Fig 1: Up-regulation of Il-17rb contributes to the aggressive malignancy phenotypes of breast cancer cell derived from tumor-draining lymph node AGene expression profiles were shown at 4T1LN to 4T1PT cells. Five genes encoding cell surface proteins were identified among up-regulated genes.BmRNA expression of each candidate gene in 4T1PT and 4T1LN cells was determined by RT–qPCR. Gapdh was used as an internal control.C, DIl-17rb, Gpr56, and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis.EWestern blotting and RT–qPCR analysis of Il-17rb, Gpr56, and Scara5 expression in 4T1 cells transduced with Il-17rb, Gpr56, Scara5, or control LacZ shRNA lentivirus, respectively.FSoft-agar colony-forming activity was examined in lentivirus-transduced shIl-17rb, shGpr56, shScara5, or shLacZ 4T1 cells (5 × 102 cells/well, n = 6 wells per group).GWestern blotting analysis was used to detect ectopic expression of Il-17rb in 4T1 cells infected with either lentiviruses carrying Il-17rb cDNA or empty vector.HSoft-agar colony-forming activity was examined using control or Il-17rb over-expressing 4T1 cells (5 × 102 cells/well, n = 6 wells per group).IWestern blotting analysis was used to examine Il-17rb in a representative Il-17rbDel 4T1 clone derived from Il-17rb-knockout 4T1 cells using a CRISPR/Cas9 system.J, KTumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5 × 102 4T1 cells from WT or Il-17rbDel clone. Tumor mass (J) and weight (K) were measured on day 28. Scale bar: 1 cm.L, MBALB/c mice (n = 3 mice per group) were injected with 5 × 105 4T1 cells from WT or Il-17rbDel clone via tail vein. Lung colonization was examined by lung morphology (L) and the numbers of tumor nodule (M) on day 21. Scale bar: 1 cm. Black arrow: Lung nodule.Data information: All experimental data were verified in at least two independent experiments. All values are presented as mean ± SD. In (B), data were presented as means ± SD (triplicate measurement). In (C, D, E, G, and I), the intensity of each band was quantified using the ImageJ software, and Gapdh was used as a loading control. Relative expression (RE) of protein levels in each figure is indicated. In (F), **P = 0.00216 for the shIl-17rb 4T1 cells. In (H), **P = 0.0000009. In (K), **P = 0.00012. In (M), *P = 0.032862. Level of significance was determined using two-tailed unpaired t-test.Source data are available online for this figure.