Laser confocal dishes

MESO257 and MESO428 cells were seeded on laser confocal dishes (Solarbio, Beijing, China), then immobilized with 4% formaldehyde at RT for 15 min. The cells were washed three times with PBS (5 min/each time) and then permeabilized using 100% methanol at −20 °C for 10 min, and blocked using 5% bovine serum albumin (BSA) (Sigma, St Louis, MO, USA) and 0.5% Triton X-100 in PBS for 1 h at RT. Next, cells were incubated with primary antibodies for AXL (sc-166269, Santa Cruz Biotechnology. CA, USA) and p53 (#2527, Cell Signaling Technology, Shanghai, China) in 5% BSA overnight at 4 °C, and then rinsed in PBS (3 × 5 min, RT). The cells were then incubated with Alexa fluor 488 or 595 secondary antibody (Invitrogen Molecular Probes, Carlsbad, CA, USA) in 5% BSA in the dark for 1 h, at RT, and then rinsed in PBS (3 × 5 min, RT). Next, the cells were stained with DAPI (Beyotime, Shanghai, China) in the dark for 5 min at RT. Finally, immunofluorescence of the cells was visualized using a laser confocal microscope (TCS SPE ll, Leica, Germany). Experiments were performed in triplicate.

We investigated the subcellular location of BmGRP78 under different stress-induced conditions using immunofluorescence staining [51 (link),52 (link)] and green fluorescence protein (EGFP)-tagged cloning [53 (link)], as in previous studies, with some modifications, respectively. Briefly, BmN cells were cultured in laser confocal dishes (Solarbio, Beijing, China) at 70–80% confluency (2 × 10⁶ cells/well) and incubated with purified anti-BmGRP78 IgG for 2 h at room temperature in the dark. After rinsing three times in PBST, the cells were incubated with Cy3-labeled goat antirabbit IgG (SA00009-2, Proteintech, Wuhan, China). For EGFP-tagged clone, the BmGRP78 open reading frame was amplified and cloned into the pIEx-1-EGFP vector. The clone containing the appropriate insert segment was identified by sequencing. The positive construct (2 µg) of the BmGRP78 gene was transfected into the BmN cells using Fugene 6 transfection reagent (Promega, Madison, WI, USA). After 24 h of incubation, the BmN cells were fixed with 4% paraformaldehyde for 15 min. Subsequently, the nuclei of the BmN cells were stained with DAPI for 10 min and observed under a confocal microscope (FV1200, Olympus, Tokyo, Japan).