Fig 2: Cideb deficiency leads to reduced SREBP processing and maturation Down‐regulation of SREBP target genes in Cideb‐deficient liver. Microarray heatmap of fatty acids/cholesterol synthesis genes that were down‐regulated in Cideb −/− mice and Scap liver‐specific knockout mice, comparing to the respective WT controls (WT VS Cideb −/−, WT VS L‐Scap −/−, n = 2 per group). The expression level of each gene was linearly normalized to range from −1 to 1 before plotting.Decreased SREBP processing and maturation in Cideb‐deficient liver. Immune blotting (IB) of the full‐length, precursor SREBP‐1/2 (SREBP FL) and the cleaved, active SREBP‐1/2 (SREBP N) from WT and Cideb −/− mice liver under ad‐lib (ND), fasting 12 h (F), and re‐feeding (R) conditions. Mice were re‐fed with high‐carbohydrate low‐fat diet for 12 h after 12‐h fasting.Defective SREBP transcriptional response to re‐feeding in Cideb‐deficient liver. Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb −/− liver under ad‐lib (ND), fasting 12 h (F), and re‐feeding (R) conditions. Mice were re‐fed with high‐carbohydrate low‐fat diet for 12 h after 12‐h fasting (n = 3 per group).Re‐introduction of Cideb rescues SREBP processing in Cideb −/− liver. WT and Cideb −/− mice were injected with AAV‐GFP or AAV‐Cideb, and sacrificed for IB with the indicated antibodies 20 days after the injection.Re‐introduction of Cideb rescues the expression of SREBP target genes in Cideb −/− liver. Transcript levels of lipogenic genes determined by qPCR from WT and Cideb −/− liver. WT and Cideb −/− mice were injected with AAV‐GFP or AAV‐Cideb, and sacrificed for qPCR 20 days after the injection (n = 3 per group).Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by 2‐tailed Student's t‐test.Source data are available online for this figure.

Fig 3: HBP regulates SREBP-1 activity by promoting SCAP N-glycosylation. (A) Western blot analysis of SCAP and nuclear SREBP-1 levels in U251 and U87 glioma cells after SCAP gene silenced by siRNA, cultured in DMEM complete medium treated with 50 ng/ml GDNF for 48 h. (B) NetNGlyc server showed that SCAP presents both N- and O-glycosylation. (C) Western blot analysis of nuclear extracts from U251 and U87 glioma cells cultured in DMEM glucose-free medium, treated with or without 50 ng/ml GDNF or in combination with tunicamycin (N-glycosylation inhibitor) (2 μg/ml) or 20 μM OSMI-1 (O-glycosylation inhibitor) for 48 h. (D) Immunofluorescence staining of SREBP-1 (red) and DAPI (blue) from U251 glioma cells cultured in DMEM complete medium, treated with 50 ng/ml GDNF or in combination with 2 μg/ml tunicamycin or 20 μM OSMI-1 for 48 h. Quantitative evaluation of SREBP-1 nuclear intensity using ImageJ (n=20). Images were captured at x200 magnification. (E) Western blot analysis of total cell lysates (SCAP N-glycosylation levels) or nuclear extracts (nSREBP-1) from U251 glioma cells cultured in DMEM glucose-free medium, treated with 50 ng/ml GDNF or/and 10 mM glucose for 48 h. Western blot analysis of total cell lysates (SCAP N-glycosylation levels) or nuclear extracts (nSREBP-1) from U251 glioma cells cultured in DMEM complete medium, treated with 50 ng/ml GDNF or in combination with (F) 20 mM RPI-1 (GDNF/RET inhibitor), (G) 20 μM azaserine (HBP inhibitor) and/or (H) 2 μg/ml tunicamycin (N-glycosylation inhibitor) for 48 h. Significance was determined by unpaired Student's t test (****P<0.0001; NS: not significant). HBP, N-acetylglucosamine; SREBP-1, sterol regulatory element-binding protein-1; SCAP, SREBP cleavage-activating protein; GDNF, glial-derived neurotrophic factor.

Fig 4: Loss of Cideb specifically inhibits loading of SREBP/SCAP into COPII vesicles Decreased SREBP‐1/2 and SCAP levels in Golgi apparatus in Cideb −/− mice. IB of organelle fractions of the indicated proteins and quantification of IB data from three independent experiments. Cideb deficiency impairs the packaging of SCAP/SREBP into COPII vesicles. Protein present in the vesicle or microsome fractions obtained from WT or Cideb −/− liver was detected by IB. Quantification of IB data from three independent experiments.Schematic of the BioID assay with Sar1A‐BirA*.Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells. Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A‐Flag‐BirA*. Cells were cultured in sterol‐depleted medium and treated with 80 μM biotin, prior to pull down with streptavidin‐conjugated beads. The biotinylated proteins were detected by IB with the indicated antibodies following SDS–PAGE.Cleaved SREBP bypasses Cideb deficiency. WT and Cideb −/− mice were injected with adenovirus expressing GFP, SREBP‐1c full length (FL), or SREBP‐1c active form (N). Mice were sacrificed 7 days after injection, and liver TAG levels and hepatic expression SREBP‐1 target genes in the indicated groups were determined (n = 5 per group).Data information: Data represent the Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by 2‐tailed Student's t‐test.Source data are available online for this figure.

Fig 5: Cideb enhances the targeting of SREBP/SCAP to ER exit sites Schematic of SCAP targeting to ER exit sites under different conditions. The red puncta represent the Sec12‐positive ER exit sites, and the yellow puncta represent the SCAP‐positive ER exit sites.Cideb enriches SCAP to ERESs via interacting with Sec12. CHO‐K1 cells transfected with GFP‐SCAP, HA‐Cideb, or HA‐Cideb‐K128A were arrested with 5 μM nocodazole for 30 min, then fixed with methanol, and stained with antibodies against Sec12 and HA. Scale bars represent 10 μm (left) and 2.5 μm (Inlay).Number of SCAP‐positive ER exit sites per cell.Normalized fluorescent intensity of SCAP at each individual ER exit site.Total fluorescent intensity of SCAP at ER exit sites per cell.Number of Sec12‐positive ER exit sites per cell.Model depicting the role of Cideb in enhancing SREBP processing. When cellular sterol level is high, SCAP binds to Insig, resulting in the retention of SREBP/SCAP on ER and low SREBP activation. The absence of sterol frees SCAP from Insig and induces the interaction between Cideb and SCAP, leading to the targeting of SREBP/SCAP to the ERESs via the interaction between Cideb and Sec12 and the enrichment of SREBP/SCAP in Golgi. Cleaved SREBP enters nucleus and activates the expression of fatty acids/cholesterol synthesis genes.Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by 2‐tailed Student's t‐test. In panel C‐F, quantification of images from three independent experiments.