Stock solutions of Epibrassinolide (BL, Sigma-Aldrich, St. Louis, MO, USA), methyl jasmonate (MeJA, TCI, Tokyo, Japen), 1-naphthylacetic acid (NAA, Sigma-Aldrich, St. Louis, MO, USA), indole-3-acetic acid (IAA, Sigma-Aldrich, St. Louis, MO, USA), and salicylic acid (SA, Sigma-Aldrich, St. Louis, MO, USA) were prepared in 100% ethanol and diluted to the appropriate concentration with sterile distilled water containing 0.1% Triton X-100 for experiments. The same volume of ethanol with 0.1% Triton X-100 was used as a mock control. To evaluate the effects of BL, NAA, IAA, MeJA, and SA on RAVs, rice seedlings were sprayed with 10 μmol BL, 5 μmol NAA, 5 μmol IAA, 100 μmol MeJA, and 500 μmol SA, respectively. Eighty seedlings per hormonal treatment were sprayed, and samples were collected for testing at 3, 6, and 12 h.
Indole 3 acetic acid iaa
Manufactured by Merck Group
Sourced in United States
Indole-3-acetic acid (IAA) is a chemical compound that serves as a plant growth regulator. It is a naturally occurring auxin, which is a class of plant hormones that promote cell elongation, cell division, and other growth-related processes in plants. IAA is a key component in various plant bioassays and experiments involving plant physiology and development.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Gibberellic acid ga3, by Merck Group (2 mentions)
Abscisic acid aba, by Merck Group (2 mentions)
Kinetin, by Merck Group (2 mentions)
Gibberellin a3 ga3, by Fujifilm (2 mentions)
Methyl jasmonate me ja, by Fujifilm (2 mentions)
Benzothiadiazole s methyl ester bth, by Fujifilm (2 mentions)
1 aminocyclopropane 1 carboxylic acid acc, by Merck Group (2 mentions)
Es fbs, by Thermo Fisher Scientific (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (2 mentions)
Hap1 cells, by Horizon Discovery (2 mentions)
Salicylic acid, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
32 protocols using indole 3 acetic acid iaa
1
Phytohormone Effects on Rice RAVs
2
Culture and Maintenance of Cell Lines
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All cell lines were grown in a humidified chamber at 37 °C in 5% CO2, and were routinely tested for mycoplasma. Human TERT-immortalized RPE-1 cells were cultured in DMEM/F12 (GIBCO) containing 10% FBS (Sigma F7524 lot BCBW6329) and 1% Pen/Strep (GIBCO). This cell line does not contain a Y chromosome. Human HEK293T cells were cultured in DMEM (GIBCO) containing 10% FBS and 1% Pen/Strep (GIBCO). This cell line does not contain a Y chromosome. Mouse F1 hybrid Cast/EiJ x 129SvJae embryonic stem cells (mESCs; a kind gift from the Joost Gribnau laboratory) were cultured on irradiated primary mouse embryonic fibroblasts (MEFs), in mESC culture media CM+/+ defined as follows: G-MEM (GIBCO) supplemented with 10% FBS (Sigma F7524 lot BCBW6329), 1% Pen/Strep (GIBCO), 1x GlutaMAX (GIBCO), 1x non-essential amino acids (GIBCO), 1x sodium pyruvate (GIBCO), 0.1 mM β-mercaptoethanol (Sigma) and 1000 U/mL ESGROmLIF (EMD Millipore ESG1107). Cells were split every 3 days and medium was changed every other day. Expression of the Dam-POI constructs was suppressed by addition of 0.5 mM indole-3-acetic acid (IAA; Sigma, I5148). This cell line does not contain a Y chromosome.
3
Aseptic Culture of E. maritimum and E. alpinum
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For the initiation of aseptic culture of E. maritimum and E. alpinum, the shoot fragments with axillary buds isolated from plantlets were used as explants. The isolated primary explants were rinsed in distilled water for 5 min and dipped in 70% (v/v) ethanol-water solution for 30 s followed by rinsing in 1.33% (E. maritimum) or 2.5% (E. alpinum) sodium hypochloride solution, containing two drops of surfactant Tween 80 for 8 min. They were finally rinsed three times in sterilized double-distilled water.
The explants of both the species were placed in a Erlenmeyer flask with 50 mL of the solidified MS medium (Murashige and Skoog [39 (link)]) with plant growth regulators—benzylaminopurine (BAP; Sigma-Aldrich, Saint Louis, MO, USA), indole-3-acetic acid (IAA; Sigma-Aldrich, Saint Louis, MO, USA), and gibberellic acid (GA3; Sigma-Aldrich, Saint Louis, MO, USA), each at the concentration of 1.0 mg/L [23 (link),40 (link)]. The culture vessels were placed in a growth chamber (21 ± 2 °C; with a 16 h light/8 h dark photoperiod; 55 µmol/m2∙s light) and subcultured every 5 weeks. Multiplication of shoots via the axillary branching method on MS medium was repeated many times, using at least 10 explants per repetition. Shoots were air dried.
