Fig. 3: CMS subtypes show distinct differences in caspase activation kinetics in response to co-treatment of Birinapant and chemotherapy.

A, B Representative time-lapse images of Venus, TMRM and CFP/FRET emission ratio in HCT116 (A) cells stably expressing IETD-FRET probe (B) treated with 2 µM oxaliplatin/10 µM 5-FU/1 µM Birinapant for 48 h. Scale bar: 20 µm. C Overview of generation and interpretation of a single-cell trace extracted from time-lapse imaging of IETD-FRET probe cleavage in an HCT116 cell showing features of apoptosis in response to combination treatment. Cleavage of IETD substrate by caspases results in a disruption in FRET signal, which can be quantified by measuring CFP/FRET emission ratio. A mild increase in CFP/FRET emission ratio (I) before the loss of TMRM signal due to MOMP (II, red dashed line) indicates caspase-8-like activity (I–II, color coded as light blue). A rapid increase in CFP/FRET emission ratio (III) after the loss of TMRM signal indicates the involvement of executioner caspases (caspase-3 and -6) (II–III, color coded as light brown). D, E Representative single-cell CFP/FRET emission ratio kinetics of HCT116 (CMS1), RKO (CMS1), GP5D (CMS2), LS1034 (CMS2), LS513 (CMS3), and MDST8 (CMS4) cell lines treated with Oxa/5-FU, Birinapant, or combination of the drugs (D). Summary of the differences in caspase activation kinetics among CMS in response to co-treatment of Oxa/5-FU and Birinapant (E). F Quantification and comparison of the differences in caspase activation kinetics among CMS in response to Oxa/5-FU (left), Birinapant (middle), or combination of the drugs (right). “Onset of IETD cleavage” indicates a point of time at which cells start cleaving IETD substrate, “Duration of IETD cleavage” refers to a period of time between onset and end of IETD cleavage. Data are shown as individual values for each cell as well as the median ± quartiles. n = 146 cells were analyzed from at least three experiments per cell line. Statistical significance was tested using one-way ANOVA followed by Tukey’s multiple comparison test. “no substrate cleavage” indicates that cells did not exhibit an increase in IETD cleavage over the course of the experiment, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.