at the indicated time-points. For synchronization to G2 and prometaphase, after 4 h release, 9 μg RO-3306 (Sigma) or 200 ng/ml nocodazole (Sigma) were added respectively. For NIPBL-AID experiments, 500 μM Indole-3-acetic acid (Sigma) was added 8 h after released. Samples were processed 16 h later. Cells were harvested by washing with PBS, followed by trypsinisation and resuspension in WT medium. Cells were then spun down, washed again with PBS, followed by fixation for 4 min in 1 % formaldehyde (Sigma). Cell pellets were stored at -20 °C or processed immediately.
Indole 3 acetic acid
Manufactured by Merck Group
Sourced in United States, Germany, France
Indole-3-acetic acid is a chemical compound that functions as a plant growth regulator. It is a naturally occurring auxin found in plants and microorganisms. Indole-3-acetic acid plays a role in various plant developmental processes, including cell elongation, root initiation, and fruit development.
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Lab products found in correlation
Formic acid, by Merck Group (6 mentions)
Indole 3 propionic acid, by Merck Group (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Abscisic acid, by Merck Group (4 mentions)
Methanol, by Merck Group (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Indole 3 lactic acid, by Merck Group (3 mentions)
Kinetin, by Merck Group (3 mentions)
Indole 3 butyric acid, by Merck Group (3 mentions)
Indole 3 acetamide, by Merck Group (3 mentions)
Ro 3306, by Merck Group (3 mentions)
Thymidine, by Merck Group (3 mentions)
Indoxyl sulfate, by Merck Group (3 mentions)
Indole, by Merck Group (3 mentions)
Glucose, by Merck Group (3 mentions)
Sucrose, by Merck Group (3 mentions)
Indole 3 carboxylic acid, by Merck Group (2 mentions)
Auxin, by Merck Group (2 mentions)
68 protocols using indole 3 acetic acid
1
Cell Cycle Synchronization Protocol
2
Cell Cycle Synchronization Protocol
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Cells were seeded into 25 cm2 flasks and grown for 3 h, then supplemented with 2 mM Thymidine (Sigma). Cells were released 16 h later by washing 2 times with prewarmed WT medium. 8 h later, cells were supplemented with 3 μg/ml aphidicolin (Sigma) and 2 mM 4sT (Carbosynth). Cells were released 16 h later by washing 2 times with PBS and addition of medium containing 2 mM 4sT (Carbosynth). For Sororin-AID experiments, 500 μM Indole-3-acetic acid (Sigma) was added 1 h prior to S-phase release. For S-phase release experiments, samples were taken at the indicated time-points. For synchronization to G2 and prometaphase, after 4 h release, 9 μg RO-3306 (Sigma) or 200 ng/ml nocodazole (Sigma) were added respectively. For NIPBL-AID experiments, 500 μM Indole-3-acetic acid (Sigma) was added 8 h after released. Samples were processed 16 h later. Cells were harvested by washing with PBS, followed by trypsinisation and resuspension in WT medium. Cells were then spun down, washed again with PBS, followed by fixation for 4 min in 1 % formaldehyde (Sigma). Cell pellets were stored at -20 °C or processed immediately.
3
Analytical Method for Indole Compounds
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Indole-3-carboxylic acid (3ICA, 99%), indole-3-acetic acid (3IAA, 98%), indole-3-propionic acid (3IPA, 99%), indole-3-lactic acid (3ILA, 99%), 5-hydroxyindole-3-acetic acid (5HIAA, ≥98%), 2,3,4,5,6-D5-benzoic acid (internal standard, D5-BA, ≥99 atom % D, ≥99%), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, contains 1% trimethylchlorosilane, 99% BSTFA), N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA, >97%), formic acid (≥95%), hexane (≥97.0%), methanol (≥99.9%) were purchased from Sigma-Aldrich (Darmstadt, Germany); sulfuric acid, acetone, diethyl ether were Laboratory Reagent grade and obtained from Khimreactiv (Staryy Oskol, Russia).
4
Auxin-Mediated Worm Manipulation
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Auxin treatment was performed by transferring worms to bacteria-seeded NGM plates containing 1 mM (if not specified) or 0.5 mM Auxin (Auxin: indole-3-acetic acid, Sigma). The preparation of Auxin stock solution (400 mM in ethanol) and Auxin-containing NGM plates was performed as previously described (Zhang et al., 2015 (link)). In all experiments, worms were also transferred to NGM plates containing 0.25% or 0.125% of ethanol (EtOH) to serve as the mock-treated control for 1 and 0.5 mM Auxin respectively.
