Journal: Journal of Lipid Research

Article Title: Phosphatidylserine transporters ORP5 and ORP8 control cholesterol trafficking from the plasma membrane to the endoplasmic reticulum

doi: 10.1016/j.jlr.2026.100989

Figure Lengend Snippet: PI(4,5)P 2 , but not PI4P, mediates cholesterol-induced PM recruitment of ORP5/8. A: Representative confocal images of HeLa cells treated with/without of 40 μg/ml Chol-CD for 30 min, followed by staining with ALOD4 (accessible pool of cholesterol) or immunofluorescence with antibody against PI4P or PI(4,5)P 2 . Scale bars = 10 μm for all images. Relative intensity was quantified from two independent experiments. ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001 (unpaired two-tailed Student's t test). B: Quantification of live-cell TIRF on sample overexpressing EGFP-EV, 2xP4M (PI4P), PHPLCδ (PI(4,5)P 2 ) upon 40 μg/ml Chol-CD loading from 3 independent experiments. Data points represent mean ± 95% CI. C: Schematics showing Pseudojanin (PJ) constructs, enzyme functions and the membrane targeted Lyn11-FRB-iRFP construct together with PJ construct before and after rapamycin treatment. D: Summarized data of EGFP-tagged ORP5, and ORP8 relative fold change when co-expressed with PJ constructs (PJ-Dead, PJ-Sac1, and PJ-INPP5E as indicated) and treated as indicated in HeLa cells. Data were quantified from three independent experiments. E: Representative TIRF and Epi images in HeLa cells overexpressing EFGP-tagged ORP5/8 or their PI(4,5)P 2 binding mutants (ORP5 R136Q and ORP8 R158C) with/without 40 μg/ml Chol-CD for 30 min. F: Relative TIRF/Epi intensity of cells shown in Figure 2E from two independent experiments. ∗∗, P < 0.01; ∗∗∗∗, P < 0.0001 (ordinary one-way ANOVA with Dunnett's multiple comparisons test, mean ± 95% CI) G: Relative fold change of live-cell TIRF for HeLa cells overexpressing EFGP-tagged ORP5/8 or their PI(4,5)P 2 binding mutants from three independent experiments. Data points represent mean ± 95% CI. PM, plasma membrane; ORP, OSBP-related protein; PI4P, phosphatidylinositol 4-phosphate; TIRF, total internal reflection fluorescence; Epi, epifluorescence; FRB, FKBP-rapamycin binding; PI(4,5)P 2 ,phosphatidylinositol 4,5-bisphosphate; Chol-CD, cholesterol—methyl-β-cyclodextrin complex.

Article Snippet: For PM PI4P/PI(4,5)P 2 detection, cells were fixed and blocked as described for the PS labeling protocol above, followed by 1 h incubation with purified PI4P antibody (Echelon Z-P004) or PI(4,5)P 2 antibody (Echelon Z-P045) and 45 min incubation with secondary antibody.

Techniques: Staining, Immunofluorescence, Two Tailed Test, Construct, Membrane, Binding Assay, Clinical Proteomics, Fluorescence

Journal: Journal of Lipid Research

Article Title: Phosphatidylserine transporters ORP5 and ORP8 control cholesterol trafficking from the plasma membrane to the endoplasmic reticulum

doi: 10.1016/j.jlr.2026.100989

Figure Lengend Snippet: Proposed model for a cholesterol-PI(4,5)P 2 -ORP5/8-GRAMD1b axis that governs PM-to-ER cholesterol transport. Cholesterol addition induces the increase of PI(4,5)P 2 levels, which is needed for the recruitment of ORP5/8 to the PM. ORP5/8 then recruit GRAMD1b through both the delivery of PS to the PM and the ORP5-GRAMD1b interaction. GRAMD1b transfers excess PM cholesterol to the ER, where cholesterol is esterified by SOAT1. ORP5/8, GRAMD1b, and SOAT1 most likely function at ER–PM contact sites. PS, phosphatidylserine; ER, endoplasmic reticulum; PM, plasma membrane; ORP, OSBP-related protein; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate.

Article Snippet: For PM PI4P/PI(4,5)P 2 detection, cells were fixed and blocked as described for the PS labeling protocol above, followed by 1 h incubation with purified PI4P antibody (Echelon Z-P004) or PI(4,5)P 2 antibody (Echelon Z-P045) and 45 min incubation with secondary antibody.

Techniques: Clinical Proteomics, Membrane

Journal: Journal of Lipid Research

Article Title: Phosphatidylserine transporters ORP5 and ORP8 control cholesterol trafficking from the plasma membrane to the endoplasmic reticulum

doi: 10.1016/j.jlr.2026.100989

Figure Lengend Snippet: PI(4,5)P 2 , but not PI4P, mediates cholesterol-induced PM recruitment of ORP5/8. A: Representative confocal images of HeLa cells treated with/without of 40 μg/ml Chol-CD for 30 min, followed by staining with ALOD4 (accessible pool of cholesterol) or immunofluorescence with antibody against PI4P or PI(4,5)P 2 . Scale bars = 10 μm for all images. Relative intensity was quantified from two independent experiments. ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001 (unpaired two-tailed Student's t test). B: Quantification of live-cell TIRF on sample overexpressing EGFP-EV, 2xP4M (PI4P), PHPLCδ (PI(4,5)P 2 ) upon 40 μg/ml Chol-CD loading from 3 independent experiments. Data points represent mean ± 95% CI. C: Schematics showing Pseudojanin (PJ) constructs, enzyme functions and the membrane targeted Lyn11-FRB-iRFP construct together with PJ construct before and after rapamycin treatment. D: Summarized data of EGFP-tagged ORP5, and ORP8 relative fold change when co-expressed with PJ constructs (PJ-Dead, PJ-Sac1, and PJ-INPP5E as indicated) and treated as indicated in HeLa cells. Data were quantified from three independent experiments. E: Representative TIRF and Epi images in HeLa cells overexpressing EFGP-tagged ORP5/8 or their PI(4,5)P 2 binding mutants (ORP5 R136Q and ORP8 R158C) with/without 40 μg/ml Chol-CD for 30 min. F: Relative TIRF/Epi intensity of cells shown in Figure 2E from two independent experiments. ∗∗, P < 0.01; ∗∗∗∗, P < 0.0001 (ordinary one-way ANOVA with Dunnett's multiple comparisons test, mean ± 95% CI) G: Relative fold change of live-cell TIRF for HeLa cells overexpressing EFGP-tagged ORP5/8 or their PI(4,5)P 2 binding mutants from three independent experiments. Data points represent mean ± 95% CI. PM, plasma membrane; ORP, OSBP-related protein; PI4P, phosphatidylinositol 4-phosphate; TIRF, total internal reflection fluorescence; Epi, epifluorescence; FRB, FKBP-rapamycin binding; PI(4,5)P 2 ,phosphatidylinositol 4,5-bisphosphate; Chol-CD, cholesterol—methyl-β-cyclodextrin complex.

Article Snippet: For PM PI4P/PI(4,5)P 2 detection, cells were fixed and blocked as described for the PS labeling protocol above, followed by 1 h incubation with purified PI4P antibody (Echelon Z-P004) or PI(4,5)P 2 antibody (Echelon Z-P045) and 45 min incubation with secondary antibody.

Techniques: Staining, Immunofluorescence, Two Tailed Test, Construct, Membrane, Binding Assay, Clinical Proteomics, Fluorescence