The explants of both the species were placed in a Erlenmeyer flask with 50 mL of the solidified MS medium (Murashige and Skoog [39 (link)]) with plant growth regulators—benzylaminopurine (BAP; Sigma-Aldrich, Saint Louis, MO, USA), indole-3-acetic acid (IAA; Sigma-Aldrich, Saint Louis, MO, USA), and gibberellic acid (GA3; Sigma-Aldrich, Saint Louis, MO, USA), each at the concentration of 1.0 mg/L [23 (link),40 (link)]. The culture vessels were placed in a growth chamber (21 ± 2 °C; with a 16 h light/8 h dark photoperiod; 55 µmol/m2∙s light) and subcultured every 5 weeks. Multiplication of shoots via the axillary branching method on MS medium was repeated many times, using at least 10 explants per repetition. Shoots were air dried.
4
Extraction and Characterization of Bioactive Compounds from Myrica gale
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Myrigalone A, myrigalone B and myrigalone D, and 2′,4′-dihydroxy-6′-methoxy-3′5′-dimethylchalcone (DMC) were extracted from Myrica gale fruits and plants as described [36 (link)] by Syngenta’s Jealott’s Hill International Research Centre (Bracknell, UK). Gibberellin A4+7 and paclobutrazol were purchased from Duchefa Biochemie (Haarlem, The Netherlands). Phloretin, dihydrochalcone, naringenin, neohesperidin dihydrochalcone, daphnetin, psoralen, angelicin, ferulic acid, acacetin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,3,5-triiodobenzoic acid (TIBA), indole-3-acetic acid (IAA), aminoethoxyvinylglycine (AVG), L-kynurenine, and hydrogen peroxide solution (30% (w/w)) were purchased from Sigma-Aldrich (St Louis, MO, USA). 5-(4-Chlorophenyl)-4H-1,2,4-triazole-3-thiol (yucasin) was purchased from Carbosynth Ltd. (Compton, Berkshire, UK). 4-(2,4-dimethylphenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid (auxinole) was purchased from Cambridge Bioscience (Cambridge, UK). All the compounds were dissolved in DMSO except for Phloretin, dihydrochalcone, naringenin and neohesperidin dihydrochalcone which were dissolved in methanol. Controls were performed with basal solvent (0.1% (v/v) DMSO or methanol) as appropriate.
5
Quantification of Auxinic Compounds by P. phymatum
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The production of auxinic compounds by P. phymatum STM815 wild-type and nifA mutant strains was quantified by a modified colorimetric assay from Gravel and colleagues [68 (link)]. Precultures were grown aerobically at 28 °C in LB without salt with the corresponding antibiotics, washed twice and inoculated into 100 mL flasks containing 20 mL of LB without salt with an initial OD600 of 0.05. Cultures were incubated at 28 °C with 180 rpm shacking for 16 h until all the strains reached an OD600 of 4. Two mL of culture was then centrifuged (5000 rpm, 5 min) and one mL of the supernatant was mixed with two mL of Salkowski’s reagent [69 (link)]. The mixture was incubated at room temperature in the dark for 20 min and the absorbance was measured at 535 nm. To quantify extracellular auxinic compounds, a 50 µg/mL of indole-3-acetic acid (IAA) (Sigma-Aldrich, Buchs, St. Gallen, Switzerland) solution was diluted in LB without salt to draw the standard curve. This experiment was conducted in three biological replicates.
6
Phytohormone Treatments on Seedlings
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Seeds of 30DPH were soaked in 70% (v/v) ethanol for 1 min followed by washing in 2.5% (v/v) sodium hypochlorite solution containing 0.1% (v/v) Tween 20 for 15 min and rinsed thoroughly with sterile distilled water. Surface sterilized seeds were grown in PhytoCon culture vessels (Phytotechnology Laboratories, Overland Park, KS, USA) containing half strength Murashige and Skoog (MS) medium for 14 days. Seedlings were kept in sucrose free liquid half strength MS medium for 24 h. Seedlings of 15DPG (days post germination) were transferred to PhytoCon culture vessels (Phytotechnology Laboratories) containing liquid half strength MS supplemented with 100 µM abscisic acid (ABA, Sigma, St. Louis, MO, USA), 50 µM brassinolide (Bra, Sigma), 50 µM gibberellic acid (GA, Sigma), 50 µM indole-3-acetic acid (IAA, Sigma), 100 µM methyl jasmonate (MeJa, Sigma), 100 µM salicylic acid (SA, Sigma), 100 µM Zeatin (Zea, Sigma) and incubated for 6 h. Leaves from a total of 72 samples from seven treatments in three biological replicates including one untreated control of three genotypes were harvested and immediately frozen as mentioned in the earlier section.
