Fig 2: AMPK-dependent phosphosites in Nrf2 enhance expression of selected endogenous Nrf2 target genes. (A) MEF Nrf2 -/- cells transfected with EGFP-WT- or EGFP-TM-Nrf2 and (B) wt or AMPKa1 -/- MEF were treated with DMSO as vehicle control or sulforaphane (Sfn, 5 µM) for 4 h. RNA was extracted and analyzed for abundance of Hmox1 and Gclc mRNA by qPCR (Hprt as reference gene). Bars depict compiled relative expression levels. (n = 3; mean +SD, unpaired Student's or Welch's t-test, a = 0.05, *P = 0.05, ns: not significant. (C) MEF Nrf2 -/- cells transfected with EGFP-WT- or EGFP-TM-Nrf2 and (D) wt or AMPKa1 -/- MEF were treated with Sfn (5 µM) for the indicated periods of time. Proteins were extracted and subjected to immunoblot analysis for HMOX1, GCLC, GFP (=EGFP-WT- or EGFP-TM-Nrf2) and actin as loading control. The depicted blots are representative for three independent biological replicates with consistent results.

Fig 3: The identified AMPK-dependent phosphosites in Nrf2 do not markedly influence half-life of Nrf2 in wt cells. (A) HEK293 were transfected with EGFP-tagged WT or TM-Nrf2 expression plasmids and treated with MG132 (20 µM) for 2.5 h (t = -2.5 to 0) to accumulate Nrf2. After washout of MG132 the translation inhibitor cycloheximide (100 µM) was added (time point 0) for another incubation period of 1–5 h as indicated. Cell lysates were subjected to immunoblot analysis for EGFP-Nrf2 (via a-GFP antibody) or actin. Representative blot and compiled densitometric evaluations (below) for EGFP-Nrf2/actin (related to signal at t = 0) are depicted. (n = 3, mean +SD, unpaired Student's t-test, two tailed, a = 0.05, *P < 0.05, ns: not significant). (B) Wt MEF were transfected with EGFP-tagged WT or TM-Nrf2 expression plasmids and treated with MG132 (10 µM) for 1 h to accumulate Nrf2. After washout, cycloheximide (30 µM) was added (time point 0) for the indicated periods of time. Cell lysates were subjected to immunoblot analysis for EGFP-Nrf2 (via a-GFP antibody) or actin. Representative blots and compiled densitometric evaluations (below) for EGFP-Nrf2/actin (related to signal at t = 0) are depicted. (n = 3, mean +SD, unpaired Student's t-test, two tailed, a = 0.05, *P < 0.05, ns: not significant).(C) Wt MEF were transfected with EGFP-tagged WT- or TM-Nrf2 expression plasmids and treated with MG132 (10 µM) and DMSO or A769662 (50 µM) for 1 h. After washout of MG132 the translation inhibitor cycloheximid (30 µM) was added (time point 0) in the presence of A769662 (50 µM) (+) or DMSO (-) for the indicated period of time. Cell lysates were subjected to immunoblot analysis for EGFP, pACC or actin. Representative blots and compiled densitometric evaluations for DMSO and A769662-treated cells (EGFP-WT- or EGFP-TM-Nrf2/actin, related to signal at t = 0) are depicted (n = 3, mean +SD).

Fig 4: The identified AMPK-dependent phosphosites in Nrf2 do not affect nuclear translocation or transactivation of reporter gene expression. MEF were transfected with EGFP-tagged WT- or TM-Nrf2 and treated with DMSO as solvent control or Sfn (2 h, 5 µM). (A) Then nuclear fractions were prepared and blotted for GFP (=EGFP-WT- or EGFP-TM-Nrf2) and lamin. Representative blots and densitometric evaluation of three independent experiments are depicted. (B) Alternatively, cells were fixed. After staining nuclei with Hoechst 33342 cells were viewed under a confocal microscope (ex/em = 405/465 nm for Hoechst dye (nuclei) and ex/em = 488/520 nm (EGFP-Nrf2)). Only Sfn-treated transfected cells are depicted. (C) MEF Nrf2 -/- cells were transfected with EGFP-tagged WT or TM-Nrf2 expression plasmid together with an ARE-dependent luciferase reporter construct (ARE-LUC). Transfected cells were treated with DMSO or Sfn (5 µM) for 40 h before luciferase activity was determined. The bar graph depicts compiled data of at least three independent experiments expressed as fold luciferase induction by Sfn compared to DMSO-treated control cells (A, C: n = 3, mean +SD, unpaired Welch's t-test, two tailed, a = 0.05, *P = 0.05, ns: not significant).

Fig 5: Mutation of the AMPK-dependent phosphosites to alanine stabilizes Nrf2 in Keap1-/- cells and impedes interaction with ß-TrCP. (A) Keap1-/- MEF were transfected with EGFP-tagged WT- or TM-Nrf2 expression plasmids, pretreated with A76996 (50 µM, 1h) as indicated and then exposed to cycloheximid (30 µM) for different periods of time. Cell lysates were subjected to immunoblot analysis for EGFP or actin. Representative blots and compiled densitometric evaluations (EGFP-WT/TM-Nrf2/actin, related to signal at t = 0) are depicted (n = 3, mean +SD, unpaired Student's t-test, two tailed, a = 0.05, different superscript letters indicate differences with p < 0.05). (B) HEK cells were transfected with an expression plasmid for myc-tagged ß-TrCP together with constructs encoding EGFP-WT-Nrf2, the triple mutated version EGFP-TM-Nrf2, or EGFP as indicated. Cells were treated with vehicle (DMSO) or A769662 (50 µM) 1 h prior to addition of MG132 (20 µM) for further 3 h to stabilize Nrf2, which was pulled down using GFP-Trap®. (Co) precipitated proteins as well as input controls were immunoblotted for myc (=ß-TrCP) or GFP (=EGFP-WT-/EGFP-TM-Nrf2 or EGFP) as indicated. Representative blot pictures and densitometric evaluations (relative myc (ß-TrCP)/GFP (Nrf2) signal) are depicted. (n = 6, mean +SD; *P < 0.05, Welch's t-test, two tailed, a = 0.05).