auxin indole 3 acetic acid, by Merck Group - Product details

1

Auxin-Induced Lifespan Modulation in C. elegans

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Auxin plates were prepared by adding auxin indole‐3‐acetic acid (Sigma‐Aldrich) from a 400 mM stock solution in ethanol into NGM at the final concentration of 1 mM (Zhang et al., 2015 (link)). For control ethanol plates, the same volume of ethanol was added to NGM. Animals were transferred on auxin or ethanol plates at the L4 stage except for dauer tests for which eggs were laid and grown on auxin or ethanol plates.
For all aging cohorts, 20 μM 5‐fluorouracil (5‐FU, Sigma‐Aldrich) was added to prevent progeny growth. Animals were transferred weekly to fresh plates, without 5‐FU after 2 weeks. The day of transition to L4 is counted as day 0 of adulthood for the cohort.

Roy C., Molin L., Alcolei A., Solyga M., Bonneau B., Vachon C., Bessereau J, & Solari F. (2022). DAF‐2/insulin IGF‐1 receptor regulates motility during aging by integrating opposite signaling from muscle and neuronal tissues. Aging Cell, 21(8), e13660.

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2

Bacterial Co-Inoculation Effects on Soybean

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Bacterial cell preparation was carried out as described in Section 2.1. Leonard’s jar experiments were performed as described in Section 2.2. The experiment was conducted to evaluate the co-inoculation effects of USDA110 with S141 or plant growth hormone on soybean. The seedlings were inoculated separately with 1 mL of 10⁶ CFU mL⁻¹ of S141 or USDA110 for the single inoculation and mixed in a ratio of 1:1 for co-inoculation treatment. The cell suspension was replaced by sterilized distilled water in the control treatment. The plants were watered regularly with N-free nutrient solution during the experiment [30 (link)]. When necessary, the N-free nutrient solution was supplemented with appropriate plant growth hormones: 1 μg mL⁻¹ cytokinin (6-Benzylaminopurine (Sigma-Aldrich, St. Louis, MO, USA) [32 ] and 10 μg mL⁻¹ auxin (Indole-3-Acetic Acid (Sigma-Aldrich) [33 (link),34 (link)]. Plant growth conditions in the laboratory were as described in Section 2.2.

Sibponkrung S., Kondo T., Tanaka K., Tittabutr P., Boonkerd N., Yoshida K.I, & Teaumroong N. (2020). Co-Inoculation of Bacillus velezensis Strain S141 and Bradyrhizobium Strains Promotes Nodule Growth and Nitrogen Fixation. Microorganisms, 8(5), 678.

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3

Tissue-specific protein degradation dynamics

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Auxin plates were prepared by adding auxin indole-3-acetic acid (Sigma-Aldrich) from a 400-mM stock solution in ethanol into NGM at the final concentration of 1 mM (Zhang et al., 2015 (link)). Tissue-specific degradation efficiency was monitored on an animal expressing GFP fused to the AID under the ubiquitous promoter Peft-3 and Prab-3::TIR1-TagBFP, Peft-3::TIR1-TagBFP, Pdpy-7::TIR1-TagBFP, or Pmyo-3::TIR1-TagBFP. Animals were grown on regular or auxin plates for 3, 36, 96, and 120 h, respectively. SDN-1-AID-mNG fluorescence was scored after tissue-specific degradation on young adult animals that had hatched on regular or auxin plates.

Zhou X., Vachon C., Cizeron M., Romatif O., Bülow H.E., Jospin M, & Bessereau J.L. (2021). The HSPG syndecan is a core organizer of cholinergic synapses. The Journal of Cell Biology, 220(9), e202011144.

