Fig 1: Phosphorylation of ETV4 at Y392 by PTK6 regulates the nuclear translocation of ETV4. A) Immunoprecipitation (with anti-FLAG) and immunoblot analysis (with anti-4G10 or anti-FLAG) of the empty vector (CON.) and ETV4 WT or Y392F overexpression vector stably transfected UM-UC-3 cells in the absence or presence of pervanadate for 30 min. B) Immunoblot analysis of cytoplasmic (C) and nuclear (N) FLAG-ETV4, GAPDH and Lamin B1 in ETV4 WT or Y392F overexpression vector stably transfected UM-UC-3 cells. C) Representative immunofluorescence images staining for FLAG-ETV4 in ETV4 WT or Y392F overexpression vector stably transfected UM-UC-3 cells. D) Immunoprecipitation (with anti-FLAG, anti-PTK6 or control IgG) and immunoblot analysis (with anti-FLAG or anti-PTK6) of the empty vector (CON.) and ETV4 WT or Y392F overexpression vector stably transfected UM-UC-3 cells. E) Immunoprecipitation (with anti-FLAG) and immunoblot analysis (with anti-4G10, anti-HA or anti-FLAG) of HEK293 cells transfected with plasmids encoding HA-PTK6 and FLAG-ETV4 in the absence or presence of pervanadate for 30 min. F) Immunoprecipitation (with anti-FLAG) and immunoblot analysis (with anti-4G10, anti-HA or anti-FLAG) of UM-UC-3 cells that stably express FLAG-ETV4 WT or Y392F, treated with NC or siPTK6 for 48 h. G) Immunoblot analysis of cytoplasmic (C) and nuclear (N) FLAG-ETV4, PTK6, GAPDH and Lamin B1 in UM-UC-3 cells that stably express FLAG-ETV4 WT or Y392F, treated with NC or siPTK6 for 48 h. H) Representative immunofluorescence images staining for FLAG-ETV4 in UM-UC-3 cells that stably express FLAG-ETV4 WT or Y392F, treated with NC or siPTK6 for 48 h. I) qRT-PCR analysis of CXCL1 and CXCL8 mRNA in ETV4 WT or Y392F overexpression vector stably transfected UM-UC-3 cells, which were treated with NC or siPTK6 for 48 h. J) Chemotactic index analysis of neutrophils attracted by indicated UM-UC-3 CM. K) qRT-PCR analysis of VEGFA and MMP9 mRNA in neutrophils incubated with indicated UM-UC-3 CM. L) Representative images of tube formation assay of HLECs incubated with CM from neutrophils with the indicated treatment for 6 h, histograms show means and SD for tube length. Statistical significance was assessed using two-tailed t tests or one-way ANOVA. NS p >0.05, *p <0.05, **p < 0.01. Data are representative of two (A–G, J,L) or three independent experiments (mean ± S.D. in I,K).
Fig 2: ETV4 is a transcriptional regulator of CXCL1/8 in BCa cells. A) qRT-PCR analysis of ETV4 mRNA in paired primary tumors and LN metastases from BCa patients (n = 12). B) Immunoblot analysis of ETV4 and GAPDH in UM-UC-3 and T24 cells transfected with siRNA for 48 h. C) qRT-PCR analysis of ETV4, CXCL1 and CXCL8 mRNA in UM-UC-3 and T24 cells transfected with siRNA for 48 h. D) ELISA analysis of CXCL1 and CXCL8 in the supernatants of UM-UC-3 and T24 cells transfected with siRNA for 48 h. E) Luciferase reporter assays analyzing the activity of wildtype (WT) and mutation (mut) CXCL1 and CXCL8 promoter. F) ChIP analysis of CXCL1 and CXCL8 genes in UM-UC-3 and T24 infected with siRNA for 48 h. G) qRT-PCR analysis of VEGFA and MMP9 mRNA in neutrophils incubated with indicated UM-UC-3 CM. H) ELISA analysis of VEGFA and MMP9 in the supernatants of neutrophils treated with the indicated supernatant for 12 h. I) Representative images of tube formation assay of HLECs incubated with CM from neutrophils with the indicated treatment for 6 h, histograms show means and SD for tube length. J) Chemotactic index analysis of neutrophils attracted by indicated UM-UC-3 CM. Statistical significance was assessed using two-tailed t tests or one-way ANOVA. *p <0.05, **p < 0.01, ***p < 0.001. Data are representative of two (B, D, E, F, H,I) or three independent experiments (mean ± S.D. in C,G).
