Fig. 2

CRPC models contain more PCSC populations with enhanced eNOS expression and higher intracellular NO levels. a, b Androgen-sensitive and castration relapse VCaP and CWR22 xenografts. q-PCR analysis showed that eNOS exhibited a progressive increase in mRNA levels in xenograft tumors at 4 days (Post-Cas) and 2 months post-castration (Relapse) as compared to tumors before host castration. Results also revealed that the castration relapse CWR22-CRPC xenograft tumors expressed significant higher levels of multiple PCSC-associated biomarkers. c, d Microscopic detection of intracellular NO by NO fluorescent probe DAF-FM in bicalutamide-resistant LNCaP-BC32 cells-derived and FACS-sorted CD133⁻/CD44⁻ and CD133⁺/CD44⁺ cell populations, grown under either adherent 2D culture or non-adherent 3D culture (spheroids) condition and upon treatments with NOS inhibitor (L-NAME, 100 μM) or substrate (L-Arginine, 0.5 mM). c Representative micrographs show the intracellular NO-activated DAF-FM signals. Bars: 50 μm. d Semiquantitative analysis of NO levels (DAF-FM fluorescence signals). Results showed that 3D culture spheroids (derived from CD133⁺/CD44⁺ cells) displayed higher intense basal NO signals as compared to adherent 2D culture CD133⁻/CD44⁻ cells without treatment with L-NAME or L-Arginine. Their NO levels as detected in both 3D culture spheroids and adherent 2D culture cells were significantly reduced or abolished upon treatment with L-NAME but significantly intensified upon L-Arginine treatment. e FACS CD133-CD44 sorting of LNCaP-BC and LNCaP cells showed that the LNCaP-BC32 cells contained more CD133⁺-CD44⁺ subpopulation than their parental LNCaP cells. The CD133⁺-CD44⁺ subpopulation was significantly reduced/lessened upon treatment with L-NAME (100 μM). Results repeated at least three times are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001