Figure 4

Costimulation of CXCR4 and HRH1 induces synergistic calcium mobilization in MDA-MB-231 cells. (A) MDA-MB-231 cells were stimulated with 3 nM CXCL12, 100 nM histamine, or both agonists at the same concentrations, and intracellular calcium flux was measured (left panel). The area under the curve of each calcium response was calculated and normalized to that of CXCL12 (right panel). Data are expressed as mean ± s.e.m. (n = 3). ***P < 0.001. (B,C) Synergistic calcium flux is mediated by CXCR4 and HRH1. Intracellular calcium flux was measured by stimulating HRH1 knockout (sgHRH1) or control (sgControl) cells with increasing concentrations of CXCL12 in the absence or presence of 100 nM histamine (B). Intracellular calcium flux was measured by stimulating CXCR4 knockout (sgCXCR4) or control (sgControl) cells with increasing concentrations of histamine in the absence or presence of 3 nM CXCL12 (C). Data are expressed as mean ± s.e.m. (n = 3–6). (D) Inhibition of the calcium response by the CXCR4 antagonist AMD3100 and the HRH1 antagonist pyrilamine. MDA-MB-231 cells were pretreated with increasing concentrations of AMD3100 or pyrilamine, and costimulated with 3 nM CXCL12 and 100 nM histamine. Data are expressed as mean ± s.e.m. (n = 3).