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Fig 6

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NF-κB–C/EBPα activation is required for S1P-induced upregulation of CAMP expression. HaCaT KC were incubated with or without C2Cer, S1P (1 μM), Tg, tunicamycin (Tm), SPHK inhibitors (DMS [2.5 μM] and SKI [1 μM]), and/or an NF-κB inhibitor (2 μM BAY11-7082) as shown in Fig. 1. (A and D) Phosphorylated forms of NF-κB or C/EBPα (either Ser-21 [Ser21] or Thr-222/226 [T222/226]) in nuclear fractions were assessed by Western immunoblot analysis. Fold changes compared with vehicle are shown in parentheses in panel A. (B) NF-κB transactivation was assessed using a luciferase reporter assay. The growth inhibition at 7 h of incubation is shown. (C) CAMP mRNA levels were determined by qRT-PCR. Similar results were obtained when the experiment was repeated (duplicated) using different cell preparations. Data are means ± SD (n = 3; P < 0.01).

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