Ad-c-Jun + Ad-Fra-1). Taken together, these results indicated that c-Jun/Fra-1 heterodimer-mediated TRE activity was critical for the proliferation of NB cells. HDACIs suppressed MEK/ERK-mediated Fra-1 expression through transcriptionally downregulating Raf1 Fra-1 accumulation has been shown to critically depend on Raf-MEK1/2-ERK1/2 activity-dependent transcriptional regulation and posttranslational stabilization [22, 23]. suppression of both MKK-7/c-Jun and Raf-1/Fra-1 activities was involved in the tumor growth inhibitory effects induced by SAHA in SH-SY5Y xenograft mice. Collectively, these findings exhibited that c-Jun/Fra-1 dimer is critical for neuroblastoma cell growth and that HDACIs act as effective suppressors of the two oncogenes through transcriptionally downregulating MKK7 and Raf1. 0.05, Figure ?Physique1B).1B). These results suggested that BRM/BRG1 ATP Inhibitor-1 HDACI treatment substantially reduced cellular viability and proliferation in NB cells, consistent with previous reports [19, 20]. Open in a separate window Physique 1 HDACI-induced transcriptional suppression of c-Jun and Fra-1 occurs before the inhibitory effects on cell proliferation(A) SH-SY5Y cells were treated with HDACIs, including 0.5 M TSA, 1 M SAHA, 2 mM VPA or 1 M M344 for 2 hours, and WB was performed to test H3, H3 K27 and H4 K5 acetylation, p21, GAPDH was reprobed to verify equal loading. LP: Long time of exposure; SP: Short time of exposure. (B) SH-SY5Y, SK-N-SH, SK-N-BE(2), KP-N-NS cells were treated with HDACIs for 4, 8, 12, 18, and 24 hours, and MTT assays were performed to determine the proliferation rates at each time point. The data are offered as Itga10 the mean S.E. = 3; Two-way ANOVA analysis, * 0.05 (Control vs. HDACI at 12 hours), $ 0.05 (HDACI at 12 hours vs. HDACI at 18 hours), # 0.05 (HDACI at 18 BRM/BRG1 ATP Inhibitor-1 hoursvs. HDACI at 24 hours). (C and D) SH-SY5Y, SK-N-BE(2) and KP-N-NS cells were treated with the four HDACIs for 12 hours, and WB was performed to detect the expression and phosphorylation of c-Jun and Fra-1. GAPDH was reprobed to verify equivalent loading. RT-PCR was performed to detect the mRNA levels of c-Jun and Fra-1. GAPDH was amplified as an equal input. (E and F) SH-SY5Y cells were treated with 0.5 M TSA for 4, 8, 12 and 24 hours, and WB was performed to detect the expression and phosphorylation of c-Jun and Fra-1. RT-PCR was performed to detect the mRNA levels of c-Jun and Fra-1. c-Jun has been shown to be an oncogene or tumor suppressor, largely depending on the cell type or stress condition [21]. Thus, we detected whether c-Jun was altered following HDACI treatment in NB cells. Interestingly, SH-SY5Y, SK-N-BE(2) and KP-N-NS cells subjected to HDACIs for 12 hours exhibited dramatic decreases in c-Jun expression and phosphorylation (the activated form) levels. Paralleling the decreased c-Jun expression, HDACI treatment also induced decreases in Fra-1 expression and phosphorylation (activated form) levels (Physique ?(Physique1C).1C). RT-PCR assays exhibited that both c-Jun and Fra-1 mRNA levels were transcriptionally downregulated by HDACIs (Physique ?(Figure1D).1D). Notably, the four HDACIs exhibited different inhibitive effects on c-Jun or Fra-1, probably due to their variable sensitivity and specificity in blocking the activity of the HDAC member(s) critical for sustaining c-Jun or Fra-1 expression. To observe the time course of the inhibitory effects of HDACIs on c-Jun and Fra-1 expression, we used 500 nM TSA to treat cells for different time durations (4, 8, 12, and 24 hours). As shown in BRM/BRG1 ATP Inhibitor-1 Figure ?Physique1E,1E, TSA treatment led to obvious decreases in c-Jun and Fra-1 phosphorylation and protein levels starting at 8 hours and lasting up to 12 hours. At 24 hours post-treatment, when common apoptosis occurred with active caspase 3, c-Jun and Fra-1 remained suppressed by TSA treatment. c-Jun and Fra-1 mRNA expression levels were BRM/BRG1 ATP Inhibitor-1 suppressed before the decrease in their protein expression levels starting at 4 hours and lasting up to 8 hours (Physique ?(Figure1F).1F). In SK-N-SH cells, HDACI also consistently led to the downregulation of c-Jun and Fra-1 protein and mRNA levels (Supplementary Data S1; Physique ?Physique1).1). Taken together, these results indicated that HDACIs caused the transcriptional downregulation of both c-Jun and Fra-1, preceding their inhibitory effect on cell proliferation. c-Jun dimerization with Fra-1 predominantly occupied the TRE site responsible for TRE activity To clarify the major dimerization partner for c-Jun or Fra-1 in SH-SY5Y cells, cell lysates were immunoprecipitated with antibodies against AP-1 users that have been shown to be able to interact with c-Jun or Fra-1 to form homo-/heterodimers. These AP-1 users include c-Fos, FosB, Fra-1, Fra2, c-Jun, JunB, JunD and ATF2..