Fig 2: Stretch induces NFAT5 palmitoylation. Exposure to biomechanical stretch promotes a slower electrophoretic migration speed of NFAT5 resulting in a band shift of about 5 kDa (see also Figures 6A and 6B) which disappears upon treatment with 2-bromopalmitate (A, *P<0.05 vs stretch, n=3). HUASMCs were incubated with fatty-acid free BSA, treated with 100 µmol/L 17-octadecynoic acid (Cayman Chemical) to label palmitoylated proteins (control was left untreated) and exposed to biomechanical stretch for 24 hours (13% elongation, 0.5 Hz) or cultured under static conditions. Afterwards, proteins were isolated and excess 17-octadecynoic acid was removed by chloroform/methanol extraction. Standard click chemistry (Martin et al, Nat Methods. 2009;6:135-138) was performed for 90 minutes utilizing 1 to 2 mg/mL protein, 100 µmol/L biotin-azide (in DMSO), 1 mmol/L Tris(2-carboxyethyl)phosphine (in water), 100 µmol/L Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (in DMSO/t-butanol, 20%/80%), and 1 mmol/L CuSO4 (in PBS). After a second chloroform/methanol extraction, biotinylated and thus palmitoylated proteins were captured by streptavidin agarose beads. After rinsing, proteins were eluted from the beads, separated by SDS-PAGE, blotted onto nitrocellulose membranes and analyzed by chemiluminescence-based immunodetection. NFAT5 was only detected in stretch-stimulated HUASMCs as evidenced by Western blot analysis indicating its palmitoylation. ß-actin was detected in the flowthrough by standard Western blot analysis to verify that comparable amounts of proteins had been applied. BSA indicates bovine serum albumin; HUASMCs, human arterial smooth muscle cells; NFAT5, nuclear factor of activated T-cells; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline: SDS, sodium dodecyl sulfate.

Fig 3: TNF production under hypertonic conditions involves p38 and NFAT5. (a) REH cells were cultured in the presence and absence of 50 mM NaCl for 16 h. Cells were washed, lysed and subsequently analyzed using western blotting with antibodies specific for the indicated proteins. (b) REH cells were challenged with 1.25 µM birinapant in the presence and absence of 50 mM NaCl for the indicated periods of time. Subsequently, cells were lysed and analyzed by western blot for the indicated proteins. The asterisks (*) in the phospho-p38, ERK and phospho-ERK blots indicate a defect in the CCD sensor of the western Blot imaging system. All samples were run on the same gel, no gels were sliced. (c–e) REH cells were challenged with 2.5 µM birinapant under isotonic (medium) or hypertonic (+50 mM NaCl) conditions in the presence and absence of the p38 inhibitors BIRB796, PH797804 and SB203580 (5 µM). Shown are data points and mean±S.E.M. from four independent experiments. (f) REH cells were challenged with 50 mM NaCl for 210 min in the presence and absence of the p38 inhibitors BIRB796, PH797804 and SB203580 (5 µM). Tnf mRNA induction was analyzed by qPCR. Data points of three independent experiments are shown. Biri, birinapant

Fig 4: ERK1/2, p38 MAPK and calcineurin are not involved in stretch-induced translocation of NFAT5. Immunofluorescence analysis of HUASMCs stretched for 24 hours and treated with DMSO (A) or 50 µmol/L PD98059 (B, scale bar: 50 µm), an inhibitor of the ERK1/2 pathway. Quantification of NFAT5-positive nuclei (C, n=3). Immunofluorescence analysis of HUASMCs stretched for 24 hours and treated with DMSO (D) or 20 µmol/L SB202190 (E, scale bar: 50 µm), a specific inhibitor of p38 MAP kinase. Quantification of NFAT5-positive nuclei (F, n=3). Immunofluorescence analysis of HUASMCs stretched for 24 hours and treated with DMSO (G and I) or 10 µmol/L cyclosporin A (H and J), an inhibitor of calcineurin. Quantification of NFAT5-positive nuclei (K, *P<0.05 vs corresponding DMSO or cyclo A control, bars represent the mean of 1 experiment ±SD performed in triplicates). DMSO indicates Dimethylsulfoxide; ERK1/2, extracellular-regulated kinase 1/2; HUASMCs, human arterial smooth muscle cells; NFAT5, nuclear factor of activated T-cells 5.

Fig 5: NFAT5 translocates to the nucleus upon hyperosmolarity. Immunofluorescence analysis of HUASMCs treated with control medium (A), 30 mmol/L NaCl (B) and 70 mmol/L NaCl (C) for 24 hours. Quantification of NFAT5-positive nuclei (D) (n=3, ***P<0.001 vs control; scale bar: 50 µm). HUASMCs indicates human arterial smooth muscle cells; NFAT5, nuclear factor of activated T-cells 5.