Figure 5.

Jak–Stat3 signaling is a critical pro-metastatic effector of Cd109. (a) Clonal growth and migration of shControl- or shCd109-expressing 889 cells without (GFP) or with expression of hyper-activatable Stat3 (Stat3C). Data from two independent experiments are presented as mean ± s.d. The n value per group is indicated. *P < 0.01 and **P < 0.001. (b) Representative fluorescence images of the lungs from mice with subcutaneous (SubQ) tumors from the indicated cell lines. Scale bars, 5 mm. (c,d) Numbers of lung (c) and liver (d) metastases from SubQ tumors. Each dot represents a mouse, and the bar denotes the mean. *P < 0.05 and **P < 0.01. (e) Lung metastasis area, as quantified by histology, in mice after intravenous (i.v.) transplantation of the indicated cell lines. Each dot represents a mouse, and the bar denotes the mean. *P < 0.05 and **P < 0.01; n.s., not significant. (f) Representative western blot analysis (of n = 2) for pStat3 in 3T3-Cd109 cells that were treated with DMSO (vehicle control), an epidermal growth factor receptor inhibitor (erlotinib), a Jak2 inhibitor (fedratinib) or the broadly acting Jak kinase inhibitors ruxolitinib, pyridone 6 and cucurbitacin I. Actin was used as a loading control. (g–i) Western blot analyses for pStat3 in 889 cells that were treated with a titration of pyridone 6 (g), ruxolitinib (h) or filgotinib (i). Actin was used as a loading control. (j) Representative western blot analysis (of n = 2) for pSTAT3 in human H460 cells that were treated with 2 μM pyridone 6 (P6). Actin was used as a loading control. (k) Clonal growth and migration ability of 889 and H460 cells that were treated with 2 μM pyridone 6. Data from two independent experiments are presented as mean ± s.e.m. **P < 0.001. All P values were calculated by an unpaired Student’s t-test.