Figure 3: IRAK1-dependent cytokine secretions are required for mammosphere formation.

(a) Mammosphere formation of MB436 shIRAK1 cells treated with and without Dox (0.5 μg ml⁻¹) for IRAK1 knockdown. CM, conditioned medium from mock-treated MB436 shIRAK1 cells. Quantifications (top) and representative images (bottom). Scale bars, 100 μm. (b) Cytokine Ab array profiling of cytokine secretions in the growth medium of MB436 cells expressing vector control or shIRAK1 in the presence of Dox (left). Quantifications of IL-6 and IL-8 and CXCL1 changes in shIRAK1 cells relative to the vector control cells (right). (c) NF-κB luciferase reporter activity in indicated MB436 cells in the presence of Dox. (d) ELISA assay showing the secretion of IL-6, IL-8 and CXCL1 in MB436 shIRAK1 cells with or without Dox or conditional medium from mock-treated cells. (e) Mammosphere formation assay in the presence of Dox or recombinant cytokines individually or in combination as indicated. (f) ELISA assay showing the secretion of IL-8 and CXCL1 in indicated TNBC cells expressing vector control or shIRAK1. (g) Mammosphere formation of MB436 shIRAK1 cells treated with Dox alone or together with CM from MDA231 shIRAK1 cells. (h) Effects of IRAK-inh (5 μM) treatment on cytokine secretions of indicated TNBC cells. Error bars represent s.e.m, n=3. *P<0.05, **P<0.01. P-values were calculated with two-tailed t test.