Figure 2.
Figure 2. DDR1 localizes at linear invadosomes and is required for their formation. (A, top) MDA-MB-231 cells transiently transfected with DDR1-Flag were cultured for 4 h on collagen I. All channels of the boxed region are shown magnified on the right. F-actin (red) colocalizes with DDR1 (green) and Tks5 (blue) at linear invadosomes. (A, bottom) MDA-MB-231 cells stably expressing DDR1-GFP were cultured for 4 h on collagen I fibrils. Tks5 (red) colocalizes with DDR1 (green) and collagen I (blue). Correlation coefficient of colocalization: actin/DDR1 r = 0.29; DDR1/Tks5 r = 0.11; n = 10. (B) MDA-MB-231 cells were transfected with control (siCT) or three independent DDR1 siRNAs. DDR1 protein expression was analyzed by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Cells transfected as in B were seeded for 4 h on collagen I. Shown are representative confocal images of MDA-MB-231 cells. Tks5 (green) and F-actin (red) are shown. Right panels show enlarged views of the boxed regions. Similar results were obtained with siDDR1 #2 and #3. (D–F) Down-regulation of DDR1 expression decreases the formation of linear invadosomes and their degradation activity. (D) Quantification of the percentage of MDA-MB-231 cells able to form linear invadosomes. Error bars represent the SEM (n > 1,000; three independent experiments; ***, P < 0.001 as compared with the control siRNA condition). (E) Quantification of the number of linear invadosomes per cell. Results are expressed as mean ± SEM (n > 500; three independent experiments; ***, P < 0.001 as compared with the control siRNA condition). (F) Bar graph shows the amount of collagen I degraded per cell over the time. Error bars represent the SEM (n = 60 fields, three independent experiments; ns, not statically significant; ***, P < 0.001; **, P < 0.005 as compared with the control siRNA condition). The right panel shows representative images of SHG collagen signals 4 h after seeding of control (siCT) or siDDR1 #1–transfected MDA-MB-231 cells. Cells are stained for Tks5 (red). Bars: (A and C, left panels) 5 µm; (A and C, magnified panels on the right) 2 µm; (E) 100 µm.

DDR1 localizes at linear invadosomes and is required for their formation. (A, top) MDA-MB-231 cells transiently transfected with DDR1-Flag were cultured for 4 h on collagen I. All channels of the boxed region are shown magnified on the right. F-actin (red) colocalizes with DDR1 (green) and Tks5 (blue) at linear invadosomes. (A, bottom) MDA-MB-231 cells stably expressing DDR1-GFP were cultured for 4 h on collagen I fibrils. Tks5 (red) colocalizes with DDR1 (green) and collagen I (blue). Correlation coefficient of colocalization: actin/DDR1 r = 0.29; DDR1/Tks5 r = 0.11; n = 10. (B) MDA-MB-231 cells were transfected with control (siCT) or three independent DDR1 siRNAs. DDR1 protein expression was analyzed by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Cells transfected as in B were seeded for 4 h on collagen I. Shown are representative confocal images of MDA-MB-231 cells. Tks5 (green) and F-actin (red) are shown. Right panels show enlarged views of the boxed regions. Similar results were obtained with siDDR1 #2 and #3. (D–F) Down-regulation of DDR1 expression decreases the formation of linear invadosomes and their degradation activity. (D) Quantification of the percentage of MDA-MB-231 cells able to form linear invadosomes. Error bars represent the SEM (n > 1,000; three independent experiments; ***, P < 0.001 as compared with the control siRNA condition). (E) Quantification of the number of linear invadosomes per cell. Results are expressed as mean ± SEM (n > 500; three independent experiments; ***, P < 0.001 as compared with the control siRNA condition). (F) Bar graph shows the amount of collagen I degraded per cell over the time. Error bars represent the SEM (n = 60 fields, three independent experiments; ns, not statically significant; ***, P < 0.001; **, P < 0.005 as compared with the control siRNA condition). The right panel shows representative images of SHG collagen signals 4 h after seeding of control (siCT) or siDDR1 #1–transfected MDA-MB-231 cells. Cells are stained for Tks5 (red). Bars: (A and C, left panels) 5 µm; (A and C, magnified panels on the right) 2 µm; (E) 100 µm.

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