Fig 3: Y392 of ETV4 is required for the pro-LN-metastatic effect. A) Quantification of published post-translational modification sites of ETV4. B) qRT-PCR analysis of ETV4, CXLC1, CXCL8 mRNA in ETV4 WT- or MUT-overexpressed UM-UC-3 cells. C) ELISA analysis of CXCL1 and CXCL8 in the supernatants of ETV4 WT- or Y392F-overexpressed UM-UC-3 and T24 cells. D) qRT-PCR analysis of VEGFA and MMP9 mRNA in neutrophils incubated with indicated CM for 3 h. E) Chemotactic index analysis of neutrophils attracted by indicated UM-UC-3 CM. F) Representative images of tube formation assay of HLECs incubated with CM from neutrophils treated with the indicated supernatant for 6 h, histograms show means and SD for tube length. G) Representative bioluminescence images (left) and histogram analysis (right) of the popliteal metastatic LNs from the indicated nude mice. The relative luminescence is 1 in empty vector (CON.). H) Images of the dissected popliteal LNs (left) from nude mice injected with the CON and ETV4 WT or Y392F overexpression vector stably transfected UM-UC-3 cells for 4 weeks. Analysis (right) of the LN volume. I) Flow cytometry analysis of CD45+CD11b+Ly6G+ neutrophils from fresh surgically excised footpad tumor tissues. J) Representative immunofluorescence images (left) staining for MPO and LYVE1 and quantification (right) of MPO+ and LYVE1 + cells infiltrated in footpad tumor tissues from indicated nude mice. Statistical significance was assessed using two-tailed t tests or one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative of two (C, E,F) or three independent experiments (mean ± S.D. in B,D).
Fig 4: BCa cell-derived CXCL1/8 promote TANs-modulated lymphangiogenesis. A) qRT-PCR analysis of VEGFA, VEGFC and VEGFD mRNA in neutrophils. B) qRT-PCR analysis of VEGFA, VEGFC, VEGFD and MMP9 mRNA in neutrophils treated with the culture media (CM) from UM-UC-3 and T24 for 3 h, respectively. C) ELISA analysis of VEGFA and MMP9 in the supernatants of UM-UC-3, T24 and neutrophils treated with the indicated supernatant for 12 h, respectively. D) Percentage of MMP9+, VEGFA+ and MMP9+ VEGFA+ cells in MPO+ cells. E) Representative immunofluorescence images staining for MPO, VEGFA and MMP9 in LN (-) (n = 20) and LN (+) (n = 16) human BCa tissues. F) Quantification of MPO+MMP9+, MPO+VEGFA+ and MPO+ MMP9+ VEGFA+ cells in LN (-) and LN (+) human BCa tissues. G) Representative images of tube formation assay of HLECs incubated with CM from UM-UC-3-induced neutrophils treated with IgG, neutralizing antibody of VEGFA (100 ng mL-1) or inhibitor of MMP9 (MMP-9-IN-1, 20 × 10-6 m) for 6 h, histograms show means and SD for tube length. H) qRT-PCR analysis of VEGFA and MMP9 mRNA in neutrophils incubated with UM-UC-3 or T24 CM and treated with inhibitor of CXCR2 (SB225002, 50 × 10-6 m) for 3 h, respectively. I) ELISA analysis of VEGFA and MMP9 in the supernatants of neutrophils incubated with above treatment for 12 h. J) qRT-PCR analysis of VEGFA and MMP9 mRNA in neutrophils incubated with CXCL1 or CXCL8 (10 ng mL-1) and treated with inhibitor of CXCR2 (SB225002, 50 × 10-6 m) for 3 h, respectively. K) ELISA analysis of VEGFA and MMP9 in the supernatants of neutrophils incubated with above treatment for 12 h. L,M) Representative images of tube formation assay of HLECs incubated with CM from neutrophils with the indicated treatment for 6 h, histograms show means and SD for tube length. Statistical significance was assessed using two-tailed t tests or one-way ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are representative of two (C,I,K,L, M) or three independent experiments (mean ± S.D. in A,B,H,J).
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