Journal: Scientific reports

Article Title: Curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at Ser351.

doi: 10.1038/s41598-021-87225-8

Figure Lengend Snippet: Figure 1. Curcumin activates Nrf2 signaling. (A) Mouse cortical cells were treated with either DMSO or 10 μM curcumin for 12 h. The mRNA levels of Nrf2-response genes were analyzed by qRT-PCR as described in the Methods. The bar graph shows the relative mRNA level of genes in the curcumin-treated group compared to the DMSO group. (B) The protein levels of Nrf2-response genes in the cells were analyzed by immunoblotting using anti-Nrf2, HO-1, NDP52, GST-mu1, NQO1, p62, and actin antibodies, respectively. Full blots are provided in Supplementary Fig. S11. (C) Mouse cortical cells were treated with either DMSO (Veh) or 10 μM curcumin (CCM) for 6 h. The cells were fixed with 4% paraformaldehyde and immunostained using anti-Nrf2 antibody. Fluorescence signals were observed using a confocal laser scanning microscope. (D) Mouse cortical cells were transiently transfected with the ARE-Luc reporter and TK-Renilla plasmids. After treatment with either DMSO (Veh) or 10 μM curcumin (CCM) for 12 h, the cells were assayed for the luciferase activity. Data shown are mean ± S.E. of three independent experiments and were analyzed using the Student’s t-test. (*p < 0.05, ***p < 0.001).

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Quantitative RT-PCR, Western Blot, Fluorescence, Laser-Scanning Microscopy, Transfection, Luciferase, Activity Assay

Journal: Scientific reports

Article Title: Curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at Ser351.

doi: 10.1038/s41598-021-87225-8

Figure Lengend Snippet: Figure 3. Curcumin-mediated Nrf2 activation is dependent on p62. (A) Mouse cortical cells were treated with DMSO (0 h) or 10 μM curcumin (CCM) for the indicated times. The levels of phosphorylated p62 (S351), p62, Keap1, and actin proteins were analyzed by immunoblotting using anti-phospho p62 (S349), p62, Keap1, and actin antibodies, respectively. (B) MEFs were treated with DMSO (Veh) or 10 μM curcumin (CCM) for 12 h. The protein levels of Nrf2, NQO1, p62, and actin were analyzed by immunoblotting using anti-Nrf2, NQO1, p62, and actin antibodies, respectively. Full blots are provided in Supplementary Fig. S11.

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot

Journal: Scientific reports

Article Title: Curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at Ser351.

doi: 10.1038/s41598-021-87225-8

Figure Lengend Snippet: Figure 4. Curcumin-mediated Nrf2 activation is dependent on the phosphorylation of p62 on S351. (A) HEK293 cells were transiently transfected with the Myc-Nrf2 expression plasmid, and then treated with DMSO (−) or 10 μM curcumin (+) for 12 h. The cell lysates were used for Nrf2 immunoprecipitation using an anti- Myc antibody. The protein level of Keap1 co-immunoprecipitated with Nrf2 was examined by immunoblotting using an anti-Keap1 antibody. Full blots are provided in Supplementary Fig. S11. (B) The bar graph shows the relative ratio of Keap1 against Nrf2 co-immunoprecipitated with the Myc antibody in cells treated with curcumin (CCM) or not. (C) HEK293 cells were transiently co-transfected with the ARE-Luc reporter and TK-Renilla plasmids along with the Myc-p62 wild-type or mutant (S349A) plasmid. After treatment with either DMSO (−) or 10 μM curcumin (+) for 12 h, the cells were assayed for the luciferase activity. Data shown are mean ± S.E. of three independent experiments and were analyzed using the Student’s t-test. (*p < 0.05, ***p < 0.001).

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Activation Assay, Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis, Luciferase, Activity Assay

Journal: Scientific reports

Article Title: Curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at Ser351.

doi: 10.1038/s41598-021-87225-8

Figure Lengend Snippet: Figure 8. Schematic diagram showing the mechanism of Nrf2 activation by curcumin. Curcumin induces the phosphorylation of p62 at S351 by PKCδ activation. Phosphorylated p62 interferes the association of Nrf2 with Keap1, stabilizing Nrf2. Accumulated Nrf2 moves into nucleus and induces the expression of its downstream genes such as GST-mu1, NQO1, HO1, and p62 etc. Increased p62 takes part in the stabilization of Nrf2 again, thus forming a positive-feedback loop.

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Activation Assay, Phospho-proteomics, Expressing

Journal: Molecular therapy. Nucleic acids

Article Title: TAZ ameliorates the microglia-mediated inflammatory response via the Nrf2-ROS-NF-κB pathway.

doi: 10.1016/j.omtn.2022.03.025

Figure Lengend Snippet: Figure 6. TAZ rescued microglia from apoptosis via Nrf2 (A and B) TAZ prevented microglia apoptosis via Nrf2. After introduction of the TAZ overexpression plasmid, cell apoptosis was evaluated in the absence or presence of the Nrf2 inhibitor ML385 by flow cytometry analysis or staining with Hoechst 33342, followed by visualization under a fluorescence microscope. N = 4. (C and D) TAZ attenuated Casp3 activity and reduced the levels of Casp3 and Bax mRNA along with recovery of Bcl2 expression through Nrf2. N = 5.

