Figure 1: BPTF is required for c-MYC transcriptional activity.
From: BPTF is required for c-MYC transcriptional activity and in vivo tumorigenesis
(a) Immunofluorescence staining of BPTF and c-MYC showing MYC-ER nuclear translocation upon 4-OHT treatment in control and BPTF-silenced HFF. Scale bar, 25 μm (b) Examples of expression of known c-MYC target genes, analysed by RT–qPCR, upon BPTF knockdown. Transcript levels were normalized against GAPDH and the vehicle-treated condition. Data are expressed as mean±s.e.m. (n≥3). P values were determined using an unpaired t-test. (c) Fold change in FPKM values of c-MYC-dependent gene sets enriched in 4-OHT-treated control cells. Values are displayed for both control and BPTF-silenced HFF MYC-ER cells. Gene sets tested and P values (Wilcoxon test): 1, Schuhmacher et al. ‘MYC targets up’ (P=3.156 × 10−15); 2, Acosta et al. ‘proliferation independent MYC targets up’ (P=2.034 × 10−08); 3, Schlosser et al. ‘MYC targets serum response up’ (P=6.62 × 10−09); 4, Schlosser et al. ‘MYC targets serum response Dn’ (P=6.11·10−09); 5, Schlosser et al. ‘MYC targets repressed by serum’ (P=2.747·10−14). (d) Fold change in mRNA levels for the set of 20 genes used for validation. Left panel: data calculated from FPKM values. Right panel: data calculated from ≥3 independent experiments assessed by RT–qPCR. P values were determined using a Wilcoxon test. (e) Examples of genes included in the validation. Transcript levels were normalized against GAPDH and the vehicle-treated condition. Data are expressed as the mean±s.e.m. P value was determined using an unpaired t-test. (f) Co-immunoprecipitation of Flag-BPTF with HA-tagged c-MYC from lysates of transiently transfected 293T cells; western blotting with the indicated antibodies. (g) Endogenous BPTF and c-MYC interact in MIA PaCa-2 cells as shown by in situ proximity ligation assay. The interaction events are visible as red dots (nuclear staining in blue) and are marked by arrowheads. The interaction of MYC with MAX is shown as a positive control. Number of dots per nuclei was quantified manually (n=70 nuclei). Scale bar, 5 μm. *P<0.05; **P<0.01; ***P<0.001.
