T1 sm

In brief, the samples fastened in 4% paraformaldehyde in phosphate‐buffered saline (PBS) (pH 7.4) were prepared. Subsequently, samples were nurtured for 10 min with PBS containing 0.25% Triton X‐100 after washing twice with ice‐cold PBS. Next, samples were washed in PBS three times for 5 min again. Next, 5% goat serum was added to cells to decrease nonspecific background for 30 min at room temperature. After that, samples were incubated at 4°C for 18 h with anti‐AQP4 antibody (rabbit, 1:100, bs‐0634R, Bioss), Tuj1 (mouse, 1:100, 2326315, Millipore), and then washed three times in 0.01 mol/L PBS for 5 min. Next, the secondary antibodies of Alexa Fluor594‐Conjugated AffiniPure (goat anti‐mouse immunoglobulin G, ZSGB‐BIO, ZF‐0512, 1:100, for Tuj1) and Dylight 488 (goat anti‐rabbit immunoglobulin G, Abbkine, A23220, 1:100, for AQP4) were used to incubate for 2 h at room temperature in darkness, followed by another three times washing with 0.01 mol/L PBS for 5 min. Lastly, samples were counterstained with DAPI for nuclei for 1 min, then rinsed with PBS slightly. Then, cells were observed and imaged under a fluorescent microscope (T1‐SM; Nikon). Average cell variety, somatic cell space, and extent of neurite in five random fields for each sample were analyzed by Image‐Pro 6.0 (MediaCybernetics).

This assay was carried out to determine the migration potential of NCIH460 cells after knockout of TIPE2 compared to the scrambled control. Initially, CRISPR/Cas9 scramble and CRISPR/Cas9 TIPE2 cells were seeded at a density of 6 × 10⁵ cells/well. When the formation of monolayer occurred, the medium was replaced with serum-free DMEM medium and incubated for 6–8 h. Subsequently, a wound was scratched in the culture well and then the migration of the cells was evaluated by observing the difference in the area of the wounds using an inverted microscope (Nikon T1-SM, Tokyo, Japan). Images were taken at different time intervals and then analyzed using Image J software. This assay was also performed to evaluate the effect of tobacco components on the migration potential of TIPE2 knockout cells. In case of that, after serum starvation followed by scratching of the wound, different tobacco components such as nicotine (1 µM), NNK (0.05 µM), NNN (0.05 µM), and BaP (0.25 µg/mL) were added to the CRISPR/Cas9 scramble as well as CRISPR/Cas9 TIPE2 cells and the migration potential of the cells was determined.

Primary neurons with various treatments were fixed in 4% paraformaldehyde for 20 min. Followed by rinsing with phosphate-buffered saline (PBS). The cells were then blocked with 40 μl of blocking solution (10 μl sheep serum, 190 μl PBS, 0.6 μl Triton-100), followed by incubation with 30 μl of β-tubulin (TUJ1) and NEUN primary antibody or Navβ2 antibody (1 : 800, Abcam, Ab138064, UK) diluted with 2% sheep serum (1 : 1000, Abcam, Ab177487, UK) overnight at 4°C. Subsequently, the cells were stained with a secondary antibody at 37°C for 1 h. Finally, the sections were stained with 4′,6-diamidino-2-phenylindole (DAPI), which contained antifluorescence quenching agent (Biyuntian Co., Ltd., C1006, China), and observed and photographed under a fluorescence microscope (NIKON T1-SM, Ti-E, Japan).