Fig 2: Ero1l overexpression in Nfib KO cells restores Vegfa expression and metastasis Bar graph representing mean Ero1l mRNA expression in the LM1, LM9, 4T1 WT with respective Nfib KO cell lines and Ero1l overexpression (OE) as indicated. Means ± s.d., n = 2 biological replicates and n = 2 technical replicates, two-tailed Student’s t-test, FC = fold change.Kaplan–Meier survival analysis of NOD/SCID mice inoculated i.v. with LM1 Nfib KO (n = 6), LM1 Nfib KO Ero1l OE (n = 6), LM9 Nfib KO (n = 6) or LM9 Nfib KO Ero1l OE (n = 6) cells. Two-tailed log-rank test.Representative bioluminescence images (left panel) and quantification (right panel) of lung metastases 5 days after i.v. injection of LM1 Nfib KO (n = 4) or LM1 Nfib KO Ero1l OE (n = 4) cells, means ± s.d., two-tailed Student’s t-test.Bar graph representing mean Vegfa mRNA expression in the LM1, LM9, 4T1 WT with respective Nfib KO cell lines and Ero1l overexpression. Means ± s.d., n = 2 biological replicates and n = 2 technical replicates, two-tailed Student’s t-test, FC = fold change.Left panel: Representative images of VEGFA-positive lung metastases. Scale bar, 1 mm. Right panel: Bar graph showing quantification of VEGFA staining in the metastatic area. Means ± s.d., n = 5 LM1/LM9 Nfib KO and LM1/LM9 Nfib KO Ero1l OE, two-tailed Student’s t-test. Source data are available online for this figure.

Fig 3: The NFIB-ERO1A-VEGFA axis promotes lung metastatic colonization through angiogenesis Representative images of VEGFA-stained tumours. Scale bar, 1 mm.Bar graph showing quantification of VEGFA staining in the tumours. Means ± s.d., n = 2, two-tailed Student’s t-test.Left panel: Representative images of VEGFA-positive lung metastases. Scale bar, 1 mm. Right panel: Bar graph showing quantification of VEGFA. Means ± s.d., n = 4 SUM159PT gNFIB shCTRL, n = 4 sh1 ERO1A, and n = 4 sh2 ERO1A, two-tailed Student’s t-test.Left panel: Representative images of VEGFA-positive lung metastases. Scale bar, 1 mm. Right panel: Bar graph showing quantification of VEGFA. Means ± s.d., n = 5 HR1g4Nfib and LM1 shCTRL, n = 5 sh1 ERO1A, and n = 5 sh2 ERO1A, two-tailed Student’s t-test.Representative image of CD31-positive endothelial structures in tumours. Scale bar, 1 mm.Bar graph showing quantification of CD31 staining in tumours. Means ± s.d., n = 2, two-tailed Student’s t-test.Left panel: Representative images of CD31-positive endothelial structures in lung metastases. Scale bar, 1 mm. Right panel: Bar graphs showing quantification of metastases as percentage of metastatic area per lung and quantification of CD31 staining in the metastatic area. Means ± s.d., n = 6 HR1 gCtrl and n = 6 HR1 g4Nfib, two-tailed Student’s t-test. Source data are available online for this figure.

Fig 4: The NFIB-ERO1A axis enhances lung metastatic colonization A, BIntersection of the top 200 differentially expressed genes (sorted by statistical significance) in tumourspheres and tumours (Nfib high-expression vs. the respective controls) and putative transcriptional targets of Nfib determined by ChIP-seq in mammary gland (Shin et al, 2016) (A) and epithelial-melanocyte stem cells (Chang et al, 2013) (B) (association rule: Basal + extension: 5,000 bp upstream, 1,000 bp downstream, 10,000 bp max extension, curated regulatory domains included).CChIP was performed in all murine cell lines (Nfib high-expression and the respective Nfib low-expression or KO controls) against Nfib followed by qPCR with primers specific for Ero1l promoter region proximal to transcription starting site (Ero1l_492 and Ero1l_493). Meg3 was used as control. Graph shows qPCR results with % input method as indicated (Nfib high-expression vs. the respective Nfib low-expression or KO controls). n = 2 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student’s t-test, n.s. = not significant.DKaplan–Meier survival analysis of NSG mice inoculated i.v. with SUM159PT gNFIB shCTRL (n = 9), SUM159PT gNFIB sh1 ERO1A (n = 7), or SUM159PT gNFIB sh2 ERO1A (n = 7) cells. Two-tailed log-rank test.ERepresentative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases 8 (SUM159PT gNFIB shCTRL) and 20 (SUM159PT gNFIB sh1 ERO1A or SUM159PT gNFIB sh2 ERO1A) days after i.v. injection of the respective cells. n = 4, means ± s.d., two-tailed Student’s t-test.FKaplan–Meier survival analysis of overall survival of mice injected i.v. with SUM159PT gNFIB shCTRL (n = 10), SUM159PT gNFIB sh1 ERO1A (n = 12), or SUM159PT gNFIB sh2 ERO1A (n = 13) cells, two-tailed log-rank test. Source data are available online for this figure.

Fig 5: Ero1l/ERO1A increases Vegfa/VEGFA expression and angiogenesis Bar graph showing the ROS levels in the NFIB models. The fluorescence signal was normalized to the blank. n = 2 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student’s t-test.Left panel: representative images of HIF1a immunofluorescence. HIF1a was stained with Alexa-633 (red) and nucleus with DAPI (blue). Scale bar, 100 µm. Right panel: bar graph showing quantification of HIF1a nuclear/cytoplasmatic ratio. n = 5 biological replicates, means ± s.d., two-tailed Mann–Whitney U-test.Left panel: Bar graph representing mean VEGFA mRNA expression in SUM159PT gCTRL and SUM159PT gNFIB breast cancer cell lines upon downregulation of ERO1A with two independent shRNAs (sh1 and sh2). Right panel: Bar graph representing mean VEGFA mRNA expression in lung extracts from animals inoculated i.v. with SUM159PT gNFIB shCTRL, SUM159PT sh1 ERO1A, or SUM159PT sh2 ERO1A cells. In both graphs n = 3 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student’s t-test, FC = fold change.Left panel: Representative images of CD31-positive endothelial structures in lungs. Scale bar, 1 mm. Right panel: Bar graphs showing lung metastases quantified as percentage of metastatic area per lung area and quantification of CD31 staining in the metastatic area. Means ± s.d., n = 8 shCTRL, n = 5 per sh1 ERO1A, and n = 6 sh2 ERO1A, two-tailed Student’s t-test.Left panel: Bar graph showing average of master segments counted after incubation of HUVEC cells with conditioned medium from LM1, HR1, 4T1 (Nfib high-expression and the respective Nfib low-expression or KO controls) and SUM159PT gNFIB shCTRL, SUM159PT gNFIB sh1 ERO1A, SUM159PT gNFIB sh2 ERO1A cells. Human VEGF121 (0.4 ng/µl) was added to SUM159PT gNFIB sh1 and sh2 ERO1A cells, n = 2 technical replicates, means ± s.d., two-tailed Student’s t-test. Right panel: Representative images showing tube formation four hours after addition of conditioned medium from HR1 gCtrl or g4Nfib. Scale bar, 100 µm. Source data are available online for this figure.