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. 2016 Feb;22(2):303–315. doi: 10.1261/rna.052571.115

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© 2016 Cho et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 5. — AXL regulates miR-34a via the JNK/ELK1 pathway. (A) The JNK pathway is involved in the negative feedback regulation. After transfection with the AXL 3′-UTR reporter for 24 h, CL1-0/AXL cells were treated with various inhibitors. The luciferase activity was measured 48 h later. An asterisk indicates a significant inhibitory effect on AXL 3′-UTR reporter activity. (B) AXL kinase activity is essential for ELK1 phosphorylation. CL1-0 cells were transfected with the full-length, kinase-dead mutant (K567R) or the intracellular domain deletion construct (del-ICD) of AXL. The protein levels of p-AXL, p-JNK, and p-ELK1 were determined by Western blot. (C) Expression of miR-34a is up-regulated by AXL. After CL1-0 cells were transfected with AXL or the vector-alone control plasmid, the primary transcript and mature forms of miR-34a were measured by real-time PCR. U6 small RNA was used as the internal control. (D) AXL-mediated down-regulation of the reporter activity can be rescued by blocking miR-34a. Luciferase reporter activity was determined in CL1-0 cells after cotransfection with pcDNA3-AXL plus miR-34a sponge (SP-A34) or pcDNA3-AXL plus control sponge (SP-C). The Renilla reporter activity was used to normalize the transfection efficiency. (E) Endogenous AXL expression is up-regulated by blocking miR-34a. CL1-0 cells were cotransfected as in D. The qRT-PCR assay was performed to determine the endogenous AXL mRNA level as described above. (F,G) The promoter activity and primary transcript expression of miR-34a are up-regulated by ELK1. CL1-5 cells were transfected with pcDNA3-ELK1, pcDNA3-ELK1 (S383D), or pcDNA3-ELK1 (S383A) plasmid along with the pGL3-AXL promoter reporter construct. The assay of luciferase reporter activity and primary transcript level of miR-34a were measured as described above. (H) Expression of AXL protein is up-regulated by ELK1. CL1-5 cells were transfected with pcDNA3-ELK1, pcDNA3-ELK1 (S383D), or pcDNA3-ELK1 (S383A) plasmid. The protein levels of AXL, ELK1 and p-ELK1 were determined by Western blot. (I) ELK1 binds to the miR-34a promoter in ChIP. CL1-5 cells were transfected with siAXL to knock down AXL expression. CL1-0 cells were transfected with AXL construct for AXL overexpression. ChIP assay was performed as described in the Materials and Methods section. At least three independent experiments were performed for each assay. Student's t-test was performed to demonstrate statistical significance. (*) P < 0.05.