Dmx1200 camera

The c-Fos immunochemistry was carried out as described previously^{20 (link)} except for the secondary antibody, the incubation time of which was 1.5 h instead of 2 h. For each animal, c-Fos immunostaining analysis was performed by counting the positive nuclei on four non-consecutive hemisections using microphotographs acquired by a tenfold lens with a DMX 1,200 camera (Nikon) coupled to ACT-1 software. The microscope was set at a specific illumination level, as was the camera exposure time. c-Fos positive nuclei were then counted on these pictures by computer-assisted morphometry using the ImageJ software as previously described^{10 (link)}.

WST-1 assay was performed to assess cell proliferation. DPCs were seeded in a 96-well plate at a density of 3000 cells per well and treated with vehicle (DMSO; Sigma-Aldrich; St. Louis, MO, USA) or various concentrations of nonanal (Sigma-Aldrich). When required, vehicle (DMSO) or SQ22,536 (Sigma-Aldrich), a cAMP inhibitor, was added 1 h before nonanal treatment. Images of the cells were captured using a Nikon Eclipse TS2 microscope equipped with the DMX1200 camera (Nikon, Tokyo, Japan). Subsequently, the treated cells were incubated for 2 h with the WST-1 reagent (Sigma-Aldrich) diluted 1:10 in culture medium. Absorbance was read at 440 nm using the Infinite M200 microplate reader (Tecan; Männedorf, Switzerland). In all the experiments, the final DMSO concentration was less than 0.1% (v/v). Furthermore, to determine whether the proliferation of Hs68 was also affected by nonanal treatment, the same method was employed as above.

SA-β-gal staining was performed using a senescence beta-galactosidase cell staining kit (Cell Signaling Technology, MA, USA), following the manufacturer’s protocol. In brief, Hs68 cells were cultured in six-well plates and treated as mentioned in Section 4.2. After that, the cells were washed with PBS and fixed with a fixative solution for 10 min at 25 °C. The cells were then rinsed with PBS and incubated with a fresh β-gal staining solution at 37 °C overnight. The images were captured from five random fields per sample using a microscope (Nikon Eclipse TS2; Nikon, Tokyo, Japan) equipped with the DMX1200 camera (Nikon). The results are expressed as the percentage of SA-β-gal-positive cells in the total cell number.