Figure 4: Matriptase is required for activation of the pro-HGF/c-Met signalling pathway and cell proliferation in primary mammary carcinoma cells, mammary tumours and human breast cancer cells.

(a) Efficient Cre-mediated ablation of matriptase in primary mammary carcinoma cells shown by western blot analysis of cells isolated from PymT-Mat^{cond KO} or PymT-Mat^control mice. Two days after plating, 10 μM 4-OHT (+) or vehicle (−) was added 24 h to ablate matriptase expression. Cells were in serum-free medium without 4-OHT for another 24 h before lysis. Skin lysates from newborn matriptase null and wild-type littermate mouse were included for antibody specificity. Anti-β-actin was used as control for equal loading. (b) Mammary carcinoma cells were isolated and treated with 4-OHT as above and exposed to active HGF (2 min) or pro-HGF (0, 10 or 20 min). Cell lysates were analysed for total c-Met, activated c-Met and its downstream targets by western blotting. (c,d) Whole lysates of mammary tumours were separated by reducing SDS–PAGE and analysed by western blotting. (c) HGF is mainly present in its active two-chain form in PymT-Mat⁺ tumours, whereas a significant level of single-chain, inactive pro-HGF is detected in PymT-Mat^hypo tumours. rHGF=recombinant pro-HGF/HGF. (d) reduced Gab1 levels of the active phosphorylated form relative to total Gab1 in PymT-Mat^hypo mammary tumours. (e) Efficient RNAi silencing of matriptase in human HCC1937 cells with three non-overlapping synthetic RNA duplexes (siM1, siM2 and siM3). A %GC matched RNA duplex was used as negative control as well as a mock (no RNA duplex). β-Actin staining ensured equal protein loading. (f) Cells were serum staved and exposed to no HGF, active HGF (2 min) or pro-HGF (15 min). Lysates were analysed for total c-Met, activated c-Met and its downstream targets. (g) Matriptase was silenced in HCC1937 cells as above and left untreated (black bars) or exposed to active HGF (grey bars) or pro-HGF (white bars) for 24 h. Bar graphs show proliferation 24 h after stimulation (*P<0.04, **P<0.005, ***P<0.0008, Student’s t-test). Data represent mean of three replicates. Error bars represent s.d. Data are representative of four independent experiments.