Journal: Cancer research

Article Title: NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction

doi: 10.1158/0008-5472.CAN-16-2204

Figure Lengend Snippet: DPP3 interacts with KEAP1 in an oxidative stress-inducible manner through a highly conserved KEAP1-binding ETGE motif. (A) Composition of KEAP1 complexes isolated under non-stress and stress conditions. HeLa S3 cells stably expressing KEAP1 with FLAG-HA double tags at the C-terminus were either untreated or treated with 10 Gy ionizing radiation (IR), 100 μM tert-butylhydroxyquinone (tBHQ), 200 μM hydrogen peroxide (H2O2), or 2 mM hydroxy urea (HU). Cells were collected 2.5 hr after IR or drug treatment, and KEAP1-containing complexes were purified from the cytoplasmic and nuclear extracts of the cells by tandem affinity purification. The “Mock” purification was carried out using HeLa S3 cells without ectopic KEAP1. (B) Alignment of amino acid sequences of the “ETGE” or “ETGE”-like motifs and their immediate surrounding regions in proteins identified in the KEAP1 complexes purified under the “untreated” condition. CYT, cytosol; NUC, nucleus. (C-D) Oxidative stress-inducible interaction between DPP3 and KEAP1. Endogenous DPP3 was immunoprecipitated (IPed) from whole cell lysates of MCF7 cells treated with indicated concentrations of H2O2 for 3 hr (C) or diquat for 24 hr (D). Proteins in the IPed materials were analyzed by western blotting. (E) Kinetics of stress-induced DPP3 binding to KEAP1. MCF7 cells were treated with 200 μM H2O2 for indicated time periods, and the complex formation between DPP3 and KEAP1 was analyzed as above. (F) Requirement of the ETGE motif of DPP3 for KEAP1 binding. The wt and mutant DPP3 proteins were IPed with anti-FLAG beads from lysates of MCF7 cells stably expressing them. The IPed DPP3 and co-IPed KEAP1 were detected by western blotting.

Article Snippet: The primary antibodies used are as follows: anti-DPP3 rabbit monoclonal (ab133671, Abcam), anti-NQO1 mouse monoclonal (sc-32793, Santa Cruz), anti-NRF2 rabbit monoclonal (ab62352, Abcam), anti-KEAP1 goat polyclonal (E20, sc-15246, Santa Cruz), anti-β-Actin mouse monoclonal (sc-69879, Santa Cruz), anti-GAPDH rabbit polyclonal (sc-25778, Santa Cruz) and anti-p62 rabbit monoclonal (ab109012, Abcam).

Techniques: Binding Assay, Isolation, Stable Transfection, Expressing, Purification, Affinity Purification, Immunoprecipitation, Western Blot, Mutagenesis

Journal: Cancer research

Article Title: NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction

doi: 10.1158/0008-5472.CAN-16-2204

Figure Lengend Snippet: Overexpression of DPP3 enhances the stability of KEAP1. (A) Levels of NRF2, DPP3, KEAP1 and p62 in MCF7 cell lines overexpressing wt and mutant DPP3 proteins. β-Actin was used a loading control. (B-C) Stabilities of KEAP in the MCF7 cell lines harboring the empty vector or overexpressing wt DPP3. Cells were either untreated or treated with 50 μg/ml of cycloheximide for 2, 4 and 6 hr, and the proteins were analyzed by western blotting. The intensities of KEAP1 bands were quantified by the ImageJ software, normalized against those of GAPDH and plotted. B shows a set of representative western blots, and C shows means of the quantified results from 3 independent experiments. Error bars represent SDs. *p<0.05. (D) Effect of DPP3 depletion in on KEAP1 levels in the stable MCF7 cell lines. The cells were treated with a control siRNA or siRNAs targeting DPP3 coding sequence (CDS) or 3′-UTR, and the proteins were analyzed by western blotting.

Article Snippet: The primary antibodies used are as follows: anti-DPP3 rabbit monoclonal (ab133671, Abcam), anti-NQO1 mouse monoclonal (sc-32793, Santa Cruz), anti-NRF2 rabbit monoclonal (ab62352, Abcam), anti-KEAP1 goat polyclonal (E20, sc-15246, Santa Cruz), anti-β-Actin mouse monoclonal (sc-69879, Santa Cruz), anti-GAPDH rabbit polyclonal (sc-25778, Santa Cruz) and anti-p62 rabbit monoclonal (ab109012, Abcam).

