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. Author manuscript; available in PMC: 2018 Jul 15.
Published in final edited form as: Cancer Res. 2017 May 4;77(14):3745–3757. doi: 10.1158/0008-5472.CAN-16-1768

Figure 3. The RAS-RAF-MEK-ERK induced expression of Orilnc1 is mediated by transcriptional factor AP1.

Figure 3

A. The expression of endogenous Orilnc1 in MCF10 (RAS and BRAF wide type) and MDA-MB-231 (KRAS mutated) cells by qRT-PCR. B. Luciferase reporter assay assessing the promoter activities of different Orilnc1 promoter fragments in MCF10A and MDA-MB-231 cells. TSS, transcription start site; the numbers ranges (i.e.−2938 to +53) are the location of each fragment relative to the TSS. The activity of SV40 promoter was set as 1 in each cell line. C. Luciferase reporter assay assessing the promoter activities of different Orilnc1 promoter fragments in MCF10A expressing KRASG12D, BRAFV600E or the control vector. Open bar, cells expressing control vector; red or orange bar, cells expressing mutant KRAS or BRAF, respectively. D. Luciferase reporter assay assessing the promoter activities of different Orilnc1 promoter fragments in MDA-MB-231 cells treated with MEK inhibitor AZD6244 or vehicle. Open bar, cells treated with vehicle; green bar, cells treated with AZD6244. E. An illustration of AP1 binding site at −132 (red indicates the consensus motif) and the sequences of the two AP1 mutations (blue indicates the mutant nucleotides). F. Luciferase reporter assay assessing the promoter activities of Orilnc1 core promoter construct and its AP1 mutant counterparts in MCF10A expressing KRASG12D, BRAFV600E or the control vector. Open bar, cells expressing control vector; red or orange bar, cells expressing mutant KRAS or BRAF, respectively. G. Luciferase reporter assay assessing the promoter activities of Orilnc1 core promoter construct and its AP1 mutant counterparts in MDA-MB-231 (left, KRASG13D, breast) and MDA-MB-435 (right, BRAFV600E, melanoma) cells treated with MEK inhibitor AZD6244 or vehicle. Open bar, cells treated with vehicle; green bar, cells treated with AZD6244. H. The locations of the primer pairs used in the ChIP assay. I. The promoter occupancy of c-Jun measured with four pairs of primers in MCF10A cells expressing control and mutant KRAS. J. The promoter occupancy measured with four pairs of primers in MDA-MB-231 cells expressing control or shRNA targeting KRAS. K. The promoter occupancy measured with four pairs of primers in MDA-MB-231 and MDA-MB-435 cells treated with vehicle or AZD6244.