Figure 1: NR4A1 is screened as an activator of TGF-β/SMAD signalling.
From: Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-β signalling
(a) A genome-wide cDNA overexpression screen in HEK 293T cells using TGF-β-induced CAGA12-luc transcriptional response as assay. Three NR4A1 cDNAs that potently stimulated the luciferase activity were identified. As expected, SMAD3 and SMAD7 stimulated and inhibited the luciferase activity, respectively. (b) Short interfering RNA (siRNA) screening chart for 5,000 druggable genes in which two NR4A1 siRNA were identified that repressed the luciferase activity. siALK5, siLuciferase and siSMAD3 that strongly repress luciferase activity served as positive controls. The x and y axes are the relative luciferase activity in two replicates in a and b. (c) Validation of NR4A1 overexpression or knockdown for SMAD3 transcriptional response induced by TGF-β (5 ng ml−1) in HEK293T cells. Data are presented as mean±s.d. (n=3 measurements). NS shRNA, non-targeting shRNA; RLU, relative luciferase units. (d) Immunoblot (IB) analysis of PC3 cells stably expressing NR4A1. (e) Immunoblot (IB) analysis of PC3 cells stably depleted of NR4A1 by shRNA (shNR4A1). (f) HaCaT cells stably expressing NR4A1 were treated with TGF-β (1 ng ml−1) or SB431542 (10 μM) for 1 h and then lysed for immunoblot analysis.
