Extended Data Fig. 2: PFKFB4, the top hit from the kinase screen, enhances the transcriptional activity of SRC-3.
From: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer
a, Effect of PFKFB4 knockdown on SRC-3 transcriptional activity in various breast cancer cell lines. n = 3 or n = 4 (siGFP plus pBIND-SRC-3) biologically independent cells. *P < 0.000009, two-way ANOVA with Tukey’s multiple comparison test. b, SRC-3 transcriptional activity in MDA-MB-231 cells expressing shRNAs targeting PFKFB4 (#09 and #20) or non-targeting control, co-transfected with pBIND or pBIND-SRC-3. n = 5, biological replicates. *P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. c, Protein expression of PFKFB4 or actin in MDA-MB-231 cells expressing shRNAs targeting PFKFB4. d, Expression of PFKFB4 and SRC-3 mRNA in indicated breast tumour cells after treatment with siRNAs targeting GFP control or PFKFB4. n = 4 or n = 3 biological replicates. See Source Data for exact P values. e, Expression of PFKFB4 and SRC-3 mRNA in MDA-MB-231 cells transduced with adenoviruses expressing GFP or PFKFB4. n = 6 biologically independent cells. ***P < 0.000001, two-way ANOVA with Tukey’s multiple comparison test. f, Left, MDA-MB-231 cells were stained with specific antibodies against SRC-3 (rabbit) and PFKFB4 (mouse) before proximity ligation assay (PLA). The PLA signals between endogenous SRC-3 and PFKFB4 are shown in the red channel, DAPI was used to stain the nuclei (blue) and the merge images show the overlay of the red and blue channels. Two representative fields from biologically independent experiments were shown from n = 5. Right, control cells were stained with either one of the antibodies against SRC-3, PFKFB4 or secondary antibody-conjugated with probes. Scale bars, 20 μm (left), 40 μm (right). Data are representative of three biologically independent experiments with similar results, and are shown as mean ± s.d.
