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. 2018 Jul 27;16(7):e2005869. doi: 10.1371/journal.pbio.2005869

Fig 5. CCL20 enhances the taxane resistance of TNBC cells through PKCζ- and p38 MAPK–mediated NF-κB activation.

Fig 5

(A) After FBS starvation for 24 hours, western blot was conducted with SUM159 and MDA-MB-231 cells. The immunoblotting bands were quantified, normalized with β-actin, and fold-changed to the first panel (similarly hereinafter). (B) Vector and CCL20v1 OE MDA-MB-231 cells were cultured in the presence or absence of specific inhibitor of p65 NF-κB activation (CAPE, 5 μM) under FBS starvation conditions for 12 hours, and western blot was performed. (C) FBS-starved CCL20v1 OE MDA-MB-231 cells were treated with PKCζ inhibitor (Go 6983, 5 μM) or p38 MAPK inhibitor (SB202190, 20 μM) for 12 hours and immunoblotted. (D) Go 6983 (5 μM) or vehicle (DMSO) was used to treat vector and CCL20v1 OE MDA-MB-231 for 12 hours under FBS starvation, and immunoblotting was performed. (E) FBS-starved vector and CCL20v1 OE SUM159 were treated (Go 6983, 5 μM; SB202190, 20 μM) for 12 hours and immunoblotted. (F) Illustration of the CCL20-PKCζ/p38-NF-κB axis. (G) Images of immunofluorescence performed with MDA-MB-231 were captured with confocal microscope. Blue, DAPI for nucleus; red, phosphorylated p65 NF-κB. Scale bars: 40 μm. (H-I) Single cells dissociated from mammospheres of MDA-MB-231 (H) or SUM159 (I) were treated with DOC (MDA-MB-231, 14.10 nM; SUM159, 5 nM) in the absence or presence of CAPE (5 μM) for 24 hours, and the chemoresistance was analyzed. (J-K) Single cells dissociated from mammospheres of MDA-MB-231 (J) or SUM159 (K) were treated with DOC (MDA-MB-231: 14.10 nM; SUM159: 5 nM) in the absence or presence of rhCCL20 (10 ng/ml), anti-CCL20 (200 ng/ml), or CAPE (5 μM) for 24 hours, and the chemoresistance was analyzed. The subsequent bars were compared to the first bar (control) to conduct statistics. (L) p65 knockdown verified by qRT-PCR in CCL20v1 OE MDA-MB-231 cells. (M) Single cells dissociated from MDA-MB-231 tumorspheres were treated with DOC (14.10 nM) for 24 hours and subjected to chemoresistance analysis. (N-O) Tumorsphere formation assay performed with MDA-MB-231 (N) and statistics (O). Scale bars: 400 μm. Data are representative of at least 3 independent experiments and shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired t test of triplicates. anti-CCL20, CCL20 neutralization antibody; CAPE, caffeic acid phenethyl ester; CCL20, C-C motif chemokine ligand 20; DOC, docetaxel; FBS, fetal bovine serum; FOV, field of view; Go, Gene Ontology; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; OE, overexpressing; PKCζ, protein kinase Cζ; qRT-PCR, quantitative real-time PCR; rhCCL20, recombinant human CCL20; TBP, TATA-box binding protein; TNBC, triple-negative breast cancer.