Figure 4: TOM1L1 regulates invadopodia and requires MT1-MMP.
From: TOM1L1 drives membrane delivery of MT1-MMP to promote ERBB2-induced breast cancer cell invasion
(a) BT-474 cells infected with the indicated shRNAs were plated onto Oregon Green 488 gelatin-coated coverslips. After 6–24 h, cells were fixed and labelled with the relevant antibodies to visualize F-actin and cortactin by confocal microscopy. Arrowheads point to actin punctiform structure (white) and degradation of the gelatin matrix (black). Inset shows higher magnification of the boxed region. Scale bar, 10 μm. (b) Same experiment as in a but cells were imaged using a confocal orthogonal (x/z) slice view. Inset shows higher magnification of the arrowhead-pointed zone. Scale bar, 10 μm. (c) Quantification of Oregon Green 488 gelatin degradation areas in μm2 (n=3). Mean±s.e.m., ***P<0.001 (Student’s t-test). (d) Western blot showing MT1-MMP expression in 12 breast cancer cell lines. Tubulin is used as loading control. Lysates used were the same as in Figs 1d and 3f. (e) Indicated proteins expression was assessed by immunoblot in BT-474 cells lysates expressing indicated shRNAs. (f) Upper panel: 48 h after transfection of control or MT1-MMP-specific siRNAs, invasion of BT-474 cells was assessed in Boyden chambers with matrigel (n=5). Mean±s.e.m., *P≤0.05 (Student’s t-test). Lower panel: WCL of BT-474 cells transfected with control (−) or MT1-MMP (+) siRNAs were immunoblotted to check MT1-MMP expression. Numbers shown under MT1-MMP represents quantification of MT1-MMP silencing compared with control condition. (g) Invasion of BT-474 cells expressing indicated shRNAs was assessed in Boyden chamber with matrigel in the presence of 2μM Lapatinib (+) or DMSO (−). (h) Western blot showing expression of the indicated proteins in seven ERBB2+ breast cancer cell lines. Blots are the same as those presented in Figs 1d, 3f and . Lower panel indicates the presence of invadopodia in the different cell lines. N=not detected, Y=yes, ?=not tested. (i) Correlation between ERBB2/TOM1L1 and invadopodia. Histogram indicates the number of cell lines exhibiting invadopodia in function of ERBB2 and TOM1L1 expression. High ERBB2/Low TOM1L1 cell lines are SKBR3 and HCC-1954. High ERBB2/High TOM1L1 cell lines are BT-474 and UACC812 and Low ERBB2/High TOM1L1 cell line is MDA-MB 361.
