A. Quiescent T98G cell extracts were immunoblotted with anti-cJun, anti-JunB and anti-JunD antibodies. Images were taken from different parts of the same autoradiography film, and therefore have identical exposures. Sizes indicated are in kDa. As expected, JunD has two protein products which run as a 40–50 kDa doublet B. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with anti-c-Jun antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for the normal rabbit IgG immunoprecipitates. The numbers in parentheses refer to the 5′-most position of the putative AP-1 binding site (see Figure 1) relative to the transcription start site. When more than one position is listed, this refers to all possible sites that would be detected within the resolution limits of the ChIP PCR amplicon (approximately ±250 nucleotides). For those genes with multiple putative AP-1 sites, only one representative site (if all were negative for a given gene) or that site which indicated binding are shown for clarity. For a complete list of all tested sites, see Table S1. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. Asterisks indicate greater than 3-fold binding as compared to MYOG in both untreated and PDGF treated samples C. Recruitment of c-Jun upon stimulation with PDGF or direct inhibition of GSK-3 with SB-216763. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated, or treated with SB-216763 for 1 hour or with DMSO vehicle control. Only those genes that initially showed c-Jun binding (panel A) are shown. Data are presented as fold change over untreated (for PDGF) or fold change over DMSO vehicle control (for SB-216763), and are averaged from 4 separate experiments ±S.E. No significant change was observed in the normal rabbit IgG or MYOG samples (not shown).

T98G cells were rendered quiescent by serum starvation for 72 hours. Cells were then left untreated (NT), or treated with SB-216763 or vehicle control (DMSO) for the times indicated. Cell extracts were immunoblotted in parallel with anti-phospho threonine 239-c-Jun, pan-c-Jun and β-actin antibodies. Data shown are representative of three separate experiments. The left and right panels of the immunoblot in (B) were taken from the same autoradiography film, and therefore are identical exposure times.

Cells were rendered quiescent and transfected with the indicated combination of siRNAs. A total of 2.5 nM of each siRNA (5 nM total) was used as compared to 5 nM of negative control siRNA. Knockdown efficiencies of c-Jun, p65 and CREB were greater than 90% under these conditions. Data are presented as % of the SB-216763 induction as compared to the induction in the presence of the negative control. All data points are averages of a minimum of n = 3 (except for the p65/CREB combination, which is n = 2), ± S.E.

Predicted AP-1 binding sites are indicated by vertical black lines in the 5-kb aligned sequences of human and mouse genes [10]. Numeric positions are relative to the transcription start site. Positions of the ChIP PCR amplicons are indicated. Refer to Table S1 for precise positions of predicted sites and of primers. Alignments are indicated as dark blue, match; orange, mismatch; white, aligned to gap.

Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with an anti-JunD antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for normal rabbit IgG. The numbers in parentheses refer to the 5′-most position of the putative AP-1 binding site (see Figure 1) relative to the transcription start site. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. * indicates greater than 3-fold binding compared to MYOG in both untreated and PDGF treated samples; # indicates greater than 3-fold binding compared to MYOG in the PDGF treated sample only.

A. Knockdown of c-Jun by siRNA. Cells were transfected with either the c-Jun siRNA or a nonspecific construct (NegCtl), or with no construct added (Mock) and then analyzed by c-Jun and β-actin immunoblots. B. Effect of c-Jun knockdown on PDGF induction. T98G cells were transfected with a c-Jun or nonspecific negative control siRNA for 24 hours, and then serum starved for 48 hours. Cells were then stimulated with PDGF for 30 minutes. Expression of the indicated genes was determined by realtime RT-PCR. Data are presented as fold-change as compared to untreated. Data are means for 4 separate experiments ± S.E. * p<0.01. C. Effect of c-Jun knockdown on SB-216763 induction. Transfection and starvation were performed as above, and then cells were treated for 1 hour with SB-216763. Data are presented as fold change as compared to vehicle control (DMSO). Data are means for 4 separate experiments ± S.E., **p<0.05.

The AP-1 data has been combined with that of CREB [10] and NFκB [12]. Both grey and black arrows indicate ChIP binding by the given factor to the gene's upstream sequence. Black arrows indicate that siRNA against the factor blocked induction of the gene in response to SB-216763 treatment greater than two-fold and statistically significant (p<0.05). The blunt-ended edge between AP-1 and NR4A1 indicates a greater than two-fold inhibition (p<0.05) of induction in the presence of AP-1. RND3 and CCL8 have been excluded from the illustration, as no ChIP binding nor transcription factor knockdown data indicated any functional connections with AP-1, NFκB or CREB.