Figure 2

DDRs do not alter tumour growth rate of HT1080 cells in vivo or in vitro. (A,B) Tumour volumes of ±DDR 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 DDRs, respectively. Then, 1 × 10⁶ cells/mouse were inoculated s.c. into mice that were fed a regular or a DOX-supplemented diet. 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 tumour extracts. Tumour extracts from representative mice (n = 4 for HT-DDR1b ± DOX and n = 5 for HT-DDR2 ± DOX) in RIPA buffer 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 same blots were then stripped and reprobed with antibodies to total DDRs (upper panels), and then reprobed for β-actin, as a loading control (lower panels). Asterisk indicates a non-specific band. Full-length blots are presented in Supplementary Fig. 10. (E,F) In vitro cell proliferation of ±DDR-expressing HT1080 cells on plastic. HT-DDR1b (E) and HT-DDR2 (F) cells were incubated with or without DOX three days and then seeded on plastic in 24-well plates (2 × 10⁴ cells/well) in triplicate in the presence or absence of DOX, in complete media. At various time points, the cells were harvested and counted with a Coulter counter. Growth rate were calculated as described in Methods. Results represent the average of three independent experiments.