E plate 96

Lymphatic malformation-lymphatic endothelial cells (LM-LECs) were maintained as described (Boscolo et al., 2015 (link)) and negative for mycoplasma at the time of these studies. Mycoplasma test was performed using the MycoAlert Mycoplasma Detection Kit (Cat # LT07-218, Lonza) following the manufacturer’s instructions. Real-time analysis of cell viability was performed by using the xCELLigence system RTCA SP (ACEA Biosciences). Briefly, 5 × 10³ LM-LECs per well were seeded in an E-Plate 96 (ACEA Biosciences) and cell proliferation was recorded hourly. When the cells reached the exponential growth phase, new media containing alpelisib at 1, 3, 10, 30, or 100 nM was added and alpelisib cytotoxic effect was recorded hourly. IC₅₀ was calculated by using the dose–response curve function available in the xCELLigence software Version 2.0. Cell index (%) reflects cell viability.

Impedance was measured at 10 kHz by an RTCA SP instrument (RTCA-SP instrument, ACEA Biosciences, San Diego, CA, USA). We have successfully tested the cellular effects of peptides and pharmaceutical excipients by impedance kinetics [23 (link),27 (link),28 (link)]. For background measurements, 50 μL cell culture medium was added to the wells. This was followed by seeding the cells at a density of 6 × 10³ cells/well to 96-well plate with gold electrodes (E-plate 96, ACEA Biosciences) coated with collagen. Cells were cultured for 5 days in a CO₂ incubator at 37 °C and monitored every 10 min until the end of experiments. Cells were treated at the beginning of the plateau phase of growth. Lira, Lira-loaded PLGA NPs, blank PLGA NPs (without cargo), Lira and PN159 solution, and PN159 peptide were diluted in cell culture medium and the effects were followed for 24 h. Triton X-100 detergent (1 mg/mL) was used as a reference compound to obtain total cell toxicity. Cell index was defined as R_n-R_b at each time point of measurement, where R_n is the cell–electrode impedance of the well when it contains cells and R_b is the background impedance of the well with the medium alone.

Primary fibroblast like synoviocytes (the kind gift of the late Professor Yrjö Konttinen, University of Helsinki, Finland)^{15 (link)} were cultured in DMEM with 10% FCS for maintaining. Plates (96-well E-Plate 96; ACEA Biosciences, San Diego, CA, USA were coated o/n with FN N-terminal 30 kDa fragment or fibronectin from bovine plasma (3 µg/cm³, Sigma-Aldrich, St. Louis, Missouri United States), fibrinogen (100 µg/ml), synthesized triple helical collagen mimetic peptide GPC-(GPP)5 -GAOGER-(GPP)5-GPC-NH₂ (10 µg/ml) or BSA (1 mg/ml) in +4 °C. The wells were blocked with BSA (1 mg/ml, RT, 1 h) and treated with PAD2 or PAD4 (1.2 U/ml, 50–150 µg/ml), 2 h - o/n, +37 °C), or PAD4 supplemented with Cl-amidine (500 µM; N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-Ornamide; Merck MilliPore) in 40 mM Tris, 5 mM CaCl₂, pH 7.4. Detached fibroblast like synoviocytes (trypsinised; P5-7 were used in the experiments; 15 000–20 000 cells per well) were seeded to the wells in serum-free DMEM. Cell spreading was measured with the xCELLigence system (Roche Applied Science).

The growth, proliferation and adhesion kinetics of Vero cells were determined using RTCA technology (ACEA Biosciences, San Diego, CA USA) as previously described with some minor modifications [18 (link)]. Briefly, 50 μl of EMEM supplemented with 10 % FBS (cell culture medium) was placed in each well of the E-plate 96 (gold-microelectrode array integrated E-plate; ACEA Biosciences, San Diego, CA USA). E-plate 96 was then connected to the system to obtain background impedance readings. This was to ensure that all wells of E-plate 96 and the connections were in good condition so as to avoid compromising the interpretation of the results. Serial dilutions of 2.0 × 10⁴, 1.8 × 10⁴ and 1.5 × 10⁴ cells in 50 μl were prepared, four replicates in each of the concentrations. These serial dilutions of cell suspensions were added to the wells containing 50 μl of culture medium. The E-plates were incubated at room temperature for 30 min in a laminar flow cabinet and then placed on the RTCA SP Station located in an incubator at 37 °C for continuous impedance recording. CI values measured by continuous impedance recordings every 2 minutes reflected the cell activities.

Cell viability was assessed using the xCELLigence real-time cell analyser (ACEA Bioscience, Agilent, San Diego, CA, USA). siRNA-transfected B4G12 cells were seeded at 4 × 10⁴ in triplicate in an E-Plate 96 (ACEA Biosciences) and left to attach for 18 hrs. Cells were left untreated or treated with indicated compounds. The electrical impedance readings of overall cell viability were recorded, using the xCELLigence real-time cell analyser, throughout the experiment for at least 8 h. The percentage of viable cells was calculated for each time point relative to time zero, which was set as the last impedance reading prior to addition of compounds. Data shown are representative of three independent experiments. Statistical analysis was performed using GraphPad software (Prism, version 7, GraphPad, San Diego, CA, USA). Groups were compared with two-way ANOVA, followed by post-hoc Bonferroni test, for multiple comparisons, with p-value < 0.05 considered significant.