Indole acetic acid iaa

LaCl₃ (purity >99.99%) was purchased from Aladdin Bio-Chem Technology Co. Ltd (Shanghai, China). FM4-64 was purchased from Thermo Fisher Scientific Co. Ltd (Shanghai, China). DPI, TyrA23, Murashige and Skoog medium (MS), methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate (Benomyl), 6-benzylaminopurine, indole acetic acid (IAA), and JA were purchased from Sigma-Aldrich Co. Ltd (Shanghai, China). Flg22 was purchased from MedChemExpress Co. Ltd (Shanghai, China). Anti-plant actin mouse monoclonal antibody (A01050) was purchased from Abbkine Scientific Co. Ltd (Wuhan, China). Goat anti-mouse IgG H&L [horseradish peroxidase (HRP)] (ab205719) and HRP anti-GFP antibody (ab6663) were purchased from Abcam Co. Ltd (Shanghai, China). Other chemicals used in this study were analytical reagents and were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

Plants were grown as for the pot trial, but one week after sowing they were harvested intact and the root systems were gently washed to remove the soil, before being submerged in 1 mM 3-O-methyl-D-glucose solution (3-O-M-glucose). 3-O-M-glucose is a non-metabolisable analogue of glucose that is routinely used to quantify the rate of sugar influx and efflux across plant and animal membranes [55 (link)-58 (link)]. The 3-O-M glucose solution was supplemented with 1 kBq ml^-1 of ¹⁴C-3-O-M-glucose (American Radiolabeled Chemicals Inc., USA). After 24 h, the root bathing solution was replaced to remove any remaining ¹⁴C-labelled 3-O-M-glucose. The root bathing solution was then replaced with either deionised water, or 1 µM of the auxin indole acetic acid (IAA; Sigma Aldrich, Poole, UK) and the roots incubated for 1 h to determine the net rate of 3-O-M-glucose efflux. Such alterations in monosaccharide efflux are generally rapid processes [56 (link)-58 (link)], as are many auxin responses [59 ], therefore one hour was deemed sufficient to assess any effect. After the plants were removed, the ¹⁴C content of the resulting root bathing solution was quantified using a Wallac 1404 scintillation counter (Wallac EG&G, Milton Keynes, UK). The root system surface areas of each plant were then determined using WinRhizo® as described above.

Mouse embryonic stem cells (mESCs) (obtained from Richard Young's lab, MIT) were cultured in knockout Dulbecco’s modified Eagle’s medium (DMEM; Gibco; 10829018-018), supplemented with 15% fetal bovine serum (FBS), PenStrep (Gibco, 15140-122), nucleoside (Sigma, ES-008), l-glutamine (Gibco, 25030-149), NEAAs (non-essential amino acids; Milipore, TMS-001-C), CHIR99021 (Selleck, S1036), PD0325901 (Selleck, S1263), β-mercaptoethanol (Sigma, M3148) and mLIF (mouse leukemia inhibitory factor; Milipore, ESG1107) at 37°C with 5% CO₂. To degrade the target proteins, we first treated the degron-tagged mESCs with 1 μg/ml doxycycline (Sigma Aldrich, D9891) for 24 h, then added 500 μM indole acetic acid (IAA; Sigma Aldrich, 115148) for the indicated times for different analyses. HEK293T (ATCC, CRL-3216) cells were cultured in DMEM (Gibco, 11995-065) supplemented with 10% FBS (Gibco, 10099-141) and PenStrep (Gibco, 15140-122) at 37°C with 5% CO₂.