Figure 1.
Collagen VI α3 expression during skin wound healing.
Frozen sections of wounds of wild type and Col6a1 null mice at 4, 7, 10 and 14 days after wounding were incubated with an affinity purified antibody against the collagen VI α3 chain followed by an Alexa 546 labeled secondary antibody (red). Nuclei were stained with DAPI (blue). d = days after wounding. d = dermis, e = epidermis, g = granulation tissue, st = scar tissue. Bar, 200 µm.
Figure 2.
Analysis of the collagen VI α3 chain in primary fibroblast cultures from wild type and Col6a1 null mouse skin.
Cells were isolated from newborn wild type and Col6a1 null mice and cultured for 4 days. (a) Immunostaining for the collagen VI α3 chain (green) and the endoplasmic reticulum marker PDI (red). Nuclei were stained with DAPI (blue). Bar, 100 µm. (b) Immunoblot analysis of collagen VI assembly in cell lysates (C) and supernatants (S). Cells were lysed with SDS-PAGE sample buffer, samples treated with 2 M urea and separated under non-reducing conditions on an agarose-polyacrylamide (0.5%/2.4%) composite gel. Immunoblots were developed with an antibody against the collagen VI α3 chain. (*) indicates the mobility of the single α3 chain, (#) indicates α3 chain dimers. (c) Higher magnification from (a). Bar, 50 µm.
Figure 3.
Collagen VI distribution in fibrotic skin lesions of wild type mice.
Mice were treated for 4 weeks with bleomycin as described in Methods. NaCl injection served as control. Frozen sections were incubated with affinity purified antibodies against the collagen VI α3 (green), α5 (green) or α6 (green) chains. The sections stained for the α5 and α6 chains were co-stained with an antibody against the endothelial marker CD31 (red). Nuclei were stained with DAPI (blue). Bar, 50 µm.
Figure 4.
Collagen VI in extracts of unwounded skin and wounds derived from wild type and Col6a1 null mice.
Extracts from unwounded skin and wounds were subjected to SDS-PAGE on 4–12% polyacrylamide gradient gels under reducing conditions, proteins transferred to a membrane and detected with affinity purified antibodies against the collagen VI α1, α2, α3, α5 and α6 chains. Boxed areas a and b on the right show a longer exposure. Arrows indicate the position of the full length proteins, asterisks indicate artefact bands.
Figure 5.
Analysis of collagen fibrils in wounds of wild type and Col6a1 null mice and of basement membranes and tensile strength in skin of Col6a1 null mice.
(a) Picrosirius red staining of day 7 wounds. d = dermis, e = epidermis, g = granulation tissue, st = scar tissue. Bar, 200 µm. (b–g) Transmission electron microscopy of day 7 wounds (b–d) and of blood vessels (e), nerves (f) and muscle (g) in unwounded Col6a1 null skin. Arrows indicate duplicated basement membranes. (h, i) Quantification of the distance between fibrils (wt = 903; Col6a1 null = 1776) (h) and of fibril diameter (wt = 890; Col6a1 null = 1091) (i) in areas from (b) and (c), (two electron micrographs from two animals per genotype were used for quantification). Load (j) and stress (k) are significantly reduced in unwounded Col6a1 null skin. *<0.05, **<0.005, ***<0.0005. Bar, 250 nm.