Figure 4.

Kruppel‐like factor 4 mediates DDX3X‐dependent regulation of cell cycle. (A) Western blot showing the protein levels of DDX3X, KLF4 and β‐tubulin in MCF7 cells transfected with either scrambled siRNA or one of two different siRNAs targeting DDX3X (#6 or #8). (B) mRNA levels of DDX3X, CCNE1, KLF4, CCNA2 and CDK2 in MCF7 cells transfected with either scrambled siRNA or KLF4 targeting siRNA, measured by qPCR. Cells were harvested 72 h after transfection. Results represent the average of three replicates. (C) mRNA levels of DDX3X,CCNE1, KLF4, CCNA2 and CDK2 in MCF7 cells transfected with increasing amounts of a hKLF4 expressing plasmid (as described in Materials and methods), measured by RT‐qPCR. Cells were harvested 48 h after transfection. Results represent the average of three replicates. (D) Cell cycle progression flow cytometric analysis in MCF7 cells transfected with increasing amount of a hKLF4 expressing plasmid (as in C). Shown is the difference in percentages for each phase (G1–S–G2M) plotted as the difference in percentages for each condition (0.5 μg hKLF4, 1 μg hKLF4, 2 μg hKLF4) to those of the 0 μg hKLF4 control values. Cells were harvested 48 h after transfection. Results represent the average of two replicates. P‐values represent statistical significance calculated with unpaired t test comparing to scrambled siRNA or 0 μg conditions (P‐values: ns. >0.05; **≤0.01; ***≤0.001). (E) Double knockdown of DDX3X and KLF4 in MCF7 cells: levels of DDX3X,KLF4, CDK2 and CCNA2 transcripts in MCF7 cells transfected with either scrambled siRNA or #6 DDX3X siRNA or #8 DDX3X siRNA alone or combined with KLF4 targeting siRNA, measured by RT‐qPCR. Cells were harvested 72 h after transfection. Results represent the average of two replicates. P‐values represent statistical significance calculated with unpaired t test comparing the double transfection conditions to the single transfection respective condition (P‐values: ns. >0.05; *≤0.05; **≤0.01; ***≤0.001; ****≤0.0001).