Expression of different ER-phagy reporters in cell line (related to Figs. 1, 2, and 3). (A) Schematic depiction of several ER-phagy reporter candidates. Each reporter incorporates the tandem fluorescent protein RFP-GFP. P1 features the RFP-GFP tandem along with the ER-lumen targeting sequence SS-KDEL. P2 consists of RFP-GFP paired with the sheet ER-membrane protein RAMP4. P3 includes RFP-GFP combined with the tubular ER-membrane protein REEP5. (B) Confocal microscopy images of HeLa cells expressing various ER-phagy reporters. HeLa cells transiently expressing each of the three reporters (P1-P3) were examined to characterize ER morphology. Scale bar: 5 μm. (C) Representative confocal microscopy images of ER-phagy in HeLa cells expressing ssYPet-TOLLES-KDEL following ER-stress inducer treatment. Cells transiently expressing ssYPet-TOLLES-KDEL were treated with 100 nM Torin1 for 4 h to induce ER-phagy. For TOLLES imaging, excitation was at 445 nm with fluorescence detection at 460–500 nm. For YPet, excitation was at 514 nm and fluorescence was detected at 530–580 nm. The pink arrowheads indicate the ER-phagy sites. Scale bar: 5 μm.