Immunofluorescence analysis (nuclear localization of ATF6)

Cells were plated on cover glasses before incubating them for 48 h in the standard culture medium described in the ‘Cell culture’ section. After cultivation for 24 h in media containing 0.5 mM PA, the cells were fixed in 4% paraformaldehyde at room temperature for 20 min and permeabilized in Triton X-100 at room temperature for 30 min. The cells were pre-incubated with 2% BSA (cat. no. C500626; Sangon Biotech Co., Ltd.) for 10 min at room temperature. Then the cells were incubated with ATF6 antibody overnight at 4°C (1:100; cat. no. ab11909; Abcam) and subsequently the Cy3^®-conjugated secondary antibody 1 h at 4°C (1:100; cat. no. BA1031, Boster Biological Technology). The nuclei were stained with DAPI for 15 min at room temperature, and the samples were mounted in glycerol and observed under a fluorescence microscope with the magnification of ×40.