Fig. 5

CCNE1 is a cell cycle-regulated JUNB target. A JUNB cDNA transfection in siJUNB-treated cells rescues CCNE1 protein expression. U2OS cells were transfected with siJUNB-792 plus or minus pCDNA3 and pCDNA3-JUNB. Forty-eight hours later, protein extracts were analyzed by immunoblot using anti-JUNB and CCNE1 antibodies. β-actin was used as a loading control. B, C CCNE1 cDNA transfection in siJUNB-transfected cells partially rescues cell cycle arrest in U2OS cells. B U2OS cells were transfected with siJUNB-792 plus or minus pCDNA3 or pCDNA3-CCNE1 and protein extracts were analyzed by immunoblot as in A. C Cell cycle of cells transfected in B was analyzed by propidium iodide (PI) staining of the cells. Flow cytometry profiles of a representative experiment are presented on the left panels. The right panel presents the mean of the percentage of cells in G0/G1, S, or G2/M phases of three independent experiments. Statistical analyses were performed using two-way ANOVA with Tukey’s multiple comparison test (**p < 0.01 and ****p < 0.0001. D JUNB overexpression leads to an increase in CCNE1 mRNA level during G1/S cell cycle progression. CCNE1 mRNA levels were analyzed by RT-qPCR after mitotic release as described in Fig. 4D. Results are the mean of two independent experiments. Statistical analyses were performed with two-way ANOVA and Tukey’s multiple comparisons test (*p<0.05, **p<0.01). E Schematic representation of CCNE1 gene. The positions of the identified JUNB binding sites, named A and B, each one containing one AP-1 site, and located close to the TSS are indicated. C is used as a control region for unspecific binding in ChIP-qPCR experiments. The arrow indicates the transcription start site. F ChIP-qPCR analysis of the enrichment of JUNB binding to the three sites analyzed in UTA6-control and UTA6-JUNB synchronized cells. Cells were synchronized in mitosis by double block with thymidine and nocodazole. Tetracycline was removed during the nocodazole block. Mitotic cells were collected, released into the cell cycle, and collected after 3 and 9 h for ChIP-qPCR analysis. ChIP was performed using a JUNB antibody or IgG antibody as control and qPCR was carried out on the regions A, B, and C. A representative experiment out of two is shown. Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ****p < 0.0001)