Fig. 5: ORP9 extraction of PI4P from the TGN helps maintain levels of accessible cholesterol in the Golgi.

a Confocal images of live WT, ORP9 KO, QKO HeLa cells expressing iRFP-P4M (PI4P biosensor), together with a TGN marker, SiT-N-mCherry. Insets show at higher magnification the regions around the TGN as indicated by white dashed boxes (green: iRFP-P4M; magenta: SiT-N-mCherry). Scale bars, 10 µm. b Quantification of the ratio of Golgi signals to the cytosolic signals of iRFP-P4M, as shown in (a) [mean ± SEM, n = 20 cells for each condition; data are pooled from two independent experiments; Dunnett’s multiple comparisons test, **P = 0.0007 (WT vs ORP9 KO), **P < 0.0001 (WT vs QKO)]. c Confocal images of the regions around the Golgi of a live QKO HeLa cell expressing ER-mCherry-FKBP-ORP9 and tagBFP-TGN38-FRB together with EGFP-GRAM-H (accessible cholesterol biosensor) and iRFP-P4M that were treated with rapamycin (200 nM) for the indicated minutes. Scale bars, 2 µm. d Time course of normalized signals of EGFP-GRAM-H and iRFP-P4M in response to rapamycin, as assessed by confocal microscopy as shown in (c) (mean ± SEM, n = 14 cells; data are pooled from three independent experiments.). e Confocal images of the regions around the Golgi of a live QKO HeLa cell expressing mCherry-tagged Sac1ΔTM (PI4P phosphatase domain of Sac1) fused with FKBP module [Sac1ΔTM-FKBP-mCherry] and tagBFP-TGN38-FRB together with EGFP-GRAM-H and iRFP-P4M treated with rapamycin (200 nM) for the indicated minutes. Scale bars, 2 µm. f Time course of normalized signals of EGFP-GRAM-H and iRFP-P4M in response to rapamycin, as assessed by confocal as shown in (e) (mean ± SEM, n = 15 cells; data are pooled from two independent experiments). Source data are provided as a Source Data file.