Fig 2: Macrophage loss of c-met repressed the capacity to degrade fibrotic ECM(A) The strategy of bone marrow-derived macrophage (BMDMs) isolation and culture.(B) Phase-contrast images of BMDM differentiation after stimulation with GM-CSF.(C) qRT-PCR analysis of tPA and uPA mRNA in BMDMs after incubation with HGF recombinant protein (20 ng/mL) for 1, 2, or 3 days. n = 3.(D) Western blot analyses demonstrating the induction of tPA in BMDMs after incubation with HGF recombinant protein (20 ng/mL).(E) Immunostaining showing HGF-induced tPA expression in macrophages. Cells were co-stained with 4',6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Scale bar, 25 µm.(F) RT-PCR analysis showed c-met gene expression in cultured c-met+/+ and c-met-/- BMDMs ex vivo.(G) Western blot analysis demonstrating reduced c-met phosphorylation after incubation with HGF recombinant protein in c-met-/- BMDMs.(H and I) qRT-PCR analysis of tPA and uPA mRNA in cultured c-met+/+ and c-met-/- BMDMs after incubation with HGF recombinant protein (20 ng/mL) for 1, 2, or 3 days. n = 4.(J and K) qPCR analysis revealing higher tPA and uPA mRNA abundance in Lyz-met+/+ than Lyz-met-/- kidneys after IRI and UUO. n = 3–5.(L–O) Western blot analyses (L, N) and quantitative data (M, O) demonstrating tPA protein abundance in Lyz-met +/+ and Lyz-met-/- mice kidneys after IRI and UUO. Dots indicate individual animals within each group. n = 3.(P–R) qRT-PCR analysis of tPA, and uPA mRNA in Ksp-met+/+ and Ksp-met-/- kidneys after IRI, FA, and UUO. n = 3–6.(S) Schematic diagram of our model for fibrotic KLM formation. Data are represented as mean ± SEM. Statistical significance was assessed using a two-tailed Student's t-test or the Rank Sum Test if data failed in normality test or one-way ANOVA, followed by the Student-Newman-Keuls test.

Fig 3: 3T3-A-EXO promoted 3LL tumor cell invasion through MMP3-mediated increase in MMP9 activation(A) 3LL cells were treated with 30 µg/ml 3T3-EXO or 3T3-A-EXO for 4 h, and then the protein levels of uPA, MMP2, MMP3, MMP9 and cathepsin B were detected by Western blot. (B) After the treatment with 30 µg/ml 3T3-EXO or 3T3-A-EXO for 4 h, 3LL cells were collected and pre-treated with 20 nM UK 356618 for 2 h. Then, the invasive ability of these cells was measured by an in vitro invasive assay (n = 5). (C) 3LL cells were treated with 30 µg/ml 3T3-EXO or 3T3-A-EXO for 4 h. Then, cells were collected and cultured in serum-free RPMI 1640 media for an additional 24 h. MMP2 and MMP9 activity in the supernatants was detected by gelatin zymography. (D) 3LL cells were treated with 30 µg/ml 3T3-A-EXO for 4 h, and then the cells were collected and cultured in serum-free RPMI 1640 media in the presence of 10 nM UK 356618 for an additional 24 h. MMP2 and MMP9 activity in the supernatants was detected by gelatin zymography. E, 3LL cells were treated with 30 µg/ml 3T3-EXO or 3T3-A-EXO with or without 10 nM MMP9 inhibitor for 4 h, and then the invasive ability of these cells was measured using an in vitro invasive assay (n = 5). (A, C, D) One representative of three independent experiments is presented. (B, E) Results are presented as the mean ± SEM of three independent experiments. (B, D, E) DMSO is solvent control. P-values were generated by Student’s t-test or one-way ANOVA followed by Tukey-Kramer multiple comparisons test; *p< 0.05; ***p< 0.001; NS, not significant. Control indicates 3LL cells treated with PBS.

Fig 4: Associations between genetically predicted PLAU and other cardiovascular conditions. (a) forest plot showed genetically-predicted protein expression of PLAU is associated with aortic aneurysms risk. (b-c) Forest plot mendelian randomization effect estimates and 95% confidence intervals for the genetic proxied antagonistic effect of PLAU and 18 cardiometabolic disorders (b) and 13 diseases (c) analysed. OR: odds ratio. 95% CI 95% confidence interval. (d) Representative Western blot analysis and quantification (e) of u-PA in aortic tissues, Mann-Whitney test, **p<0.01 (e) Quantification of u-PA in human plasma, Mann-Whitney test, ***p<0.001.

Fig 5: Autocrine PLAUR activation regulates PAQR11 expression.(A and B) qPCR analysis of cells treated for 2 days with 1 µM JAK inhibitor (P6) (A) and in shSTAT3-transfected cells (B). (C) Pearson correlation between PLAU and PAQR11 mRNAs in TCGA LUAD cohort. (D and E) qPCR analysis of siPLAU-transfected cells treated for 2 days with recombinant PLAU (10 ng/ml) (D) and in cells cotransfected with siPLAU and empty vector (Vec) or constitutively active mutant STAT3 (STAT3-CA) (E). (F) MMD gene promoter (schema). Luciferase assays on H1299 cells cotransfected with MMD promoter constructs and STAT3-CA or Vec. Empty reporter (pGL3). (G) Luciferase assay on H1299 cells cotransfected with wild-type (-500) or E-box–mutated MMD promoter and indicated expression vectors. (H) Chromatin immunoprecipitation assays on the MMD promoter (-500) using immunoglobulin G (IgG) or anti-ZEB1 antibodies. PAQR11 3'UTR (negative control). (I and J) qPCR analysis of H1299 cells treated for 2 days with 1 µM P6 (I) or cotransfected with STAT3-CA or Vec and siZEB1 or siCTL (J). (K) Heatmap depiction of ZEB1 and PAQR11 mRNA levels; pSTAT3/total STAT3 ratio; and secreted PLAU levels (sPLAU) in murine LUAD cell line panel. (L) Heatmap depiction of Pearson correlation (R values) of results in (K). Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments. P values, two-tailed Student’s t test (two-group comparisons) and one-way ANOVA test (multiple comparisons).