Figure 5.

Activation of EGFR signalling induced by PMPA was suppressed by treatment of antiplatelet agent or podoplanin-neutralizing antibody in vivo. (a,b) PC-10 cells (5 × 10⁶ cells) were subcutaneously injected in BALB/c-nu/nu mice. After 15 days, BALB/c-nu/nu mice bearing PC-10 tumour were dosed orally with 50 mg/kg erlotinib daily. Tumour volume was measured every 3 days (a). All data are shown as means ± SD. (N = 4) *P < 0.05 by Mann–Whitney U-test. After 3 weeks, the tumours were extracted and homogenized in SDS lysis buffer. The phosphorylated-EGFR signal in the tumours was analysed by Western blot (b). Multiple exposure images of full-length blots were presented in Supplementary Fig. S8. (c–e) PC-10 cells (5 × 10⁶ cells) were subcutaneously injected in BALB/c-nu/nu mice. After 15 days, clopidogrel dissolved in drinking water with 0.003% HCl (equal to orally dosed 25 mg/kg/daily) was given to BALB/c-nu/nu mice bearing PC-10 tumours. Tumour volume was measured every 3 days (c). All data are shown as means ± SD. (N = 5) *P < 0.05 by Mann–Whitney U-test. After 18 days, the tumours were extracted and homogenized in SDS lysis buffer. The phosphorylation of EGFR signal in the tumours was analysed by Western blot (d) and the signal intensity of EGFR phosphorylation was quantified by Image J software (NIH) (e). *P < 0.05 by Mann–Whitney U-test (f,g). PC-10 cells (5 × 10⁶ cells) were subcutaneously injected into NOD/SCID mice. After 18 days, these mice were treated with 100 μg/mouse control human IgG or chimeric MS-1 (ChMS-1) antibody every week by intravenous administration as previously described⁵. After 15 days, the tumours were extracted and homogenized in SDS lysis buffer. The phosphorylated-EGFR signal in the tumours was analysed by Western blot (f), and the signal intensity of EGFR phosphorylation was quantified by Image J software (NIH) (g). (N = 4) *P < 0.05 by Mann–Whitney U-test.