Figure 2: TOM1L1 regulates ERBB2-driven cell invasion.

(a) TOM1L1 expression was assessed by western blotting in SKBR3 cells infected with empty retroviruses (mock) or coding TOM1L1. (b) SKBR3 cells infected as in a were seeded in the upper compartment of a Boyden chamber containing 1 mg ml⁻¹ matrigel. After 48 h, cells present in the lower chamber were visualized by Hoechst staining. (c) Quantification of b. The histogram shows the invasion ratio normalized to control conditions (n=3). (d) SKBR3 cells infected as indicated were embedded as spheroids in a collagen and matrigel matrix for 75 h and then imaged by phase contrast microscopy. Yellow dotted line shows the sphere size at t=0 and the red dotted line shows invasive cells or invasive fronts at the end of the experiment. Invasive fronts were twice larger in TOM1L1-expressing cells than in controls cells (n=4). Scale bar, 100 μm. (e) TOM1L1 expression was assessed by western blotting in BT-474 cells infected with viruses containing control shRNA (Ctrl) or two different TOM1L1 shRNAs (TOM1L1 #1 and #2). (f) Invasion of the BT-474 infected cells was assessed in Boyden chambers with matrigel as in b. Cells in the lower chamber were stained with Hoechst and counted as in c (n=3) (g,h) Infected BT-474 cells were embedded as spheroids in a collagen and matrigel matrix for 5 days and imaged by phase contrast microscopy. Yellow dotted line shows sphere size at t=0, green dotted line shows sphere size at t=120 h and red dotted line shows invasive cells or invasive fronts. The surface explored by the cells was decreased by 1.5-fold in TOM1L1 silenced cells compared with controls (n=3). Scale bars, 400 μm. (i) SKBR3 cells infected as indicated were seeded in Boyden chambers as in b,c and incubated with DMSO, 12.5 μM GM6001 (MMP inhibitor), 1 μM lapatinb (an EGFR/ERBB2 inhibitor) and 2 μM SU6656 (SRC inhibitor) for 48 h. The histogram shows the invasion ratio normalized to control conditions (n=10). All histograms in Fig. 2 show mean±s.e.m. *P≤0.05 (Student’s t-test).