Figure 4: CXCL1 and CXCR1/2 signalling blockade inhibits ASC migration.

(a) Upper: quantitative RT–PCR analysis of CXCL1 messenger RNA (mRNA) expression in RM1 cells transduced with control shRNA (control-sh) or with shRNA silencing CXCL1 (CXCL1-sh), normalized to 18S RNA. Lower: CXCL1 protein concentration in medium conditioned by control-sh- or CXCL1-sh-transduced RM1 cells measured by ELISA. **P<0.01 (Student’s t-test). (b) Mouse ASCs were subjected to migration through 8-μm pores to a transwell chamber with conditioned medium (CM) from control-sh- or CXCL1-sh-RM1 cells. CM was supplemented with indicated concentrations of CXCL1, antibodies (abs) blocking CXCR1 or CXCR2, or CXCR1/CXCR2 inhibitor, reparixin, where indicated (+). Shown below are representative bright-field micrographs of crystal violet-stained transwell membranes corresponding to each treatment group. Scale bar, 100 μm. (c) ASCs isolated from SC and PP WAT of an obese and a non-obese prostate cancer patient were subjected to migration through 8-μm pores to a transwell chamber with CM from control-sh- or CXCL1-sh-RM1 cells. CM was supplemented with indicated concentrations of CXCL1, ab blocking CXCR1 or CXCR2, or reparixin where indicated (+). In b and c, plotted are relative migrated cell numbers normalized to migration of ASCs towards 10% FBS (set at 100%). For all panels, experiments were performed in technical triplicate. Graphs show mean±s.e.m. *P<0.05 versus control-sh CM (Student’s t-test).