Lsm 510 meta and 800

To detect single-stranded DNA (ssDNA), the cells were fixed on ice with 4% PFA for 20 min and then with 80% methanol in PBS at − 20 °C overnight. The next day, the cells were washed and treated for 4 h at 37 °C with 200 µg/mL RNase (QIAGEN) and S1 nuclease (Promega) as indicated. Then, the cells were washed and blocked with a blocking solution (PBS containing 0.5% FBS), and then incubated overnight at 4 °C with the anti-ssDNA primary antibody. The following day, the cells were incubated with the secondary antibody for 2 h and then DAPI before mounting. Images of ssDNA were blindly captured by using confocal microscopy (LSM 510 Meta and 800; Zeiss, Göttingen, Germany) with a × 100 objective.

The cells were fixed for 20 min with 4% paraformaldehyde and 0.1% Triton X-100 in PBS buffer. After washing with PBS, the fixed cells were incubated in a blocking solution (PBS containing 0.5% FBS) for 1 h at room temperature to reduce nonspecific antibody binding. The cells were treated with diluted primary antibodies in a blocking solution for 1 h at room temperature or overnight at 4 °C. After washing three times with PBS, the cells were incubated with Alexa Fluor-conjugated secondary antibodies (1000:1). The nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Vector Laboratories; Burlingame, CA, USA; H-1200). The cells were imaged by using a Nikon Eclipse TE2000 fluorescence microscope. As indicated in the text, the neuronal cells were also imaged using confocal microscopy (LSM 510 Meta and 800; Zeiss, Göttingen, Germany) with a × 2 × , × 40, and × 100 objective, and the images were printed. The antibodies used for immunocytochemistry are listed in Supplementary Table 4.

A proximity ligation assay (PLA) experiment was performed using a Duolink® in situ red kit according to the manufacturer’s instructions (Sigma-Aldrich). Briefly, the cells were fixed for 10 min with 4% paraformaldehyde and 0.1 Triton X-100 in PBS buffer. After washing with PBS, the fixed cells were blocked using Duolink® blocking solution for 30 min at 37 °C. Primary antibodies against GFP and BiP/GRP78 were diluted with Duolink® antibody diluent, applied to each cell line and incubated overnight at 4 °C. The cells were washed three times for 5 min each time with in Duolink® wash buffer A and incubated with Duolink® anti-mouse MINUS and anti-rabbit PLUS for 1 h at 37 °C. After washing three times, ligation and amplification reactions were performed for 30 min and 100 min at 37 °C, respectively. The cells were washed twice with Duolink® wash buffer B and then mounted with Duolink® mounting medium. The cells were examined using confocal microscopy (LSM 510 Meta and 800; Zeiss, Göttingen, Germany).