Figure 7: MT1-MMP suppresses VEGF-C expression via regulation of PI3Kδ signalling.

(a) Western blot analyses of VEGF-C in WT and Mmp14^−/− BMMs with ectopic expression of control vector, full-length MT1-MMP, the cytosolic domain-deleted MT1-MMP or the E/A240 mutant MT1-MMP after stimulation with 1 μg ml⁻¹ LPS for 24 h. (b) qPCR analyses of Vegf-c mRNA expression in WT and Mmp14^−/− BMMs with ectopic expression of a control vector, full-length MT1-MMP, the cytosolic domain-deleted or the E/A240 mutant MT1-MMP. Cells were stimulated with 1 μg ml⁻¹ LPS for 6 h before the analyses (**P<0.01, n=3, two-tailed t-test). (c) qPCR analyses of Vegf-c mRNA in WT and Mmp14^−/− BMMs with ectopic expression of a control vector, full-length MT1-MMP or the E/A240 mutant MT1-MMP that have been incubated with or without 20 μM IC87114 for 6 h before LPS stimulation (**P<0.01, n=3, two-tailed t-test). (d) Western blot analyses of VEGF-C in WT and Mmp14^−/− BMMs with ectopic expression of a control vector, WT p110δ or p110δ (D910A) mutant cDNA. Cells were stimulated with LPS (1 μg ml⁻¹ for 24 h) before protein analyses. (e) The proliferative rate of pulmonary LECs co-cultured with LPS-activated WT or Mmp14^−/− BMMs expressing a control vector, WT p110δ or p110δ (D910A) mutant cDNA measured by MTT assay (**P<0.01, n=3, two-tailed t-test). (f) Transcription of Vegf-c in WT and Mmp14^−/− BMMs by qPCR analyses. Cells were treated with or without IC87114 and JSH-23 for 4 h before LPS challenging. (g) Western blot analyses of IκBα and IκBβ in WT and Mmp14^−/− BMMs in response to LPS stimulation. Quantification of IκBα and IκBβ expression was shown in Supplementary Fig. 14b (*P<0.05, **P<0.01, ***P<0.001; n=3, two-tailed t-test). (h) Western blot analyses of IκBα and IκBβ in WT BMMs, WT BMMs treated with IC87114, Mmp14^−/− BMMs expressing a control vector or full-length MT1-MMP or E/A240 MT1-MMP, or WT p110δ cDNA or p110δ (D910A) mutant cDNA, in response to LPS stimulation for 15 min. Quantification of IκBα and IκBβ expression was shown in Supplementary Fig. 14c. (i) Western blotting analyses of p65 in the nuclear fractions of WT, Mmp14^−/− BMMs expressing either a control vector or full-length MT1-MMP, or E/A240 MT1-MMP, or WT p110δ or p110δ (D910A) mutant. Cells were stimulated with or without 1 μg ml⁻¹ LPS for 1 h before protein extraction. Quantification of nuclear p65 was shown in Supplementary Fig. 14d (*P<0.05, n=3, two-tailed t-test). (j) Binding of p65 at the Vegf-c promoter in WT BMMs, Mmp14^−/− BMMs and Mmp14^−/− BMMs treated with a combination of LPS, IC87114 and JSH-23 were examined by qPCR of ChIP assays using a specific antibody against p65 (*P<0.05, n=3, two-tailed t-test). Data represent the average±s.e.m. The experiments were repeated at least three times.