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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Mol Cancer Res. 2014 Sep 25;13(2):250–262. doi: 10.1158/1541-7786.MCR-14-0385

Figure 2. TRIML2 is a p53 target gene.

Figure 2

(A) The human colorectal cancer cell line Hct116 containing wild-type p53 (WT) or the somatic cell knock-out for p53 (p53−/−) were treated with etoposide (100μM) and monitored after twenty-four hours. Whole cell lysates and extracted RNA were subjected to western blot analysis (top panel) and qRT-PCR (bottom panel), respectively. Densitometry quantification of TRIML2 levels, normalized to GAPDH, is depicted. Expression levels of mRNA were normalized to cyclophilin A. The data depicted are the averaged results from three independent experiments; error bars mark standard error. The asterisk denotes a p-value <0.05.

(B) Left panel: Chromatin immunoprecipitation of Hct116 cells treated with DMSO or 5-FU (5μM) for 72 hr. Immunoprecipitated DNA was eluted and analyzed by q-PCR and regular PCR using primers flanking p53 response elements (RE) on the known p53 target genes MDM2 and CDKN1A (p21) as positive controls. Upper right panel: diagram of the TRIML2 gene, along with three potential consensus p53 response elements (RE). The start site of transcription (TSS) is denoted +1, and the location of p53 REs is shown relative to this. Ex= exon. Bottom right panel: Immunoprecipitated DNA was eluted and analyzed by PCR using primers flanking the p53 REs (Table S2). In each case the percent of DNA bound, normalized to input, is depicted. The data depicted are the averaged results from three independent experiments; error bars mark standard error. The asterisk denotes a p-value <0.05.

(C) Left panel: western blot analysis for p53, p21, TRIML2 and loading control (GAPDH) in H1299 cells containing doxycycline-inducible P72 and R72 forms of p53 following doxycycline treatment (0.75μg/ml) for 24 hr. Right panels: Chromatin immunoprecipitation analyses in H1299 cells expressing doxycycline-inducible P72 or R72 forms of p53 using primers that span the p53 binding site in CDKN1A (p21), BTG2, or RE1 and RE2 of TRIML2 following doxycycline treatment (0.75μg/ml) for 24 hr. The data depicted are the averaged results from three independent experiments; error bars mark standard error. The asterisk denotes a p-value <0.05.