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: Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association

Article Title: Antiferroptotic properties of allicin and related organosulfur compounds-diallyl disulfide and diallyl trisulfide-from Garlic.

doi: 10.1016/j.fct.2024.115124

Figure Lengend Snippet: Fig. 4. The antiferroptotic effect of allicin is independent of Nrf2-regulated phase II enzyme expression. A. Western blot analysis showing that allicin upregulated expression of the phase II enzymes GCLM, GCLC, and HO-1 in WT HT22 cells but not Nrf2-KD HT22 cells. Cells were incubated with allicin (1 or 10 μM) for 16 h (Upper) Representative immunoblot. (Lower) Band intensities as quantified using ImageJ. Results are presented as the mean ± SD. *P < 0.05; ****P < 0.0001; ns, not significant. B. Allicin protected both WT and Nrf2-KD HT22 cells against erastin-induced death. Cells were treated with the indicated concentrations of allicin in the absence or presence of erastin for 24 h. Cell death was measured using an LDH assay. Results are presented as the mean ± SD. ###P < 0.001; ####P < 0.0001 compared to the corresponding erastin alone by ANOVA with post hoc Tukey’s test.

Article Snippet: Nrf2-knockdown (KD) HT22 cells were generated using the Nrf2 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, Dallas, TX, USA, cat# sc-421869) and the Nrf2 HDR plasmid (Santa Cruz Biotechnology, cat# sc-421869-HDR) as previously described (Hirata et al., 2020).

Techniques: Expressing, Western Blot, Incubation, Lactate Dehydrogenase Assay

Journal: Pharmaceutics

Article Title: Colon-Targeted Poly(ADP-ribose) Polymerase Inhibitors Synergize Therapeutic Effects of Mesalazine Against Rat Colitis Induced by 2,4-Dinitrobenzenesulfonic Acid.

doi: 10.3390/pharmaceutics16121546

Figure Lengend Snippet: Figure 3. AQSA-Glu potentiates the anticolitic activity of 5-AIQ. Three days after colitis induction by DNBS, 5-AIQ (5 mg/kg) and AQSA-Glu [7.5 mg/kg, equivalent to 2.5 mg/kg of 5-AIQ (L) and 15 mg/kg, equivalent to 5 mg/kg of 5-AIQ (H)] were administered orally to rats once per day, and the rats were euthanized 24 h after the sixth treatment. (A) Left panel: photos of the distal colons of rats in which serosal and luminal sides are shown separately. Right panel: overall colonic damage was scored for each group and presented as colonic damage score (CDS). * α < 0.05 vs. DNBS control. (B) H & E staining was performed with the colonic tissue sections of rats subjected to various treatments. Upper panel: representative images of 100× magnification. Lower panel: representative images of 200× magnification for the dotted boxes in the upper panel. In the inflamed distal colons (4.0 cm), (C) myeloperoxidase (MPO) activity was measured in addition to determining the levels of (D) CINC-3 and (E) iNOS and COX-2 using an Elisa kit and Western blotting. A loading control (α-Tubulin) was used for Western blot analysis of COX-2 and iNOS. NM: not measurable. The data are represented as mean ± SD (n = 5). * p < 0.05 vs. DNBS control # p < 0.05.

Article Snippet: Nrf2, HO-1, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) proteins were detected using the following antibodies: anti-Nrf2 (sc-365949, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HO-1 (sc-136961, Santa Cruz Biotechnology), anti-COX-2 (sc-365374, Santa Cruz Biotechnology), anti-iNOS (NOS-2) antibody (sc-7271, Santa Cruz Biotechnology), and secondary antibodies corresponding to the primary antibodies (Santa Cruz Biotechnology).

