Figure S5.
Assessment of ER-phagy activity in different tissues in inducible ER-TRG mice and in different CA-ER-TRG mice (related to Figs. 9 and 10). (A) Representative image of ER-phagy levels in cerebellum of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. The Rosa CreERT2−/−; LSL-RFP-GFP-KDEL+/−was used as control. (B–D) Analysis of ER-phagy levels in the liver of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice by immunoblotting under ad libitum feeding and starvation conditions, respectively. Mice underwent a 16-h fast with free access to water, while control mice had ad libitum access to both food and water. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. The immunoblots were quantified in panel D. Values were stated as mean ± SEM (n = 4 per group). Statistical differences were evaluated using Student’s t test. **P < 0.01. Scale bar: 20 μm. (E) Representative confocal images and quantitative analysis in the liver of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice under starvation. Quantitative analysis revealed the ratio of RFP: total area (RFP+GFP) and the fluorescence ratio of RFP: total RFP intensity by ImageJ on a pixel-by-pixel basis. Values were stated as mean ± SEM (n = 4 per condition). Statistical differences were evaluated using Student’s t test. **P < 0.01, ***P < 0.001. Scale bar: 20 μm. (F) Assessment of ER-phagy in mature cerebellar neurons of CA-ER-TRG (+/−) mice. ER-TRG expression was induced in mature neurons using AAV-hSyn-Cre (n = 4 per group). RFP signals were detected in the soma and neuronal processes. Top: Combined RFP and GFP signals in the molecular layer. Bottom: Combined signals in the granule cell layer. Scale bar: 20 μm. (G) Quantitative analysis of ER-phagy levels in neurons and glia cells from AAV-hSyn-Cre-activated ER-TRG mice. The number of RFP-only puncta per soma or pocesses in hippocampal neurons and glia cells in brain was calculated using ImageJ and quantified for each treatment. n = 50–80 cells per group from three mice. (H) Confocal microscopy images of RFP and GFP fluorescence in the liver of AAV-TBG-Cre-activated ER-TRG (+/−) mice (n = 4 per group). The overlay of RFP and GFP signals corresponds with liver section images in ER-TRG mice. Scale bar: 50 μm. (I) Immunoblot analysis of ssRFP-GFP-KDEL in the brains of CA-ER-TRG mice revealed specific expression and ER-phagy signals in neurons induced by AAV-hSyn-Cre. Tissue homogenates from a control mouse and a CA-ER-TRG (+/−) transgenic mouse were examined using specific antibodies. Full-length RFP-GFP, double red asterisk (**) markers, were detected with an anti-RFP antibody. GAPDH and Tubulin were used as internal controls. Source data are available for this figure: SourceData FS5.

Assessment of ER-phagy activity in different tissues in inducible ER-TRG mice and in different CA-ER-TRG mice (related to Figs. 9 and 10). (A) Representative image of ER-phagy levels in cerebellum of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice. The Rosa CreERT2−/−; LSL-RFP-GFP-KDEL+/−was used as control. (B–D) Analysis of ER-phagy levels in the liver of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice by immunoblotting under ad libitum feeding and starvation conditions, respectively. Mice underwent a 16-h fast with free access to water, while control mice had ad libitum access to both food and water. Mice were treated with tamoxifen for 2 or 3 days, and samples were analyzed after 5 or 7 days. The immunoblots were quantified in panel D. Values were stated as mean ± SEM (n = 4 per group). Statistical differences were evaluated using Student’s t test. **P < 0.01. Scale bar: 20 μm. (E) Representative confocal images and quantitative analysis in the liver of inducible ER-TRG (Rosa CreERT2+/−; LSL-RFP-GFP-KDEL+/−) mice under starvation. Quantitative analysis revealed the ratio of RFP: total area (RFP+GFP) and the fluorescence ratio of RFP: total RFP intensity by ImageJ on a pixel-by-pixel basis. Values were stated as mean ± SEM (n = 4 per condition). Statistical differences were evaluated using Student’s t test. **P < 0.01, ***P < 0.001. Scale bar: 20 μm. (F) Assessment of ER-phagy in mature cerebellar neurons of CA-ER-TRG (+/−) mice. ER-TRG expression was induced in mature neurons using AAV-hSyn-Cre (n = 4 per group). RFP signals were detected in the soma and neuronal processes. Top: Combined RFP and GFP signals in the molecular layer. Bottom: Combined signals in the granule cell layer. Scale bar: 20 μm. (G) Quantitative analysis of ER-phagy levels in neurons and glia cells from AAV-hSyn-Cre-activated ER-TRG mice. The number of RFP-only puncta per soma or pocesses in hippocampal neurons and glia cells in brain was calculated using ImageJ and quantified for each treatment. n = 50–80 cells per group from three mice. (H) Confocal microscopy images of RFP and GFP fluorescence in the liver of AAV-TBG-Cre-activated ER-TRG (+/−) mice (n = 4 per group). The overlay of RFP and GFP signals corresponds with liver section images in ER-TRG mice. Scale bar: 50 μm. (I) Immunoblot analysis of ssRFP-GFP-KDEL in the brains of CA-ER-TRG mice revealed specific expression and ER-phagy signals in neurons induced by AAV-hSyn-Cre. Tissue homogenates from a control mouse and a CA-ER-TRG (+/−) transgenic mouse were examined using specific antibodies. Full-length RFP-GFP, double red asterisk (**) markers, were detected with an anti-RFP antibody. GAPDH and Tubulin were used as internal controls. Source data are available for this figure: SourceData FS5.

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