Article Snippet: After total and nuclear proteins were isolated and quantified using the kit, western blotting was performed with primary antibodies against TAZ (1:1,000, Thermo Fisher Scientific), phospho-TAZ (Ser89, 1:1,000, Cell Signaling Technology), Nrf2 (1:1,000, Proteintech), histone H3 (1:5,000, Proteintech), Gapdh (1:5,000, Proteintech), IkBa (1:1,000, Proteintech), phospho-IkBa (1:1,000, Cell Signaling Technology), andNF-kB subunit p65 (1:1,000, Cell Signaling Technology), as described previously.42 Measurement of antioxidant enzyme activity and GSH content After different treatments, proteins were extracted to assess the activity of the antioxidant enzymes SOD, CAT, GPX, and GR and resolve GSH content as well as the GSH/GSSG ratio in accordance with the corresponding assay kit (Beyotime).

Techniques: Over Expression, Plasmid Preparation, Cytometry, Staining, Microscopy, Activity Assay, Expressing

Journal: Molecular therapy. Nucleic acids

Article Title: TAZ ameliorates the microglia-mediated inflammatory response via the Nrf2-ROS-NF-κB pathway.

doi: 10.1016/j.omtn.2022.03.025

Figure Lengend Snippet: Figure 7. TAZ repressed NF-kB by enhancing antioxidant capacity dependent on Nrf2 (A) Western blot analysis of IkBa, phosphorylated IkBa, and p65 expression after transfection with the TAZ overexpression plasmid, followed by addition of the corresponding inhibitor for antioxidant enzymes, GSH synthesis, and Nrf2. p-IkBa, phosphorylated IkBa. N = 3. (B) Visualization of nc p65 after introduction of the TAZ overexpression plasmid and EGFP-p65 vector, followed by nc staining with Hoechst 33342 in the presence or absence of different inhibitors or the NF-kB activator BA. N = 3. (C) Determination of NF-kB transcriptional activity after introduction of the TAZ overexpression plasmid and pNFkB-luc vector in the presence or absence of different inhibitors or the NF-kB activator BA. N = 5. (D) Effects of the NF-kB activator BA on expression of IkBa, phosphorylated IkBa, and p65 after introduction of the TAZ overexpression plasmid. N = 3. (E) Activation of NF-kB by BA resisted the recruitment of TAZ on the levels for IL-1b, IL-6, and TNF-a mRNA. N = 5. (F) Activation of NF-kB by BA counteracted the rescue of TAZ on the release of IL-1b, IL-6 and TNF-a. N = 5.

Article Snippet: After total and nuclear proteins were isolated and quantified using the kit, western blotting was performed with primary antibodies against TAZ (1:1,000, Thermo Fisher Scientific), phospho-TAZ (Ser89, 1:1,000, Cell Signaling Technology), Nrf2 (1:1,000, Proteintech), histone H3 (1:5,000, Proteintech), Gapdh (1:5,000, Proteintech), IkBa (1:1,000, Proteintech), phospho-IkBa (1:1,000, Cell Signaling Technology), andNF-kB subunit p65 (1:1,000, Cell Signaling Technology), as described previously.42 Measurement of antioxidant enzyme activity and GSH content After different treatments, proteins were extracted to assess the activity of the antioxidant enzymes SOD, CAT, GPX, and GR and resolve GSH content as well as the GSH/GSSG ratio in accordance with the corresponding assay kit (Beyotime).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Staining, Activity Assay, Activation Assay

Journal: Molecular therapy. Nucleic acids

Article Title: TAZ ameliorates the microglia-mediated inflammatory response via the Nrf2-ROS-NF-κB pathway.

doi: 10.1016/j.omtn.2022.03.025

Figure Lengend Snippet: Figure 8. Schematic of TAZ regulation in the inflammatory response After shuttling into the nucleus, TAZ might drive transcription of Nrf2 by interacting with the TEAD transcription factor and induce nc translocation of Nrf2 to enhance antioxidant capacity with reduction of intracellular ROS, resulting in impediment of NF-kB activation to ameliorate the microglia-mediated inflammatory response. Simul- taneously, TAZ rescued microglia from apoptosis by preventing mitochondrial dysfunction, followed by blockage of mPTP opening and Cyt C release from mitochondria into the cytosol.

Article Snippet: After total and nuclear proteins were isolated and quantified using the kit, western blotting was performed with primary antibodies against TAZ (1:1,000, Thermo Fisher Scientific), phospho-TAZ (Ser89, 1:1,000, Cell Signaling Technology), Nrf2 (1:1,000, Proteintech), histone H3 (1:5,000, Proteintech), Gapdh (1:5,000, Proteintech), IkBa (1:1,000, Proteintech), phospho-IkBa (1:1,000, Cell Signaling Technology), andNF-kB subunit p65 (1:1,000, Cell Signaling Technology), as described previously.42 Measurement of antioxidant enzyme activity and GSH content After different treatments, proteins were extracted to assess the activity of the antioxidant enzymes SOD, CAT, GPX, and GR and resolve GSH content as well as the GSH/GSSG ratio in accordance with the corresponding assay kit (Beyotime).

Techniques: Translocation Assay, Activation Assay