Figure 1. The depletion of VAPs, ORP10,... | Rockefeller University Press

Figure 1.

$Figure 1. The depletion of VAPs, ORP10, and ORP9 in combination with OSBP1, or of FAPP1 increases Golgi PI4P. (A) Immunodetection of PI4P with a monoclonal anti-PI4P antibody in control (CTRL), FAPP1-KD, ORP10-KD, and VAP-KD HeLa cells fixed and costained with anti–Golgin-97 antibody. Bar, 10 µm. (B and C) Quantification of Golgi PI4P levels (the ratio of PI4P and Golgin-97 fluorescence intensity at the Golgi complex; see Materials and methods) in cells transfected with the indicated single (B) or double (C) siRNAs. Means ± SD, three independent experiments, n > 200 cells per experiment; Student’s t test. Bar, 10 µm. (D) PI4P levels in control and FAPP1-KD HeLa cells and in WT and FAPP1-KO MEFs with (+FAPP1) or without GFP-FAPP1 overexpression. Top panel: Representative images of the MEFs. The arrowhead in a lower panel indicates a cell overexpressing GFP-FAPP1. Graph: Quantification of Golgi PI4P levels in control and FAPP1-KD HeLa cells and in WT and FAPP1-KO MEFs, with (+FAPP1) or without GFP-FAPP1 overexpression. Means ± SD, three independent experiments, n = 60–80 cells per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in B–D; Student’s t test. (E) Distribution of the PI4P GFP-P4C probe in control, FAPP1-KD, ORP10-KD, and VAP-KD fixed HeLa cells. Bar, 10 µm. (F) The ratio between GFP-P4C fluorescence intensity in the Golgi and PM area. Means ± SD, three independent experiments, n = 30–43 cells/condition. (G and H) Evaluation of Golgi PI4P levels by mass assay. (G) Golgi fractions were isolated from control (CTRL), FAPP1-KD, and ORP10-KD HeLa cells (see Materials and methods). Western blot showing the distribution of a TGN protein (TGN46) and a PM protein (Na+/K+ ATPase) after sucrose gradient separation. Dashed rectangle, fractions enriched for Golgi markers. (H) Golgi fractions were incubated with 32P-ATP and, where indicated, with recombinant PIP5K1C. 32P-PI4,5P2 produced by PIP5K1C incubated with 3 µM porcine brain PI4P (PI4P, right lane) was taken as a reference for PI4,5P2. The amount of 32P-PI4,5P2 produced by PIP5K1C using PI4P present in Golgi fractions as a substrate was assessed by TLC and expressed as a percentage of control (graph). Means ± SD of three independent experiments.$

The depletion of VAPs, ORP10, and ORP9 in combination with OSBP1, or of FAPP1 increases Golgi PI4P. (A) Immunodetection of PI4P with a monoclonal anti-PI4P antibody in control (CTRL), FAPP1-KD, ORP10-KD, and VAP-KD HeLa cells fixed and costained with anti–Golgin-97 antibody. Bar, 10 µm. (B and C) Quantification of Golgi PI4P levels (the ratio of PI4P and Golgin-97 fluorescence intensity at the Golgi complex; see Materials and methods) in cells transfected with the indicated single (B) or double (C) siRNAs. Means ± SD, three independent experiments, n > 200 cells per experiment; Student’s t test. Bar, 10 µm. (D) PI4P levels in control and FAPP1-KD HeLa cells and in WT and FAPP1-KO MEFs with (+FAPP1) or without GFP-FAPP1 overexpression. Top panel: Representative images of the MEFs. The arrowhead in a lower panel indicates a cell overexpressing GFP-FAPP1. Graph: Quantification of Golgi PI4P levels in control and FAPP1-KD HeLa cells and in WT and FAPP1-KO MEFs, with (+FAPP1) or without GFP-FAPP1 overexpression. Means ± SD, three independent experiments, n = 60–80 cells per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in B–D; Student’s t test. (E) Distribution of the PI4P GFP-P4C probe in control, FAPP1-KD, ORP10-KD, and VAP-KD fixed HeLa cells. Bar, 10 µm. (F) The ratio between GFP-P4C fluorescence intensity in the Golgi and PM area. Means ± SD, three independent experiments, n = 30–43 cells/condition. (G and H) Evaluation of Golgi PI4P levels by mass assay. (G) Golgi fractions were isolated from control (CTRL), FAPP1-KD, and ORP10-KD HeLa cells (see Materials and methods). Western blot showing the distribution of a TGN protein (TGN46) and a PM protein (Na⁺/K⁺ ATPase) after sucrose gradient separation. Dashed rectangle, fractions enriched for Golgi markers. (H) Golgi fractions were incubated with ³²P-ATP and, where indicated, with recombinant PIP5K1C. ³²P-PI4,5P₂ produced by PIP5K1C incubated with 3 µM porcine brain PI4P (PI4P, right lane) was taken as a reference for PI4,5P₂. The amount of ³²P-PI4,5P₂ produced by PIP5K1C using PI4P present in Golgi fractions as a substrate was assessed by TLC and expressed as a percentage of control (graph). Means ± SD of three independent experiments.