A confocal microscope

FISH analysis using a biotin-labeled probe specific for lncRNA NR-133666 (GenePharma Co. Ltd. Shanghai, China) was performed. CIA FLS cells were fixed with 4% paraformaldehyde for 20 min, then the cells were washed twice with phosphate buffer saline (PBS). We then added protease K (20 μg/ml) to digest for 1–5 min and incubated with the FISH probe for 1 h. The nucleus was counterstained with 4,6-diamidino-2-phenylindole (DAPI). A confocal microscope (Leica Microsystems, Mannheim, Germany) was used to acquire images.

The cells were seeded into 24-well culture plates, and lentiviral vectors were then used to silence circRYK (shcircRYK) or function as a control (shCONT), fix the cells in 4% paraformaldehyde for 15 min, then permeabilize the cells with 0.3% Triton X-100 for 30 min at 37 °C. The cells were treated with FITC-conjugated secondary antibody and 5% BSA in PBS for one hour at room temperature in the dark, after which they were blocked for thirty minutes with 10% goat serum. Fluorescent Abs were then incubated at 4 °C for an additional night. The cells were then stained with DAPI for 5 min. Pictures were obtained utilizing a confocal microscope (Leica Microsystems) [30 (link)].

The assumption that mitochondria serve as the major intracellular source of ROS was based on experiments with isolated mitochondria, rather than on direct measurements in living cells. The MitoSOX™ Red mitochondrial superoxide indicator is a novel fluorogenic dye for the highly selective detection of superoxide in the mitochondria of viable cells. The MitoSOX™ Red reagent is a viable-cell permeant that rapidly and selectively targets the mitochondria. Once in the mitochondria, the reagent is oxidized by superoxide and exhibits red fluorescence. The reagent is readily oxidized by superoxide, but not by other ROS or reactive nitrogen species-generating systems, and the oxidation of the probe is prevented by superoxide dismutase. The oxidation product becomes highly fluorescent upon binding to nucleic acids. Mitochondrial superoxide is generated as a byproduct of oxidative phosphorylation. Briefly, cells were stained by MitoSOX™ Red reagent (5 μM) at 37 °C for 30 min, and the fluorescence intensity was measured using a confocal microscope (Leica Microsystems, Mannheim, Germany) [41 (link)].

In brief, cells were washed with PBS, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked in 2% BSA before staining with anti-phalloidin and anti-FAK antibodies (both from Cell Signaling). Nuclear DNA was visualized with 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma). Images were collected with a confocal microscope (Leica Microsystems).

P3 cardiomyocytes were plated on gelatin-coated coverslips, transfected with the corresponding siRNA at day one and grown in cardiomyocyte medium supplemented with 0.2% horse serum and AraC for three additional days. Depleted cells were transfected with the FAPP-PH-GFP plasmid 20–24 hr before the end of the experiment, and analyzed by live-imaging in a confocal microscope (Leica Microsystems). Individual regions of GFP fluorescence in cardiomyocytes were monitored and followed over time after treatment with vehicle or 100 nM ET-1 for 40 min.