Figure S2.
Identification of ER-phagy reporter mouse (related to Fig. 4). (A) Representative images of mouse morphology of wild-type and heterozygous ER-TRG adult mice. Scale bar: 2 cm. (B) Graphs illustrating the body weights of male and female wild-type and heterozygous ER-TRG mice. Statistical analyses of body weights were performed on 8-wk-old male and female mice. Values were stated as mean ± SEM (n = 5–8 per group). Statistical differences were analyzed using Student’s t test. *P <0.05. (C) Quantification of RFP+ puncta colocalization with lysosomes in primary MEF cells under mTOR inhibition. The number of RFP+ puncta that colocalized with LysoTracker or LAMP1 signals was calculated using ImageJ for each treatment. Data were collected from 20 to 30 cells for each condition. Values were stated as mean ± SEM. Statistical differences were analyzed using two-way ANOVA. ***P < 0.001. (D) Confocal microscopy images showing the colocalization of RFP-GFP fluorescence with lysosomes in primary MEF cells under mTOR inhibition. MEF cells, derived from E13.5 ER-TRG (+/−) embryos, were treated with 100 nM Torin1 for 4 h. CTSD, a lysosomal enzyme, was employed as a lysosomal marker. Enlarged views reveal RFP-only signals colocalizing with lysosomes. The pink arrowheads indicate the ER-phagy sites. Scale bar: 5 μm. (E) Immunoblot analysis of PERK and p-PERK in primary MEF cell extracts. Cells were isolated from E13.5 embryos of ER-TRG (+/−) pregnant mice. Samples labeled #1, #2, and #3 represent cells from different mice. Cells #3 from the ER-TRG (+/−) group were used as a positive control, treated with Tg (1 μM) for 0, 1, 2, 3 h, respectively. “SE” denotes short exposure, while “LE” indicates long exposure. (F) Immunoblot analysis of PERK and phosphorylated PERK (p-PERK) in various tissues of wild-type and heterozygous ER-TRG mice. Tissue homogenates were probed using specific anti-PERK and p-PERK antibodies. Tubulin served as an internal control. (G) Quantitative analysis of ER-phagy levels in primary cells from ER-TRG (+/−) mice. The number of RFP+ puncta per cell in sensory neurons, BMDMs and hepatocytes was calculated using ImageJ and quantified for each treatment. Values were stated as mean ± SEM. Statistical differences were analyzed using one-way ANOVA. ***P < 0.001. (H and I) Quantitative analysis of immunoblotting showing ER-phagy activities in BMDMs and hepatocytes isolated from ER-TRG (+/−) mice. The band intensity of RFP fragments relative to full-length RFP-GFP indicates the presence of ER-phagy products, signifying ER-phagy activity. Values were stated as mean ± SD (n = 3). (J) Fluorescence quantification methods. The level of ER-phagy was defined from two aspects: (a) ER-phagy is defined as the number of pixels of RFP-only puncta divided by the total pixels (RFP + GFP), which is used to delineate the total area of tissue or cells because some section images did not completely fill the field of view. (b) ER-phagy is also defined as the fluorescence intensity ratio of lysosomal signals (RFP-only puncta) to total RFP signals. The method is described in detail in Materials and methods. Source data are available for this figure: SourceData FS2.

Identification of ER-phagy reporter mouse (related to Fig. 4 ). (A) Representative images of mouse morphology of wild-type and heterozygous ER-TRG adult mice. Scale bar: 2 cm. (B) Graphs illustrating the body weights of male and female wild-type and heterozygous ER-TRG mice. Statistical analyses of body weights were performed on 8-wk-old male and female mice. Values were stated as mean ± SEM (n = 5–8 per group). Statistical differences were analyzed using Student’s t test. *P <0.05. (C) Quantification of RFP+ puncta colocalization with lysosomes in primary MEF cells under mTOR inhibition. The number of RFP+ puncta that colocalized with LysoTracker or LAMP1 signals was calculated using ImageJ for each treatment. Data were collected from 20 to 30 cells for each condition. Values were stated as mean ± SEM. Statistical differences were analyzed using two-way ANOVA. ***P < 0.001. (D) Confocal microscopy images showing the colocalization of RFP-GFP fluorescence with lysosomes in primary MEF cells under mTOR inhibition. MEF cells, derived from E13.5 ER-TRG (+/−) embryos, were treated with 100 nM Torin1 for 4 h. CTSD, a lysosomal enzyme, was employed as a lysosomal marker. Enlarged views reveal RFP-only signals colocalizing with lysosomes. The pink arrowheads indicate the ER-phagy sites. Scale bar: 5 μm. (E) Immunoblot analysis of PERK and p-PERK in primary MEF cell extracts. Cells were isolated from E13.5 embryos of ER-TRG (+/−) pregnant mice. Samples labeled #1, #2, and #3 represent cells from different mice. Cells #3 from the ER-TRG (+/−) group were used as a positive control, treated with Tg (1 μM) for 0, 1, 2, 3 h, respectively. “SE” denotes short exposure, while “LE” indicates long exposure. (F) Immunoblot analysis of PERK and phosphorylated PERK (p-PERK) in various tissues of wild-type and heterozygous ER-TRG mice. Tissue homogenates were probed using specific anti-PERK and p-PERK antibodies. Tubulin served as an internal control. (G) Quantitative analysis of ER-phagy levels in primary cells from ER-TRG (+/−) mice. The number of RFP+ puncta per cell in sensory neurons, BMDMs and hepatocytes was calculated using ImageJ and quantified for each treatment. Values were stated as mean ± SEM. Statistical differences were analyzed using one-way ANOVA. ***P < 0.001. (H and I) Quantitative analysis of immunoblotting showing ER-phagy activities in BMDMs and hepatocytes isolated from ER-TRG (+/−) mice. The band intensity of RFP fragments relative to full-length RFP-GFP indicates the presence of ER-phagy products, signifying ER-phagy activity. Values were stated as mean ± SD (n = 3). (J) Fluorescence quantification methods. The level of ER-phagy was defined from two aspects: (a) ER-phagy is defined as the number of pixels of RFP-only puncta divided by the total pixels (RFP + GFP), which is used to delineate the total area of tissue or cells because some section images did not completely fill the field of view. (b) ER-phagy is also defined as the fluorescence intensity ratio of lysosomal signals (RFP-only puncta) to total RFP signals. The method is described in detail in Materials and methods. Source data are available for this figure: SourceData FS2.

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