SO-mediated c-Jun depletion does not occur at the transcrip-tional level among LPS-stimulated RAW264.7 cells. (A, Left panel; B, D-H) RAW264.7 cells (1 × 106 cells/ml) were pre-treated with SO (400 μM) for 30 minutes, followed by LPS (1 μg/ml) incubation for the indicated times, both in the absence or presence of actinomycin D (Actino D), U0, SP, SB, or NSC. The levels of c-Jun, p-c-Jun, p-ERK, ERK, p-p38, p38, p-JNK, JNK, and β-actin from cellular factions [nuclear fraction (NF) and cytosolic fraction (CF)] or whole-cell lysates (WL) were detected through immunoblotting analysis. (A, Middle panel) The immunofluorescence of c-Jun after pre-treatment with SO among LPS-stimulated RAW264.7 cells was observed through confocal microscopy. (A, Right panel) RAW264.7 cells were transfected with AP-1-Luc, as well as β-gal (as a transfection control) plasmid constructs, and were treated with SO in the presence or absence of PMA (100 nM) for 12 hours. Luciferase activity was determined through luminometry. (C) c-Jun mRNA expression was determined through semiquantitative reverse transcriptase (RT)-PCR analysis after pre-treatment, with or without Actino D/SO among LPS-stimulated RAW264.7 cells. #P < 0.05 or ##P < 0.01 compared to the normal group and *P < 0.05 or **P < 0.01 compared to control groups. Normal group: no-treatment group. Control group: LPS or PMA alone group. Blue notation: compared to LPS + SO or NSC group.
