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cmi12150-sup-0001-si.tif335K

Fig. S1. Heparin does not affect cell growth or susceptibility for infection.

A. HaCaT cells were incubated with the indicated concentrations of heparin for 2 h, after which unbound heparin was washed away. 24 h after heparin addition, cells were stained with propidium iodide, and the DNA content was analysed by flow cytometry. Depicted is the proportion of cells with a DNA content reflecting G1 phase (DNA = 1), S phase (DNA = 1–2), and G2/M phase (DNA = 2). Results correspond to the mean of three independent experiments ± SD.

B. HaCaT cells were treated with heparin as for (A) 24 h post addition of heparin, the cell populations were counted and normalized for buffer treated cells. Shown are the mean values of three independent experiments ± SD.

C. HaCaT cells were preincubated with the indicated concentrations of heparin. Unbound heparin was washed away and cells were infected with HPV-16 PsVs. Infection was quantified by flow cytometry and normalized to buffer treated cells. Results represent the mean values for at least three independent experiments ± SD.

D. Recombinant HSV-1 expressing GFP under the CMV promoter was preincubated with heparin. HaCaT cells were infected with preincubated virus and infection was determined by flow cytometry. Results were normalized for buffer-treated virus and depicted as the mean values for three independent experiments ± SD.

E. 1 mg ml−1 biotinylated heparin was separated on a Optiprep step gradient (5%/39%). Fractions were collected from the interface between the two Optiprep concentrations and from the top of the gradient. Presence of heparin was analysed by ELISA using HRP-conjugated streptavidin. Shown is the mean of triplicates ± SD.

cmi12150-sup-0002-si.tif364K

Fig. S2. Preincubation with sulfated polysaccharides inhibits infection. HPV-16 PsVs were preincubated with the indicated concentrations of polysaccharides followed by infection of HaCaT (top row) and HeLa (bottom row) cells. Infection was analysed as in Fig. 1A. Shown are the median values for at least three independent experiments ± SD.

cmi12150-sup-0003-si.tif1002K

Fig. S3. Lyase efficiency for removal of HS and CS from cells and ECM.

A. HaCaT or HeLa cells were treated with buffer (untreated), heparinase I, chondroitinase or both, chondroitinase and heparinase I. Cells were immunostained for HS as described in Experimental procedures. Depicted are microscopy pictures of HaCaT cells (left) with HS (green) and nuclear signal (blue). For quantification of HS removal by lyases (right), cells were removed using EDTA and immunostained for HS. HS fluorescence/cell was analysed by flow cytometry and normalized to untreated (buffer treated) cells. Results correspond to the mean of at least three independent experiments ± SD. n.d., not determined. Scale bar correspond to 10 μm.

B. Experiments were carried out as in (A), but cells were immunostained for CS and not treated with a combination of chondroitinase and heparinase. Depicted are microscopy pictures of HeLa cells (left) with CS (green) and nuclear signal (blue). Since HaCaT cells did not exhibit detectable CS signal (not shown), the quantification of CS removal by lyases (right) was carried out for HeLa cells only.

C. ECM derived from HaCaT cells was treated with buffer (untreated) or heparinase I and stained for HS as in (A). Depicted are images obtained by epifluorescence microscopy. Scale bars correspond to 10 μm.

cmi12150-sup-0004-si.tif765K

Fig. S4. HPV-16 can bind to heparinase treated ECM.

A. ECM coated coverslips were treated with buffer (untreated) or heparinase I and subsequently infected with AF-labelled HPV-16 PsV. For ECM detection, a laminin-332 staining was used. Given are fluorescence microscopy pictures of a representative area. Scale bar corresponds to 10 μm.

B. Quantification of virus binding after heparinase digestion. Depicted is the mean for at least three independent experiments ± SD.

C and D. ECM obtained from HaCaT cells was treated with buffer (untreated), chondroitinase ABC, heparinase I, a mixture of heparinase I and III (cocktail) or double digested with chondroitinase ABC and heparinase cocktail. HPV-16 PsVs were allowed to bind to the treated ECM. Subsequently, HaCaT cells were seeded onto ECM bound virus. Infection was analysed by flow cytometry. Depicted are the mean values ± SD of three independent experiments normalized to untreated ECM.

cmi12150-sup-0005-si.tif1021K

Fig. S5. Heparin reduces ECM binding on mouse-derived ECM.

A. AF-labelled PsVs were preincubated with heparin or HA (both 10 mg ml−1). Viruses were bound to ECM derived from mouse keratinocytes. The upper panels show the HPV-16 signal. For ECM visualization, laminin-332 staining was performed. Scale bar corresponds to 10 μm.

B. Quantification of virus binding to mouse-derived ECM for the given concentrations of heparin and HA. The results correspond to the mean values of three independent experiments ± SD, and were normalized to mock incubated virus.

C. HPV-16 PsVs were preincubated with the indicated concentrations of polysaccharides and bound to mouse-derived ECM. Mouse keratinocytes were seeded on top, and infection analysed by flow cytometry 48 h post cell seeding. Depicted are the mean values for at least three independent experiments ± SD.

cmi12150-sup-0006-si.tif140K

Fig. S6. NaClO3 reduces sulfation of HSPG. HaCaT cells (treated overnight with 50 mM NaClO3) were fixed and stained using an heparan sulfate antibody that recognizes certain sulfated HS patterns (A) or were infected with HSV-1 (B). Quantification of HS signal as in Fig. S3. Quantification of HSV-1 infection as described in Experimental procedures. Shown are the mean values for at least three independent experiments ± SD, normalized for untreated cells.

C. HSV-1 preincubated with heparin or buffer were bound to ECM produced by HaCaT cells. HaCaT cells treated with NaClO3 or not (untreated) were seeded on top of the ECM-bound virus were seeded. Cells were fixed and infection was analysed by flow cytometry as described in Fig. 5. Represented are the mean of three independent experiments ± SD, normalized for infection of buffer incubated virus of untreated HaCaT cells.

cmi12150-sup-0007-si.tif134K

Fig. S7. Heparin preincubated virus are susceptible to inhibiton of growth factor receptors. HPV-16 PsVs were preincubated with 10 mg ml−1 or buffer treated. Preincubated viruses were bound to ECM and HaCaT cells were seeded on top of the ECM-bound virus. Cells were treated with the indicated concentrations of iressa (A) or treated with 50 mM NaClO3 and the indicated concentrations of iressa (B). Samples were analysed by flow cytometry and normalized as described for Fig. 5.

cmi12150-sup-0008-si.pdf118K

Table S1. GAG oligosaccharide probes investigated in the microarrays.

cmi12150-sup-0009-si.pdf60K

Table S2. Antibodies directed against HPV-16 L1 used in ELISA and dot blot experiments (compare Fig. 7).

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