Figure 1.
APOE localizes to cytoplasmic LDs in astrocytes during lipogenesis. (A) Representative fields of TRAE3-H cells untreated (− OA) or treated with 400 µM oleic acid for 5 h (+ OA). Cells were then fixed and stained for endogenous APOE with an anti-APOE antibody, an anti-GM130 antibody to label the Golgi, and LDs with BODIPY 493/503. In the merged image, APOE is yellow, GM130/Golgi is cyan, and LDs are magenta. White dotted lines outline the plasma membranes of individual cells in the field. Scale bar, 50 µm. (B) Individual cells from A (labeled by white inset boxes) show the increase in LDs and the enrichment of APOE on the LD surface upon OA loading. Scale bar, 10 µm. (C) Inset of images from B. The top panels show the colocalization of APOE with the Golgi and the lack of LD-associated APOE in untreated cells. The bottom panels show APOE coating the surface of LDs and the concomitant reduction in Golgi colocalization upon OA treatment. Scale bar, 1 µm. (D) Quantification of the total area of BODIPY 493/503-labeled LDs per cell ± OA. N = 150 cells per condition, with 50 cells from each independent experiment. Each data point represents one cell, and each color represents data collected from a separate, independent experiment. (E) Percentage of cells that have APOE on the surface of LDs ± OA. Each data point represents the percentage of 50 randomly selected cells from one independent experiment with APOE on the surface of LDs. 61.1% ± 11.5 of cells have APOE on the surface of LDs after 5 h 400 µM OA treatment. (F) Quantification of APOE enrichment on the surface of LDs in TRAE3-H cells ± OA. LD enrichment is equal to the mean intensity of APOE signal surrounding LDs divided by the mean intensity of the entire cell minus the LDs. N = 150 cells per condition, with 50 cells from each independent experiment. Each data point represents one cell, and each color represents data collected from a separate, independent experiment. (G) Quantification of colocalization of APOE with the Golgi marker GM130 as measured by the Mander’s coefficient in TRAE3-H cells ± OA. The Mander’s coefficient was calculated by dividing the area of overlap between APOE and GM130 masks by the total area of the APOE mask. N = 150 cells per condition, with 50 cells from each independent experiment. Each data point represents one cell, and each color represents data collected from a separate, independent experiment. (H) Immunogold electron micrographs of endogenous APOE in TRAE3-H cells treated with 400 µM OA for 5 h. The primary APOE antibody was included in images labeled “+ Primary,” and not included in the negative control images labeled “− Primary.” Silver-enhanced gold particles localize directly to the surface of LDs at the interface between the LD monolayer surface and the cytoplasm. Scale bars: 200 nm (left), 20 nm for zoom (right). P values for D, F, and G were calculated using a clustered Wilcoxon rank sum test via the Rosner-Glynn-Lee method. **** P < 0.0001. P value for E calculated via unpaired two-tailed t test. * P < 0.05.

APOE localizes to cytoplasmic LDs in astrocytes during lipogenesis. (A) Representative fields of TRAE3-H cells untreated (− OA) or treated with 400 µM oleic acid for 5 h (+ OA). Cells were then fixed and stained for endogenous APOE with an anti-APOE antibody, an anti-GM130 antibody to label the Golgi, and LDs with BODIPY 493/503. In the merged image, APOE is yellow, GM130/Golgi is cyan, and LDs are magenta. White dotted lines outline the plasma membranes of individual cells in the field. Scale bar, 50 µm. (B) Individual cells from A (labeled by white inset boxes) show the increase in LDs and the enrichment of APOE on the LD surface upon OA loading. Scale bar, 10 µm. (C) Inset of images from B. The top panels show the colocalization of APOE with the Golgi and the lack of LD-associated APOE in untreated cells. The bottom panels show APOE coating the surface of LDs and the concomitant reduction in Golgi colocalization upon OA treatment. Scale bar, 1 µm. (D) Quantification of the total area of BODIPY 493/503-labeled LDs per cell ± OA. N = 150 cells per condition, with 50 cells from each independent experiment. Each data point represents one cell, and each color represents data collected from a separate, independent experiment. (E) Percentage of cells that have APOE on the surface of LDs ± OA. Each data point represents the percentage of 50 randomly selected cells from one independent experiment with APOE on the surface of LDs. 61.1% ± 11.5 of cells have APOE on the surface of LDs after 5 h 400 µM OA treatment. (F) Quantification of APOE enrichment on the surface of LDs in TRAE3-H cells ± OA. LD enrichment is equal to the mean intensity of APOE signal surrounding LDs divided by the mean intensity of the entire cell minus the LDs. N = 150 cells per condition, with 50 cells from each independent experiment. Each data point represents one cell, and each color represents data collected from a separate, independent experiment. (G) Quantification of colocalization of APOE with the Golgi marker GM130 as measured by the Mander’s coefficient in TRAE3-H cells ± OA. The Mander’s coefficient was calculated by dividing the area of overlap between APOE and GM130 masks by the total area of the APOE mask. N = 150 cells per condition, with 50 cells from each independent experiment. Each data point represents one cell, and each color represents data collected from a separate, independent experiment. (H) Immunogold electron micrographs of endogenous APOE in TRAE3-H cells treated with 400 µM OA for 5 h. The primary APOE antibody was included in images labeled “+ Primary,” and not included in the negative control images labeled “− Primary.” Silver-enhanced gold particles localize directly to the surface of LDs at the interface between the LD monolayer surface and the cytoplasm. Scale bars: 200 nm (left), 20 nm for zoom (right). P values for D, F, and G were calculated using a clustered Wilcoxon rank sum test via the Rosner-Glynn-Lee method. **** P < 0.0001. P value for E calculated via unpaired two-tailed t test. * P < 0.05.

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