The decrease in PA levels resulted in an accumulation of lysosomes in the juxtanuclear region.

<p>A) Cells were labeled with LysoTracker and then untreated or treated with 1-ButOH for the indicated times. The treatment with 1-ButOH resulted in an increase in the number of lysosomes in the juxtanuclear region. After 15 min of treatment, the lysosomes seem to be fused with each other. The figures are volume reconstructions of the micrographs following LysoTracker staining. Bar, 16 µm. B) Quantification of CI-M6PR volume by pixel intensity of Lysotracker staining-thresholded areas (p<0.05, Dunnett’s Multiple Comparison Test).</p

PLD has a role in TGN-46 trafficking.

<p>Cells were untreated or treated with 1-ButOH and <i>tert-</i>ButOH for the indicated times and immunostained using TGN-46 antibody. In control untreated cells, TGN-46 is associated with the plasma membrane and concentrated in the perinuclear region. After the treatment with 1-ButOH, TGN-46 is no longer associated with the plasma membrane and is concentrated in the perinuclear region. Cells treated with <i>tert-</i>ButOH have the same characteristics of control cells. All micrographs are projected Z-series images. Bar, 16 µm. B) Quantification of TGN-46 pixel intensity in perinuclear-thresholded areas (p<0.05, Dunnett’s Multiple Comparison Test).</p

Co-localization between PLD1, PLD2, clathrin and TGN-46 in HSY cells.

<p>PLD1 and PLD2 (green) are distributed in the cytoplasm and concentrated in the juxtanuclear region, where they are extensively co-localized with clathrin (red) and TGN-46 (blue). Images are single focal planes. Bar, 16 µm.</p

Treatment with 1-ButOH alters the morphology of the Golgi apparatus.

<p>A) Cells were untreated or treated with 0.5% 1-ButOH or 5 µg/mL BFA for the indicated times and the Golgi apparatus immunostained for GM-130. With treatment with 1-ButOH, the Golgi apparatus becomes disorganized and the saccules are dilated, while the treatment with BFA resulted in a vesiculation and spreading of the Golgi apparatus. Images are Z-series projections. Bar, 16 µm. B) Quantification of the Golgi volume by pixel intensity of GM-130 staining-thresholded areas. After 15 min of incubation with 1-ButOH there is a significant increase in the volume of the Golgi apparatus (p<0.05, Dunnett’s Multiple Comparison Test). C) Ultrastructural analysis shows control untreated cells with a characteristic Golgi apparatus composed of stacks of flattened cisternae. In cells treated with 0.5% 1-ButOH for 5 min the Golgi saccules are slightly swollen and by 15 min the Golgi apparatus is disorganized and the saccules are dilated. Numerous coated vesicles (arrows) are evident in close proximity to the swollen cisternae after 5 min of treatment with 0.5% 1-ButOH. Treatment with 0.5% <i>tert-</i>ButOH for 5 min or 15 min does not modify the structure of Golgi apparatus. (G, Golgi apparatus). Bar, 500 nm.</p

Treatment with specific PLD2 inhibitor alters the morphology of the Golgi apparatus.

<p>A) Cells were untreated or treated with 1 µM of PLD1 inhibitor or PLD2 inhibitor for 30 min and the Golgi apparatus immunostained for GM-130. The treatment with PLD2 inhibitor alters the organization of the Golgi apparatus and it becomes more compact. Treatment with PLD1 inhibitor did not affect the morphology of Golgi apparatus which is similar to the untreated cells. The images are Z-series projections. Bar, 10 µm. B) Quantification of the Golgi area by GM-130 staining-thresholded areas. The PLD2 inhibitor treatment significantly decreases the area of the Golgi apparatus when compared to untreated cells (p<0.05, Dunnett’s Multiple Comparison Test).</p

PLD1 and PLD2 are present in the Trans-Golgi Network.

<p>A) Cells were immunostained for PLD1 and PLD2 (green) and TGN-46 (red). Both isoforms of PLD have a punctuate distribution throughout the cytoplasm. PLD1 is highly expressed in the nucleus (N, nucleus) and PLD2 is associated with the plasma membrane (Inset, arrowhead). The perinuclear distribution of TGN-46 co-localized with PLD1 and PLD2. Images are single focal planes. Bar, 16 µm. B) Quantification of the colocalization between TGN and PLD1 and TGN and PLD2. Data is expressed as % colocalization±SEM from 3 experiments.</p

PLD regulates CI-M6PR trafficking.

<p>HSY cells were transfected with CI-M6PR-GFP. Transfected cells were untreated or treated with 1-ButOH and <i>tert-</i>ButOH for the indicated times. In control untreated cells, CI-M6PR-GFP was distributed throughout the cytoplasm, but concentrated in the perinuclear region. After the treatment with 1-ButOH, CI-M6PR-GFP concentrated extensively in the perinuclear area. Cells treated with <i>tert-</i>ButOH have the same characteristics of control cells. All micrographs are projected Z-series images. Bar, 16 µm. B) Quantification of CI-M6PR area in perinuclear-thresholded areas (p<0.05, Dunnett’s Multiple Comparison Test).</p

Inhibition of PA production alters the distribution of clathrin.

<p>Cells were untreated or treated with 1-ButOH and <i>tert-</i>ButOH for the indicated times and labeled using an anti–clathrin antibody. In control untreated cells, clathrin is dispersed throughout the cytoplasm in a punctuate manner. After the treatment with 1-ButOH for 5 min and 15 min, the intracellular distribution of clathrin was changed, concentrating in the perinuclear region, however the treatment with <i>tert</i>-ButOH did not alter the intracellular distribution of clathrin. All micrographs are projected Z-series images. Bar, 16 µm. B) Quantification of clathrin pixel intensity in perinuclear-thresholded areas (p<0.05, Dunnett’s Multiple Comparison Test).</p