Fig. 4

eNOS can promote EMT of PCSCs and in vivo prostate tumor growth and metastasis. a, b RT-qPCR analysis of stemness-associated genes in adherent 2D culture cells and 3D spheroids with eNOS transgene overexpression. Results showed that eNOS overexpression induced significant upregulation of stemness genes in adherent 2D culture DU145 cells and also 3D culture LNCaP spheroids. c RT-qPCR analysis of four EMT-associated markers [mesenchymal markers: CHD2 (N-cadherin), EMT-inducing factors: transcription factor ZEB1 and CLDN1 (claudin-1); epithelial marker: CDH1 (E-cadherin], in LNCaP-eNOS cells. Results showed that eNOS overexpression could induce significant upregulation of CDH2, ZEB1 and CLDN1 but downregulation of CDH1 in 3D culture spheroids formed by LNCaP-eNOS cells as compared to spheroids formed by the LNCaP vectors cells. d, e Wound healing assay. d Representative images of PC-3 M-vector/-eNOS/-sheNOS-transduced cells taken at 0 h and 41 h time point. Bar: 200 μm. e Semiquantitative analysis of wound closure determined by measurement of width of wounds. Results showed that PC-3 M-eNOS cells showed significant higher migration capacity, whereas PC-3 M-sh-eNOS cells showed reduced capacity, as compared to PC-3 M-vector cells. f Luciferase-based bioluminescence in vivo imaging. Representative bioluminescence pictures of mice at 5 weeks post-inoculation of PC-3 M-vector/eNOS/sh-eNOS cells. Intense bioluminescence tumor growth signals were detected in the prostates of mice which had received orthotopic inoculation of PC-3 M-eNOS cells. Moderate and very weak bioluminescence signals were detected in mice bearing inoculations of PC-3 M-vector or PC-3 M-sh-eNOS cells, respectively. g Photograph shows the dissected prostate tumors formed by the inoculated PC-3 M-vector/eNOS/sh-eNOS cells in mice. Significant larger tumors were formed by PC-3 M-eNOS cells. h Table summarizes the tumor weights and detected metastasis to para-aortic lymph nodes in host mice bearing xenografts of PC-3 M-vector/eNOS/sh-eNOS cells. Results repeated at least three times are expressed as mean ± SD, *P < 0.05, **P < 0.01