Fig. 4

Cytokine production by r0190 via MAP kinase and NF-κB pathways. a BMDMs were treated with r0190 (1 μg/ml) for the indicated time periods. At the end of the stimulation periods, the cells were lysed, and cell lysates were subjected to Western blot analysis to determine the intracellular levels of phosphorylated or unphosphorylated forms of ERK, p38, and JNK. b BMDMs were pretreated with the indicated concentrations of MAP kinase inhibitors including ERK (U0126), p38 (SB203580), or JNK (SP600125) for 1 h, followed by stimulation with r0190 (1 μg/ml) for an additional 24 h. Secreted TNF-α in culture supernatant was quantified by ELISA. Values are the mean ± SD of triplicate samples. ** P < 0.01 as compared with the r0190-treated group. c BMDMs were treated with r0190 (1 μg/ml) for the indicated time periods. At the end of the stimulation periods, the cells were lysed, and cell lysates were subjected to Western blot analysis to determine the intracellular levels of IκB α. d BMDMs were stimulated with r0190 (1 μg/ml) for 1 h and translocation of p65 was determined by immunofluorescence staining with anti-p65 antibody, followed by Alexa-488–conjugated secondary antibody and DAPI. Images were obtained by Olympus FV1000 confocal microscope. e BMDMs were pretreated with the indicated concentrations of parthenolide for 1 h and then stimulated with r0190 (1 μg/ml) for an additional 24 h. TNF-α expression was measured by ELISA. Values are the mean ± SD of triplicate samples. ** P < 0.01 as compared with the r0190-treated group