XPO5 KD during cell cycle entry causes defective proliferation associated with delayed G1/S transition. (A) Common functional annotation obtained from microarray data and miRNA target genes predicted using TargetScan (darker shade) or PITA (lighter shade) software. Bars, P-values of Gene Ontology (GO) terms in the cDNA array; the line shows the number of miRNAs with the indicated GO terms. For details, see the Supplementary Materials and Methods. (B) Heat map of miR-17, 20a, 93, 106a, and 19a target genes predicted using TargetScan, which were up-regulated by XPO5 KD. The genes known to be involved in cell cycle and cell proliferation are shown in red. (C) Reporter assay with luciferase reporter bearing 3′ UTRs of Pten and Cyld was quantified 48 h after cotransfection with an XPO5 expression vector (*P < 0.01). (D) Pten mRNA and protein levels following transfection of a negative control or Xpo5 siRNA, and a negative control or inhibitor for miR-19a (60 nM, 48 h). The RNA level was determined by real-time PCR, and the protein level was determined by Western blotting. β-Actin was used as internal control for both analyses. (E) Heat map of differentially expressed cell cycle genes in XPO5 KD and control MEF cells. GO category of “GO:0007049 cell cycle.” (F) Cell cycle analysis of XPO5 KD MEF cells. For details, see the Supplementary Materials and Methods. (G) Bromodeoxyuridine (BrdU) incorporation assay of XPO5 KD MEF cells (n = 3, mean ± SD, *P < 0.01).
