Fig. 3: TAK1-degradation leads to RIPK1-dependent apoptosis.

A Schematic cartoon of TAK1 depletion using a dTAG-TAK1 degradation system. B Western blots of time course experiment treating dTAG-TAK1 GSCs with 100 nM dTAG^V-1 ligand for indicated amount of time. C Barplot of total % Annexin V positive cells quantified by flow cytometry after treatment with 100 nM dTAG^V-1 ligand. 2 biological replicates at each time point are shown. Early apoptotic cells are defined as Annexin V + /DAPI- and late apoptotic cells as Annexin V + /DAPI+. D Western blot of apoptosis markers 24 h after treatment with 100 nM dTAG^V-1 ligand. E Barplot of total % Annexin V positive cells quantified by flow cytometry after treatment with 100 nM dTAG^V-1 ligand for 4 days in dTAG-TAK1 degron cells after knockout of indicated gene. F Barplot of competitive growth assay of dTAG-TAK1 cells expressing BFP and parental GSCs. Fold change of %BFP-positive cells in population after treatment with dTAG^V-1 ligand for 7 days is shown relative to DMSO-treated control. G Cumulative growth assay in dTAG- TAK1 degron cells upon knockout of the second indicated gene by CRISPR and treatment with dTAG^V-1 ligand. H Barplot depicting fold change of %BFP-positive dTAG-TAK1 cells in population after treatment with dTAG^V-1 ligand for 7 days relative to DMSO-treated control and treatment with increasing concentrations of TNF ligand blocking antibody Etanercept. I Western blot of RIPK1 phosphorylation events after treatment with TNFα with or without TAK1 protein depletion. *denotes unspecific band. J Cartoon of molecular response to TAK1 inhibition in TAK1-dependent GSCs.