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. 2020 May 6;10:7662. doi: 10.1038/s41598-020-64713-x

Figure 6.

Figure 6

Interaction of miR-145 with ATF6 and ATF6 feedback with AFAP1-AS1 in breast cancer cells. (A) Dual fluorescent reporter assay. MDA-MB-231 cells were transiently transfected with pmirGLO/ATF6 3′-UTR, miR-145 mimics, negative control, ASO-miR-145, or ASO-NC for 48 h and subjected to Luciferase assay. The right panel shows the results of the Luciferase assay with mutated pmirGLO/ATF6 3′-UTR transfection. (B) qRT-PCR. MDA-MB-231 cells were transiently transfected with miR-145 mimics, negative control, ASO-miR-145, or ASO-NC for 48 h and subjected to qRT-PCR analysis of ATF6 expression. (C) Western blot. MDA-MB-231 cells were transiently transfected with miR-145 mimics, negative control, ASO-miR-145, or ASO-NC for 48 h and subjected to Western blot analysis of ATF6 protein expression. (D) qRT-PCR. MDA-MB-231 cells were transiently transfected with ATF6 siRNA or negative control for 48 h and subjected to qRT-PCR analysis of ATF6 expression. (E) Western blot. MDA-MB-231 cells were transiently transfected with ATF6 siRNA or negative control for 48 h and subjected to Western blot analysis of ATF6 protein expression. (F) qRT-PCR. MDA-MB-231 cells were transiently transfected with ATF6 siRNA or negative control for 48 h and subjected to qRT-PCR analysis of AFAP1-AS1 expression. (G) Luciferase assay. MDA-MB-231 cells were transiently transfected with pGL3-AFAP1-AS1, pRL-TK, pSilencer-NC, and/or pshR-ATF6 for 48 h and subjected to Luciferase assay to measure AFAP1-AS1 luciferase activity. ***p < 0.05 and ***p < 0.001.