Labtek 2 chambers

Intracellular Ca²⁺ variations ([Ca²⁺]_i) in cultured SMCs were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2 as previously described49 (link)50 (link). SMCs sub-cultured for 4 days in Lab-Tek II® chambers (Nunc, USA) were loaded with 2.5 μM Fura-2AM plus 0.02% Pluronic F-127. Cells rinsed with PSS were maintained in basal buffer during a 15-min waiting period for the de-esterification of Fura-2AM and chambers were mounted on a microscope stage (Axiovert, Zeiss, Germany; 20x objective). Buffer and drugs were then applied by perfusion to the cells as indicated in the figure legends. Cells were illuminated by excitation with a dual UV light source at 340 nm and 380 nm using a lambda DG-4 excitation system (Sutter Instrument Company, CA, USA). Images were captured digitally every 0.35 seconds with a cooled CCD camera (Photometrics, Roper scientific, France) at 510 nm emission. Changes in [Ca²⁺]_i were deduced from variations in the F340/F380 ratio after correction for background and dark currents (Metafluor software, Universal Imaging Corporation, USA). Data were averaged (at least 25 cells per field chosen randomly; one field per cover glass; 4 cover glasses for each experimental condition), with n representing the number of cell cultures.

CH7C17 cells (15 × 10⁶) were allowed to settle in LabTek II chambers (Nalge Nunc International, Sigma-Aldrich Quimica SL, Madrid, Spain) and maintained at 37 °C in a 5% CO₂ atmosphere in phenol red-free RPMI medium 1640 containing 25 mM HEPES and 2% FBS in an incubator coupled to a Leica TCS SP2 confocal microscope (Leica Microsistemas S.L.U.). T cells transiently co-transfected with YFP-RBD-Raf-1 and CFP-H-Ras were serum starved at 37 °C for 2 h before stimulation with DMSO, PGA₁ (30 μM/15 min), or anti-human CD3ɛ T3b mAb (5 μg/ml). Time-lapse confocal images were collected with an HCX PL APO × 40 NA 1.32 oil-immersion objective lens (Leica), and fluorescence images were captured every 1 min. Six confocal Z-sections were necessary to capture the entire fluorescent signal at each time. The ratio of fluorescence intensity between the cellular regions of interest was calculated in a single Z plane, corresponding with the maximum fluorescence plane, by using Leica Confocal Software, version 2.61 (Leica Microsystems, Leica Microsistemas S.L.U.). At least 100 cells were analyzed for each sample. For fluorescence profile analysis, 8 μm cross-sections were drawn (white bar).

To perform the NRF2 translocation analyses, HREC were seeded in fibronectin-coated LabTek II chambers (Nalge Nunc International, Rochester, NY, USA) and treated with Pter 5 uM (DMSO as the vehicle was used at a concentration of 0.1%) 24 h later. Cells were incubated for 0, 5, 6 or 8 h and were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Then cells were incubated with 1% Triton X-100 in PBS for 10 min to permeabilise cell membranes, followed by incubation with 3% bovine serum albumin (BSA) and 5% FBS in PBS for 1 h. Cells were incubated with 1: 250 dilution of primary antibodies against NRF2 (Cell Signaling, Danvers, MA, USA) overnight at 4 °C, followed by the Alexa Fluor^® secondary antibody (Fisher Scientific, Madrid, Spain) for 1 h at room temperature in the dark. Nuclei were stained with 4,6-diamidino-2-phenyindole, dihydrochloride (DAPI) (Fisher Scientific, Madrid, Spain) and then examined under a Leica TCS SP2 confocal microscope (Leica, Wetzlar, Germany).