Figure 3: TOM1L1 pro-invasive activity requires ERBB2-induced interaction of its GAT domain with TOLLIP.

(a) Schematic showing TOM1L1 and the different mutants used in the study. (b) Upper panel: invasion of SKBR3 cells infected with the indicated viruses was assessed in Boyden chambers with matrigel. The histogram shows the invasion ratio normalized to the control value after 48 h of invasion (n=4). Mean±s.e.m *P<0.05, **P<0.01, NS, no significant (Student’s t-test). Lower panel: western blots showing the expression of the various TOM1L1 mutants in SKBR3 cells. (c) Lysates from BT-474, 3T3-SRCY527F and SKBR3 cell lines expressing the indicated constructs were immunoprecipitated with an anti-TOM1L1 antibody and blotted as indicated. Note that only the 3T3-SRCY527F cell lysate shows a band corresponding to p-tyr above the Ig band. (d,e) In vivo metastasis assay: luciferase-expressing HCC-1954 cells infected as indicated were injected in mice hearts (left ventricle). At the indicated times, mice were injected intraperitoneally with luciferine and imaged. (d) Mice imaging show higher bioluminescence signal in the brain of mice injected with TOM1L1-expressing cells compared with mock or ΔGAT-expressing cells (day 21 post injection). Right panel shows representative images of mice brains in each condition. (e) Quantification in time of the number of mice exhibiting bioluminescence signals in brain or legs. Note that bioluminescence signals are detected earlier and in more mice (day 15) when injected with cells that express TOM1L1 than with control cells (mock) or the ΔGAT mutant. (f) Western blot analysis of TOLLIP expression in 12 breast cancer cell lines. Note that TOLLIP is preferentially expressed in ERBB2+ cell lines. Lysates used were the same as in Fig. 1d. (g) Upper panel: invasion of SKBR3 cells infected as indicated and transfected twice with control (−) or TOLLIP siRNAs (+) was assessed and quantified as in b (n=5). Mean±s.e.m *P<0.05 (Student’s t-test). Lower panel: the efficiency of TOLLIP silencing was confirmed by western blot analysis of TOLLIP expression in the transfected cells. (h) Whole cell lysates of SKBR3 cells infected with indicated viruses were immunoprecipitated with an anti-TOLLIP antibody and immunoblotted as shown.