Figure 4: Identification and characterization of GPCR antagonists.

a, Fold change of cellular cAMP concentration of MMTV-LINK-A and MDA-MB-231 cells treated with indicated GPCR antagonists (n.s., p>0.05, *p<0.05, **p<0.01, ***p<0.001). Results are mean ± s.e.m. of n=3 independent experiments. P values were determined by unpaired two-tailed Student’s t test. b, Immunoblotting detection using indicated antibodies of tissue lysates of MMTV-Tg(LINK-A) tumor lysates subjected to vehicle or Rauwolscine treatment. Three independent experiments were performed and yielded similar results. c, Measurement of viability index of 3-dimentional spheroid formation assay of NMuMG and MMTV-LINK-A cells (left), or MCF10 A or MDA-MB-231 cells (right) in the presence of a serial 2-fold dilution of Rauwolscine. Results are mean ± s.d. of n=3 independent experiments. d, Representative images at day 0 to 5 (left) and measurement of proliferation index (right) of 3-dimentional spheroid formation assay of NMuMG and MMTV-LINK-A cells (top), or MCF10 A or MDA-MB-231 cells (bottom) in the presence of vehicle or Rauwolscine 30 nm (top) or 40 nM (bottom) (***p<0.001). Results are mean ± s.d. of n=3 independent experiments. P values were determined by unpaired two-tailed Student’s t test. e and f, Representative images of day 8 (e) or measurement of proliferation index (f) of indicated normal or breast cancer cells in the presence of vehicle or Rauwolscine (Rau, 40 nM) (n.s., p>0.05, ***p<0.001). Results are mean ± s.d. of n=8 spheroids per experimental condition, P values were determined by one-way ANOVA. g, Measurement of proliferation index of indicated normal or breast cancer cells in the presence of scramble or LINK-A LNAs (5 nM) (n.s., p>0.05, ***p<0.001). Results are mean ± s.d. of n=8 spheroids per experimental condition, P values were determined by one-way ANOVA.