Extended Data Fig. 9: Data Figure 9. Ang2 and Claudins signaling and Claudin2/10 suppression.

(a) Relative mRNA expression of Cldn5 in parental NIH-3T3 cells and NIH-3T3 cells treated with Adeno-CMV-luc (Ad-Ctrl) or Ad-CMV-CLDN5v2 (Ad-Claudin 5), n = 3 biologically independent experiments (with each 2 technical replicates). (b) Representative western blot and quantification of his-tagged Claudin 5 (His), Claudin 5, and β-actin in parental NIH-3T3 cells and NIH-3T3 cells treated with Adeno-CMV-luc (Ad-Ctrl) or Ad-CMV-CLDN5v2 (Ad-Claudin 5), n = 3 biologically independent experiments. Uncropped blots are shown in the Image Source Data File. (c) Relative mRNA expression of Cldn5 in NIH-3T3 cells with the indicated treatment. Dox on/off indicates doxycycline exposure, n = 4 biologically independent experiments (with each 2 technical replicates). (d) Orthotopic 4T1 tumor volume over time in the indicated groups, n = 5 mice per group. (e) CLDN5 mRNA levels in metastatic lungs of patients with breast cancer (n = 9) and healthy lungs (n = 6) (GSE193103)⁶². (f, g) Relative mRNA expression of Cldn2 (f) and Cldn10 (g) in kidney endothelial cells treated with the indicated guide RNAs, n = 3 biologically independent experiments (with each 2 technical replicates). (h) Relative mRNA expression of Cldn2 and Cldn10 in sorted E-cadherin⁺ epithelial cells and CD31⁺ endothelial cells, n = 3 mice per group. Gating strategy is shown in Supplementary Fig. 9. (i, j) Relative mRNA expression levels of Cldn2 and Cldn10 in sorted CD31⁺ endothelial cells (i) and E-cadherin⁺ epithelial cells (j) after treatment with the indicated guide RNAs, n = 3 mice per group. (k) Experimental design for the evaluation of the downregulation of Claudin 2 and Claudin 10 in the kidney of mice with 4T1 orthotopic tumors. (l) Orthotopic 4T1 tumor volume over time in the indicated groups. On day 14, LentiCRISPRv2 control vector, n = 5; LentiCRISPRv2 Cldn2 and Cldn10, n = 5; Day 18, LentiCRISPRv2 control vector, n = 5; LentiCRISPRv2 Cldn2 and Cldn10, n = 6; Day 22, LentiCRISPRv2 control vector, n = 8; LentiCRISPRv2 Cldn2 and Cldn10, n = 6 mice. (m) Representative H&E images of the lung of mice in the indicated groups. Scale bar, 25 µm. (n) Quantification of lung metastatic area. LentiCRISPRv2 control vector, n = 15; LentiCRISPRv2 Cldn2 and Cldn10, n = 20 mice. (o) Representative images for GFP immunolabeling and quantification of percent of the GFP positive area in lung. Scale bar, 50 µm, n = 9 mice per group. Data are presented as mean values +/− SEM, Min-to-Max in E. For a: one-way ANOVA test with Tukey’s multiple comparisons test. For d: two-way ANOVA with Sidak’s multiple comparisons test. For b, c, f, g: one-way ANOVA test with Dunnett’s multiple comparisons test. For e: two-tailed Mann-Whiney test. For i, j, n, o: Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test).

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