Il 1β

U251‐MG cells were seeded in 24‐well plates at a density of 1 × 10⁵ cells/well. 24 h later, the cells were transfected with PEI reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The total amount of 0.6 μg of plasmid DNA per well was used, including 0.4 μg of the luciferase‐coding reporter plasmid and 25 ng of Reg‐1, Reg‐2, TTP, corresponding RNase‐inactive mutant or the empty control plasmid. The quantity of DNA/well was normalized using an empty pcDNA3.1/mycHisA vector (Invitrogen). 24 h post‐transfection cells were stimulated with IL‐1β (Invivogen) or left untreated. 24 h after stimulation cells were lysed and firefly and Renilla luciferase activity were measured using Dual‐Luciferase Reporter Assay System (Promega), according to the manufacturer's instructions. For transfection efficiency normalization the Renilla luciferase, encoded on the same plasmid as the firefly luciferase (pmirGLO), was used. Luciferase data are presented as relative luciferase activity (firefly/Renilla) and normalized to 1.

HT-29/kb-seap-25 cells were seeded on 96-well plates at 3 × 10⁴ cells/well and incubated for 24 h at 37°C under 5% CO₂ prior to stimulation (Lakhdari et al., 2010 (link)). Monolayer confluence was checked under the microscope before and after every stimulation. TNFα (1 ng mL^–1; PeproTech), IL-1β (1 ng mL^–1; Invivogen), and LPS from E. coli O111:B4 (1 ng mL^–1; L3024-5MG, Sigma-Aldrich) were used to induce inflammation. The cells were stimulated with the samples (controls and EV preparations) and inflammation inducers for 24 h. The supernatants from all the wells were then revealed with Quanti-Blue^TM reagent (Invivogen) to assess SEAP activity. Cell proliferation was evaluated under all conditions using the CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS, Promega), according to the manufacturer’s instructions. Absorbance was read at 655 nm for the SEAP activity assay and at 490 nm for the MTS assay using a Xenius (SAFAS Monaco) microplate reader. For surface protein assays, 10⁹ EV ml^–1 were applied directly or after incubation at 37°C for 1 h in the presence or absence of proteinase K (20 μg mL^–1; Qiagen), in order to evaluate a possible role for EV surface proteins in immunomodulation.

50,000 hPDL-MSCs were seeded per well in 3 mL DMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 50 µg/mL streptomycin at collagen type I coated 6-well BioFlex^® plates (FlexCell International Cooperation, Burlington, VT, USA). After 24 h incubation, cell cycles were synchronized by changing medium to DMEM without any FBS, supplemented with only 100 U/mL penicillin and 50 µg/mL streptomycin. After overnight-starvation, hPDL-MSCs were stimulated with 1 ng/mL or 5 ng/mL IL-1β (Invivogen, San Diego, CA, USA) using DMEM medium supplemented with either only antibiotics or with antibiotics and 2% FBS. Appropriate unstimulated hPDL-MSCs served as control. Simultaneously, STS was applied to hPDL-MSCs using the FlexCell FX-5000T™ Tension System (FlexCell International Cooperation, Burlington, VT, USA) with 6% equibiaxial elongation. IL-1β stimulated and unstimulated hPDL-MSCs, which were seeded on the same cell culture plate without applying STS served as references. 6 and 24 h later, MMP-1, MMP-2, TIMP-1 and IL-1β gene and protein expression levels were measured by quantitative polymerase chain reaction (qPCR) and ELISA, respectively. Additionally, cell viability was determined by live/dead staining followed by fluorescence microscopy 24 h after stimulation.