L glutamin

Cell lines Hs746T and H596 were obtained from ATCC (Manassas, VA). Cell lines E98^{28 (link)}, Hs746T, H596, U87-EV and U87-EGFRvIII (obtained from dr. Web Cavenee, Ludwig Cancer Inst., USA^{27 (link)}) were cultured at 37 °C in the presence of 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L glucose and 4 mM L-glutamin (Lonza, Basel, Switserland), 10% fetal calf serum (FCS, Gibco, Waltham, MA, USA) and 40 µg/ml gentamycin (Centrafarm, Ettenleur, The Netherlands). The MET^Δ7-8-expressing astrocytoma cell line E98 and the EGFR-expressing astrocytoma line E468, as well as the generation of orthotopic xenografts thereof, has been described before^{8 (link),28 (link)}.

Human bone marrow-derived MSCs of two healthy donors (Donor 4266 and 3520, 33 and 20 year old males, respectively) were purchased from Lonza (PT-2501). MSCs were differentiated into osteoblasts as described previously^{24 (link)}. Briefly, MSCs were cultured in αMEM medium (Gibco, Thermofisher) supplemented with 10% heat-inactivated fetal calf serum a (FCS, Sigma), 20 mM Hepes (Sigma), 100 U/mL penicillin (Lonza) and 100 µg/mL streptomycin (Thermofisher) and 1.8 mM CaCl₂ (Sigma) at 37 °C and 5% CO₂ in a humidified atmosphere. Medium on day 3 post-seeding was supplemented with 100 mM dexamethasone (dex) and 10 mM β-glycerophosphate (osteogenic medium) resulting in differentiation into mineralizing osteoblasts within 2–3 weeks¹⁴ . The media were refreshed twice per week. Vero cells (African green monkey kidney epithelial cells, ATCC CCL-81) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, the Netherlands) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Greiner Bio-One, Austria), 2 mM L-glutamin (Lonza), 1% sodium bicarbonate (Lonza), 1% Hepes (Lonza), 100 U/mL penicillin (Lonza) and 100 µg/mL streptomycin (Lonza) at 37 °C and 5% CO₂ in a humidified atmosphere.

The pellets were cultured after a pre-plating step (Sharifiaghdas et al. 2011 (link)), in 75 cm² flasks at a seeding cell density of 1800 cells/cm², coated with Matrigel™ (BD Biosciences, The Netherlands) diluted 1:200 in DMEM culture medium containing 1% PSA, until 85–90% confluency in Advanced DMEM (Life technologies, USA) containing 20% Fetal Bovine Serum (FBS, Life technologies, USA), 10% Horse Serum (HS, Life technologies, USA), 4 mM l-Glutamin (Lonza, Germany) and 1% PSA (growth medium: GM). Subsequently, medium was aspirated and the cells were washed with PBS. Next, cells were passaged by trypsinization with 0.05% trypsin-EDTA (Life technologies, USA) for 5–8 min at 37 °C, 5% CO₂ in a humidified incubator and neutralization with TNS (Life technologies, USA). The resulting suspension was spun down at 460 g for 5 min at ambient temperature. After removal of the supernatant, cells were resuspended in freezing medium (Life technologies, USA) and stored in liquid nitrogen. Phenotype of the cells was confirmed by CD56 immunostaining and ability to form myotubes upon serum withdrawal (data not shown).

To screen the secretome of SVF cells isolated from the thigh region of healthy subjects and lipedema patients, 5 × 10⁵ freshly isolated SVF cells were seeded in a T-25 cell culture flask with 2.5 mL EGM-2 medium, which was replaced with serum-free DMEM low glucose with 2 mM of L-glutamin (Lonza) after 2 h. After 24 h, the CM was collected and stored at −80 °C until further use. The CM was subjected to a customized membrane-based sandwich immunoassay (RayBio^® Membrane-Based Antibody Arrays (C-Series)) according to the manufacturer’s instructions (RayBiotech, Peachtree Corners, GA, USA). Imaging of the membranes was performed on a Vilber Lourmat Fusion-SL-3500 WL (Vilber, France) and analyzed using FusionCapt Software (Vilber). The images were then analyzed with the Fiji Plugin “Protein Array Analyzer” (ImageJ) and normalized according to the manufacturer’s provided analysis table.

