Fig. 5

Rho signaling defines the therapeutic outcome. A Effect of Rho signaling inhibitors on MLN-induced apoptosis in monolayer cultures of PCa cells. The cells were treated for 1 day with increasing doses of MLN and 10 µM Y27632 or 20 µg/ml C3E. The percentage of apoptotic cells was evaluated by automated fluorescence microscopy using CellEvent™ Caspase-3/7 Green Detection Reagent (CE). For all shown drug vs. control effects: p < 0.001 (B) Effect of 10 µM Y27632 on MLN-induced apoptosis in LNCaP spheroids. The histogram shows the stimulation of apoptosis by MLN in small (< 250 µm) spheroids measured by CE fluorescence. Statistical significance: ***-p < 0.001. On the right: 10 µM Y27632 prevents the mechanical rupture of large (> 400 µm) spheroids induced by 250 nM MLN (the hypoxic apoptotic core of spheroids is stained with CE). Scale bar = 200 µm. C Effect of the inhibition of Rho signaling on LNCaP (top) and PC3 (bottom) tumoroid growth. Color histograms: Preformed tumoroids were treated for 7 days with 10 µM Y27632 (Y), 20 µg/ml C3E, or 3 µM CCG1423 with or without (control) 100 nM MLN. The viability was assessed by measuring ATP with ViaLight™ reagent. Black histograms: Tumoroids were grown from Rho-depleted cells for 10 days and assessed for viability. E Biphasic MLN dose response curves for LNCaP and PC3 tumoroids. The biphasicity is indicated by the green circle. F MLN dose response curves for PC3 tumoroid invasion (measured by tumoroid spread area) and Rho activation. Statistical significance: *- p < 0.05, **-p < 0.01 and ***-p < 0.001. G Dose-dependent Rho stimulation by MLN analyzed by western blot and quantified in Fig. 5F. The active GTP-bound isoforms are hash-tagged