Six well plate

Proliferation was determined by manual cell count using a Neubauer hemocytometer. Fibroblasts were seeded at 80% confluence (10,000 cells per cm² in six-well plate (Sarstedt, Sevelen, Switzerland) and allowed to adhere overnight in growth medium. Fibroblasts were serum deprived overnight before being stimulated in the presence or absence of treprostinil over 48 h. Fibroblasts were harvested by trypsinization and counted.

For expression analysis, 5×10⁵ cells per well were plated and grown on six-well plates (Sarstedt) for 4 days or taken after 24 h after nucleofection. Total RNA was isolated with miRVana Isolation Kit (Life Technologies) and quality controlled as described earlier.^{45 (link)} MiRNA as well as lncRNA-specific RT-qPCR was performed as described previously^{20 ,45 (link),46 (link)} considering SNORD44 and 47 as references for miRNA and GAPDH, SDHA and SNORD47 for lncRNA, respectively.

HK2 cell lines were obtained from American Type Cell Collection (ATCC). Cells (passages 6–17) were maintained in six- well plates (Sarstedt, Germany) in Keratinocyte-Serum Free (KSF, Invitrogen 17005–075, USA) Medium cultured at 37°C in a humidified atmosphere of 5% CO₂. These cells were exposed to 100ng/ml latent TGFβ1 (R&D Systems 299-LT-005) with or without 10 μM PXS64 (Courtesy of Pharmaxis ltd) for 48 hours. PXS64 was dissolved in DMSO and diluted in PBS buffer. Plain KSF medium (containing 5 mM D-glucose) plus the same amount of DMSO were used as the control. The supernatant and cell lysate were collected after 48 hours treatment.

HeLa cells were grown at 5% CO₂ at 37°C in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were passaged at a 1:4 or 1:6 ratios at 80–90% confluence, using trypsin digestion. For transfection, cells were seeded in six-well plates (Sarstedt). Using Lipofectamine 3000 (Thermo Fisher), cells were transfected according to the manufacturers protocol. Concisely, a mixture of 125 µl Opti-MEM (Thermo Fisher) with 2 µg of the respective plasmid DNA and 4 µl P3000 reagents was prepared. After 5 min of incubation, the mixture was transferred to another solution containing 125 µl Opti-MEM supplemented with 4 µl Lipofectamine3000 reagent. Cells were incubated for 6 h after the addition of transfection mixture to the cellular growth medium at 5% CO₂, 37°C. The cells were passaged using trypsin digestion and seeded in 35 mm glass bottom dishes (Fluorodish, World Precision Instruments). Cells were grown for 2 days at standard cell culture conditions before imaging.

Cell death was determined through the permeability of the plasmatic membrane; for this purpose, six-well plates (Sarstedt, Nümbrecht, Germany) were used. After the treatment time, cells were harvested by trypsinization and the Muse^® Count & Viability Kit (Luminex^®, Austin, TX, USA) was added following the manufacturer’s instructions. Cell death was determined using the Muse^® Cell Analyzer (Luminex^®, Austin, TX, USA).

After reaching confluence in the primary culture, BM-MSCs were trypsinized and seeded at a density of 2 × 10⁵ MSCs/well, in six-well plates (Sarstedt, USA). After 48 hours, the maintenance medium was removed and media for osteogenic or adipogenic differentiation StemPro (Gibco Invitrogen, USA) were added to the subcultures, in triplicate, according to the manufacturer’s recommendation with modifications. The adipogenic medium was supplemented with 5% rabbit serum. The media were changed every 2 to 3 days and confirmation of osteogenic and adipogenic differentiation was obtained, respectively, by demonstrating the deposition of a calcium-containing matrix using the histological method of staining with 2% Alizarin red, pH 4.2 and the presence of intracytoplasmic lipid droplets by staining with 0.5% oil red O (Sigma-Aldrich Corp., USA).
For chondrogenic differentiation, the BM-MSCs were cultivated at a density of 10⁶ MSCs/mL in a 3D pellet in a Falcon tube (15 mL) for 21 days in StemPro chondrogenic differentiation medium that was changed every three days. To confirm that chondrogenic differentiation had occurred, the pellets were stained with Alcian Blue, pH 2.5, and toluidine blue, pH 1, which detect proteoglycans [39 (link)].