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. 2015 Aug 17;35(18):3145–3162. doi: 10.1128/MCB.00332-15

FIG 5.

FIG 5

Ezrin regulates the DDX3 protein level. (A) Expression of ezrin protein was inhibited by siRNA in human OS (MG63.3), hepatocellular cancer (HepG2), and lung cancer (H1944) cell lines, which resulted in reduced DDX3 expression. Protein levels were detected by Western blotting of total cell lysates. (B) K12 is a mouse OS cell line, and K7M2 is a subclone of K12 with high ezrin expression and high metastatic potential. Ezrin expression in K7M2 was inhibited by stable antisense oligonucleotides (labeled as AS1.46 cells). The expression levels of ezrin, DDX3, and actin proteins were determined by Western blotting. (C) Ezrin and DDX3 mRNA levels were measured by real-time PCR in MG63.3 cells from panel A, and the results were normalized to 18S rRNA levels and expressed as fold change over the control group (*, P < 0.0001; Student's t test). The values are presented as the means and standard deviations of the results of triplicate experiments. (D) Low-ezrin-expressing K12 mouse OS cells were transfected with either an empty vector (EV) or a cDNA coding for wild-type ezrin containing a Myc-His tag. The protein levels of ezrin, DDX3, and actin were detected by Western blotting after 48 h. (E) MG63.3 cells were transfected with a cDNA coding for DDX3 containing a FLAG tag or a negative-control EV. Ezrin expression was inhibited by siRNA, and the level of expression of DDX3 from the vector was determined by Western blotting using anti-FLAG antibody. (F) K7M2 cells were treated with 2.0 μM NSC305787 or vehicle control for 4 days, and the expression levels of ezrin, DDX3, and actin were determined by Western blotting. (G) The expression levels of DDX3 in MG63.3 cells were determined at 0 h, 4 h, 8 h, 18 h, 24 h, 48 h, and 72 h after transfection of cells with siRNA for ezrin. (H) MG63.3 cells were transfected with either an siRNA targeting endogenous DDX3 (left) or a cDNA coding for DDX3 containing a FLAG tag (right). The expression level of ezrin was determined by Western blotting using antiezrin antibody. The asterisk indicates the Flag-tagged exogenous DDX3.