Techniques: Over Expression, Mutagenesis, Plasmid Preparation, Western Blot, Software, Sequencing

Journal: Cancer research

Article Title: NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction

doi: 10.1158/0008-5472.CAN-16-2204

Figure Lengend Snippet: DPP3 is overexpressed human breast cancer and correlates with increased NRF2 target gene expression and poor prognosis. (A) Box-and-whisker plots indicating the median score (horizontal line), the interquartile range (IQR, box boundaries) and 1.5 times the IQR (whiskers) demonstrate significantly higher DPP3 mRNA expression in 94 human breast tumors compared to 94 matched adjacent normal tissue samples (p=1.84×10-40, paired t-test). (B-C) A Spearman rank correlation demonstrating that DPP3 mRNA expression is positively correlated with DNA copy number status in (B) 1,031 TCGA breast tumor samples (p=1.6×10-96; r=0.5871) and (C) 1,992 samples from the METABRIC cohort (p=2.8×10-77, r=0.4019). (D) A Spearman rank correlation demonstrating that DPP3 and KEAP1 expression are positively correlated (p=7.6×10-11, r=0.2009) in the TCGA cohort. DPP3 expression is positively associated with NRF2 target gene expression (p=5.4×10-14, r=0.2314) despite a negative correlation with NRF2 mRNA expression (p=2.8×10-08, r= [-0.1719]). Tumors with mutations in DPP3, KEAP1, NRF2, FH and KRAS are indicated with vertical bars. (E) Similar results as in D were observed in the METABRIC cohort (n=1,992). Breast cancer samples in D and E are ranked based on DPP3 mRNA expression; high KEAP1, NRF2 and NRF2 target gene expression is shown in red while low expression is indicated in blue. (F-H) Kaplan-Meier plots comparing disease-specific survival in human breast tumors from the METABRIC cohort based on high (top quartile) versus low (bottom quartile) DPP3 expression in (F) all tumors, (G) ER+ tumors, or (H) ER- tumors. (I-K) Kaplan-Meier plots comparing disease-specific survival in human breast tumors from the METABRIC cohort based on high (top quartile) versus low (bottom quartile) NRF2 target gene expression in (I) all tumors, (J) ER+ tumors, or (K) ER- tumors.

Article Snippet: The primary antibodies used are as follows: anti-DPP3 rabbit monoclonal (ab133671, Abcam), anti-NQO1 mouse monoclonal (sc-32793, Santa Cruz), anti-NRF2 rabbit monoclonal (ab62352, Abcam), anti-KEAP1 goat polyclonal (E20, sc-15246, Santa Cruz), anti-β-Actin mouse monoclonal (sc-69879, Santa Cruz), anti-GAPDH rabbit polyclonal (sc-25778, Santa Cruz) and anti-p62 rabbit monoclonal (ab109012, Abcam).

Techniques: Expressing, Whisker Assay

Journal: Cell chemical biology

Article Title: Non-covalent NRF2 Activation Confers Greater Cellular Protection Than Covalent Activation

doi: 10.1016/j.chembiol.2019.07.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Goat polyclonal anti-KEAP1 , Santa Cruz Biotechnology , Cat# sc-15246, RRID:AB_2132638.

Techniques: Virus, Recombinant, Reverse Transcription, Reporter Assay, Software, Imaging, Modification

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells were transfected with HA-tagged Keap1 or with lipofectamine alone (sham) as described in Methods. (A) Treatment was with 500 µM spermine NONOate or CSNO for 10 minutes in HHBSS. Cellular lysates were immunoprecipitated with either anti-HA or anti-Keap1 and were immunoblotted with anti-HA as described in Methods. Keap1 has an apparent molecular weight of about 70 kDa. (B) Lysate was pre-incubated with Keap1 blocking peptide prior to immunoprecipitation and immunoblotting (lane1) and compared with control lysates (lane 2).

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Transfection, Immunoprecipitation, Molecular Weight, Incubation, Blocking Assay, Western Blot

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells were transfected with HA-tagged Keap1 and treated with spermine NONOate or CSNO in HHBSS. Lysates were prepared according to the MPB protocol, as described in Methods, followed by immunoprecipitation using anti-HA and immunoblotting with NeutrAvidin-HRP. (A) Time-response to 500 µM spermine NONOate or CSNO for 0–15 min. (B) Dose-response to 0–500 µM CSNO for 10 min.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells were treated with CSNO. Lysates were prepared according to the MPB protocol, followed by immunoprecipitation using anti-Keap1 and immunoblotting with NeutrAvidin-HRP or anti-Keap1. (A) Dose response to 0–500 µM CSNO for 10 min. (B) Time response to 500 µM CSNO for 0–60 min.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells which were transfected with HA-tagged Keap1 were treated with 500 µM CSNO for 0–120 minutes. Cytosolic and nuclear extracts were prepared, as described in Methods, and immunoblotted using anti-Keap1, anti-α-tubulin and anti-Lamin A. The cytosolic protein α-tubulin and the nuclear protein Lamin A were used as loading controls. The molecular weight of α-tubulin is 55 kDa.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Transfection, Molecular Weight

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells which were not transfected were treated with 0 or 500 µM CSNO. Half of the cells were pre-treated with 5 ng/ml leptomycin B for 2 hours prior to the addition CSNO. After 90 minutes, lysates were prepared and immunoblotted using anti-Keap1, anti-Nrf2 and anti-Lamin A. The nuclear protein Lamin A was used as a loading control.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Transfection

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: Key thiols in Keap1 are modified by nitrosative stress as well as by oxidative stress and electrophiles. This alters the interaction of the Keap1-Nrf2-Cul3 complex in the cytosol and results in decreased degradation of Nrf2 and possibly of Keap1. Nrf2 accumulates in the nucleus where it forms a heterodimer with MafG and activates the ARE leading to transcriptional upregulation of cytoprotective and antioxidant genes.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Modification