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. 2019 Mar 23;20(6):1471. doi: 10.3390/ijms20061471

Figure 3.

Figure 3

In hepatocytes, c-Jun transcription factors bound to the apoE promoter and upregulated its activity. (A) PMA stimulation of HepG2 cells induced phospho-c-Jun expression (lane 4 vs. 3) and increased total c-Jun protein levels (lane 6 vs. 5), which led to an increased apoE protein expression (lane 2 vs. 1), as demonstrated by Western blot. An increase in apoE protein level was noticed also when the cells were transfected to overexpress c-Jun (lane 10 vs. 9). A similar increase in apoE protein level (lane 14 vs. 13) was induced by overexpression of the dominant negative form of c-Jun (c-Jun DN). The expression of β-actin was used as an internal control (lanes 7, 8, 11, 12, 15, and 16); (B) From a series of apoE promoter deletion mutants used for HepG2 cell transfection, only the fragment −55apoE was not affected by c-Jun or c-Jun DN overexpression, and the smallest apoE fragment activated was -100apoE; (C) DNAP assays using nuclear extracts from HepG2 cells showed that c-Jun proteins bound to −300apoE and −100apoE (lanes 1 and 2, respectively). The positive control represents the c-Jun level in the nuclear extract from HepG2 cells (lane 4). In the negative control, a nonspecific biotinylated DNA fragment was used in the DNAP assays (lane 3); (D) Chromatin immunoprecipitation (ChIP) assay using chromatin from HepG2 hepatocytes (control cells or cells overexpressing c-Jun or c-Jun DN) and anti-c-Jun antibodies showed that c-Jun bound to the apoE promoter. Higher amounts of apoE promoter DNA were immunoprecipitated from HepG2 cells overexpressing c-Jun (lane 3) compared to control cells (lane 1) or hepatocytes transfected with c-Jun DN (lane 5). In the negative controls, nonspecific IgG was used to precipitate chromatin from control cells (lane 2), HepG2 cells transfected with c-Jun (lane 4) or c-Jun DN (lane 6), and no PCR products were identified. The input, representing 1% from each type of chromatin sample obtained from the control or c-Jun/c-Jun DN transfected hepatocytes, produced the expected band (lanes 7, 8, and 9, respectively).