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Detailed Protocols for Cell Culture Treatments

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Treatments were generally carried out in log-phase cultures at OD ~0.4–0.6, depending on the length of treatment, such that cultures remained in log growth during the course of the experiment. For drugs dissolved in DMSO, vehicle-only controls contained the same final volume of DMSO. Auxin (indole-3-acetic acid, Sigma-Aldrich) was prepared fresh daily at 100 mM in ethanol and added at a final concentration of 100 µM. Diazaborine (DZA, Calbiochem) was prepared at 15 mg/ml in DMSO (stored at −20°C, protected from light) and used at the indicated concentration. Cycloheximide (Sigma-Aldrich) was purchased as a 100 mg/ml DMSO stock and added at a final concentration of 100 µg/ml (for sucrose gradients) or 200 µg/ml (for stress experiments). AZC (L-azetidine-2-carboxylic acid, Sigma-Aldrich) was prepared at 1 M in water and used at the indicated concentration. Diamide (Sigma-Aldrich) was prepared at 1 M in water and added at a final concentration of 1.5 mM. Rapamycin (LC Laboratories) was prepared fresh daily in ethanol and used at a final concentration of 200 ng/ml (to inhibit r-protein synthesis) or 1 µg/ml (for anchor-away, in a Rapamycin-resistant tor1-1 background). Beta-estradiol (Sigma-Aldrich) was prepared as a 1000X stock for each experiment in ethanol and added to the indicated final concentration.

Tye B.W., Commins N., Ryazanova L.V., Wühr M., Springer M., Pincus D, & Churchman L.S. (2019). Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness. eLife, 8, e43002.

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5

Auxin-Dependent AID Assays

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AID assays were conducted as described previously (36 ). Briefly, age-synchronized animals were grown on biotin auxotroph E. coli strain MG1655-seeded NGM plates in the presence or the absence of 1 mM auxin (indole-3-acetic acid; Sigma).

Artan M., Hartl M., Chen W, & de Bono M. (2022). Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans. The Journal of Biological Chemistry, 298(9), 102343.

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6

Immunoblotting and Immunofluorescence Analysis

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Antibodies used for immunoblotting and indirect immunofluorescence analysis were: rabbit anti-SMC2 (1:500) (Saitoh et al., 1994 (link)), guinea pig anti-GgTopo 2α (1:1000) (Earnshaw et al., 1985 (link)), rabbit anti-GgCENP-T (1:1000) (Hori et al., 2008b (link)), and rabbit anti-MAD2 (1:100, gift of Tatsuo Fukagawa, Graduate School of Frontier Biosciences, Osaka University, Japan), rabbit-anti GgcyclinB2 (gift of Eric Nigg, retired from Biozentrum, University of Basel, Switzerland), mouse anti-α-tubulin DM1A (1:1000, lot no. 066M4870V, Sigma-Aldrich), mouse anti-Cdk1 monoclonal A17 (1:200–1:500, lot no. GR133813-4, Abcam), and rabbit anti- phospho-histone H3 (Ser 10) (D2C8) (1:1600, lot no. 3, Cell Signaling Technology).
Nocodazole dissolved in DMSO was added to a final concentration of 0.5 µg/ml (Sigma-Aldrich). Doxycycline dissolved in water was added to a final concentration of 0.5 µg/ml (BD) unless stated otherwise. 1NMPP1 dissolved in DMSO was added to a final concentration of 2 µM. Indole-3-acetic acid (auxin) dissolved in ethanol was added to a final concentration of 125 µM unless stated otherwise (Fluka, Sigma-Aldrich).

Samejima K., Booth D.G., Ogawa H., Paulson J.R., Xie L., Watson C.A., Platani M., Kanemaki M.T, & Earnshaw W.C. (2018). Functional analysis after rapid degradation of condensins and 3D-EM reveals chromatin volume is uncoupled from chromosome architecture in mitosis. Journal of Cell Science, 131(4), jcs210187.