Techniques: Activity Assay, Control, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Pharmaceutics

Article Title: Colon-Targeted Poly(ADP-ribose) Polymerase Inhibitors Synergize Therapeutic Effects of Mesalazine Against Rat Colitis Induced by 2,4-Dinitrobenzenesulfonic Acid.

doi: 10.3390/pharmaceutics16121546

Figure Lengend Snippet: Figure 4. Colon-targeted PARP inhibitors synergize the anticolitic effects of mesalazine. (A) RAW264.7 cells pretreated with 5-ASA (20 mM), 3-AB (1 mM), and 5-AIQ (10 µM) for 1 h were challenged with LPS for 24 h. The levels of iNOS and COX-2 proteins were analyzed using West- ern blotting. (B) SSZ (50 mg/kg) and ABSA (36 mg/kg, equimolar to 50 mg/kg of SSZ) suspended in PBS (1 mL) were administered orally to rats. The rats were killed 2, 4, and 8 h after oral administration. The concentrations of 5-ASA in the cecum were analyzed using HPLC. (C) Three days after colitis induction by DNBS, SSZ (50 mg/kg), AQSA-Glu (15 mg/kg), a mixture of AQSA-Glu (15 mg/kg) + olsalazine (OSZ, 19 mg/kg, half-equimolar to 50 mg/kg of SSZ), and ABSA (36 mg/kg, equimolar to 50 mg/kg of SSZ) were administered orally to rats once per day, and the rats were euthanized 24 h after the sixth treatment. (C) Left panel: photos of the distal colons of rats where serosal and luminal sides are shown separately. Right panel: overall colonic damage was scored for each group and presented as colonic damage score (CDS). * α < 0.05 vs. DNBS control. (D) H & E staining was performed with the colonic tissue sections of rats subjected to various treatments. Upper panel: rep- resentative images of 100× magnification. Lower panel: representative images of 200× magnification for the dotted boxes in the upper panel. In the inflamed distal colons (4.0 cm), (E) myeloperoxidase (MPO) activity was measured in addition to determining the levels of (F) CINC-3 and (G) iNOS and COX-2 using an Elisa kit and Western blotting. A loading control (α-Tubulin) was used for Western blot analysis of COX-2 and iNOS. NM: not measurable. The data are represented as mean ± SD (n = 5). * p < 0.05 vs. DNBS control # p < 0.05.

Article Snippet: Nrf2, HO-1, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) proteins were detected using the following antibodies: anti-Nrf2 (sc-365949, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HO-1 (sc-136961, Santa Cruz Biotechnology), anti-COX-2 (sc-365374, Santa Cruz Biotechnology), anti-iNOS (NOS-2) antibody (sc-7271, Santa Cruz Biotechnology), and secondary antibodies corresponding to the primary antibodies (Santa Cruz Biotechnology).

Techniques: Control, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 2. Keap1 protein decreases in differentiated adipocytes, while steady-state mRNA expression for Nrf2 and Keap1 increases. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 4, 8, 11, and 14. (A) Nrf2 and (C) Keap1 (70 kDa) protein expression was examined by immunoblotting. Black bars, full-length (FL) Nrf2 (56 kDa); gray bars, degraded (Deg) Nrf2 (40 kDa); white bars, total (Tot) Nrf2; the sums of full-length Nrf2 and degraded Nrf2 were determined to obtain relative total Nrf2 protein expression. (B) Relative steady-state Nrf2 and (D) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Expressing, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 3. NQO1 protein increases during limited clonal expansion and postmitotic growth arrest of differentiation. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 1, 2, 3, and 4. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 4. NQO1 and Keap1 proteins, but not mRNA, decreases as adipocyte differentiation progresses. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 4, 5, 6, 7, and 8. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 4. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 6. LiCl treatment increases NQO1 protein, but not NQO1 mRNA. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of LiCl at Day 8 and harvested on Day 11. A 40 mM NaCl was used as a vehicle control (VC). (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Control, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 7. Sulforaphane increases NQO1 protein and mRNA levels. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of sulforaphane (SFN). A 0.3% DMSO was used as a VC. (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot