Clariostar apparatus

Cells (25 mL; OD_600nm = 0.3) growing exponentially in MC were carefully collected by centrifugation (7 min; 4,922 RFC; room temperature) and resuspended in 75 μL of lysis buffer (Na₂HPO₄ 2H₂O: 60 mM; NaH₂PO₄ 4H₂O: 4 mM; KCl: 10 mM; MgSO₄ 7H2O: 1 mM; DTT: 1mM; Lyzozyme: 0.1 mg/mL; DNase I: 40 U/mL). They were then incubated 20 min on ice, 5 min at 37 °C, and 15 min at room temperature. Crude extracts were then collected by centrifugation (10 min; 14,000 rpm; 4 °C). The PykA activity was determined using the colorimetric/fluorometric assay kit K709-100 (CliniScience, Nanterre, Fr) and fluorescence (Ex/Em = 535/587 nm) was assessed using a ClarioStar apparatus (BMG Labtech, Champigny-sur-Marne, Fr). Protein concentration was monitored using the standard Bradford assay.

DPPIV enzyme activity of the STB-EVs was determined using a DPPIV-Glo Protease Assay (Promega, Southampton, UK). Briefly, the luminescent DPPIV-Glo Reagent was incubated at R/T for 30 min with either clinical samples (STB-EVs isolated from perfused placentae from both GDM and normal pregnancy), Tris-BSA (blanks) or a purified DPPIV enzyme in Tris-BSA as standard (Recombinant human CD26 protein, Abcam, Cambridge, UK). Luminescence was measured using a FLUOstar Omega apparatus (BMG Labtech, Aylesbury, UK).
For the DPPIV enzymatic inhibition studies, MEDIUM/LARGE STB-EVs and SMALL STB-EVs were pre-treated with vildagliptin (DPPIV-specific inhibitor) (Sigma Aldrich, Gillingham, UK) (0–100 nM) for 1 h, and DPPIV residual enzymatic activity was determined.
STB-EV-bound DPPIV ability to break GLP-1 was measured using a GLP-1 active assay according to manufacture instructions (Cisbio, Codolet, France). MEDIUM/LARGE STB-EV and SMALL STB-EV pools of three GDM patients in series dilution were mixed with 1400 pg/ml of GLP-1, respectively, and incubated with anti-Active GLP-1-Tb3+Cryptate Antibody and with anti-Active GLP-1-d2 antibody overnight at RT. Recombinant DPPIV (Recombinant human CD26 protein, Abcam, Cambridge, UK) was used as positive control, and PBS was used as negative control. HTRF measurement was performed using a CLARIOstar apparatus (BMG Labtech, Aylesbury, UK).

Optical rotations were determined with a PerkinElmer Polarimeter 341 (Boston, MA, USA). UV spectra were recorded on a Milton Roy Spectronic 3000 Array spectrophotometer (Rochester, Monroe, NY, USA). IR spectra were obtained with a PerkinElmer FT-IR 1760X spectrophotometer (Boston, MA, USA). Mass spectra were measured using a Bruker MicroTOF mass spectrometer (ESI-MS) (Billerica, MA, USA). NMR spectra were recorded on a Bruker Avance DPX-300FT NMR spectrometer or a Bruker Avance III HD 500 NMR spectrometer (Billerica, MA, USA). Yeast α-glucosidase enzyme and p-nitrophenol-α-d-glucopyranoside were obtained from Sigma Chemical, Inc. (St. Louis, MO, USA), and acarbose was purchased from Fluka Chemical (Buchs, Switzerland). Microtiter plate readings were carried out with a CLARIOstar apparatus (BMGLABTECH, Ortenberg, Germany).