Mounting solution

Immunocytochemistry assessments were accomplished as described previously.²³, ⁴⁵ Briefly, cells of WiDr or SW48‐Dox lines were pretreated with Genz‐161 or transfected with siRNA‐Gb3 synthase (100 nM) for 48 hours, then placed (8000/chamber) in four‐chamber slides and cotreated with 100 nM doxorubicin for 48 hours in 5% FBS medium. After wash and fixation, cells were blocked with 5% goat serum in PBS for 30 minutes at room temperature, and then incubated with primary antibodies, anti‐p21 (1:200), ‐phosphorylated p53 (pp53), and ‐METTL3 (1:1500) in blocking solution, at 4ºC overnight. Corresponding Alexa Fluor 488‐conjugated anti‐mouse IgG and Alexa Fluor 555‐conjugated anti‐rabbit IgG (1:1000) were applied for further incubation to recognize the corresponding primary antibodies. After washing, cell nuclei were counterstained with DAPI (4̍,6‐diamidino‐2‐phenylindole) in mounting solution (Vector laboratories, Burlingame, CA, USA). Images (200 × magnification) were captured with an Olympus BX63 automated fluorescence microscope with monochrome CCD camera (Olympus, Tokyo, Japan). Alexa Fluor 488‐conjugated anti‐mouse IgG and Alexa Fluor 555‐conjugated anti‐rabbit IgG were purchased from Thermo Fisher Scientific.

To perform an indirect immunofluorescence assay (IFA), SARS-CoV-2 samples obtained from the Korea Centers for Disease Control and Prevention were used to infect Vero E6 cells. To prepare the SARS-CoV-2 antigen slide, cells infected for 3 days were cultured on Teflon-coated well slides overnight at 37°C in a 5% CO₂ environment and fixed with 80% acetone the next day. The patient’s serum was diluted using a twofold serial dilution from 1:16 and then reacted with SARS-CoV-2 antigens in a moist chamber for 30 min at 37°C. After washing, slides were incubated with secondary antibodies at a 1:400 dilution (fluorescein isothiocyanate-conjugated anti-human IgG; MP Biomedicals, OH, USA). The slides were examined under a fluorescence microscope (Olympus IX73, magnification: 400×) after dispensing the mounting solution (Vector Laboratories). An IgG antibody titer of ≥1:32 was established as the cut-off value using IFA on clinical samples from 15 individuals who underwent health screening.

3×10⁵ cells were seeded onto cover glass in 6-well plate and treated with PTX the next day. After 48 h, cells were harvested, washed once with PBS, and then fixed with 3.7% paraformaldehyde (Sigma) in PBS for 30 min at room temperature. The fixed cells were washed three times with PBS, and stained with DAPI solution (2.5 μg/ml) for 20 min at room temperature. Cells were washed twice with PBS and mounted with a mounting solution (Vector Laboratories, Burlingame, CA, USA). The morphologic changes in the nucleus were analyzed by Nuance fluorescence microscope (Perkin-Elmer, Waltham, MA).