4.5. Immunofluorescent Staining and Cell Analysis

For immunofluorescence staining, SCs were fixed with 3.7% formaldehyde at room temperature for 15 min and permeabilized with 0.3% Triton X-100 for 5 min at 4 °C. Cells were then incubated with 3% BSA and 10% goat serum in PBS for 1 h at room temperature. Cells were incubated with either c-Jun (1:400), YAP/TAZ (1:500), or p75NTR (1:500) primary antibody overnight at 4 °C, followed by treatment with appropriate secondary antibody and rhodamine-phalloidin (R415, Thermo Fisher) for 1 h at room temperature. Cells were mounted to glass cover slides using antifade mounting solution with DAPI ({"type":"entrez-protein","attrs":{"text":"P36971","term_id":"544448","term_text":"P36971"}}P36971, Thermo Fisher) and sealed with nail polish. Coverslips were imaged using a Nikon Eclipse Ti2 inverted microscope with a Nikon DS-Qi2 camera.

Cellular properties were analyzed using Nikon NIS Elements software (v. 5.02.00). SC spreading area, nuclear elongation, and mean pixel fluorescent intensity of p75NTR were automatically detected and analyzed using Nikon analysis software. Mean pixel fluorescent intensities of c-Jun and Sox-2 were detected using the “ROI” (region of interest) and “Threshold” functions within Nikon NIS Elements software, as described elsewhere [17]. To measure and quantify YAP/TAZ activity, mean pixel YAP/TAZ fluorescent intensities of the nuclear area and the entire cellular area were measured by Nikon NIS Elements software. The ratio between nuclear and cytoplasmic mean pixel YAP/TAZ fluorescent intensity was calculated using Equation (1) (FITC: Fluorescein isothiocyanate):