Extended Data Fig. 1: Kinome-wide screen identified potential kinases regulating SRC-3 intrinsic transcriptional activity.
From: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer
a, HeLa cells expressing varying concentrations of the pBIND or pBIND-SRC-3 constructs were used to measure SRC-3 activity. n = 4 biologically independent samples. *P < 0.000001, one-way ANOVA with Sidak’s multiple comparison test. RLU are normalized by protein content. b, HeLa cells expressing pBIND or pBIND-SRC-3 were treated with siRNA targeting GFP or PRKCZ at the indicated dose followed by luciferase assay to measure SRC-3 activity. n = 3 biologically independent samples. *P < 0.000001, one-way ANOVA with Tukey’s multiple comparison test. c, Different control siRNAs targeting GFP or luciferase (Luc) were used to measure SRC-3 activity in HeLa cells expressing pBIND or pBIND-SRC-3. n = 3 biologically independent samples. The GFP control siRNAs in the red box were used in the library screen as controls. d, Effect on SRC-3 transcriptional activity by three sets of siRNA (sets A, B and C) targeting 636 human kinases in HeLa cells. Effect of GFP control siRNA was set at 1 (dotted line), the cut-off fold for increased activation was set at 2, and reduced activity at 0.75 following z-score analysis. n = 3 siRNAs per /kinase, n = 6 siGFP per plate; total n = 1,908 (siRNAs targeting kinases) n = 144 (siGFP control) independent samples. e, SRC-3 activity in HeLa cells across 24 kinome-library plates in the presence of control siRNA targeting GFP. n = 6 biologically independent replicates for each plate. f, A secondary screen was performed in HeLa cells to confirm the primary screen hits using a pooled siRNA targeting the kinases followed by SRC-3 transcriptional activity. n = 3 biologically independent samples. Boxes are as in Fig. 2e. g, Relative proliferation of MDA-MB-231 cells 4 days after treatment with siRNAs targeting GFP (control), SRC-3 or the indicated kinases. n = 3 biological replicates. *P < 0.0001. two-way ANOVA with Dunnett’s multiple comparisons test. Unless stated otherwise, data are mean ± s.d.