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7

Knockdown of NIPBL, WAPL, and CTCF in HCT-116 Cells

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HCT-116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO₂. The following siRNAs (Dharmacon) were used: Non-targeting control, NIPBL, WAPL, CTCF. siRNA sequences can be found in Supplementary Table 2. Duplex siRNA were incubated for 20 minutes at room temperature with RNAiMAX transfection reagent (Thermo Fisher) in Opti-MEM reduced serum medium (Thermo Fisher) and seeded into wells. HCT-116 were trypsinized and resuspended in antibiotic-free medium, then plated with medium containing siRNA for a final siRNA concentration of 50 nM (non-targeting control, NIPBL, or WAPL) or 150 nM (CTCF). For CTCF knockdowns, cells were retreated with 150nM CTCF siRNAs 24 hours after initial treatment. After 72 h (NIPBL, WAPL, non-targeting control) or 96 h from the initial RNAi treatment (CTCF), cells were harvested for experiments. For the CTCF and RAD21 double-knockdown experiments, cells were grown in medium supplemented with 500 μM indole-3-acetic acid (auxin; Sigma-Aldrich I5148) for 6 hours following RNAi treatments, and then harvested for experiments.

Luppino J.M., Park D.S., Nguyen S.C., Lan Y., Xu Z., Yunker R, & Joyce E.F. (2020). Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes. Nature genetics, 52(8), 840-848.

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8

Yeast Auxin Response Assay

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Yeast cells were grown at 30°C in YPD to an OD₆₀₀ ~0.3. Indole-3-acetic acid (auxin; Sigma-Aldrich) was added to a final concentration of 500 μM (Morawska et al, 2008). After 120 min, cells were prepared for live-cell imaging in SD with 2% glucose supplemented with 500 μM of auxin. For western blotting, 20 ml of culture was harvested at an OD₆₀₀ of ~0.6 with or without 500 μm auxin, and protein extraction and western blotting were carried out as described above.

Bensidoun P., Reiter T., Montpetit B., Zenklusen D, & Oeffinger M. (2022). Nuclear mRNA metabolism drives selective basket assembly on a subset of nuclear pore complexes in budding yeast. Molecular cell, 82(20), 3856-3871.e6.

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9

Knockdown of NIPBL, WAPL, and CTCF in HCT-116 Cells

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HCT-116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO₂. The following siRNAs (Dharmacon) were used: Non-targeting control, NIPBL, WAPL, CTCF. siRNA sequences can be found in Supplementary Table 2. Duplex siRNA were incubated for 20 minutes at room temperature with RNAiMAX transfection reagent (Thermo Fisher) in Opti-MEM reduced serum medium (Thermo Fisher) and seeded into wells. HCT-116 were trypsinized and resuspended in antibiotic-free medium, then plated with medium containing siRNA for a final siRNA concentration of 50 nM (non-targeting control, NIPBL, or WAPL) or 150 nM (CTCF). For CTCF knockdowns, cells were retreated with 150nM CTCF siRNAs 24 hours after initial treatment. After 72 h (NIPBL, WAPL, non-targeting control) or 96 h from the initial RNAi treatment (CTCF), cells were harvested for experiments. For the CTCF and RAD21 double-knockdown experiments, cells were grown in medium supplemented with 500 μM indole-3-acetic acid (auxin; Sigma-Aldrich I5148) for 6 hours following RNAi treatments, and then harvested for experiments.

Luppino J.M., Park D.S., Nguyen S.C., Lan Y., Xu Z., Yunker R, & Joyce E.F. (2020). Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes. Nature genetics, 52(8), 840-848.

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Auxin indole 3 acetic acid

Lab products found in correlation

9 protocols using auxin indole 3 acetic acid

Auxin-Induced Lifespan Modulation in C. elegans

Bacterial Co-Inoculation Effects on Soybean

Tissue-specific protein degradation dynamics

Detailed Protocols for Cell Culture Treatments

Auxin-Dependent AID Assays

Immunoblotting and Immunofluorescence Analysis

Knockdown of NIPBL, WAPL, and CTCF in HCT-116 Cells

Yeast Auxin Response Assay

Knockdown of NIPBL, WAPL, and CTCF in HCT-116 Cells

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