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. 2010 Aug 27;5(8):e12452. doi: 10.1371/journal.pone.0012452

Figure 3. Silencing CREB increases AP-2α expression and promoter activity.

Figure 3

(A) Western Blot analysis showing silencing of CREB in both A375SM and C8161-c9 cells stably transduced with CREB shRNA as compared to non-targeting control (NTshRNA). Approximately 80–90% CREB downregulation was observed in the CREB shRNA transduced cells as compared to NTshRNA as determined by densitometry. Following silencing of CREB, expression levels of AP-2α were upregulated in both A375SM and C8161-c9 cell lines as detected by Western Blot. α-Actin was used as a loading control for total lysate samples. Lamin is used as a loading control for nuclear fractions. (B) The AP-2α promoter region (nucleotides −1,500 to +50 from the transcription initiation site) was amplified from genomic DNA and cloned into the pGL3-basic firefly luciferase vector. Schematic representation of the promoter region shows the predicted CREB and E2F-1 binding sites. The luciferase activity driven by the AP-2α promoter was significantly increased by 90- and 20-fold after CREB silencing in both A375SM and C8161-c9 cell lines respectively, as compared to NT transduced cells. *, p<0.001. SB2 cells were used as a positive control. (C) Rescue of CREB expression in CREB-silenced cells resulted in down-regulation of AP-2α expression. α-Actin was used as a loading control. (D) The luciferase activity driven by the AP-2α promoter decreased significantly (*, p<0.001) after rescue of CREB expression in both A375SM and C8161-c9 cell lines. NTshRNA/EV, nontargeting control cells. CREBshRNA/EV, CREB-silenced control cells. CREBshRNA/RESCUED, CREB-silenced cells transduced with CREB nontargetable expression vector. (E) Chromatin Immunoprecipitation (ChIP) studies showed decreased binding of CREB to the promoter of AP-2α in both CREB-silenced cell lines (A375SM and C8161-c9) as compared to NT transduced cell lines. IgG antibodies were used as negative controls. Input DNA was used to determine equal amounts of chromatin. (F) Colony formation assay performed in soft agar layer depicts a significant decrease in clonogenicity after silencing CREB in both A375SM and C8161-c9. *, p<0.001.