Two photon super resolution point scanning confocal microscope

We grew HepG2 cells on cell slides inside a 24-well plate for 24 h. The medium was then decanted and the wells were washed three times with cold PBS. The cells were then fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 5 min. After washing three times with PBS, the cells were blocked for 1 h at 25 °C in PBS with 5% bovine serum albumin. The primary antibodies were diluted by 1:100 in PBS with 1% bovine serum albumin (antibody dilution buffer) and incubated overnight at 4 °C. After washing three times with PBS, Alexa Fluor 488 anti-rabbit and Alexa Fluor 647 anti-mouse antibodies (Cell Signaling, USA) were added to the antibody dilution buffer at 1:500 and 1:1,000 dilutions, respectively. We then added DAPI to the slides, and incubated them for 1 h at room temperature. After washing the slides five times with PBS, we mounted them using ProLong Gold antifade reagent (Invitrogen, USA). We acquired images using a Two-photon super-resolution point scanning confocal microscope (Nikon, Japan) and selected representative images for each sample.

We grew HEK293T cells overnight on cleaned coverslips inside a 24-well plate at 37 °C in a cell culture incubator. The PKR-VN173 and SPHK1-VC155 constructs and the mutants were transfected with Lipofectamine 2000 according to the manufacturer’s instructions. After transfection for 24 h, the nuclear DNA of the living cells was stained with Hoechst 33342. We acquired images using a Two-photon super-resolution point scanning confocal microscope (Nikon, Japan) and selected representative images for each sample. Twenty cells from three independent biological experiments were randomly set for statistical analysis.