Fig 1.
Expression of Lox family members murine and human dermis.
A,B: qRT-PCR analysis of Lox, Loxl1, Loxl2, Loxl3 and Loxl4 mRNA expression during mouse development (A) and in FACS-sorted fibroblast subpopulations (papillary fibrbroblasts, CD26+ SCA1-; reticular fibroblasts, CD26- SCA1+) (B) from PDGFRαH2BeGFP mice. C: Expression of LOX family members in human foetal and adult dermis. Details of tissue samples are shown in (S1 Table). ND = no signal detectable.
Fig 2.
Loxl2 ablation and overexpression are not compensated by Lox family members and do not alter dermal histology, thickness or cell density.
A: qPCR analysis of Lox family member mRNA expression in Loxl2-KO and Loxl2-KI mice at P2. (n = 4 for control and Loxl2-KO; n = 2 for Loxl2-KI). B: Hematoxylin and eosin (H&E) staining. Scale bars: 100 μm. C,D: Dermal thickness from basal membrane to hypodermis (C) and of the hypodermis (D). E,F: Total dermal cell number in upper, papillary (E) and lower, reticular dermis (F). (n = 2 for P2 and P120 Loxl2-KI, n = 3 for all others).
Fig 3.
Fibroblast subpopulations are not affected by Loxl2 deletion or overexpression.
A: Immunostaining for α-SMA (green), DLK1 (red) and CD26 (white). B: Immunostaining for LRIG1 (red) and SCA1 (white). Nuclei are labelled with DAPI (blue). Scale bars: 100 μm. C: Mean immunofluorescence quantification of DLK1 (left panel) and LRIG1 (right panel) in the upper and lower dermis (n = 2 for P2 Loxl2-KI, n = 3 for all others). Data are means ±SD. Note the high expression of DLK1 in the lower, reticular dermis and LRIG1 in the upper, papillary dermis at P2, which are down regulated with age.
Fig 4.
Dermal stiffness but not collagen maturation is altered in the dermis of Loxl2-depleted and overexpressing transgenics.
A: Herovici staining, enabling visualisation of immature collagen fibres (blue) and mature collagen (pink/purple). B: Collagen density quantification of Loxl2-KO, Loxl2-KI and control skin samples at the indicated postnatal ages. Collagen density values were calculated as collagen pixel per total tissue. C: Quantification of B-CHP signal. The data shown are means ± SD; n = 7 (Control), n = 5 (Loxl2-KO), n = 2 (Loxl2-KI) at P21 and n = 4 (Control), n = 6 (Loxl2-KO), n = 2 (Loxl2-KI) at P120. ns; not significant. D,E: AFM measurments of P21 old dorsal dermis. Experimental strategy with a representative 50 μm2 scan area showing the Young’s Modulus E distribution (D). Quantification of Young’s modulus E distribution in control, Loxl2-KO and Loxl2-KI dermis at P21. The mean is shown below (n = 4 biological replicates per genotype with 3 scan areas per sample).
Fig 5.
Loxl2 deletion or overexpression do not affect skin response to TPA.
A,B: Quantification of dermal cells in the upper, papillary (A) and lower, reticular (B) dermis upon TPA treatment. The data shown are means ± SD; n = 2 (untreated control), n = 6 (Control TPA), n = 2 (Loxl2-KO TPA), n = 3 (Loxl2-KI TPA). C: Hematoxylin and eosin (H&E) staining. D: Herovici staining. E: Picrosirius red staining visualised in polarised light. Scale bars: 100 μm.
Fig 6.
Loxl2 overexpression or deletion does not influence the tumour stroma composition of DMBA/TPA-induced papilloma.
A-C: Hematoxylin and eosin (H&E) staining (A), Herovici staining (B), Picrosirius red staining (C) of DMBA/TPA induced papillomas from Loxl2-KO, Loxl2-KI and control mice. D: Immunostaining for α-SMA (green) in papillomas from Loxl2-KO, Loxl2-KI and control mice. Nuclei are labelled with DAPI (blue). E: Quantification of tumour stroma cell density. The data shown are means ± SD; (n = 3 control, n = 2 Loxl2-KO, n = 2 Loxl2-KI). F: Transepidermal perforation in an SCC stained with Elastica van Gieson Staining. Representative image for all genotypes is shown. Scale bars: 100 μm.