Figure 5
Figure 5. C/EBPα p42 and p30 oppositely modulate Trib2 expression via binding of the promoter. (A) Putative binding sites of C/EBP family of transcription factors in the Trib2 promoter. Several binding sequences are shared among members α, β, and δ of the C/EBPα family. E2F putative binding sites are also indicated (long dash line), and the region is targeted for ChIP-PCR analysis. (B) Nuclear extracts prepared from K562-C/EBPα-p42-ER or K562-C/EBPα-p30-ER cells induced with β-estradiol for 24 hours were assayed for C/EBPα binding by EMSA. Arrows indicate C/EBPα binding. FP, free probe; comp, cold competition. (C) 3T3 cells were cotransfected with empty control, E2F1 alone, or in combination with increasing amount of (left) C/EBPα-p42 or (right) C/EBPα-p30 and Trib2 luciferase reporter constructs (pGL3 control or 2.6-kb promoter), and luciferase activity was measured. Data presented are mean ± SD of duplicate cultures and representative of 2 independent experiments. *P < .05 and **P < .005. (D) K562-C/EBPα-p42-ER and K562-C/EBPα-p30-ER cells induced with β-estradiol or vehicle control for 24 hours were measured for Trib2 mRNA expression by real-time PCR analyses. Data are presented relative to vehicle control and representative of 2 independent experiments. Error bars denote ±SD of each sample measured in triplicate. **P < .005 and ***P < .001. (E) Nuclear extracts prepared from (upper) K562-C/EBPα-p42-ER and (lower) K562-C/EBPα-p30-ER cells transfected with E2F1 and induced with β-estradiol or vehicle control for 24 hours were assayed for E2F1 binding for WT sites B and C by EMSA. FP, free probe. (F) Supershift EMSA assay was performed in nuclear extracts prepared from K562-C/EBPα-p30-ER cells transfected with E2F1 and induced with β-estradiol or vehicle control for 24 hours using antibody specific for E2F1 or C/EBPα. Oligonucleotide for WT site B was used as a probe. FP, free probe. (G) K562 cells were cotransfected with empty control, E2F1 alone, or E2F1 in combination with C/EBPα-p42 or C/EBPα-p30 and Trib2 luciferase reporter constructs (pGL3 control or 2.6-kb promoter region), and luciferase activity was measured. Data presented are mean ± SD and representative of 2 independent experiments. ***P < .001 using an unpaired Student t test. (H) Normal murine GMP cells were chromatin immunoprecipitated using anti-C/EBPα or normal IgG control antibodies. PCR was performed using primers against the region indicated in A. Error bars denote ±SD of each sample measured in triplicate. ***P < .001. (I) Real-time PCR analysis of Trib2 mRNA levels in WT (+/+) GMP and −/p30 GMP cells. **P < .005.

C/EBPα p42 and p30 oppositely modulate Trib2 expression via binding of the promoter. (A) Putative binding sites of C/EBP family of transcription factors in the Trib2 promoter. Several binding sequences are shared among members α, β, and δ of the C/EBPα family. E2F putative binding sites are also indicated (long dash line), and the region is targeted for ChIP-PCR analysis. (B) Nuclear extracts prepared from K562-C/EBPα-p42-ER or K562-C/EBPα-p30-ER cells induced with β-estradiol for 24 hours were assayed for C/EBPα binding by EMSA. Arrows indicate C/EBPα binding. FP, free probe; comp, cold competition. (C) 3T3 cells were cotransfected with empty control, E2F1 alone, or in combination with increasing amount of (left) C/EBPα-p42 or (right) C/EBPα-p30 and Trib2 luciferase reporter constructs (pGL3 control or 2.6-kb promoter), and luciferase activity was measured. Data presented are mean ± SD of duplicate cultures and representative of 2 independent experiments. *P < .05 and **P < .005. (D) K562-C/EBPα-p42-ER and K562-C/EBPα-p30-ER cells induced with β-estradiol or vehicle control for 24 hours were measured for Trib2 mRNA expression by real-time PCR analyses. Data are presented relative to vehicle control and representative of 2 independent experiments. Error bars denote ±SD of each sample measured in triplicate. **P < .005 and ***P < .001. (E) Nuclear extracts prepared from (upper) K562-C/EBPα-p42-ER and (lower) K562-C/EBPα-p30-ER cells transfected with E2F1 and induced with β-estradiol or vehicle control for 24 hours were assayed for E2F1 binding for WT sites B and C by EMSA. FP, free probe. (F) Supershift EMSA assay was performed in nuclear extracts prepared from K562-C/EBPα-p30-ER cells transfected with E2F1 and induced with β-estradiol or vehicle control for 24 hours using antibody specific for E2F1 or C/EBPα. Oligonucleotide for WT site B was used as a probe. FP, free probe. (G) K562 cells were cotransfected with empty control, E2F1 alone, or E2F1 in combination with C/EBPα-p42 or C/EBPα-p30 and Trib2 luciferase reporter constructs (pGL3 control or 2.6-kb promoter region), and luciferase activity was measured. Data presented are mean ± SD and representative of 2 independent experiments. ***P < .001 using an unpaired Student t test. (H) Normal murine GMP cells were chromatin immunoprecipitated using anti-C/EBPα or normal IgG control antibodies. PCR was performed using primers against the region indicated in A. Error bars denote ±SD of each sample measured in triplicate. ***P < .001. (I) Real-time PCR analysis of Trib2 mRNA levels in WT (+/+) GMP and −/p30 GMP cells. **P < .005.

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