Figure 5. SF-3-026 and analogues inhibit PMA-induced expression of Fos family IEGs.

(A) HeLa cells treated with PMA (200 nM) for up to 120 min show transient phosphorylation of Elk-1 (pElk-1 S³⁸³) and subsequent expression of c-Fos, FosB, FosB2, Fra1 and c-Jun. (B) Expression of phosphorylated c-Fos (p-c-Fos Ser³⁷⁴, Thr²³² or Ser³²), total c-Fos, FosB, FosB2, Fra1 or c-Myc in HeLa cells pre-treated for 30 min with the indicated dose of SF-3-030 and then treated with PMA for 120 min. Phosphorylated ERK1/2 and MEK1/2 levels are shown in (A) and (B). (C) Cells were pre-treated as in (B) with the indicated dose of SF-3-030 or 10 μM U0126 and the levels of phosphorylated c-Fos (p-c-Fos Ser³⁷⁴ and Thr²³²), total c-Fos, FosB, FosB2, Fra1 or the negative feedback ERK1/2 phosphorylation sites (Ser²⁸⁹, Ser²⁹⁶ and Ser³⁰¹) on Raf-1 were evaluated. Phosphorylated ERK1/2 and total ERK2 are shown for controls. Controls for (B–D) include cells treated only with PMA (PMA lane) or PMA treatment in the presence of U0126 (U0126 lane). (D) Cells treated as in (C) were immunoblotted for phosphorylated RSK1 (pRSK1 Thr³⁵⁹/Ser³⁶³), total RSK1, c-Jun or Egr1. (E) HeLa cells were pre-treated with or without SF-3-030 as in (D) or 10 μM SB239063 and then stimulated in the absence or presence of anisomycin (Anis.) for 1 h followed by analysis of phosphorylated ATF2 (pATF2 Thr⁷¹), MK2 (pMK2 Thr²²²) or p38α/β MAPK (pp38α/β). Total ATF2, MK2 or p38α MAPK expression levels are shown for loading controls.