A proinflammatory chemokine, CCL3, sensitizes the heat- and capsaicin-gated ion channel TRPV1

Zhang et al. 10.1073/pnas.0406030102.

Supporting Information

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Supporting Figure 6
Supporting Figure 7
Supporting Figure 8

Supporting Figure 6

Fig. 6. The inhibitory effects of capsazepine (CZP). The calcium responses of CCR1:TRPV1/HEK293 cells upon stimulation by 0.15 m M capsaisin (CAP) at 50 second were recorded by ratiometric fluorimeter. CZP was added to the cells at 0 second in the dotted gray line and at time 170 second in the solid gray line. The solid dark line represents the cells without the influence of CZP.

Supporting Figure 7

Fig. 7. Analysis of the expression of CCR1 and TRPV1 in rat dorsal root ganglion (DRG) neurons by Immunohistochemical staining (IHC). Ten independent IHC images were analyzed for each marker. The cells with a median fluorescent intensity higher than 130 in the TIFF images were counted as positive cells. These data were plotted in Fig. 2B.

Fig. 8. (A) CCL5 and CXCL8 enhance calcium response of TRPV1. Rat DRG neurons were pretreated with medium, CCL5, CXCL8, or CXCL8/CCL3 at 100 ng/ml each for 20 min, then stimulated by 0.15 m M CAP. The images were recorded by microscope-based ratio imaging. The statistical significance was analyzed by the software PRISM 3.0 (GraphPad, San Diego). (Each bar represents an average of four experiments, » 8-20 responding cells; *, **, P < 0.05.) (B) CCL3 enhances the response of mouse DRG neurons to CAP. Mouse lumbar DRG neurons were isolated and cultured in vitro for 6 h. These cells were then pretreated with medium or CCL3 at 100 ng/ml for 20 min and stimulated with CCL3 or CAP at 0.15 m M, as indicated. Images were acquired by microscope-based ratio imaging. Statistical significance was analyzed by PRISM 3.0. (Each bar represents an average of four experiments, » 8-20 responding cells; ***, P < 0.05.)