Hoechst 33342

To extract proteins that freely diffuse in cells, the cells were permeabilized as described previously (Kimura et al., 2006 (link)). Cells that had been plated on a 35-mm glass-bottom dish 1 day before were chilled on ice, and washed twice with ice-cold PBF [100 mM potassium acetate, 30 mM KCl, 10 mM Na₂HPO₄, 1 mM dithiothreitol, 1 mM MgCl₂, 1 mM adenosine triphosphate (Thermo Fisher Scientific), and 5% Ficoll (Nacalai Tesque)] followed by incubation with ice-cold PBF containing 0.1% Triton X-100 for 5 min. The cells were washed twice with cold PBF and incubated with Cy5-conjugated γ-H2AX Fab and Alexa Fluor 488-conjugated H4K20me2 Fab in PBF for 3–4 h on ice. The laser irradiation assay and observation were performed at 29°C using a confocal microscope (FV-1000), as described above. To compare the levels of endogenous proteins that remained after permeabilization, cells without or with permeabilization were fixed, treated with Triton X-100, and incubated with mouse anti-ATM (0.2 μg/ml; Santa Cruz Biotechnology; G-12) or mouse anti-Ku86 (1 μg/ml; Santa Cruz Biotechnology; B-1) antibody, and then with Cy3-conjugated goat anti-mouse IgG (H+L) (0.5 μg/ml; Jackson ImmunoResearch) and Hoechst 33342, as described above.

Between 50 and 60 × 10³ fibroblasts were plated onto 13 mm poly L-lysine glass coverslips (Corning) in DMEM-F12 culture media containing 10% FBS and grown overnight in a 37°C incubator with 5% CO₂. The next day, serum-free media was added for 24, 48, or 72 hours to induce ciliation. Cells were placed on ice for 10 minutes before fixation in 1% paraformaldehyde for 5 minutes followed by 15 minutes in ice-cold methanol. Cells were then rinsed, permeabilized for 5 minutes with 0.1% SDS in PBS, rinsed, and blocked with 0.1% Triton-X, 5% normal donkey serum, and 5% BSA in PBS. Primary antibodies diluted in block were incubated overnight at 4°C, rinsed, and incubated with secondary antibodies for 1 hour at 22°C.
For all conditions, 10 μg/ml Hoechst 33342 was added with donkey secondary antibodies conjugated to Alexa Fluor 488, 568, or 647 (Jackson ImmunoResearch). Coverslips (no. 1.5, Electron Microscopy Sciences) were mounted with Prolong Glass (Thermo Fisher Scientific). Images were acquired using a Zeiss Observer 7 inverted microscope with a 63 × oil-immersion objective (1.40 NA) and a LSM 800 confocal with Airyscan detector controlled by Zen 5.0 (Carl Zeiss Microscopy). Image manipulation was limited to adjusting the brightness level, image size, rotation, and cropping using FIJI (ImageJ).

In this study, an immunofluorescence staining technique was conducted to investigate the PPARγ nuclear translocation state. Briefly, cells were seeded, treated, and fixed as mentioned in the lipid staining section and then permeabilized with 0.25% Triton X-100 in PBS for 2 min at room temperature. After permeabilization, cells were washed in PBS, blocked with 1% BSA and 0.3 M glycine in PBS for 30 min at room temperature, and then stained with anti-PPARγ antibody (H-100, Santa Cruz Biotechnology) overnight at 4°C. On the next day, cells were washed three times with PBS for 5 min each to remove excessive primary antibodies. After the wash, Hoechst 33342- (2 μg/mL) and DyLight 488-conjugated goat-anti-rabbit IgG antibodies (Jackson ImmunoResearch Laboratories) were added and incubated in the dark for 1 hour at RT. After fluorescent labeling, slides were washed, mounted, and imaged under an Olympus IX-70 microscope as described before.