Figure 3

Recruitment of syntenin-1 to endosomes after HPV16 infection.

(a,b) Endosomal syntenin-1 increased according to HPV incubation time. Western blot (a) and label-free quantitative mass spectrometry (b) of L1, syntenin-1 and Rab5/Rab5a in HeLa cells infected with HPV16 PsV for the indicated time points. Rab5 marks early endosomes and serves as loading control. Fraction 4 in the Western blot shows endosomal fractions used for mass spectrometry. Fractions 9 to 11 represent post-nuclear supernatant (PNS) rich in soluble forms of Rab5. (c–e) Syntenin-GFP expressing HeLa cells show significantly increased colocalisation of CD63 and L1 at 7 hours post infection (7h p.i.). (c,d) Representative pictures show immunofluorescence of L1 (blue), CD63 (red) and syntenin-GFP (green) expressing HeLa cells. (e) Quantification of syntenin-GFP-colocalising CD63 pixels was performed by analysis of at least 10 images (3-5 cells per image) using Colocalisation Software 4.7 (Zeiss). *P < 0.05 compared to control. (f,g) Syntenin-1 recruitment to HPV L1 positive endosomes is significantly decreased after CD63 depletion. (f) Shows representative pictures of syntenin-GFP (green) and L1 (red) after 7 h infection in control or CD63 siRNA treated HeLa cells. (g) Quantification of syntenin-GFP-colocalising L1 pixels was performed by analysis of at least 20 images (3-5 cells per image) using Colocalisation Software 4.7 (Zeiss). *P < 0.05 compared to control. (h) CD63 positive endosomes contain both L1 and L2. Representative pictures show HeLa cells transfected with CD63-GFP expression plasmid and stained with pAb K75 and mAb L2-1. CD63-GFP is shown in green, L2 in red, L1 in blue and the nucleus in grey.