The integrity of the ERTGoCS requires the PS transfer activity of ORP10. (A) Subcellular localization of mCherry-tagged WT-ORP10 and of the Golgi marker Golgin-97. Scale bar, 10 µm. (B) Residues in ORP5/ORP8 involved in PS/PI4P exchange are conserved in the ORP10 ORD domain. (C) Subcellular localization of ORD-domain mutant forms (L418D and H535/536A) of mCherry-tagged ORP10. Scale bar, 10 µm. (D) Quantification of ERTGoCS in WT and ORP10-KD cells and in ORP10-KD cells expressing siRNA-resistant GFP-tagged WT-ORP10 or ORD-domain mutant forms of ORP10. The experiment was performed in HeLa cells stably expressing the TGN46-FRB-HA and mCherry-T2A-FKBP-Cb5 reporter proteins as shown in Fig. S3 A and specified in the corresponding legend (see also Materials and methods). Means ± SD of three independent experiments; n > 150; **, P < 0.01; ***, P < 0.001; Student’s t test. (E) The PS distribution in control and ORP10-KD cells using the C2 domain of lactadherin (C2-Lact-GFP, a marker for PS). Insets: Golgi marker Golgin-97. Scale bar, 10 µm. Right: Fluorescence intensity ratio of C2-Lact-GFP measured in an equivalent area of the Golgi and the PM in mock and ORP10-KD cells. Data are means ± SD. n = 50. ***, P < 0.001; Student’s t test. (F and G) Quantification of ERTGoCS in control cells and in ORP10-KD cells untreated or treated with PS (DOPS; F), or in cells expressing PS synthase 1 (PTDSS1) in its WT and constitutively active (P269S) forms (G). Means ± SD of three independent experiments; n > 150. *, P < 0.05; ***, P < 0.001; Student’s t test. (H) Quantification of ERTGoCS in HeLa cells and in ORP9-KD HeLa cells treated with itraconazole. n > 150; three independent experiments. Data are means ± SD. *, P < 0.05; ns, not significant with respect to untreated control cells; Student’s t test.