Figure 3.

DDRs and COL1 promote the growth rate of HT1080 cells in vivo but not in vitro. (A,B) Volumes of ±DDR/+COL1 tumours as a function of time. HT-DDR1b (A) and HT-DDR2 (B) cells were incubated for two days with or without DOX to repress or induce DDR expression, respectively. Then, the cells were harvested and mixed with an ice-cold solution of COL1 (2 mg/ml, final concentration), as described in the Methods section. Mice fed a regular or a DOX-supplemented diet were inoculated s.c. with 1 × 10⁶ cells/mouse in 100 μl of the COL1/cell suspension. Number of mice for each group is provided in Supplementary Table 1. Tumours were measured every 2–3 days, and tumour volumes were calculated. Tumour growth rates were determined, as described in the Methods section. (C,D) Expression and activation of DDRs in ±DDR/+COL1 tumour extracts. Tumour extracts derived from representative mice harbouring either HT-DDR1b (C) and HT-DDR2 (D) ±DOX tumours (n = 4 in each group) were resolved by reducing SDS-PAGE followed by immunoblot analyses using antibodies against phosphorylated DDR1b (Y513) (C, middle panel) or DDR2 (Y740) (D, middle panel). The blots were then stripped and reprobed with antibodies against total DDRs (upper panels), and then reprobed for β-actin (lower panels). pCTF, phosphorylated C-terminal fragment of DDR1b. Asterisk indicates a non-specific band. Full-length blots are presented in Supplementary Fig. 11. (E,F) In vitro cell proliferation of ±DDR -expressing HT1080 cells in 2D COL1. HT-DDR1b (E) and HT-DDR2 (F) cells were incubated with or without DOX for three days, and then 2 × 10⁴ cells/well were seeded on 24-well plates coated with fibrillar COL1 (100 μg/well), in triplicates, in complete media. At various time points, the cells were detached and counted with a Coulter counter. Results represent the average of three independent experiments.