Characterization of transcription factor response kinetics in parallel

Abstract

Background: Transcription factors (TFs) are effectors of cell signaling pathways that regulate gene expression. TF networks are highly interconnected; one signal can lead to changes in many TF levels, and one TF level can be changed by many different signals. TF regulation is central to normal cell function, with altered TF function being implicated in many disease conditions. Thus, measuring TF levels in parallel, and over time, is crucial for understanding the impact of stimuli on regulatory networks and on diseases.

Results: Here, we report the parallel analysis of temporal TF level changes due to multiple stimuli in distinct cell types. We have analyzed short-term dynamic changes in the levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription 3 (Stat3), cAMP response element-binding protein (CREB), glucocorticoid receptor (GR), and TATA binding protein (TBP), in breast and liver cancer cells after tumor necrosis factor-alpha (TNF-α) and palmitic acid (PA) exposure. In response to both stimuli, NF-kB and CREB levels were increased, Stat3 decreased, and TBP was constant. GR levels were unchanged in response to TNF-α stimulation and increased in response to PA treatment.

Conclusions: Our results show significant overlap in signaling initiated by TNF-α and by PA, with the exception that the events leading to PA-mediated cytotoxicity likely also include induction of GR signaling. These results further illuminate the dynamics of TF responses to cytokine and fatty acid exposure, while concomitantly demonstrating the utility of parallel TF measurement approaches in the analysis of biological phenomena.

Keywords: HepG2 cells; Kinetics; MDA-MB-231 cells; Palmitic acid treatment; Parallel; Transcription factors.

Fig. 1

Quantification of TF levels in nuclear extracts of MDA-MB-231 cells after TNF-α stimulation. a Single TF detection by bead assay. The percentage of radiolabeled TF probe remaining on the beads (relative to signal that did not bind or was washed from the beads) was calculated. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). b Single TF detection by EMSA. The fractions of bound and unbound DNA probe were quantified, and the fraction of bound signal was calculated relative to the total signal from the lane. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). c Correlation between detection by bead assay and EMSA. The 1:1 line (blue) is included for reference. d Parallel TF detection by bead assay. Signals were normalized with respect to an internal standard and then the ratio with respect to control was calculated. (n = 3, * indicates p < 0.05). e Correlation of single and parallel bead assay measurements. The 1:1 line (blue) is included for reference

Fig. 2

Quantification of TF levels in nuclear extracts of HepG2 cells after TNF-α stimulation. a Single TF detection by bead assay. The percentage of radiolabeled TF probe remaining on the beads (relative to signal that did not bind or was washed from the beads) was calculated. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). b Single TF detection by EMSA. The fractions of bound and unbound DNA probe were quantified, and the fraction of bound signal was calculated relative to the total signal from the lane. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). c Correlation between detection by bead assay and EMSA. The 1:1 line (blue) is included for reference. d Parallel TF detection by bead assay. Signals were normalized with respect to an internal standard and then the ratio with respect to control was calculated. (n = 3, * indicates p < 0.05). e Correlation of single and parallel bead assay measurements. The 1:1 line (blue) is included for reference

Fig. 3

Quantification of TF levels in nuclear extracts of HepG2 cells after palmitic acid treatment. a Single TF detection by bead assay. The percentage of radiolabeled TF probe remaining on the beads (relative to signal that did not bind or was washed from the beads) was calculated. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). b Single TF detection by EMSA. The fractions of bound and unbound DNA probe were quantified, and the fraction of bound signal was calculated relative to the total signal from the lane. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). c Correlation between detection by bead assay and EMSA. The 1:1 line (blue) is included for reference. d Parallel TF detection by bead assay. Signals were normalized with respect to an internal standard and then the ratio with respect to control was calculated. (n = 3, * indicates p < 0.05). e Correlation of single and parallel bead assay measurements. The 1:1 line (blue) is included for reference