DDR1 is required for invasion of invadosome-bearing cells in 3D collagen matrix. (A) MDA-MB-231 cells were placed for 4 h into a 3D collagen gel (red), fixed, and processed for immunofluorescence staining (Tks5, green). The z-cut section is shown on the right. The broken line on the left panel corresponds to the focal plane shown in the z-cut section. (B) DDR1 expression in MDA-MB-231 cells transfected with control siRNA (siCT) or two siRNAs targeting DDR1 (siDDR1 #1 and #2) was monitored 3 d after cell seeding (anti-DDR1 [D1G6]; Cell Signaling Technology). (C–E) MDA-MB-231 cells transfected as in B were seeded for an invasion assay and allowed to invade the collagen matrix for 1 h or 3 d. Z confocal optical sections were taken. (C) Assays were quantified by measuring an invasion index. The bar graph represents the ratio of the number of MDA-MB-231 cells penetrating the collagen gel/number of seeded cells. Data are expressed as mean ± SEM (three independent experiments). ***, P < 0.001 as compared with control siRNA (siCT). (D and E) Representative confocal images of MDA-MB-231 cells transfected with control siRNA (siCT) or siRNA targeting DDR1 seeded on collagen I fibrils. F-actin, green; collagen I fibrils, red. Images were taken 1 h (left) or 3 d after seeding (middle and right). Top panels correspond to focal planes whereas bottom panels represent matched z-cut sections. The broken lines on the bottom panel correspond to focal planes. 1 h after seeding, MDA-MB-231 cells transfected with control or siDDR1 #1 are on the top of the gel. 3 d later, control MDA-MB-231 cells deeply invaded the collagen plug (D, right), whereas DDR1-depleted cells were still on the top of the collagen plug (E, right). Bars: (A) 5 µm; (D and E) 50 µm.