Figure 6: Role of IRAK1 signalling in acquired resistance to paclitaxel.

(a) Western blotting showing the p-IRAK1 of immnoprecipitated IRAK1 in SUM159 cells treated with 5 nM paclitaxel (PTX) for 24 and 48 h. (b) Qantitative reverse transcriptase–PCR analysis of indicated cytokine mRNAs in MDA231 cells treated with 5 nM PTX for 24–72 h. (c) Mammosphere formation assay of MDA231 cells expressing vector or shIRAK1 after PTX (10 nM) pretreatment for 96 h in monolayer. PTX-treated cells were then washed and the viable cells were seeded for mammosphere assay. (d) IHC analysis of p-IRAK1 (S376) in primary and recurrent breast tumour samples. Left, representative IHC images. Scale bar, 100 μm. Right, quantification of p-IRAK1 levels in 18 paired tissue samples (paired two-tailed t-test). (e) Western blotting shows the indicated proteins in MDA231 and SUM159 parental and paclitaxel resistant (PR) sublines. (f) Cell viability of parental and paclitaxel resistant sublines were treated with respective doses of PTX, IRAK-inh (5 μM) or both. PTX, 0.43 and 75 nM for MDA2131 and MDA231-PR, respectively; 0.73 nM and 1 μM for SUM159 and SUM159-PR, respectively. (g) Apoptosis as determined by FACS analysis of Sub-G1 cells in SUM159-PR (top) and MDA231-PR (bottom) cells treated with paclitaxel, together with or without indicated small molecule inhibitors of IRAK1, p38, JNK or IKKβ/NF-κB (PS1145 and Bay117082). (h) Western blotting showing the indicated molecular signalling events in SUM159-PR cells treated in g. (i) Schematic representation of the roles of IRAK1 in driving metastasis and chemoresistance in TNBC. Error bars represent s.e.m, n=3. *P<0.05, **P<0.01, ***P<0,001. P-values were calculated with two-tailed t-test.