Figure 7 | Scientific Reports

Figure 7

From: Comparative analysis of lysyl oxidase (like) family members in pulmonary fibrosis

Figure 7

FMT is dependent on the enzymatic activity of LOXL2. (a) Comparison of collagen I crosslinking capacity of recombinant LOXL2 and LOXL3. Human lung collagen I coated microplates were incubated with LOXL2 or LOXL3, respectively, for 2 hours followed by aldehyde detection using O-(biotinylcarbazoylmethyl) hydroxylamine as a probe. Relative fluorescence units (RFU) indicate the amount of aldehyde intermediate in collagen I produced by LOXL2 and LOXL3, respectively. White bars indicate resulting fluorescence after incubation with 20 nM LOX/L, whereas black bars represent 100 nM of the respective enzyme. Data is shown as mean ± SD of three independent experiments. **p < 0.01; ***p < 0.001. (b) NHLF cells were transfected with 16.6 nM of control or LOXL2 siRNA. After 12 hours, cells were incubated in conditioned medium derived from HEK293 cells transfected with LOXL2 wild type, LOXL2 enzymatic dead or control plasmids (mock). 24 h after transfection, cells were stimulated with TGF-β1 (5 ng/ml) for 48 hours in presence of the respective conditioned medium. Gene expression changes of acta2 mRNA were determined using Taqman gene expression assays. Data are normalized to RNA-polymerase 2 expression and control treatment and presented as mean ± SD of three independent experiments. *p < 0.05, relative to siControl treatment. (c) LOXL2 protein content detected by LOXL2-specific Western Blotting, 72 hours after HEK293 transfection with the respective plasmids (10 \({\mu }{\rm{l}}\) supernatant per lane).

Back to article page