Fig 1.
Andrographolide increases NRF2 protein expression and transcriptional activity.
(A) Structure of andrographolide. The α and β carbons of the Michael acceptor group are indicated. (B) HEK293T cells were treated with increasing andrographolide concentrations, as indicated, for 4 hours. DMSO was added as a negative control and the Cullin E3 RING ligase (CRL) inhibitor MLN4924 (1 μM) served as positive control. Cells were lysed and cell lysates analyzed by Western blotting using NRF2 and actin antibodies. (C) To measure the time dependence of andrographolide dependent NRF2 activation, HEK293T cells were treated with 7.5 μM andrographolide or 1 μM MLN4924 for the indicated times. Subsequently, cell lysates were analyzed by Western blotting with the indicated antibodies. The CRL substrate p27Kip1 served as a positive control for the CRL inhibitor MLN4924. (D) To measure Nrf2 transcriptional activity, the ARE luciferase reporter assay was performed as described under Materials and Methods. The cells were transfected for 24 hours before treatment with andrographolide (7.5 μM) or sulforaphane (10 μM) for 6 hours. The results shown are representative of two independent experiments.
Fig 2.
Western blot analysis of NRF2 protein concentrations in HEK293T cells after treatment with andrographolide.
(A, B) HEK293T cells were transfected with HA-tagged NRF2 and either wild type FLAG-tagged KEAP1 or C151S KEAP1 mutant for 48 hours before treatment with andrographolide (7.5 μM), arsenite (20 μM), PMX290 (2 μM), or sulforaphane (10 μM) for 6 hours. The cell lysate samples were subjected to SDS-PAGE and analyzed using Western Blotting with HA and FLAG antibodies.
Fig 3.
Effect of andrographolide on the interaction between KEAP1 and CUL3.
(A, B) The cells were transfected with the indicated expression plasmids for two days, followed by drug treatment with 7.5 μM andrographolide (A) or 2 μM PMX290 (B) for 4 hours or 6 hours, respectively. Cells were then lysed and subjected to FLAG immunoprecipitation, as described under Materials and Methods. The data in (C) represent the mean and S.E.M. of 5 independent repeats of the immunoprecipitation in (A).
Fig 4.
At high concentration, andrographolide functions independently of Cys151 in KEAP1.
(A, B) Cells were transfected with the indicated expression plasmids for 2 days, followed by drug treatment with 100 μM andrographolide for 4 hours, as indicated. Cells were then lysed and subjected to immunoprecipitation with V5 antibody, coupled to protein G-sepharose (A) or FLAG M2 agarose (B), as described under Materials and Methods. Cell lysates and immunoprecipitates were analyzed by Western blotting with the indicated antibodies. (C) Cells were transfected with HA-tagged NRF2 and either wild type FLAG-tagged KEAP1 or C151S KEAP1 mutant for two days and then treated with 7.5 μM or 100 μM andrographolide for 4 hours. The cell lysate samples were analyzed using Western Blotting with the indicated antibodies.
Fig 5.
The role of the KEAP1 BTB domain in mediating NRF2 ubiquitination.
Cells were transfected with the indicated expression plasmids for 2 days, followed by cell lysis and Western blotting with FLAG, HA and α-tubulin antibodies. Details of the used expression plasmids are included in Materials and Methods.
Fig 6.
Effects of organic extracts isolated from the endophytes on NRF2.
(A) To screen the 49 organic extracts isolated from the endophytes for NRF2 transcriptional activation, the ARE reporter assay was performed as described under Materials and Methods. HEK293T cells were transfected with ARE-pGL2 plasmid for 24 hours before treatment with the organic extracts (0.5%) or sulforaphane (SF; 10 μM) for six hours before analysis. (B) Dose response of the effect of organic extracts on NRF2 transcriptional activation. The assay was carried out as in (A). (C) Morphology, Lacto-phenol Cotton Blue staining and identity (based on ≥99% sequence similarity of BLAST results) of the fungal endophyte from which ORX 41 was derived. (D) Western blot analysis of the effect of organic extract ORX 41 on the NRF2 protein concentration. HEK293T cells were transfected with HA-NRF2 for 2 days before treatment with vehicle (methanol treatment) or ORX 41 for 6 hours at the indicated concentrations, followed by Western blotting with HA and β-actin antibody.