Vectashield medium containing dapi

Immunohistochemical analysis was performed as previously described in [12 (link)]. Briefly, the testes were removed and fixed in 4% paraformaldehyde. The tissues were frozen and cut into 25 µm sections, which were mounted on slide glasses. Suspended spermatozoa were treated with 4% paraformaldehyde and washed in 10 mM glycine in PBS, and then, they were mounted on glass slides and air-dried. The tissues and cells were treated with the appropriate blocking buffer and incubated with primary antibodies (chorein, PGK2, LDHC, and IDH3A) and the appropriate secondary antibodies. The immunoreactions were visualized using the avidin-biotinylated enzyme complex method (VECTASTAIN ABC kit, Vector Laboratories, Burlingame, CA, USA) or the immunofluorescence technique using secondary antibodies (Alexa Fluor 555 Donkey anti-goat IgG (H + L) and/or Alexa Fluor 480 donkey anti-rabbit IgG (H + L), Invitrogen, Eugene, OR, USA). For immunofluorescence, the coverslips were washed and mounted with Vectashield medium containing DAPI (Vector Laboratories) and then viewed with a BZ-X 710 fluorescence microscope system (Keyence, Osaka, Japan).

To detect NRF2 translocation induced by IQOS CSE, ATI-like cells cultured on a 12 well hanging insert (control cells and cells exposed to 20% IQOS CSE for 0.5, 1, 2 and 4 h) were fixed in 4% paraformaldehyde. After blocking with 3% normal goat serum (Rockland Immunochemicals, Inc., Limerick, PA, USA) in PBS, the cells were incubated with rabbit anti-NRF2 (N2C2) antibody (GeneTex, Inc., Irvine, CA, USA) overnight. Next, cells were incubated with the secondary antibody, Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) for 1 h, and subsequently, the cells were mounted with Vectashield medium containing DAPI (Vector laboratories, Burlingame, CA, USA).

For imaging experiments, 1 × 10³ cells were plated on glass cover slides (VWR, West Chester, PA) 24 h before drug treatment. Cells with 80% confluency were used for the following experiments. Pre-incubation of cells with 50 μM PD98059 for 1 h was followed by treatment with 20 μM of clotrimazole in serum-free medium. Immunostaining was done using the following primary antibodies after clotrimazole treatment: anti-EIF3α pAb (SGs marker), or anti-hnRNP K mAb at dilutions of 1:100, 1: 50, and 1:100, respectively. Cells were fixed with 4% PFA formaldehyde for 15 min followed by ice-cold methanol for 5 min. Cells were then washed once with ice-cold PBS, and non-specific binding sites were blocked in PBS/0.1% BSA for 1 h at room temperature prior to incubation with primary antibodies. The immune reaction for each primary antibody was detected by Cy5 (blue; for EIF3α), or FITC-(green; for hnRNPK) conjugated secondary antibodies (1:250) for 30 min at room temperature. Slides were mounted in Vectashield medium-containing DAPI (Vector Laboratories, Inc., Burlingame, CA) and analyzed using AxioVision under a microscope (Carl Zeiss Inc.).