5
Auxin-Induced Degradation of UBC9
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HT1080 cells, stably expressing UBC9 fused to an auxin-inducible degron were used (manuscript in preparation). Degradation of the expressed UBC9-auxin degron fusion, leading to impaired sumoylation, was induced by adding auxin, indole-3-acetic acid (Sigma; 200 µM final), for 24 hr to the cell culture medium (DMEM + Glutamax, Gibco).
6
Plant Growth Regulation: A Comprehensive Protocol
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6-Benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), kinetin (KIN), Murashige and Skoog (MS) basal medium, and mercury (II) chloride (HgCl2), were acquired from Sigma-Aldrich, Germany. Dimethyl sulfoxide, acetone, and additional solvents and reagents were bought from VWR International, Belgium.
7
Indole-3-Acetic Acid Assimilation in Caballeronia glathei
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All IAA derivatives used in this study (indole-3-acetic acid, 3-(2-hydroxyethyl)indole, ethyl-3-indole-acetate, 3-(3-hydroxypropyl)indole, 3-indoleacetonitrile, indole-3-acetamide, indole-3-butyric acid, DL-indole-3-lactic acid, indole-3-propionic acid, tryptamine, indole-3-acrycil acid and indole-3-carboxylic acid) and H218O were purchased from Sigma-Aldrich. All cloning and protein expression reagents were from ThermoFisher Scientific (Vilnius, Lithuania). All other reagents used in this study were of analytical or higher grade.
Caballeronia glathei strain DSM50014 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany. This strain was routinely cultivated in M1 medium (5 g L−1 peptone, 3 g L−1 meat extract, pH 7). For the IAA assimilation experiments, C. glathei DSM50014 was cultivated in M9 medium (3.5 g L−1 Na2HPO4, 1.5 g L−1 KH2PO4, 2.5 g L−1 NaCl, 0.2 g L−1 MgSO4, 0.01 g L−1 CaCl2). Escherichia coli strains used in this study are listed inSupplementary Table S2 . All E. coli strains were cultivated in LB medium. Ampicillin (50 μg mL−1) and streptomycin (30 μg mL−1) were added when necessary.
Caballeronia glathei strain DSM50014 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany. This strain was routinely cultivated in M1 medium (5 g L−1 peptone, 3 g L−1 meat extract, pH 7). For the IAA assimilation experiments, C. glathei DSM50014 was cultivated in M9 medium (3.5 g L−1 Na2HPO4, 1.5 g L−1 KH2PO4, 2.5 g L−1 NaCl, 0.2 g L−1 MgSO4, 0.01 g L−1 CaCl2). Escherichia coli strains used in this study are listed in
8
Arabidopsis Protoplast Transformation and Auxin Assay
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Protoplast isolation, transformation and auxin treatment were performed as described previously [18 (link)]. In brief, protoplasts were isolated from Arabidopsis thaliana by plasmolysis and flotation. For each sample, 105 protoplasts were transformed with 20 μg of plasmid DNA using a PEG1500-based method with a total volume of 120 μL of protoplast/DNA mixture and the addition of 120 μL PEG1500-Ca(NO3)2 solution. Volumes were adjusted to 2 mL with PCA regeneration medium after transformation and protoplasts were incubated in the dark for 24 h. Auxin (indole-3-acetic acid, Sigma, cat. no. 128869) was dissolved in PCA medium and added to a final concentration of 1 μM where appropriate and renilla and firefly bioluminescence was determined after 1 h simultaneously using a BioTec Synergy 4 and Tecan infinite M200 pro plate reader, respectively.
9
Quantitative Analysis of Phytohormones
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Tissue extraction was carried by a modified method from Gómez-Cadenas et al. (2002) (link) and Durgbanshi et al. (2005) (link) Frozen tissue (0.5 g FW-1) was extracted with 80% methanol (5 mL) using mortar and pestle, after centrifugation (5.000 g × 10 min) supernatant was dried using N2 gas. After re-suspension using 2 mL water:pure diethyl-ether (1:1) samples are shaken for 1 min. The diethyl-ether fraction was subsequently dried using N2 gas and re-suspended in 10% methanol (200 μL). A 10 μL volume was injected to a UHPLC-mass spectrometer (Orbitrap, Thermo Scientific, Waltham, MA, United States). After 20 min run, quantification was carried out using pure standards of indole-3-acetic acid, (±)-abscisic acid (ABA), (±)-jasmonic acid, and salicylic acid (Sigma–Aldrich, St. Louis, MO, United States) using Xcalibur v.2.13 (Thermo Scientific, Waltham, MA, United States).
10
Colorimetric Assay for IAA Production
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The IAA production of S141 and S141 IAA-related mutant strains were grown in LB medium broth supplement with 0.1% tryptophan for 48 h at 30 °C. After incubation, the IAA production was colorimetrically determined as described by Costacurta et al. [36 ]. The standard was prepared using pure indole-3-acetic acid (Sigma-Aldrich).
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