Fig. 1

Induced by ADT, CREB activation is critical for neuroendocrine phenotype of NEPC cells. a Western blotting for p-CREB (S133, activated) and NE markers ENO2 and TUBB3 in AR-positive LNCaP and VCaP cells treated with 10 μM ADT drug MDV3100 (enzalutamide) for 72 h, without or with androgen 5 nM DHT during the last 24 h before cell lysate was collected. The upper band in p-CREB western blots is pS133-CREB and the lower band is p-ATF1. b Western blotting for CREB activation level (p-CREB) in NEPC NE1.3, NCI-H660, and 144-13 cells, as compared to LNCaP cells. c RT-qPCR for NE markers CHGA, CHGB, and ENO2 in prostate cancer cells overexpressing CREB wild-type (WT) cDNA. Y-axis shows relative fold changes in expression, normalized to GAPDH. d RT-qPCR for NE markers CHGA and CHGB in NEPC 144-13 and NE1.3 cells treated with Protein Kinase A inhibitor PKI (10 μM), beta adrenergic antagonists ICI (10 μM) or propranolol (PRO, 10 μM) for 48 h. Error bars in PCR results represent standard deviation (s.d.) of triplicate experiments. e LNCaP cells carrying Dox-inducible-shCREB were grown in regular FBS medium, or CSS medium without or with 1 μg ml⁻¹ of Dox for 6 days, followed by western blotting. f LNCaP cells carrying Dox-inducible ACREB (encoding a CREB inhibitory peptide) were treated with DMSO, 10 μM MDV3100, or 10 μM MDV3100 plus 1 μg ml⁻¹ of Dox for 24 and 72 h, followed by western blotting