Fig 2: SA accumulation leads to covalent modification of KEAP1 lysines.A) Structure of succinic anhydride alkyne (SA-alkyne). B) Relative luminescence values of ARE-LUC reporter activity from IMR32 cells treated with SA or SA-alkyne (12 mM) for 4 hrs (n=5, ****P<0.0001, **P<0.01, one-way ANOVA). C) Representative fluorescence scan of rhodamine azide labeling of anti-FLAG immunoprecipitated material after treatment of HEK293T cells expressing KEAP1-FLAG with the indicated concentrations of SA-alkyne (10 mM) for 1 hr. D) Representative fluorescence scan of rhodamine azide labeling of anti-FLAG immunoprecipitated material after pretreated with the indicated concentrations of SA followed by a 1 hr labeling with SA-alkyne (10 mM) in HEK293T cells expressing KEAP1-FLAG. E) Relative ARE-LUC activity from IMR32 cells expressing the indicated KEAP1 mutant and MYC-NRF2 and then treated with 1 mM SA for 4 hrs (n=5, ***P<0.001, two-way ANOVA). F) Western blotting for anti-N-succinyl lysine positivity of anti-FLAG immunoprecipitated material from HEK293T cells expressing KEAP1-FLAG after 1 hr treatment with the indicated concentrations of SA. G) Western blotting for anti-N-succinyl lysine positivity of anti-FLAG immunoprecipitated material from HEK293T cells expressing KEAP1-FLAG and shRNAs targeting SUCLG2, stabilized by the desuccinylase inhibitor ET-29 (10 µM).

Fig 3: Loss of succinyl coA synthetase activates NRF2 driven transcription.A) Schematic of the cellular screening platform using ARE-GFP-LUC K562 reporter cells. Shown is a representative plot indicating positivity for GFP after transduction with an shRNA targeting KEAP1. B) Schematic depicting the enzymes of central carbon metabolism targeted by the shRNA screen (boxes). C) Replicate fold increases in percent GFP positive ARE-GFP-LUC K562 cells after a 72 hr exposure to the indicated shRNAs of the screen. D,E) Relative transcript levels of SUCLG2 (D) and NQO1 (E) after 48 hr transient transfection of HEK293T cells with shRNAs targeting SUCLG2 (n=3, ****P<0.0001, **P<0.01, one-way ANOVA). F) Relative luminescence values of ARE-LUC reporter activity 72 hr after transduction of lentiviruses encoding shRNAs to SUCLG2 and NRF2 in ARE-GFP-LUC K562 (n=3, ****P<0.0001, two-way ANOVA).