Fig. 2

MNX1-AS1 is a direct target of c-Myc. a P493–6 cells carrying a Tet-Off c-Myc expression system treated with 1 μM doxycycline were subjected to qPCR and Western blot analysis, respectively. b and c MNX1-AS1 levels were measured by qPCR after infection of HepG2 cells with lentiviruses containing control shRNA (sh-ctrl) or two independent shRNAs targeting c-Myc (B), or after transfection of Huh7 cells with either empty Flag (control), Flag-c-Myc or Flag-P53 (negative control) plasmids (C). d Schematic of the putative c-Myc binding domains (BD) in the proximal promoter of MNX1-AS1 illustrating the design of mutant constructs as luciferase reporters. e and f ChIP assays were conducted in HepG2 cells using c-Myc antibodies or control IgG. Bound DNA fragments were subjected to semi-quantitative RT-PCR (E) or qPCR (F) using the specified primers. g Luciferase reporter assays in HepG2 cells after co-transfection of the plasmids from (D) for 24 h. The expression of c-Myc was confirmed by Western blotting. A is mean ± SD; n = 3 independent experiments, two-tailed paired Student’s t test, ***p < 0.001. B, C and G are mean ± SD; n = 3 independent experiments, one-way ANOVA with Tukey’s multiple comparison post-test, ns, not significant, ***p < 0.001. E is represent of three independent experiments. F is mean ± SD; n = 3 independent experiments, two-way ANOVA with Bonferroni’s multiple comparison post-test, ns, not significant, ***p < 0.001