Fig. 6

Effects of sildenafil treatment and PDE6H KO on cell cycle arrest, glycogen levels, and spheroid necrosis in HCT116 cells. a Cell cycle distribution of mock and PDE6H KO HCT116 cells following treatment with sildenafil, cGMP analogue 8-Br-cGMP (10 μM), and PKG inhibitor KT5823 (1 μM). b Levels of mitochondrial ROS and percentage of dysfunctional mitochondria in mock and PDE6H KO HCT116 cells, n = 5. Median MitoTOX signal fluorescence intensity indicated mitochondrial ROS levels. Percentage dysfunctional mitochondria were denoted as the fraction of live cells with high MitoTracker Green and low MitoTracker DeepRed signal. c Seahorse mitochondrial test was conducted after 20-h glutamine depletion. d Glycogen enzymatic assay results and glycogen granule immunofluorescent staining of mock and PDE6H KO HCT116 following sildenafil treatment, in normoxia and hypoxia. Red, glycogen granules; blue, DAPI. e PDE6H depletion effects on the necrotic/viable HCT116 spheroid volume ratio, N = 10. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001