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. 2005 Apr;166(4):985–998. doi: 10.1016/S0002-9440(10)62320-6

Figure 6.

Figure 6

Identification of TRPV1 in cultured ORS and HaCaT keratinocytes, the activation of which elevates [Ca2+]i and inhibits proliferation. A: TRPV1-ir (green fluorescence) in cultured ORS keratinocytes. Nuclei were counterstained by DAPI (blue fluorescence). Inset, negative control of TRPV1 staining. B: Western blot analysis of TRPV1 expression on cell lysates of ORS and HaCaT keratinocytes, and of HDFs. Arrow indicates predicted molecule size (95 kd). C: Q-PCR analysis of TRPV1 expression in cultured cells and in the human HFs (for the human HFs, data are identical to those shown in Figure 1H). Data of TRPV1 expression were normalized to the expression of GAPDH of the same sample and are expressed as relative mRNA amounts as a function of startup cDNA (relative amount of 1 was defined as the detection threshold). NHEK, normal human epidermal keratinocytes. Values are mean ± SEM of three independent determinations. D: Fura-2 AM-loaded cells were challenged with various concentrations of capsaicin or with 10 μmol/L capsaicin and 100 nmol/L iodo-RTX (I-RTX) together in Tyrode’s solution containing 1.8 mmol/L calcium, and [Ca2+]i was determined as described in Materials and Methods. Data are expressed as mean ± SEM obtained on 15 to 20 cells per group. *, Significant (P < 0.05) differences compared to the resting [Ca2+]i. **, Significant (P < 0.05) effect of I-RTX to prevent the action of 10 μmol/L capsaicin to increase [Ca2+]i. E: A BrdU proliferation assay. Cells were plated in 96-well multititer plates in quadruplicates and were treated with various concentrations of capsaicin (Caps) or with 30 μmol/L capsaicin and 100 nmol/L iodo-RTX (I-RTX) together for 72 hours. For determining the extracellular calcium dependence, experiments were performed in low-Ca2+ SFM (0.4 mmol/L) and high-Ca2+ SFM (2 mmol/L). Data are expressed as mean ± SEM as a percentage of the matched control values regarded as 100%. *, Significant (P < 0.05) differences compared to control (vehicle-treated) proliferation. **, Significant (P < 0.05) effect of I-RTX to prevent the action of 30 μmol/L capsaicin to inhibit proliferation. Original magnification, ×630 (A).