Figure 4.
Figure 4. FAPP1 negatively controls ApoB100 export from the TGN in a PI4P-dependent manner. (A) Immunoblot of the medium and cell lysates from Mock, FAPP1-KD, or ORP10-KD cell cultures using the anti-ApoB100 antibody (see Materials and methods). (B) Quantification of the Western blot shown in A. Secretion of ApoB100 (ratio between the amount in the medium and in the cell lysates) expressed as a percentage of control (CTRL). Means ± SD, n = 3. *, P < 0.05. (C) ApoB100 secretion evaluated by ELISA from control (CTRL) or FAPP1-KD cells, expressed as the amount (ng) of ApoB100 in the medium normalized for the protein content. *, P < 0.05. n = 3. (D) ApoB100, albumin, and α1-anti-trypsin secretion evaluated by pulse-chase. Control or FAPP1-KD cells were incubated as described in Materials and methods. Secretion is expressed as a ratio between the amount of labeled cargo in the medium and the total labeled cargo (medium plus cell lysate). *, P < 0.05. n = 3. (E) Control, FAPP1-KD, and ORP10-KD HepG2 cells were stained with anti-PI4P (green) and anti–Golgin-97 (red) antibodies. Bar, 10 µm. (F) Quantification of PI4P levels shown in E. n = 3; ***, P < 0.001. (G) Golgi-to-PM transport of ApoB100 in control and FAPP1-KD HepG2 cells. Representative images of the distribution of ApoB100 (anti-ApoB100 staining) under steady-state conditions, after a temperature block at 20°C, and after 15 and 60 min of the release from the 20°C temperature block. Insets, Golgin-97 staining. Bar, 10 µm. (H) Golgi-to-PM transport of ApoB100 in control, FAPP1-KD, ORP10-KD, FAPP1+PI4KIIIβ-KD, and ORP10+PI4KIIIβ-KD HepG2 cells. Quantification of Golgi emptying was performed by measuring the ratio between the ApoB100 signal and the Golgin-97 signal at the indicated times after release of the temperature block. Data are means ± SD expressed as a percentage of the ApoB100 signal in the Golgi compared with the signal at time 0 (i.e., at the end of the temperature block). n > 100; three independent experiments. **, P < 0.01; ***, P < 0.001; Student’s t test. (I) Control and FAPP1-KD HepG2 cells were treated as described in G, and analyzed after 15 min of the release of the 20°C block. Cells were stained for ApoB100 (gray), Golgin-97 (G97 inset), and COPI (blue). Bar, 10 µm. The arrowheads indicate ApoB100-positive peripheral structures that are probably post-Golgi carriers since they lack a COPI coat.

FAPP1 negatively controls ApoB100 export from the TGN in a PI4P-dependent manner. (A) Immunoblot of the medium and cell lysates from Mock, FAPP1-KD, or ORP10-KD cell cultures using the anti-ApoB100 antibody (see Materials and methods). (B) Quantification of the Western blot shown in A. Secretion of ApoB100 (ratio between the amount in the medium and in the cell lysates) expressed as a percentage of control (CTRL). Means ± SD, n = 3. *, P < 0.05. (C) ApoB100 secretion evaluated by ELISA from control (CTRL) or FAPP1-KD cells, expressed as the amount (ng) of ApoB100 in the medium normalized for the protein content. *, P < 0.05. n = 3. (D) ApoB100, albumin, and α1-anti-trypsin secretion evaluated by pulse-chase. Control or FAPP1-KD cells were incubated as described in Materials and methods. Secretion is expressed as a ratio between the amount of labeled cargo in the medium and the total labeled cargo (medium plus cell lysate). *, P < 0.05. n = 3. (E) Control, FAPP1-KD, and ORP10-KD HepG2 cells were stained with anti-PI4P (green) and anti–Golgin-97 (red) antibodies. Bar, 10 µm. (F) Quantification of PI4P levels shown in E. n = 3; ***, P < 0.001. (G) Golgi-to-PM transport of ApoB100 in control and FAPP1-KD HepG2 cells. Representative images of the distribution of ApoB100 (anti-ApoB100 staining) under steady-state conditions, after a temperature block at 20°C, and after 15 and 60 min of the release from the 20°C temperature block. Insets, Golgin-97 staining. Bar, 10 µm. (H) Golgi-to-PM transport of ApoB100 in control, FAPP1-KD, ORP10-KD, FAPP1+PI4KIIIβ-KD, and ORP10+PI4KIIIβ-KD HepG2 cells. Quantification of Golgi emptying was performed by measuring the ratio between the ApoB100 signal and the Golgin-97 signal at the indicated times after release of the temperature block. Data are means ± SD expressed as a percentage of the ApoB100 signal in the Golgi compared with the signal at time 0 (i.e., at the end of the temperature block). n > 100; three independent experiments. **, P < 0.01; ***, P < 0.001; Student’s t test. (I) Control and FAPP1-KD HepG2 cells were treated as described in G, and analyzed after 15 min of the release of the 20°C block. Cells were stained for ApoB100 (gray), Golgin-97 (G97 inset), and COPI (blue). Bar, 10 µm. The arrowheads indicate ApoB100-positive peripheral structures that are probably post-Golgi carriers since they lack a COPI coat.

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