Apoptosis induction by dimerization of iCasp9 is independent of Bax/Bak and XIAP. a–c HCT116 control cells and HCT116 cells deficient for Bax and knockdown for Bak (DKO) were treated with 500 μg/ml 5-FU for 24 h, after which they were analyzed by FACS for PI-exclusion (a), apoptotic nuclei (b), and by western for cleaved caspase 3 and cleaved PARP (c). Tubulin was used as a loading control. d–f WT HCT116 and DKO HCT116 cells were transduced with either pMX-iCasp9-IRES-GFP or control vector pMX-IRES-YFP after which they were treated with 10 nM of dimerizer AP20187 for different time points, cells were analyzed by FACS for GFP-positivity (d), PI-exclusion (e) and apoptotic nuclei (f). WT HCT116 and DKO HCT116 cells iCasp9-IRES-GFP or pMX-IRES-YFP were analysed for g 5-FU and h dimerizer-induced caspase-3 activity after 16 and 1 h, respectively or i by western for cleaved caspase 3 and cleaved PARP after dimerizer treatment at different time points. Tubulin was used as a loading control

Dimerization of inducible caspase-9 induces apoptosis in vivo. A colon spheroid culture transduced with pMX-iCasp9-IRES-GFP was injected subcutaneously in Balb/c nude mice. a Tumors were grown until 200 mm³ before the mice were treated either once or twice with 1 mg/kg dimerizer AP20187. One group was left untreated. After treatment, the tumors regressed but re-growth occurred rapidly, after which the half of the group was retreated again. The arrows indicate the time point of treatment. b Tumors were removed 48 h after the last treatment, embedded in paraffin and cut into slides. H&E, Ki67 and cytodeath M30 staining showed that cell death occurs in the treated tumors, but still, living proliferating cells are present. c, d FACS analysis of these tumors show that treatment reduces both the GFP⁺ and Epcam⁺ and CD133⁺ fraction of the tumors cells, although Epcam⁺CD133⁺ cells are still present, both in tumors that were treated once (c) or twice (d). e Spheroid cells transduced with pMX-iCasp9-IRES-GFP were injected subcutaneously in nude mice and treated either directly after cell injection or after the tumors reached 200 mm³ for 5 days with 1 mg/kg dimerizer. The arrows indicate the start time point of treatment. f FACS analysis of tumors after outgrowth, analyzing GFP, Epcam and CD133 expression

Dimerization of iCasp9 induces apoptosis in CSCs in vitro. a–d Spheroid culture derived from a primary colon carcinoma was transduced with pMX-iCasp9-IRES-GFP and single-cell cloned by FACS. Two clones were treated with 10 nM dimerizer AP20187 for different time points. Cells were analyzed by FACS for GFP-positivity (a), PI-exclusion (b), apoptotic nuclei (c) and by western for cleaved caspase 3 and cleaved PARP (d). Tubulin was used as a loading control. e, f CSCs with iCasp9 cells were treated for 48 h with 500 μg/ml 5-FU or for 1 h with 10 nM dimerizer. Next, early markers of apoptosis, Annexin-V staining (e) and activity of caspase-3 were measured (f). g, h The clonogenic capacity of the C002-iCasp9 after dimerizer treatment was analyzed by LDA, showing that CSCs are not resistant to apoptosis induction by dimerization of iCasp9. The graphs represent either the outgrowth of spheroids (g) or the clonogenic fraction calculated by ELDA (h)