Stachyose

Labrafac lipophile WL-1349
and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE) were kindly gifted from Gattefosse Germany and Lipoid Germany,
respectively. Kolliphore HS15, polyethylenimine (PEI) (average M_w ∼ 0.8 and 25 kDa), Oleic acid, Dulbecco’s
modified Eagle’s medium (DMEM), Nile red, penicillin/streptomycin
solution, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), trypsin-EDTA (1×), poly(vinyl alcohol)
(PVA), tricine, trehalose, sucrose, stachyose, and glycerol were purchased
from Sigma-Aldrich (St. Louis, MO, USA). polyethylenimine (PEI) (average M_w ∼ 1.8 kDa) was purchased from Alfa
Aesar, USA. N,N′-dicyclohexylcarbodiimide
(DCC), and N-hydroxysuccinimide (NHS) were purchased
from Thermo Fisher Scientific (Suwanee, GA, USA). Ethidium bromide
(EtBr), Hoechst 33342, and H₂DCFDA were purchased from
Invitrogen (Thermo Fisher Scientific, Suwanee, GA, USA), USA. Agarose
molecular biology grade was purchased from IBI Scientific. Firefly
Luciferase (FLuc) mRNA was purchased from TriLink Biotechnologies
(San Diego, California, USA). Other chemicals and solvents used in
the work were analytical grade and were used as procured.

Soy molasses was provided by Shandong Rongzheng Chemical Co., Ltd. (Linyi, China). Sucrose, raffinose, stachyose, and manninotriose standards were purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). Fungal genomic DNA extraction kit was purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China). Other reagents used were domestically produced biological or analytical reagents. Strain YT312 was obtained by our research team from the environment of Baijiu brewing. Yeast extract peptone dextrose (YPD) medium, Wallerstein laboratory nutrient (WL) medium, and other identification media for strains were prepared according to the methods described by Fan et al., Kurtzman et al., Buchana et al., and Dong et al. [38 (link),39 (link),40 ,41 ]. The fermentation medium was processed according to our previous method, as follows: soy molasses was diluted with distilled water 8-fold, and the pH was adjusted to 6.0; then, it was divided into 30 mL per bottle (250 mL) and sterilized at 110 °C for 10 min [37 ].

Fast Blue BB (FBBB) [4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi-(zinc chloride) (Rebello et al., 2016 (link)), bile extract porcine, pancreatin from porcine pancreas, pepsin from porcine gastric mucosa, α-amylase from human saliva, 2,2′-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2′-diazobis-(2-aminodinopropane)-dihydrochloride (AAPH), fluorescein, 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich, Co. (St. Louis, MO, United States). Standards of chlorogenic acid, 2-coumaric acid, 2,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, vanillic acid, and vanillin were provided by Extrasynthese (Lyon, Genay Cedex, France). Standards of 6-hydroxy-2,5,7, 8-tetramethyl-2-carboxylic acid (Trolox), gallic acid, caffeic acid, ferulic acid, vitexin, D-(+)-glucose, D-(+)-galactose, D-(+)-xylose, D-(–)-arabinose, and stachyose were acquired from Sigma-Aldrich, Co. (St. Louis, MO, United States). Standards of 3³-α-L-plus 2³-α-L-arabinofuranosyl-xylotetraose (XA³XX/XA²XX), 2³,3³-di-α-L-arabinofuranosyl-xylotriose (A^{2, 3}XX), and xylotriose were obtained from Megazyme (Wicklow, Ireland). Raffinose and cellobiose standards were supplied by Merck (Darmstadt, Germany).

The high-performance liquid chromatography (HPLC)-evaporative light scattering detection (ELSD) method was used to determine soluble sugars in Hevea leaves and phloem exudate. HPLC analysis was performed on a Waters e2695 separations module (Waters, Milford, MA, United States) equipped with an Alltech 3300 ESLD detector (Alltech, Deerfield, IL, United States). Separation was achieved on a XBridge^TM Amide column [4.6 mm × 250 mm i.d., 3.5 μm particle size (Waters, Milford, MA, United States)]. All samples and standards were filtered through 0.45 μm Millipore filters before loading samples of 10 μL onto the machine. The HPLC-ELSD conditions were optimized following Ma et al. (2014) (link) with a solvent ratio of 85 acetonitrile:15 water (v/v), a flow rate of 1 mL/min, the column and drift tube temperatures set at 45 and 82°C, respectively, and the nebulizer gas flow rate set at 2 L/min. Peaks were quantified using calibration standards of HPLC grade sugars, viz. glucose, fructose, sucrose, raffinose, and stachyose (Sigma–Aldrich, Shanghai, China), and sorbitol and quebrachitol (Shanghai Yuan ye Bio-Technology Co., Ltd., Shanghai, China).

CV-1, a green monkey kidney cell line (ATCC CCL 70), and HaCaT, a spontaneously transformed human keratinocyte cell line37 (link), were obtained from the American Type Culture Collection and the Cell Lines Service (CLS 300493, Eppelheim, Germany), respectively. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 1.8 mM CaCl₂ supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humid atmosphere of 5% CO₂. NHEKs were obtained from Lonza (Walkersville, MD) and maintained serum free in the keratinocyte basal medium KBM Gold (Lonza) containing 0.3 mM calcium supplemented with KGM-SingleQuot (Lonza). NHEKs were used for experiments within passage 2. Cells at 60 to 70% confluency were treated with vehicle, RFOs, or TO901317. Isomaltotriose and raffinose-series oligosaccharide family members including stachyose, verbascose, and d-(+)-raffinose·5H₂O were purchased from Sigma-Aldrich (St Louis, MO). The synthetic LXR agonist T0901317 was purchased from Cayman Chemical (Ann Arbor, MI).

Media constituents were acquired from HiMedia Laboratories, India. ρ-Nitrophenyl-a α-d-galactopyranoside (ρNPGal), Stachyose, Phenyl Methyl Sulphonyl Flouride (PMSF) and k-Carrageenan were procured from Sigma-Aldrich Inc, USA. Ion exchange matrix-Q-Sepharose fast flow and gel filtration matrix-Sephacryl S-300 were brought from GE Healthcare, USA.

Seven different levans were used in this study: 1) levan synthesized from 1.2 M (410.8 g/L) sucrose by levansucrase Lsc3 of Pseudomonas syringae pv. tomato; 2) levan synthesized from 0.3 M (151.3 g/L) raffinose by Lsc3 of P. syringae; 3) levan synthesized from 1.2 M (410.8 g/L) sucrose by the Lsc3Asp300Asn (D300N) mutant of P. syringae; 4) levan of Zymomonas mobilis 113S (kindly provided by Dr. A. Vigants, University of Latvia, Latvia); 5) levan (L8647) of Erwinia herbicola (Pantoea agglomerans) from Sigma-Aldrich (Germany); 6) levan from Halomonas smyrnensis (a gift from Prof. E.T. Öner, Marmara University, Turkey; isolated as in [36 ]); 7) levan from timothy grass (a gift from Dr. A. Kasperowicz, Polish Academy of Sciences, Poland; isolated as in [37 ]). Levans of Z. mobilis, E. herbicola and H. smyrnensis are all produced from sucrose. 1-kestose, nystose, stachyose and inulin from dahlia were from Sigma-Aldrich (Germany), FOS-preparations P95 and Synergy1 were from Beneo (Belgium), xylooligosaccharide mixture was from Sweet Town Biotech (Taiwan) and raffinose was from Naxo (Estonia).