Figure 1. Overexpression of active ADAMTS6 inhibits focal adhesions, but not ADAMTS10.

(a) Immunofluorescence microscopy of ARPE-19A cells overexpressing ADAMTS6 or ADAMTS10. Focal adhesions were visualised by immuno-staining of vinculin (B/W, green) and phalloidin (B/W, red) on glass coverslips after 3 days. Shown are cells overexpressing ADAMTS6 wild type (ATS6 WT), an ADAMTS6 active site mutant (ATS6 ASM), an ADAMTS6 furin cleavage site mutant (ATS6 FM) and cells overexpressing ADAMTS10 wild type (ATS10 WT). Control cells were infected with lentivirus containing an empty vector. ATS6 WT cells had no detectable focal adhesions; however more focal adhesions per cell were detected with ATS6 ASM, ATS6 FM and ATS10 WT. Examples of focal adhesions are indicated by arrows. The number of focal adhesions (FA) per cell and length was calculated by manual annotation using ImageJ (FA number indicated (n = 5 cells)). Images were taken with a 40x objective. Scale bars indicate 50 μm. (b) FA lengths were categorised into 3 groups (0–4, 4–8 and 8–12 μm). The graph shows % of the total FA per cell of the 3 length groups. Cells overexpressing ATS6 ASM, ATS6 FM and ATS10 WT had significant increases in longer FA (4–8 μm). (focal adhesion measurements per cell n > 20). (c) Western blot of media taken from ARPE-19A cells overexpressing ADAMTS6 variants. ATS FM cannot be cleaved by furin so still contains the pro-domain. (d) ARPE-19A cells overexpressing ADAMTS6 (ATS6 WT) had greatly reduced pFAK in ARPE-19A. Western blotting analysis for total FAK and pFAK is shown. Quantification of band intensity is shown as a ratio of the control band intensity (n = 3). The western blot shown is from a representative experiment. Statistical significance for deviation from the control values was calculated using 1-way ANOVA (2-way ANOVA for FA length comparisons) with a Bonferroni’s multiple comparisons test using GraphPad Prism V6. Asterisk indicate P values where *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.