7
Phytohormone Responses in Rice Seedlings
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All stock solutions, except brassinolide (BR), were prepared at a concentration of 100 mM as described previously (Jiang et al., 2009 (link)). BR was prepared at 20 mM concentration. Indole-3-acetic acid (IAA; Sigma, St. Louis, MO, United States), gibberellin A3 (GA3; Wako, Osaka, Japan), ABA [(±)-cis–trans, Sigma], methyl jasmonate (ME-JA; Wako), and brassinolide (BR; Wako) were dissolved in absolute ethanol. Kinetin (Sigma) and benzothiadiazole S-methyl ester (BTH; Wako) were dissolved in dimethyl sulfoxide (DMSO); and 1-aminocyclopropane-1-carboxylic acid (ACC; Sigma) and sodium salicylate (SA; Nacalai Tesque, Tokyo, Japan) in H2O.
For plant treatments, rice seedlings at four-leaf stage (three true leaves) were transferred to a container containing each of the phytohormone solutions at 50 μM for IAA, GA3, CK, ACC, ABA, JA, and BTH, and at 10 μM for BR. Water was used as mock control. The rice seedlings were further grown for 4 weeks, and leaf lengths of the fifth leaf were measured.
Measurement of JA and ABA content were performed as described previously (Kojima and Sakakibara, 2012 (link)).
For plant treatments, rice seedlings at four-leaf stage (three true leaves) were transferred to a container containing each of the phytohormone solutions at 50 μM for IAA, GA3, CK, ACC, ABA, JA, and BTH, and at 10 μM for BR. Water was used as mock control. The rice seedlings were further grown for 4 weeks, and leaf lengths of the fifth leaf were measured.
Measurement of JA and ABA content were performed as described previously (Kojima and Sakakibara, 2012 (link)).
8
Analysis of Arabidopsis ref3 Mutants
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The wild-type lines used were Columbia (Col-0) and Landsberg erecta (Ler) which are the backgrounds for the ref3-3 and ref3-1 mutants, respectively. [6 ] Both ref3 mutant lines were obtained from the ABRC Seed Stock Center. All plants were grown in sterile conditions on half-strength Murashige and Skoog medium (pH 5.7) containing 1% sucrose and 0.8% PhytoAgar (MS/2 medium). The following chemicals were used for treatments: indole-3-acetic acid (IAA; Sigma) and trans-cinnamic acid (t-CA; Sigma). All rosette growth response analyses were done as previously described. [2 ] The descriptive statistics, plotting and hypothesis testing were done using Prism 6 software (GraphPad Software Inc). All data are presented as means ± SD of at least three independent experiments. When means of more than two samples were compared, we used ANOVA followed by post-test to find a significant difference between pairs of means. The significance levels, indicated by asterisks in the figures, illustrate the results of the post-test.
9
Conditional Degradation of Mcm2 in Yeast
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All yeast strains used in this study are listed in Supplementary Table S1.
The mcm10–1-aid strains were obtained from National BioResource Project-Yeast (NBRP-Yeast) and mcm2-td degron strain was obtained from Karim Labib (44 (link)).
In all experiments, cells were grown at 25°C to slow down replication dynamics as described before (45 (link)). Medium used in this study is YP medium (1% yeast extract, 2% peptone) supplemented with 2% of glucose (YPD) or galactose (YPG). To induce degradation of aid tagged proteins in auxin inducible degron, exponentially growing cells in YPD were transferred to YPG to induce the expression of OsTIR1–9Myc for 45 min. A natural auxin, indole-3-acetic acid (IAA) (Sigma), was then added to medium at a final concentration of 500 μM. mcm2-td strains carrying plasmids for exogenous expression of wild-type mcm2, mcm2–2Aand mcm2–2D were grown overnight in minimal medium with 2% raffinose at 25°C. Wild-type and mutant Mcm2 proteins were overexpressed in YPG from galactose-inducible promoter at 37°C to induce the degradation of endogenous MCM2 gene.
The mcm10–1-aid strains were obtained from National BioResource Project-Yeast (NBRP-Yeast) and mcm2-td degron strain was obtained from Karim Labib (44 (link)).
In all experiments, cells were grown at 25°C to slow down replication dynamics as described before (45 (link)). Medium used in this study is YP medium (1% yeast extract, 2% peptone) supplemented with 2% of glucose (YPD) or galactose (YPG). To induce degradation of aid tagged proteins in auxin inducible degron, exponentially growing cells in YPD were transferred to YPG to induce the expression of OsTIR1–9Myc for 45 min. A natural auxin, indole-3-acetic acid (IAA) (Sigma), was then added to medium at a final concentration of 500 μM. mcm2-td strains carrying plasmids for exogenous expression of wild-type mcm2, mcm2–2Aand mcm2–2D were grown overnight in minimal medium with 2% raffinose at 25°C. Wild-type and mutant Mcm2 proteins were overexpressed in YPG from galactose-inducible promoter at 37°C to induce the degradation of endogenous MCM2 gene.
10
In Vitro Colon Cancer Cell Culture
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