Fig. 5

eNOS-derived NO scavenging significantly impairs the CSC phenotype in human colon cancer cells. a Formation of tumorspheres from HCT-116 and Caco-2 cells pre-treated for 24 hours with c-PTIO (100 μM) with or without DETANONOate or CSNO (100 μM). Representative images 1–2 weeks after seeding are shown. b Formation of organoids from cells pre-treated for 24 hours with c-PTIO (100 μM) with or without NO donors (100 μM). Representative images 1–2 weeks after seeding are shown. c Immunoblot of β-catenin, Bmi1, Notch1, and Sox2 expression in HCT-116 and Caco-2 tumor cells treated for 24 hours with c-PTIO (100 μM) with or without DETANONOate or CSNO (100 μM). d Organoid immunofluorescence of β-catenin from control or Caco-2 organoids treated with c-PTIO (100 μM) for 24 hours. e RT-qPCR of iNOS-induced expression (IL-1β 3 ng/ml, IFN-γ 200 U/ml and TNF-α 75 ng/ml, for 6 hours) from control and iNOS-shRNA expressing HCT-116 cells. f Immunoblot of previously mentioned proteins in iNOS-knockdown HCT-116 cancer cells treated with c-PTIO (100 μM). Stain-free technology was used as loading control in immunoblot experiments. * P < 0.05 compared with the control. Scale bars: 100 μm. CSC cancer stem cell, CSNO S-nitrosocysteine, eNOS endothelial NO synthase, iNOS inducible NO synthase, RT-qPCR quantitative real-time reverse-transcriptase polymerase chain reaction