Figure 2

LL-37 synergistically enhances Heregulin-induced mitogen-activated protein kinase (MAPK) phosphorylation through the ERBB2 receptor. (a) Phosphorylation of ERBB2 (upper panel) and MAPK (lower panel) by treatment of ZR75-1 cells with LL-37 and Heregulin β3 (HRG) on their own and in combination. The diagram shows the mean phosphorylation level as evaluated by Western blot analysis, normalised against Ponceau staining and relative to untreated samples. On the left side is shown one representative of the triplicates used for evaluation. The lower rows show Western blots against total ERBB2 and MAPK, demonstrating an unaltered protein level, and Ponceau staining of the corresponding sections of the blot. (b) Inhibition of MAPK activation in ZR75-1 by the ERBB inhibitor PD153035 but not by pertussis toxin or metalloprotease inhibitor GM6001. The arrow points to the histogram for PD153035 treatment. (c) LL-25 inhibits the LL-37 dependent activation of MAPK. Experiments were performed in the MJ1105 control cell line using HRG at 2 ng/ml, and by varying the concentrations of both LL-37 and LL-25 as displayed. The diagram shows the levels of MAPK phosphorylation relative to the conditions of 2 μM LL-37. (d) Synergistic effect between HRG and endogenous production of transgenic hCAP18 in the MCF-7 derivative MJ1105. For all experiments, the Ponceau staining used for normalisation is shown. All conditions were run in triplicates (n = 3), and in addition repeated at least on one independent occasion.