Fig. 1

Depletion of JUNB blocks the cell cycle in G1/S and reduces entry into S phase. A JUNB mRNA levels upon transfection of siRNA against JUNB in U2OS cells. JUNB mRNA levels were analyzed by RT-qPCR following transfection of U2OS cells for 72 h with either a control siRNA (siControl) or JUNB-specific siRNAs (siJUNB-792, siJUNB-803, siJUNB-848). Data are shown as means with SEM from three independent experiments. Statistical analyses were performed by one-tailed paired t-test (**p<0.01, ****p<0.0001). B Jun family protein levels upon transfection of siRNAs against JUNB. JUNB, JUN, and JUND protein levels were assayed by immunoblotting in U2OS cells processed as in A. GAPDH was used as a loading control. C Immunofluorescence analysis of JUNB in U2OS cells upon siJUNB-792 and siJUNB-848 transfection. RNAi transfection conditions were the same as in A. The green color indicates positive JUNB staining, blue color indicates nuclear staining by DAPI (scale bars, 50 μm). D Distribution of U20S cells in the cell cycle and DNA synthesis upon transfection of siJUNB. RNAi transfection conditions were the same as in A. EdU incorporation and total DNA stained with propidium iodide were analyzed for cell cycle analysis by two-parametric flow cytometry under the conditions described in “Materials and methods.” Fluorescence intensity of cells stained with the Alexa Fluor 647 azide (AF-647) and PI is shown. Upper panel: distribution of the cells labelled with EdU in G0/G1, G2/M, and cells in S phase of a representative experiment is indicated. Lower panel: EdU unlabelled cells were included as negative controls for EdU staining. E Graph showing the mean of the percentage of cells in G0/G1, S, and G2/M phases of three independent experiments in D. Statistical analyses were performed using two-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, ****p < 0.0001). F Cell proliferation assay upon siJUNB- or JUNB expression plasmid transfection. Cell proliferation was assayed using the MTS assay. Left panel: RNAi conditions were the same as in A. Right panel: U2OS cells were transfected with either an empty vector (pCDNA3) or a JUNB expression plasmid (pCDNA3-JUNB) for 48 h. Statistical analyses were performed by one-tailed paired t-test (**p < 0.01, ****p < 0.0001). Error bars represent SEM of triplicate independent experiments