Figure 1: RTK-dependent ERRα activity in breast cancer cells.

(a) Box plot depicting the % of nuclei positive ERRα by immunohistochemical staining in human breast tumour samples classified in various subtypes. Significance determined by one-way analysis of variance (ANOVA; P=0.0487). (b) Heatmap showing the intensities of ERRα binding of peaks determined by ChIP-seq in SKBr3 cells on 90 min stimulation with EGF (100 μM) or HRG (100 μM) or vehicle (veh), and clustered according to overlapping or growth factor-specific peaks by Homer. Below: western blot of ERRα and RPLP0 as loading control. (c) Average binding intensity of ERRα recruitment in the region ±500 bp around the centre of the peaks bound in all conditions (common sites) or induced on growth factor treatment (GF-induced). Statistical significance calculated by one-way ANOVA. (d) Enrichment of canonical pathway analysis classified according to –log(P value) generated by IPA for target genes displaying ERRα recruitment at ±20 kb from their transcriptional start site, in serum-starved conditions or on growth factor treatment. (e) Gene set enrichment of KEGG pathways enriched on depletion of ERRα in SKBr3 cells and specifically observed on treatment of cells with growth factors (false discovery rate<25%). siRNA-mediated depletion of ERRα is monitored by western blot analysis in all the conditions studied (upper left panel). (f) Standard ChIP analysis of genomic enrichment of ERRα recruitment to growth factor-reprogrammed sites in SKBr3 cells on treatment with vehicle or lapatinib. (g) Western blot depicting the expression of ERRα in SKBr3, BT-474 and NIC-5231 breast cancer cells on treatment with lapatinib. (h) Relative mRNA expression of ESRRA, a known target gene of ERRα in SKBr3 cells on lapatinib treatment. (i) Expression of ERRα and ubiquitin in SKBr3 cells treated with lapatinib or veh in the presence or not of the proteasome inhibitor MG-132. All statistical significance is calculated for results in lapatinib-treated cells relative to vehicle (veh) using three independent replicates. Error bars represent s.e.m., statistical significance is calculated using two-tailed unpaired t-test; *P<0.05; **P<0.01.