Fluostar omega apparatus

LPS micelle formation was studied using LPS-BODIPY-FL as described.^{36 (link)} The fluorescence of LPS-BODIPY-FL increases upon disaggregation of LPS micelles. Increasing concentrations of granzyme (0–20 μg/ml) were added to LPS-BODIPY-FL (7.5 μg/ml) in 250 μl PBS. LPS-BODIPY-FL alone was used as a negative control and LPS-BODIPY-FL plus GrK served as a positive control. Disaggregation of LPS micelles upon treatment of LPS-BODIPY-FL with 2% SDS was set at 100%. Fluorescent intensity of all samples was measured kinetically for 2 h at 37 °C at 520 nm using the FluoStar Omega apparatus (BMG Labtech, Ortenberg, Germany).

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).

Bicistronic assays were performed using the Renilla/Firefly expression plasmid (pRF) in which were cloned either wt MELOE‐1 IRES, defined as 275 nucleotides upstream of MELOE‐1 (ORF 1491–1631), or the IRES from EMCV virus used as a positive control [9 (link)]. The empty pRF vector was used as negative control. Melanoma (M113) cells were seeded in RPMI‐10% FCS one day prior to transfection to reach 50–70% confluency and were transfected with the different pRF constructs (200 ng per well in 96‐well plate) using 0.4 µL of LTX Lipofectamine (Thermo Fisher Scientific). Cells were lysed 48 h post‐transfection, and luminescence activities (Renilla and Firefly) were measured with a FLUOstar Omega apparatus (BMG LabTech, Champigny sur Marne, France) using the dual reporter assay as instructed (Promega). Results are expressed as the ratio Renilla/Firefly*100. Silencing of hnRNP‐A1 was performed by cotransfecting 50–100 nm of hnRNP‐A1‐specific siRNA (100 nm of sc‐270345, Santa‐Cruz Biotechnology (Heidelberg, Germany) or 50 nm of Hs‐HNRPA1‐1, functionally validated FlexiTube siRNA, Qiagen, Courtaboeuf, France) or a universal siRNA as negative control (sc‐37007, Santa‐Cruz Biotechnology and AllStars Negative control FlexiTube siRNA Qiagen).