Loganin

RSC96 cells (rat Schwann cell line, BCRC No. 60507) were purchased from Bioresource Collection and Research Center, Food Industry Research and Development Institute (Hsinchu, Taiwan). RSC96 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) containing 5.6-mM glucose, 4-mM L-glutamine plus with 100 U/mL penicillin, 100-µg/mL streptomycin and 10% (v/v) fetal bovine serum (Thermo Fisher Scientific) at 37 °C in 5% CO₂ humidified atmosphere. The medium was changed every 2–3 days.
At 70% confluence cells were synchronized by serum starvation for 4 hr. RSC cells were pretreated with various concentrations (0.1, 1, 10, 25, 50 μM) of loganin (#19997, Cayman Chemical, Ann Arbor, MI, USA) or 1-mM N-acetyl-L-cysteine (NAC, A9165, Sigma-Aldrich, St. Louis, MO, USA) for 2 h and then treated with normal glucose (NG; 5.6-mM glucose), NG plus mannitol as an osmotic control (5.6-mM glucose + 19.4-mM mannitol) and high glucose (HG; 25-mM glucose) for 24, 48 and 72 h. All experiments were performed within 10 cell passages.

PCR reagents, plasmid purification, restriction enzymes, and T4 DNA ligase were used as recommended by New England Biolabs (NEB) unless otherwise noted. Vectors used in this study included the bacterial expression vectors pCWori (43 (link)) and pET28a (Novagen). E. coli cell strains used in this study included Top10 (Invitrogen), DH5α, and BL21(DE3) (NEB). All reagents were used as received from commercial sources. Loganin (Cayman Chemical) and loganic acid (Arctom Chemicals) were 98+% pure.
LC-MS analyses of the Camptotheca SLAS mutants were conducted using a Shimadzu LC-MS2010EV system at the Institute for Genomic Biology, University of Illinois Urbana-Champaign. High resolution LC-MS analyses of the WT Camptotheca SLASs and the SLS, SLAS common ancestor were performed by Dr Alexander Ulanov in the Metabolomics Laboratory of the Roy. J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign using a Dionex Ultimate 3000 series HPLC system (Thermo Scientific) with Q-Exactive MS system (Thermo Scientific). Spectroscopic measurements were recorded with a Cary UV-Vis Bio100 dual beam spectrophotometer or Molecular Devices SpectraMax M-series plate reader as appropriate.