Figure 2: Nuclear AURKA activates MYC transcription and induces a shift in MYC promoter usage. | Nature Communications

Figure 2: Nuclear AURKA activates MYC transcription and induces a shift in MYC promoter usage.

From: Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype

Figure 2

(a) DBD/AURKA-DBD, DBD-reporter and pRL-TK were co-transfected into 293T for 24 h. Immunoblotting (IB; left panel) and dual-luciferase reporter (right panel) analysis were performed. (b) DBD/AURKA-DBD, DBD-reporter and pRL-TK were co-transfected into 293T cells. After 12 h, cells were treated with VX-680, MLN8237 or Aurora-A inhibitor I for 12 h. IB (left panel) and dual-luciferase reporter assay (right panel) were performed. (c) Diagram showing potential TADs in AURKA. (d) DBD/AURKA-DBD, DBD-reporter and pRL-TK were co-transfected into 293T cells for 24 h. IB (left panel) and dual-luciferase reporter assay (right panel) were performed. (e) STRING analysis showing connections among AURKA-regulated genes. (f) ER/AER, MYC promoter/basic reporter and pRL-TK were co-transfected into MDA-MB-231 cells. After 6 h, AURKA nuclear translocation was induced by treating cells with 200 nM OHT for 18 h. Dual-luciferase reporter assay was performed. (g) Treatment was the same as in f. Expression of indicated genes was determined via real-time PCR and IB analysis. (h) Treatment was similar to d, except for using MYC promoter reporter instead of DBD reporter. IB (left panel) and a dual-luciferase reporter (right panel) analysis were performed. (i) AURKA or empty vector was co-transfected into MDA-MB-231 cells with truncated MYC promoter (MYC) or basic reporter (Vec) along with pRL-TK. After 24 h, dual-luciferase reporter assays were performed. (j) MDA-MB-231 cells were subjected for ChIP analysis of MYC promoter occupancy. Results were normalized with the input. (k) VX-680-treated MDA-MB-231 cells (48 h) were subjected for ChIP analysis of MYC promoter occupancy. (l) Cells were cultured in suspended or adherent condition with same medium for 7 days. S1 nuclease protection assay (SNPA) was performed to examine MYC P1 and P2 transcripts. Control gene, β-2-microglobulin (B2M), mRNA in parallel identical samples were also determined. (m) BCSC and non-BCSC populations were isolated according to CD24 expression. SNPA was performed. (n) ER/AER plasmids were transfected into 293T cells. After 6 h, AURKA nuclear translocation was induced by treating cells with 200 nM OHT for 18 h. SNPA was performed. (o) DBD/AURKA-DBD were transfected into 293T cells for 24 h. SNPA was performed. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; *P<0.05, **P<0.01, ***P<0.001).

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