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Figure 4


Fig. 4. Dynamin-2 regulates LOX-1-mediated OxLDL internalisation. HeLa cells cotransfected with tagged LOX-1 and Myc-tagged wild-type (WT) dynamin-2 or Myc-tagged K44A dominant-negative dynamin-2 were incubated with (A) rhodamine-conjugated transferrin (red) for 15 minutes or (B) pulsed with OxLDL (red) for 5 minutes followed by chase at 37°C (for 55 minutes) before fixation and labelling with mouse anti-Myc and FITC-conjugated anti-mouse IgG antibodies (green). Panels in A represent whole cell projections and nuclei are stained with DAPI (blue). Images in B represent 0.5 µm optical sections through the cell nucleus (middle) or at the cell apex (top). Asterisks indicate cell nuclei. (C) HeLa cells cotransfected with tagged LOX-1 and Myc-tagged WT dynamin-2 or Myc-tagged dominant-negative dynamin-2 were pulsed with labelled OxLDL (red, i) followed by fixation and incubation with sheep anti-LOX-1 and Cy5-conjugated anti-sheep IgG antibodies (displayed as green, ii) and mouse anti-Myc and FITC-conjugated anti-mouse IgG antibodies (not shown). (iii) Merged images with colocalisation of labelled OxLDL and LOX-1 shown as yellow. (iv) Z-axis image view of cells shown in panel iii. Images represent projected stacks of whole cells. (D) Labelling of cell surface glycoproteins and LOX-1 on HeLa cells transfected with LOX-1 and dominant-negative dynamin-2 using FITC-conjugated Con A (Con A, green, i) followed by sheep anti-LOX-1 and Alexa Fluor 594-conjugated anti-sheep IgG antibodies (red, ii) and mouse anti-Myc and Alexa Fluor 633-conjugated anti-mouse IgG antibodies (not shown). Con A labelling was performed on ice prior to fixation and processing for microscopy. (iii) Merged image with colocalisation appearing yellow. (iv) Z-axis image view of cells shown in panel iii. Images represent projected stacks of whole cells. Scale bars: 10 µm. (E) The percentage of WT or DN dynamin-2 transfected cells with internalised labelled OxLDL was evaluated. Data represent the mean ± s.e.m. (n=3 experiments). Comparison to control to calculate P values, *P<0.001.





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