Fig. 6

ERRα and ERG can act to co-activate the eNOS-NO signaling in prostate cancer cells. a RT-qPCR analysis of ERRα and eNOS expression in prostate 3D culture spheroids versus adherent 2D culture cells. Left: spheroids (Sp) grown from DU145 or LNCaP cells exhibited higher ERRα expression levels as compared to their corresponding 2D cultures. Right: spheroids exhibited significant higher eNOS expression as compared to their parental cells. Overexpression of ERRα in 2D-cutured cells could significantly upregulate eNOS expression (blue bars). b Immunoblot analysis of eNOS, ERG and ERRα in DU145 and LNCaP cells. The blots were cropped around the bands at 133, 55, 50 and 42 kDa molecular weight markers from different membrane blots. Results showed that transient overexpression of either ERG or ERRα could significantly enhance protein expression of eNOS. c Measurement of cGMP levels in 3D culture spheroids derived from DU145-ERG-transduced cells. Analysis showed that DU145-ERG spheroids contained higher cGMP concentration levels than DU145-vector spheroids, and with such increased cGMP levels being eliminated by L-NIO treatment. d Immunoblot analysis of eNOS, ERG and ERRα in VCaP cells upon treatments with ERRα inverse agonist XCT790 (5 μM), ERG inhibitory peptide EIP1 (50 μM) or its negative control muEIP1 (50 μM). The blots were cropped around the bands at 133, 55, 50 and 42 kDa molecular weight markers from different membrane blots. Results showed that inhibition of ERRα by XCT790 and also ERG by EIP1 could moderately reduce the eNOS levels in VCaP cells. e 3D culture spheroid formation assay performed on ERRα-overexpressed prostate cancer cells. Results showed that overexpression of ERRα could significantly promote the spheroid formation capacities of both DU145 and LNCaP cells. However, their spheroid formation capacities could be weakened by treatments with inhibitors of NOS (L-NIO, 100 μM) or sGC (ODQ, 20 μM). Results repeated at least three times are expressed as mean ± SD, *P < 0.05, **P < 0.01. f Schematic diagram illustrates the contribution of regulatory loop between ERRα and ERG in the activation of eNOS-NO-sGC-cGMP-PKG signaling pathway in the regulation of PCSCs and CRPC