Fig. 2

Transcriptomic analysis of JUNB knockdown in the human U2OS osteosarcoma cell line reveals that JUNB is involved in many cancer cell properties. A Gene Set Enrichment Analysis (GSEA) of gene expression upon JUNB depletion. The JUNB-regulated genes were pre-rank-ordered according to their fold change (log2) between siControl, siJUNB-792, and siJUNB-848, and analyzed based on MSigDB hallmark gene set collection [34]. The table shows gene sets upregulated in siControl vs siJUNB-792 and siJUNB-848. Gene sets are significantly enriched at FDR < 0.05. The graphs show the enrichment score (ES) plotted on the y-axis. The plots represent the enrichment score (ES) of the genes included in the gene sets indicated in each title, where the relative expression of each individual gene appears as a black line. The shift of genes to the left (higher expression in siControl cells) or to the right (higher expression in siJUNB-792 and siJUNB-848 cells) indicates the degree of enrichment of the signature. B Heatmap hierarchical clustering of 246 consistent differentially expressed JUNB-regulated genes (FDR < 0.05). C RT-qPCR validation of a panel of JUNB target genes involved in cell cycle regulation and cell proliferation. The mRNA steady-state levels of the indicated genes were analyzed in U2OS cells 48h after transfection with either siControl, siJUNB-792, or siJUNB-848. The data shown are means with SEM from 3 independent experiments. Statistical analyses were performed using the one-tailed paired t-test (*p<0.05, **p<0.01, ***p<0.001). D Depletion of JUNB leads to a decrease in CCNE1 and pRB protein levels, and to an increase in that of TGFB2. U2OS cells were transfected with siControl, siJunB-792, or siJunB-848 72 h and protein extracts were analyzed by immunoblotting using the indicated antibodies. E Schematic representation of the G1/S transition regulation by E2F/pRb and JUNB