Assessment of ER-phagy in physio-pathological perturbations (related to Fig. 8 ). (A) Quantitative analysis indicated increased RFP⁺ and RFP⁺LAMP1⁺ signals at liver during starvation. The fluorescence intensity of RFP-only, RFP⁺LAMP1⁺ and total area were calculated respectively by ImageJ and the fluorescence ratio of RFP-only (or RFP⁺LAMP1⁺): the total RFP was quantified. Values were stated as mean ± SEM (n = 5 per condition). Statistical differences were analyzed using Student’s t test. **P < 0.01, ***P < 0.001. (B and C) Analysis of ER-phagy levels in the liver and skeletal muscle of transgenic mice during starvation by immunoblotting. Tissue homogenates from both regularly fed and starved mice were examined through immunoblotting. Full-length RFP-GFP and RFP fragments were detected with an anti-RFP antibody and the band intensity of RFP fragments relative to full-length RFP-GFP indicates the presence of ER-phagy products, signifying ER-phagy activity. GAPDH and Tubulin served as internal controls. Adult male wild-type and ER-TRG (+/−) mice were used in experiments. Values were stated as mean ± SEM (n = 4 per condition). Statistical differences were analyzed using two-way ANOVA. ***P < 0.001. (D) Confocal imaging of RFP and GFP signals in the skeletal muscles (gastrocnemius, soleus) of ER-TRG (+/−) mice under normal feeding and starvation. Scale bar: 50 μm. (E) Hematoxylin and Eosin (HE) staining of liver sections from PBS-treated and hepatic cancer model mice in ER-TRG (+/−) mice. Control mice received PBS injections, while model mice were injected with plasmids expressing C-Myc, N-Ras, and SB11 for 4 wk. Scale bars: 1 cm (top), 100 μm (bottom). (F) Quantitative analysis indicated reduced RFP⁺ and RFP⁺LAMP1⁺ signals at tumor sites compared with control liver or adjacent tissue. The fluorescence ratio of RFP-only (or RFP⁺LAMP1⁺): the total RFP was quantified. Values were stated as mean ± SEM (n = 4 per condition). Statistical differences were analyzed using Student’s t test. **P < 0.01. (G and H) ER-phagy levels and quantitative analysis in the liver of ER-TRG (+/−) mice with hepatic cancer. Confocal imaging reveals diminished ER-phagy at tumor sites relative to control liver or adjacent tissues, as indicated by CTSD co-staining. The pixel number, fluorescence intensity of RFP-only, RFP+CTSD+ and total area were calculated by ImageJ. The ratio of RFP-only (or RFP+CTSD+): the total area (RFP+GFP) and the fluorescence ratio of RFP-only (or RFP+CTSD+): the total RFP were quantified. Values were stated as mean ± SEM (n = 4 per condition). Statistical differences were assessed using Student’s t test. **P < 0.01. Scale bar: 20 μm. (I and J) Analysis of ER-phagy levels in the liver of ER-TRG (+/−) mice with hepatic cancer by immunoblotting. Values were stated as mean ± SEM (n = 4 per condition). Statistical differences were analyzed using Student’s t test. **P < 0.01. (K) Quantitative analysis indicated altered RFP⁺ and RFP⁺CTSD+ signals in muscle cells at injury area compared to PBS-treated tissue. The fluorescence ratio of RFP-only (or RFP⁺LAMP1⁺): the total RFP was quantified. Values were stated as mean ± SEM (n = 4 per condition). Statistical differences were analyzed using one-way ANOVA and Kruskal–Wallis test. *P < 0.05, **P < 0.01, ***P < 0.001. (L) Analysis of ER-phagy levels in the skeletal muscle of ER-TRG (+/−) mice with CTX injury by immunoblotting. The band intensity of RFP fragments relative to full-length RFP-GFP indicates the presence of ER-phagy products, signifying ER-phagy activity. Coomassie blue staining confirmed protein loading. Adult male wild-type and ER-TRG (+/−) mice were used in experiments. Source data are available for this figure: SourceData FS4.