Colocalization analysis between Pyr-met-Chol and protein markers of intracellular membrane compartments in PC-3 cells.

<p>TPE microscopy imaging of fixed PC-3 cells was performed after a 48 h incubation with 0.1 mg/ml of Pyr-met-Chol-labelled purified LDL (left panels) or Pyr-met-Chol-labelled purified HDL (right panels), followed by permeabilization with Triton X-100 and immunostaining using specific antibodies against either EEA-1 (early endosomes), CD63 (prostasome-precursor vesicles), caveolin-1 (caveolae), Lamp-1 (late endosomes), calnexin (endoplasmic reticulum) or CD13 (plasma membrane). <i>Panels A and B</i>: Manders coefficient were calculated as indicators of the proportion of Pyr-met-Chol signal in the intensely stained structures that colocalized with the signal of each of the antibodies (secondary antibodies labelled by Cy3, except Alexa-546 for caveolin-1 detection). <i>Panels C and D</i>: Merge images of the colocalization of Pyr-met-Chol (cyan channel) with Lamp-1 (red channel); superimposition is in white. <i>Panels E and F</i>: Merge images of the colocalization of Pyr-met-Chol (cyan channel) with CD63 (red channel); superimposition is in white. Scale bar corresponds to 10 μm.