Figure 4
(A) Endocytic uptake of Tat (FAM)-P22-MVIIA VLPs by RBMVE cells.
Spinning disc microscopy observations of RMBVE cells after 20 min incubation with conjugated VLPs using: (a,d) DAPI filter. (b,e) RhoB filter for lysotracker (Excitation/ Emission: 490/525 nm). (c,f) Overlay of DAPI, RhoB and FITC channels showing the co-localization of conjugated VLPs with acidic organelles. Cells with hypertonic solution (0.4 M sucrose) show reduced uptake of conjugated VLPs, but still the absolute presence of lysosomes (e–f). Scale bar = 12 μm. (g) The reduced cellular uptake of conjugated VLPs plotted by measuring the green fluorescence intensities for the conjugated VLPs of the cells incubated 1 without and 2 with hypertonic solution. Ten different regions were selected and fluorescence intensities were measured. 2 were corrected relative to the value obtained from the image 1 (n ~ 100). (B) Tat (FAM)-P22-MVIIA VLPs in recycling pathway as indicated by Rab11 Immunostaining. (a–f) Distribution of Rab11, which is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. Immunoreactivity in fluorescent VLPs treated cells stained for Rab11 are indicated as follows: (a,d) = DAPI and FITC filter. (b,e) = RhoB and DAPI. And (c,f) = overlay images with all channels on (DAPI, RhoB and FITC). Arrows in images (c) and (f) show the concentrated VLPs in recycling endosomes due to co-localization with Rab11. (g) Quantitation of images for green (FITC) and red (RhoB) filters shown in (A–F). *p = 0.55 or p > 0.05. Scale bar: 12 μm.