The human breast adenocarcinoma cell line MCF-7 was obtained from the American Type Culture Collection (ATCC) and maintained in Eagle’s Minimum Essential Medium (MEM) (Gibco by Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Gibco), 10 μg/mL insulin (SAFC Biosciences by Sigma-Aldrich), 2 mM l-glutamin and 50 U/mL of a penicillin–streptomycin solution (Lonza). The human non-cancerous mammary epithelial cell line MCF-10A was obtained from the ATCC and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F-12 (1 : 1) (Gibco) supplemented with 5% horse serum (HS) (Gibco), 10 μg/mL insulin (SAFC Biosciences), 0.5 μg/mL hydrocortisone (Sigma-Aldrich), 20 ng/mL epidermal growth factor (EGF) (Gibco), 10 μg/mL cholera toxin (Sigma-Aldrich) and 50 U/mL of a penicillin–streptomycin solution (Lonza). All cell lines were validated in the Genomics Core Facility at Alberto Sols Biomedical Research Institute (Madrid, Spain).

The human cervix carcinoma cell line HeLa and a genome-edited clonal HeLa LMNA knockout (LMNA-KO) cell line [47 (link)] were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with l-glutamin (Lonza, BE12-604F/12), 10% fetal bovine serum (Gibco, 10270-106), and 1% penicillin/ streptomycin (Gibco, 15140-122), according to standard procedures. Proliferative capacity was monitored by cell counting with every passage, and cultures were tested for mycoplasma infection using a PCR test kit (Bioconnect, PK-CA91-1024) every 2 months. Cells were grown on different substrates depending on the type of analysis that was done post-photoporation: µ-Slide Angiogenesis slides (Ibidi, 81506) for immunofluorescence staining, glass bottom dishes (Greiner Bio-one, 627870) for live cell imaging and 96-well plates (VWR) for whole transcriptome analysis.

Peripheral blood was collected from chronic HBV patients or healthy controls in sodium-heparin tubes, and PBMC were isolated by Ficoll-Paque (GE Healthcare, Uppsala, Sweden) gradient centrifugation, and frozen. PBMC (1×10⁶ cells/ml) were thawed and stimulated in 96-well plates in 250 µl X-VIVO culture medium (Lonza, Verviers Sprl, Belgium) containing penicillin/streptomycin (Gibco, Paisley, UK), L-glutamin (Lonza), and HEPES (Lonza) as described previously [7] (link): the cells were cultured either unstimulated or stimulated with 100 ng/ml Toll-like receptor (TLR) 2 ligand Pam3CSK4, 100 ng/ml TLR4 ligand LPS (both from Invivogen), 1 µg/ml TLR7/8 ligand R848 (Enzo Life Sciences, Antwerp, Belgium), or human plasma-derived pHBsAg Ay (American Research Products (ARP), Waltham, MA, USA) at a concentration of 5 µg/ml unless mentioned otherwise. In the experiment shown in Figure S1, in parallel to plasma-derived HBsAg Ay (5 µg/ml) stimulation, PBMC were also stimulated with recombinant HBsAg (1 µg/ml; Prospec, Rehovot, Israel) for 5 hours. In the IL-10 inhibition experiment, 10 ng/ml IL-10 (Miltenyi Biotec, Bergisch Gladbach, Germany) or medium as a negative control, was added at the same time as pHBsAg (1 µg/ml; ARP). After a total of 18 hours of culture, supernatants were harvested, and cytokine production (IL-6 and TNF) was determined by ELISA (all kits from eBioscience).