*2.6. Immunofluorescence*

Protein expression of FasL, caspase-3 and TGF-beta1 were studied by fluorescence microscopy (Biorevo BZ-9000, Keyence, Japan) on cover slips with cells after stimulation with IL-1 α (1.0 ng/mL) in the presence or absence of compounds after 48 h. After treatment, cell permeability was increased by Triton-x (5%). Afterwards the primary antibody was added (1:100 in 5% donkey serum), caspase-3, FasL and TGF-beta1 overnight. After washing, the secondary antibody was added (1:100; FITC/Cy3, Agilent Dako, Santa Clara, CA, USA). Nuclei were stained with Hoechst (1:4000) (Merck Sigma-Aldrich, Darmstadt, Germany).

#### *2.7. IncuCyte Live-Cell Analysis*

IncuCyte Live-Cell Analysis System (IncuCyte ® S3, Sartorius, Göttingen, Germany) was used to observe cell death and proliferation. The experiments were performed with VSMCs and H9c2 cells. Cells were transferred into 96-well plates and treated with the

compounds or vehicle under same conditions (medium with 1% or 10% FBS) for 48 h (up to 5 days). Cell proliferation was monitored by analyzing the occupied area (% confluence) of cell images over time. Analysis of the IncuCyte images was performed with Incucyte® Analysis Software.

#### *2.8. Glucose, Lactate, Electrolytes Concentrations*

The concentrations of glucose, lactate, sodium, calcium and potassium were measured in cell supernatant 48 h after treatments. Na, K and Cl were quantified by an indirect ion selective electrode (ISE, Gen. 2 Roche® Diagnostics GmbH) on a Roche® cobas ISE module. Lactate dehydrogenase activity was determined applying Roche® cobas lactate dehydrogenase according to IFCC version 2 (LDHI2, #05169330 190). Glucose and lactate were quantified photometrically on a Roche® cobas analyzer.
