Mannitol

Human coronary artery smooth muscle cells (Lonza, Basel, Switzerland) were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (Nacalai, Kyoto, Japan) (glucose; 5.5 mM) supplemented with 15 % fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 IU/ml penicillin and 100 g/ml streptomycin (Nacalai) (normal glucose-containing media). High glucose-containing media (final 25 mM) was made by addition of 19.5 mM glucose to normal glucose-containing media. In like manner, 19.5 mM mannitol (Sigma-Aldrich, St. Louis, MO, USA) was added to normal glucose-containing media as an osmolality control (mannitol-containing media). Cells between passages 8 and 14 were used for the experiments. Unless otherwise noted, cells were cultured for the indicated time periods without changing media. In some experiments, HCASMCs were cultured with rosuvastatin (10 µM), mevalonate (Sigma-Aldrich) (100 µM), FPP (Sigma-Aldrich) (10 µM), GGPP (Sigma-Aldrich) (10 µM), fasudil (Wako) (10 µM) and Y-27632 (Wako) (10 µM). Human umbilical vein endothelial cells (HUVECs) (Lonza) were cultured at 37 °C in the EGM-2 BulletKit (Lonza).

From 1 to 14 d of age, animals were fed their respective experimental diets. At day 14, birds from all treatments, except those of CNF, were feed deprived for 15.5 h. Intestinal permeability was measured by the lactulose/mannitol (L/M) test following the protocol described by Gilani et al. [22 (link)]. To this end, following the 15.5 h fasting period (15 d of age), two birds per pen (12 chicks per treatment) were randomly selected and administered an oral gavage of lactulose and mannitol. Lactulose and mannitol solution contained 25 g lactulose and 5 g of mannitol (Sigma Aldrich, Alcobendas, Madrid, Spain) dissolved in 100 mL Milli-Q water at 25 °C. Birds were slaughtered (CO₂ atmosphere asphyxiation) and sampled 90 min after the oral gavage. After this short-term fasting period, 16 chickens per pen were fed their respective diets ad libitum until the end of the trial at 32 d of age.

HCECs (Bena Culture Collection Biotechnology Co, Ltd) were cultured in EMEM (Eagle's Minimal Essential Medium, Gibco) supplemented with 10% fetal bovine serum (Lonsera) and 100 U mL⁻¹ penicillin (Phygene). At 60% confluence, the cells were starved for 24 h in a serum-free medium, followed by stimulation with 35 mM glucose or mannitol (Sigma-Aldrich) (35 mM mannitol as osmotic control) in the same serum-free medium for 24 h, with or without rhFGF-21.

Homozygous T₂ transgenic rice seeds were used in further studies. Dehusked wild-type and transgenic rice seeds were sterilized with 2% NaOCl for 50 min, thoroughly rinsed with sterile distilled water, and sown on half-strength Murashige and Skoog (MS) medium under cool daylight fluorescent lamps (60 μmol m⁻² s⁻¹) (Philips, Amsterdam, The Netherlands) under a 14 h light/10 h dark photoperiod at 28 °C/24 °C (day/night). Seven-day-old seedlings were used in further experiments. For mannitol (Sigma-Aldrich, St. Louis, MO, USA) treatment, surface-sterilized rice seeds were sown and grown on half-strength MS medium containing various concentrations of mannitol. Melatonin contents were measured in frozen samples (0.1 g) that were pulverized to a powder in liquid nitrogen using the TissueLyser II (Qiagen, Tokyo, Japan). The sample powders were extracted with 1 mL chloroform, followed by centrifugation for 10 min at 12,000× g, and the supernatants (200 µL) were evaporated and dissolved in 0.1 mL 40% methanol. The resulting 10 µL aliquots were subjected to high-performance liquid chromatography (HPLC) with a fluorescence detector system (Waters, Milford, MA, USA) as described previously [19 (link)]. Melatonin was eluted after about 31 min under the HPLC conditions. The measurements were performed in triplicate.

Rat glomerular MCs were obtained from China Center for Type Culture Collection (no. HBYZ-1; CCTCC, WuHan, CN). Cells were cultured in DMEM low glucose medium (Hyclone, Logan, UT) with 10 % heat-inactivated FBS (Hyclone, Logan, UT) and 1 % antibiotic solution (Sigma-Aldrich, St. Louis, MO). Cells from passages three to five were used for all the experiments. Monolayer rat glomerular MCs were rendered quiescent in DMEM low glucose medium containing 0 % fetal bovine serum for 24 h. After treated with or without 30 mM D-glucose, mannitol or L-glucose (Sigma-Aldrich, St. Louis, MO), the cells were harvested for the extraction of protein and RNA. The ERK specific inhibitor PD98059 (Sigma-Aldrich, St. Louis, MO) was added to the cells 30 min before treated with 30 mM D-glucose, mannitol or L-glucose. All treatments were performed in DMEM low glucose media containing 0 % FBS. Each experiment was performed in triplicate.

Wild-type plants (Col-0) were grown in vitro at 21 °C under a 16 h day (110 µmol m⁻² s⁻¹) and 8 h night regime. Sixty-four wild-type seeds were sown on a 14 cm diameter Petri dish with solid 1/2 Murashige and Skoog (MS) medium (Murashige and Skoog, 1962 ) (6.5 g l⁻¹ agar, Sigma), overlaid with a nylon mesh (Prosep) of 20 μm pore size. During growth, plates were randomized. For the proteome (30 min and 4 h mannitol stress), phosphoproteome, and expression analyses, half of the plants were transferred to solid 1/2 MS medium (with 6.5 g l^-1 agar) and the other half to solid 1/2 MS medium containing 25 mM mannitol (Sigma) at 15 days after stratification (DAS). The third leaf was harvested 20 min, 40 min, and 4 h (for expression analysis), 30 min (for proteomics and phosphoproteomics), or 4 h (for proteomics) after transfer. In total three or four biological replicates were performed for the 4 h proteomics and transcriptomics or the 30 min proteomics and phosphoproteomics, respectively. Approximately 100 mg of leaf material was harvested per sample. All experiments were performed independently.