Penicillin streptomycin

To examine the presence of NME1 and NME2 in EVs, we used human breast adenocarcinoma MDA-MB-231T cell lines, which were stably transfected with one of the following constructs: pcDNA3 (Co), pcDNA3/FLAG-NME1 (F::NME1) or pcDNA3/MYC-NME2 (M::NME2) (the clones are a kind donation of Maja Herak Bosnar (Rudjer Boskovic Institute, Zagreb, Croatia)). These cell lines were cultured in DMEM high glucose media (Biosera, Nuaille, France) supplemented with 10% FBS (Biosera), 2 mM L-glutamine (Biosera), 100 µI/mL penicillin–streptomycin (Biosera), 7.5% NaHCO3 (Biosera) and 50 mg/mL geneticin (Sigma) at 37 °C with 5% CO₂.
To monitor the effect of EVs derived from MDA-MB-231T cells, we applied a human skin derived cell line, fibroblast 203-9. They were cultured in DMEM high glucose media (Biosera) supplemented with 10% FBS (Biosera), 2 mM L-glutamine (Biosera), 100 µI/mL penicillin–streptomycin (Biosera), 1% sodium pyruvate (Biosera) at 37 °C with 5% CO₂.

Human K562 cells were cultured in RPMI-1640 media at 37 °C, supplemented with 10% heat-inactivated FBS (Biosera, UK), 1% l-glutamine and 1% penicillin–streptomycin. Cells were washed in low conductivity DEP buffer medium containing 8.5% sucrose and 0.3% dextrose and resuspended in CPM at a final concentration of 10⁶ cells/ml. HeLa cells were cultured in MEM with Earle’s salts and non-essential amino acids (Biosera, UK) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich, UK), 1% l-glutamine and 1% penicillin–streptomycin (Sigma Aldrich, UK). At about 80% confluency, cells were trypsinized and washed twice in DEP buffer medium and resuspended in CPM at a final concentration of 10⁶ cells/ml. HL-1 cells were cultured in Claycomb media supplemented with 10% heat-inactivated FBS, 1% penicillin–streptomycin, 1% l-glutamine and Norepinephrine (0.1 mM) (Biosera, UK). The cells were trypsinised at 100% confluence and washed twice in DEP buffer medium before resuspending in CPM at a final concentration of 10⁶ cells/ml. Viability of cell suspensions was confirmed on control samples prior to experiment using a LIVE/DEAD Staining Kit (Sigma Aldrich, UK). Further viability was monitored with trypan blue (Sigma Aldrich, UK).

Human HeLa cells (kindly provided by Melchior F., Universität Heidelberg, Germany), HEK293 Flp-In T-REx cells (Thermo Fischer Scientific, Waltham, MA, USA), and HeLa Flp-In T-REx cells (kindly provided by Mayer T., Universität Konstanz, Germany) were cultured at 37 °C with 5% CO₂ in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and penicillin–streptomycin (Biosera, Nuaille, France). For hypoxic treatment, cells were exposed to 1% O₂, and 5% CO₂ in an INVIVO2 200 hypoxia workstation (Ruskinn Life Sciences, Pencoed, UK) for the indicated times. When required, cells were treated with 2 μΜ rapamycin for 6 h (Sigma-Aldrich, St Louis, MO, USA) or DMSO at the appropriate concentration as a solvent control.
HeLa Flp-In T-REx cells were transfected with pcDNA5-2xFlag-His6-siinsEXOSC10 (WT or K583R) or pcDNA5-2xFlag-His6 (empty vector) plasmids using XtremeGene9^® transfection reagent (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer’s guidelines. Transfected cells in which the transgene was integrated into the Flp-In locus were selected with 1 µg/mL of blasticidin and 200 µg/mL of hygromycin.

U87MG human malignant glioma, SK-MEL-28 human melanoma, SK-OV-3 human ovarian cancer, and A549 human lung cancer cell lines were purchased from ATCC. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Biosera, Nuaille, France), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; Biosera), and with 1% Penicillin/Streptomycin (Biosera). Cells were cultured in sterile T75 flasks with ventilation cap (Sarstedt, Nümbrecht, Germany) at 37 °C in a humidified atmosphere with 5% CO₂ in ESCO CelCulture Incubator (ESCO, Friedberg, Germany). Manipulations with the cells were performed in biosafety cabinet (laminar) ESCO Sentinel Gold class II model AC2-4E8 (ESCO).

In this study, we used human dental pulp stem cell line (HDPSCs; Royan Institute, Tehran, Iran). HDPSCs were cultured and expanded in low-content glucose Dulbecco’s modified Eagle medium (DMEM/LG; Gibco), with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Biosera). The culture plates were maintained at 37ºC in a humidified atmosphere with 5% CO₂. The cells between passages 3 to 6 were subjected to various experiments.

FCLs were established from skin biopsies of two patients with SA (PF1-2), after informed consent. Sex and age matched adult human primary dermal fibroblasts were purchased from Lonza in order to be used as non-SA controls (CF1-2). The cell lines were further cultured and expanded in DMEM medium (GIBCO, Thermo-Fisher), supplemented with 10% FBS (Thermo-Fisher), and 10U/mL Penicillin/Streptomycin (Biosera).