Fig 2: DDR1b enhances cell migration on collagen I and localizes to focal adhesions. (A) Adhesion of Vector- DDR1a-, or DDR1b-expressing HEK cells to collagen I (0–3 µg/ml). Values are the mean ± SEM of 3 independent experiments performed at least in triplicate. **p < 0.05 relative to HEK-Vector. (B) Migration of Vector- DDR1a-, or DDR1b-expressing HEK cells towards collagen I (20 μg/ml). Values are the mean ± SEM of 3 independent experiments with at least 4-6 fields/microscopic field counted. Statistical analysis in (A,B): One-way ANOVA followed by Tukey’s multiple comparison test. (C) Total internal reflection fluorescence (TIRF) microscopy of HEK-DDR1a and HEK-DDR1b cells plated on collagen I (20 µg/ml) for 1 h and then co-stained with anti-DDR1 and integrin beta 1 (12G10), FITC-phalloidin, or anti-paxillin antibodies. Overlay images reveal co-localization of DDR1b with activated integrin β1, actin or paxillin. Co-localization of DDR1a, DDR1b with 12G10 (D), actin (E), paxillin (F) at the cell membrane of cells plated on collagen I was determined using the ImageJ/JACoP analysis and the Manders’ overlap coefficient (Bolte and Cordelieres, 2006). Values are the mean ± SEM of 3-4 FAs calculated for 10-13 cells and represent the fraction of DDR1 signal overlapping with 12G10 phalloidin and paxillin. Statistical analysis: unpaired two-tailed t-test. (G) Migration of Vector- DDR1a-, and DDR1b-expressing HEK cells transfected with Cnt or Itgβ1 siRNA towards collagen I (20 μg/ml). Values are the mean ± SEM of 3 independent experiments with at least 7 fields/microscopic field counted. Statistical analysis was done with One-way ANOVA followed by Tukey’s multiple comparison test.

Fig 3: Aggregated and phosphorylated DDR1 is present in the double walled anti-DDR1 structures. (A) COS-7 cells transiently expressing DDR1 were stimulated with collagen I for 60 minutes at 37 °C, then incubated on ice with anti-DDR1 mAb 7A9 and anti-collagen I mAb, before fixation, permeabilisation and immunostaining for phospho-tyrosine 513 (pY-DDR1). Intensity of the three stains was measured across the three lines shown (with a line width of 200 nm), the data were normalised so that the lowest and highest value from each stain was 0 and 100 A.U. (B,C) COS-7 cells transiently expressing DDR1-SNAP were incubated with SNAP-Surface Alexa Fluor-546 for 60 minutes at 37 °C, then stimulated with collagen I for 60 minutes (B) or for 5, 10, or 60 minutes (C) at 37 °C. Cells were then incubated on ice with anti-DDR1 mAb 5D5, before fixation, and secondary Ab staining (B), or fixed and mounted (C). 3D-SIM images were acquired using a Zeiss ELRYA microscope. Images are from a maximum intensity projection of all 15 slices (B) or from a single slice (A,C). White boxes indicate corresponding areas shown at higher magnification in lower images (B). Scale bars, 5 µm (A), 30 µm (upper image in B), 2 µm (enlarged images in B) or 3 µm (C). White arrows indicate examples of anti-DDR1 mAb binding at the edges of aggregated DDR1-SNAP signal (B). At least 10 cells were imaged for each condition.

Fig 4: Correlation between BIK and DDR1 mRNA expression levels in breast cancer patients. (A) from the TCGA dataset; and (B) from the bc-GenExMiner dataset.

Fig 5: Collagen binding and collagen-induced DDR1 redistribution of signalling defective DDR1 mutants. COS-7 cells transiently expressing WT-DDR1 or the indicated DDR1 mutant were stimulated with collagen I as detailed below. (A) Cells were stimulated with collagen for 10 minutes at 37 °C or left unstimulated, then incubated on ice with mAb 7A9, before fixation and secondary Ab staining. White boxes in left columns indicate corresponding areas shown at higher magnification in right columns. (B) Cells were stimulated with collagen for 60 minutes at 37 °C or left unstimulated, then incubated on ice with mAb 7A9 (shown in green) and anti-collagen I mAb (shown in magenta), before fixation, and secondary Ab staining. Graph shows the quantified anti-collagen signal with the exclusion of collagen not colocalised with DDR1. The mean collagen-immunostain intensity was calculated for each condition then normalised so that WT-DDR1 collagen-unstimulated and stimulated values were 0 and 100 A.U., respectively. Error bars are SEM (N = 65–121 cells from 3 independent experiments). (C) Cells expressing WT-DDR1 were incubated with either collagen I, a mixture of collagen I and anti-DDR1 mAb 7A9, or left unstimulated for 60 minutes at 37 °C. Cells were then immunostained as in B. Arrows indicate collagen aggregates not colocalising with DDR1. Cells were imaged using a widefield microscope. Scale bars, 30 µm or 5 µm (enlarged images in A). For each condition, at least 20 cells were imaged in A, and at least 30 cells were imaged in C.