Fig. 5: NanoTAC degraders trigger MLKL degradation and block necroptotic cell death. | Nature Communications

Fig. 5: NanoTAC degraders trigger MLKL degradation and block necroptotic cell death.

From: Development of NanoLuc-targeting protein degraders and a universal reporter system to benchmark tag-targeted degradation platforms

Fig. 5

a MLKL−/− HT29 cells stably expressing the doxycycline-inducible untagged MLKL or the C-terminal fusion protein MLKL- NanoLuc were induced with 40 ng/mL doxycycline overnight, then stimulated with NC4 (NanoLuc-CRBN) for 5 h before the addition of TNF (100 ng/mL) + Smac mimetic (compound A; 500 nM) + caspase inhibitor; z-VAD-fmk (10 μM), for 24 h. Cell death was assessed by flow cytometric analysis of PI exclusion. N = 3 independent experiments (symbols), EB represent mean ± SD. b–d MLKL−/− HT29 cells expressing doxycycline-inducible untagged MLKL or the C-terminal fusion protein MLKL-FKBPF36V were induced with 40 ng/mL doxycycline overnight, then stimulated with FC1, FC2 or FV1 for 5 h before the addition of TNF (100 ng/mL) + Smac mimetic (compound A; 500 nM) + caspase inhibitor; z-VAD-fmk (10 μM), for 24 h. Cell death was assessed by flow cytometric analysis of PI exclusion. N = 3 independent experiments (symbols), EB represent mean ± SD. e, f MLKL−/− HT29 cells expressing doxycycline-inducible untagged MLKL or the C-terminal fusion protein MLKL-Halo were treated with 40 ng/mL doxycycline overnight to induce the constructs, then stimulated with the indicated concentrations HV1 or HV2 for 5 h before the addition of TNF (100 ng/mL) + Smac mimetic (compound A; 500 nM) + caspase inhibitor; z-VAD-fmk (10 μM), for 24 h. Cell death was assessed by flow cytometric analysis of PI exclusion. e N = 4, f N = 3 independent experiments (symbols), EB represent mean ± SD. g Western from MLKL−/−CASP8−/− HT29 cells stably expressing the doxycycline-inducible C-terminal fusion protein, MLKL-NanoLuc (MLKL-NLuc) and constitutively expressing the C-terminal fusion protein caspase-8-FKBPF36V. Cells were induced with 40 ng/mL doxycycline overnight, then stimulated with FV1 and NC4 for 5 h. h Cells from g were treated with 40 ng/mL doxycycline and stimulated with 125 nM of FV1, NC4, or NC* for 5 h before the addition of TNF (100 ng/mL) + Smac mimetic (compound A; 500 nM) for 19.5 h. Cell death was assessed using an IncuCyte and Cytotox-Red uptake. N = 3 independent experiments, one representative experiment shown, EB represent mean ± SD. Source data are provided as a Source Data file.

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