Fig. 4. Necroptotic traits induced by DTT-304 but not by DTT-205.

Human colon carcinoma HT-29 cells knockout for MLKL and stably expressing RIP3 coupled to green fluorescent protein (GFP) were treated with 0.65 to 10 µM DTT peptides for the indicated time and following assessed for the aggregation of RIP3 indicative for necroptosome formation by epifluorescence microscopy. The combination of TNFα (T), SMAC/DIABLO mimetic peptide B6 (S), and the pan-caspase inhibitor z-VAD-fmk (Z) was used as positive control for the induction of necroptosis. Representative images (a, c) and quantifications of RIP3 aggregates (b, d) are depicted (size bar equals 10 µm; mean ± SD of triplicate assessments, Student’s t test, *p < 0.5, **p < 0.01, ***p < 0.001). Downstream MLKL activation was visualized with phosphoneoepitope-specific antibody (e, f) Of note, exclusively DTT-304 but not DTT-205 depicted traits of necroptosis. Murine lung cancer TC-1 cells that were CRISPR gene edited in RIP3 and MLKL were treated with 0.65 to 10 µM of DTT-205 or DTT-304 for 6 h and viability was assessed by means of an exclusion dye (g, h; mean ± SD of triplicate assessments, Student’s t test, *p < 0.5, **p < 0.01, ***p < 0.001). Knockouts of RIP3 and MLKL were partially resistant to DTT-304 yet not to DTT-205-induced cell death