Figure 2. Signal transduction in receptor-negative (CXCR6-, CX3CR1-) tumor cells after stimulation with soluble chemokines (1 nM s-CXCL16 or s-CX3CL1).
A) As shown by Western blots after SDS-PAGE separation, receptor-negative but hence responsive cell lines like the glioma cell lines U343, A764, T98G and primary glioma cultures from different patients display a time- and dose-dependent phosphorylation of the kinase ERK (extracellular signal-regulated kinase p42/p44) after stimulation with s-chemokines for the indicated times (compare also Figure 2 - figure supplement 1). The responsiveness coincidences with the presence or absence of the corresponding tm-chemokines; compare Figure 1. Stimulation with epidermal growth factor (EGF; 2 nM) serves as a positive phosphorylation control. Re-blot against non-phosphorylated kinase ERK2 ensures equal loading of the lanes. (B) Immunostaining of s-chemokine-stimulated U343 or A764 glioma cells confirms the time-dependent phosphorylation of ERK (rabbit anti-pERK 1/2 with secondary antibody Alexa-Fluor 555 (red)-anti-rabbit IgG; blue nuclear counterstaining with DAPI). Bars indicate 20 µm. (C): The tm-chemokine negative LOX melanoma cells are not responsive to 1 nM s-chemokines, ERK-phosphorylation is only observed in positive control samples stimulated with epidermal or fibroblast growth factors (EGF or FGF-2, 2 nM). All shown data are representative results from 2-3 independent experiments, respectively, for biological replicates please refer to Figure 2—figure supplements 1 and 2.
Figure 2—figure supplement 1. Biological replicates of western blot experiments showing time- and dose-dependent activation of ERK1/2 upon stimulation with s-chemokines in responsive glioma cell lines (compare Figure 2A).
Figure 2—figure supplement 2. Biological replicates of western blot experiments showing stimulations of non-responsive LOX melanoma cells with s-chemokines (compare Figure 2C).
