Figure 2
Figure 2. C/EBPα directly regulates nfkb1 gene expression. (A) Parental Ba/F3 cells and clones expressing MT-C/EBPα or MT-C/EBPαLZ were cultured with zinc chloride, and RNA was extracted after 16 hours. p50 transcripts were measured and normalized to mS16 levels with the use of quantitative reverse transcriptase PCR. Mean and SE from 3 independent experiments are shown. (B) Ba/F3 cells carrying the indicated C/EBPα-ER variant were cultured with (+) or without (-) estradiol (E2) for 7 hours, and normalized p50 mRNA levels were assessed by quantitative reverse transcriptase PCR. p50 transcripts levels were similarly assessed in the presence of cycloheximide. The average of RNA ratios with or without E2 from 3 independent experiments is shown. (C) Parental Ba/F3 cells and clones expressing MT-C/EBPα or MT-C/EBPαLZ were cultured with zinc chloride for 16 hours and subjected to ChIP analysis. After immunoprecipitation with C/EBPα antiserum, enrichment of DNA fragments corresponding to the proximal or the distal regions of the nfkb1 promoter were determined with quantitative real-time PCR relative to input. Averages from 3 repetitions are presented. (D) A representative gel of ChIP of C/EBPα of C/EBPαLZ on the nfkb1 promoter is shown. (E) U937 lysates were subjected to ChIP analysis with the use of antiserum against C/EBPα (α) C/EBPβ (β), NF-κB p50 (p50), or normal rabbit IgG (Ig). PCR was used to amplify a DNA fragment centered at 20 bp or an upstream fragment starting at −2284 bp of the NFKB1 gene. (F) U937 cells were transduced with vectors expressing shRNA against C/EBPα. Total cellular RNA was extracted from parental cells and from 2 clones expressing different shRNAs that resulted in effective knockdown of CEBPA. Expression of the indicated genes was measured with quantitative real-time PCR. *P ≤ .001 for the comparison between parental cells and either of the clones expressing an shRNA targeting CEBPA. (G) mRNA levels of NF-κB p65 were evaluated in Ba/F3 cells expressing ER fusions of C/EBPα or its indicated variants, 7 hours after induction with E2. The average normalized transcript level ratios with or without E2 from 3 independent repetitions are shown.

C/EBPα directly regulates nfkb1 gene expression. (A) Parental Ba/F3 cells and clones expressing MT-C/EBPα or MT-C/EBPαLZ were cultured with zinc chloride, and RNA was extracted after 16 hours. p50 transcripts were measured and normalized to mS16 levels with the use of quantitative reverse transcriptase PCR. Mean and SE from 3 independent experiments are shown. (B) Ba/F3 cells carrying the indicated C/EBPα-ER variant were cultured with (+) or without (-) estradiol (E2) for 7 hours, and normalized p50 mRNA levels were assessed by quantitative reverse transcriptase PCR. p50 transcripts levels were similarly assessed in the presence of cycloheximide. The average of RNA ratios with or without E2 from 3 independent experiments is shown. (C) Parental Ba/F3 cells and clones expressing MT-C/EBPα or MT-C/EBPαLZ were cultured with zinc chloride for 16 hours and subjected to ChIP analysis. After immunoprecipitation with C/EBPα antiserum, enrichment of DNA fragments corresponding to the proximal or the distal regions of the nfkb1 promoter were determined with quantitative real-time PCR relative to input. Averages from 3 repetitions are presented. (D) A representative gel of ChIP of C/EBPα of C/EBPαLZ on the nfkb1 promoter is shown. (E) U937 lysates were subjected to ChIP analysis with the use of antiserum against C/EBPα (α) C/EBPβ (β), NF-κB p50 (p50), or normal rabbit IgG (Ig). PCR was used to amplify a DNA fragment centered at 20 bp or an upstream fragment starting at −2284 bp of the NFKB1 gene. (F) U937 cells were transduced with vectors expressing shRNA against C/EBPα. Total cellular RNA was extracted from parental cells and from 2 clones expressing different shRNAs that resulted in effective knockdown of CEBPA. Expression of the indicated genes was measured with quantitative real-time PCR. *P ≤ .001 for the comparison between parental cells and either of the clones expressing an shRNA targeting CEBPA. (G) mRNA levels of NF-κB p65 were evaluated in Ba/F3 cells expressing ER fusions of C/EBPα or its indicated variants, 7 hours after induction with E2. The average normalized transcript level ratios with or without E2 from 3 independent repetitions are shown.

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