Figure 7: Enhanced CELF1 expression in human breast cancer correlates with increased metastasis.

(a) Heat map depicting log₂-fold difference in mRNA expression of indicated genes between tumour-normal matched pairs for 111 TCGA patients. Topmost coloured bar indicates PAM50 assignments. Columns are ordered by PAM50 subtype. Top: heat map shows exemplary set of genes correlated with subtype, and bottom: heat map depicts genes translationally regulated by CELF1 during EMT and CELF1. For each heat map, rows are ordered by decreasing variance from top to bottom. Columns are ordered by PAM50 assignment and within PAM50 assignment by increasing mean log₂-fold change of genes from left to right. (b) qRT-PCR-based determination of change in steady-state and polyribosomal-bound CELF1 mRNA using total (T) and polyribosomal (P) mRNA obtained from untreated and TGF-β-treated MCF10A cells. Data are represented as mean±s.e.m. All panels are representative of a minimum of three experimental replicates. (c) CELF1 is hyperphosphorylated in mesenchymal MCF10A cells. Immunoprecipitates obtained using anti-CELF1 antibody or mouse IgG from MG-132 treated (8 h) MCF10A±TGF-β (72 h) cells were probed with indicated antibodies. (d) Inhibition of proteasome-mediated degradation results in increased CELF1 protein levels in epithelial MCF10A cells. Cells were incubated with MG-132 for the indicated times and immunoblotted for the indicated proteins. HSP90 serves as a loading control. (e) Representative image of CELF1 protein expression in mammary carcinoma and tumour adjacent normal mammary gland tissue, as analysed by IHC staining. Image was obtained with × 20 objective. Brown staining indicates antigen, while blue staining represents counterstain. Scale bar, 100 μm. (f) Relative CELF1 protein expression as assessed by the per cent score in the 29 matched breast tumour and adjacent normal tissue (P<0.0001; Wilcoxon’s signed-rank test). (g) Intracellular CELF1 staining was scored in breast tumour samples from patients with tumour size <20 mm and no lymph node involvement (T1,N0) (n=10), and with lymph node involvement along with tumour size between 20–50 mm (T2,N>0) (n=53) and >50 mm (T3,N>0) (n=13), and the P-value of the trend was determined using the Jonckheere–Terpstra test. Box plots represent the 25th to 75th quartiles with the bold horizontal line representing the median value. (h) Trend between CELF1 expression and tumour grade I (n=12), grade II–III (n=97) and grade III (n=7). The P-value of the trend was determined using the Jonckheere–Terpstra test. Box plots represent the 25th to 75th quartiles with the bold horizontal line representing the median value. See also Supplementary Fig. 6. Full scans of blots are shown in Supplementary Fig. 12.