Uorescence microscopy

Cells were seeded onto poly-L-lysine coated coverslips, xed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked using standard 5% bovine serum albumin in PBS blocking solution and incubated with primary antibody against Drp1 (1:1000, Abcam, UK) for 1 h at room temperature. After washing three times using PBS, the coverslips were incubated with Alexa Fluor 488-labeled goat antirabbit secondary antibody (1:1,000, abcam, UK) for 1 h at room temperature. The antibody was removed by washing three times with PBS and the samples were mounted for visualization and imaged using confocal microscopy.
Fluorescence in situ hybridization (FISH)
End-labeled 6-carboxy uorescein (FAM) probes were synthesized for PVT1 and ZFP36L2 (Invitrogen).
Cells on coverslips were xed using 10% neutral-buffered formalin and resuspended in hybridization buffer [0.7 M NaCl, 0.1 M Tris (pH 8.0), 0.1% SDS, and 10 mM EDTA] containing the probes. Samples were heated at 55°C for 30 min and unbound probes were washed using probe-free hybridization buffer. Cells were counterstained and mounted using mounting medium containing 4 , 6-diamidino-2-phenylindole (DAPI). Coverslips were analyzed by uorescence microscopy (Carl Zeiss, Jena, Germany).

HN6 transfected cells were cultured on the sterile glass-coverslips in 24-well plates for 12 hours. Cells were then xed with 4% PFA and permeabilized with 1% Triton and blocked with goat serum for 30 minutes. In the shaded condition, the cells were incubated with the eIF6 (diluted 1:100, Abcam) or AKT (diluted 1:100, CST) or p-AKT (diluted 1:200, CST) antibodies at 4℃ overnight and then stained with goat anti-rabbit IgG antibody Cy3 (Proteintech, China) for 1 hour at 37℃. After staining with DAPI (Sigma, St Louis, MO), cells were analyzed using uorescence microscopy (ZEISS, Germany).