Carl zoom inverted florescence microscope

Cells were seeded in confocal dishes (Thermo Fisher Scientific), grown to 30% confluence, and treated with aspirin for 24 h with or without Compound C pre-treatment for 4 h. The media was removed, and the cells were fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were washed with PBS three times for 5 min each, and then permeabilized in 0.5% Triton X-100 at 37 °C for 15 min. After washing a further three times in PBS for 5 min each, primary antibodies targeting c-myc (1:200; Santa Cruz Biotechnology, Houston, TX, USA) and β-catenin (1:250) were diluted in PBS, added to the cells, and incubated at 4 °C overnight. Thereafter, the cells were washed three times in PBS for 5 min each, and incubated with secondary CY3-conjugate anti-mouse IgG antibodies (Sigma Aldrich) at a dilution of 1:100 for 2 h at room temperature in the dark. After washing again, the cells were incubated with DAPI (Sigma Aldrich; 5 μg/ml) in the dark for 5 min to stain the nuclei. All images were captured using an Axiovision camera with a Carl Zeiss zoom inverted florescence microscope (Carl Zeiss, Oberkochen, Germany) at a magnification of 60 ×.

Cells were seeded in 6-well plate in triplicate and treated with 5 mmol/L aspirin with or without Compound C pre-treatment. After 24 h, the culture media was removed and cells were washed three times with PBS. Next, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and rinsed with PBS once for 1 min. Thereafter, the cells were washed with 60% isopropanol for 15 s. Freshly diluted Oil Red O working solution (Hat Biotechnology, Xian, China) was then added, and the cells were incubated for 60 min at room temperature. Then, cells were rinsed again with 60% isopropanol for 15 s and washed three times with PBS for 5 min each. All images were captured using an Axiovision camera with a Carl Zeiss zoom inverted florescence microscope at a magnification of 60 ×. For the quantitation of lipid loading, the cells were seeded in 96-well plates and treated as described above. After removing the staining solution, the dye retained in the cells was eluted with isopropanol and the optical density at 540 nm (OD₅₄₀) was determined with a spectrophotometer (Bio-Rad Laboratories).