Figure 3.
Comparison of two ER-phagy reporter strategies. (A) Schematic representation of two ER-phagy reporters. Left: the key elements of reporters include the tandem fluorescent proteins RFP-GFP, as well as YPet-TOLLES, paired with ER-lumen targeting sequence, KDEL. Both RFP and TOLLES demonstrate increased resistance to acidity. Right: ssRFP-GFP-KDEL is cleaved by lysosomal enzymes to yield the RFP fragment, while the GFP signal is quenched in lysosomes. (B–D) Confocal microscopy images of HeLa cells expressing the ER lumen-targeting, tandem RFP-GFP (ER-TRG, ssRFP-GFP-KDEL) tag under various conditions. HeLa cells expressing ER-TRG were subjected to treatments with EBSS (14 h), Tunicamycin (1 μg/ml, 16 h), or Torin1 (100 nM, 4 h) to induce ER stress. Scale bar: 5 μm. (E) Quantitative analysis of RFP+ puncta per cell following ER stress inducer treatment as depicted in Fig. 1, B–D. HeLa cells expressing ER-TRG were treated with EBSS (14 h), Tunicamycin (1 μg/ml, 16 h), or Torin1 (100 nM, 4 h) to induce ER stress. Data were collected from 50 to 80 cells for each condition. Values were stated as mean ± SEM. Statistical differences were analyzed using Mann–Whitney test. ***P < 0.001. (F) Gene expression levels of different ER-phagy receptors in various mouse tissues. Data were retrieved from the online GEO database (https://www.ncbi.nlm.nih.gov-/geo/query/acc.cgi?acc=GSE10246). Tissue labels include adipose brown (Adipose b), adipose white (Adipose w), cerebellum (Cereb), cerebral cortex (Cereb C), hippocampus (Hippoca), large intestine (Intest l), small intestine (Intest s), osteoclasts (Osteo), pancreas, placenta, and skeletal muscle (Skeletal m). (G) Representative confocal microscopy images showing ER-phagy induced by ER-phagy receptors. HeLa cells stably expressing ER-TRG were transfected with expression plasmids for ER-phagy receptors for 24 h, followed by fixation for confocal microscopy analysis. Scale bar: 5 μm. See also Fig. S1.

Comparison of two ER-phagy reporter strategies. (A) Schematic representation of two ER-phagy reporters. Left: the key elements of reporters include the tandem fluorescent proteins RFP-GFP, as well as YPet-TOLLES, paired with ER-lumen targeting sequence, KDEL. Both RFP and TOLLES demonstrate increased resistance to acidity. Right: ssRFP-GFP-KDEL is cleaved by lysosomal enzymes to yield the RFP fragment, while the GFP signal is quenched in lysosomes. (B–D) Confocal microscopy images of HeLa cells expressing the ER lumen-targeting, tandem RFP-GFP (ER-TRG, ssRFP-GFP-KDEL) tag under various conditions. HeLa cells expressing ER-TRG were subjected to treatments with EBSS (14 h), Tunicamycin (1 μg/ml, 16 h), or Torin1 (100 nM, 4 h) to induce ER stress. Scale bar: 5 μm. (E) Quantitative analysis of RFP+ puncta per cell following ER stress inducer treatment as depicted in Fig. 1, B–D. HeLa cells expressing ER-TRG were treated with EBSS (14 h), Tunicamycin (1 μg/ml, 16 h), or Torin1 (100 nM, 4 h) to induce ER stress. Data were collected from 50 to 80 cells for each condition. Values were stated as mean ± SEM. Statistical differences were analyzed using Mann–Whitney test. ***P < 0.001. (F) Gene expression levels of different ER-phagy receptors in various mouse tissues. Data were retrieved from the online GEO database (https://www.ncbi.nlm.nih.gov-/geo/query/acc.cgi?acc=GSE10246). Tissue labels include adipose brown (Adipose b), adipose white (Adipose w), cerebellum (Cereb), cerebral cortex (Cereb C), hippocampus (Hippoca), large intestine (Intest l), small intestine (Intest s), osteoclasts (Osteo), pancreas, placenta, and skeletal muscle (Skeletal m). (G) Representative confocal microscopy images showing ER-phagy induced by ER-phagy receptors. HeLa cells stably expressing ER-TRG were transfected with expression plasmids for ER-phagy receptors for 24 h, followed by fixation for confocal microscopy analysis. Scale bar: 5 μm. See also Fig. S1.

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