Fig. 6

E2-induced activation of TrkB and EGFR signaling converge to promote invasion and mammosphere formation in TNBC. a 231BR and b F2-7 cells were starved in 5% charcoal-stripped FBS media for 12 h, treated with vehicle (DMSO), 1 µM Lapatinib for 8 h, 1 µM ANA-12 for 2 h, and then stimulated with vehicle, 100 ng/ml BDNF, CM-OH, or CM-E2 for 10 min. Alpha tubulin was used as loading control. Numbers indicate ratio of protein/α-tubulin relative to control. Graphs show fold changes of p-TrkB/Tubulin, p-PLCγ/total PLCγ and p-AKT/Total AKT relative to vehicle-treated cells from 2–4 independent experiments. c 231BR cells expressing shNC (shNC) or an shRNA targeting TrkB (hu-shTrkB1) were plated in matrigel-coated plates and serum-starved for 16 h. After scratch wound, invasion well were coated with matrigel and then CM-OH or CM-E2 used as chemoattractants. Graphs show average RWD ± SEM. The data were analyzed using repeated measures ANOVA with Tukey’s multiple comparison test. Adjusted P values *P < 0.05, **P < 0.01, ***P < 0.001 at 24 h. d shNC or hu-shTrkB 231BR cells were plated on organotypic brain slices from E2-treated mice and invasion assessed using Sholl analysis. Left: distribution of the median # of intersections away from initial sphere edge 48 h after seeding. Right: Total # of new intersections. e 231BR cells were plated in matrigel-coated plates, serum-starved for 16 h and pre-treated with vehicle, 1 µM Lapatinib, 1 µM ANA-12, or both. Invasion on matrigel-filled scratch wound was measured as in c. f 231BR cells were pretreated with ANA-12, Lapatinib or a combination, and plated on organotypic brain slices from E2-treated mice. Left: distribution of the median # of intersections away from initial sphere edge 48 h after seeding. Right: Total # of new intersections. Kruskal–Wallis with Dunn’s multiple comparisons test. g F2-7 cells were plated in EGF-free mammocult media (500 cells/well) and treated with vehicle, CM-OH, CM-E2 alone or in combination with 1 µM ANA-12, 1 µM Lapatinib, or both. Mammospheres were quantified at 14 days. Graph shows the number of mammospheres per well (n = 10 per treatment) in one representative experiment (from 2 repetitions). Data were analyzed using Kruskal–Wallis test with Dunn’s multiple comparison correction. For all graphs adjusted P values are shown. For all WBs α-tubulin was used as loading control and numbers indicate ratio of protein/α-tubulin relative to control or time zero