Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A ) Immunofluorescence microscopy. Primary cultured hepatocytes isolated from 5-week-old Atg7 flox/flox and Atg7 flox/flox ;Alb- Cre mice were immunostained with anti-p62 antibody. Scale bars: 10 μm. ( B ) Electron microscopy. Representative electron micrographs of cytoplasmic regions in primary cultured hepatocytes from 5-week-old Atg7 flox/flox ;Alb- Cre mice. Boxed region is displayed at higher magnification. Typical p62 bodies, along with concentric membranous structures associated with the endoplasmic reticulum (ER), accumulated in Atg7 -deficient hepatocytes. Arrow indicates the concentric membranous structure, while arrowheads indicate p62 bodies. Scale bars: 5 μm; 500 nm. ( C ) Immunoblot analysis. Wild-type ATG7 or the active-site mutant ATG7 C572S was introduced into Atg7 -knockout primary cultured hepatocytes (isolated from 5-week-old Atg7 flox/flox ;Alb- Cre mice) using an adenoviral system. Cell lysates were collected at the indicated time points after infection and subjected to immunoblot analysis with the specified antibodies. The bar graphs present the quantitative densitometric analysis of p62, Ser351-phosphorylated p62, and KEAP1, normalized to GAPDH ( n = 3). Data are presented as means ± s.e. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s test. ( D ) Immunofluorescence microscopy. Wild-type ATG7 or the active-site mutant ATG7 C572S was introduced into Atg7 -knockout primary cultured hepatocytes (isolated from 5-week-old Atg7 flox/flox ;Alb- Cre mice) using an adenoviral system. Immunostaining was performed at the indicated time points after infection with a p62 antibody. The size and number of p62 bodies per cell were quantified ( n = 500 cells). Horizontal bars indicate medians, boxes represent the interquartile range (25th–75th percentiles), and whiskers extend to 1.5× the interquartile range; individual outliers are shown as points. Statistical analysis was performed using Welch’s t test. Scale bars: 10 μm (main panels), 1 μm (inset panels). Scale bars: 10 μm. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques: Immunofluorescence, Microscopy, Cell Culture, Isolation, Electron Microscopy, Western Blot, Mutagenesis, Knock-Out, Infection, Immunostaining

Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A , B ) Immunohistofluorescence analysis. Liver sections from Atg7 flox/flox , Atg7 flox/flox ; p62 S351A/S351A , Atg7 flox/flox ;Alb- Cre , and Atg7 flox/flox ;Alb- Cre ; p62 S351A/S351A mice ( A ) and from Atg7 flox/flox , Atg7 flox/flox ; p62 T352A/T352A , Atg7 flox/flox ;Alb- Cre , and Atg7 flox/flox ;Alb- Cre ; p62 T352A/T352A mice ( B ) at 3 month-old were immunostained with anti-p62 (left panels) and anti-KEAP1 (middle panels) antibodies. The right panels show the merged images of p62 (red) and KEAP1 (green). Arrowheads indicate examples of p62 bodies. Bars: 10 μm. The graph shows the mean intensity of KEAP1 in p62 bodies in the livers of each genotype ( n = 4) mice. Vertical bars are means ± s.e. Statistical analysis was performed by Welch’s t test. ( C ) Schematic diagram of KEAP1 retention within p62 bodies in three mouse lines: Atg7 flox/flox ;Alb- Cre ( Atg7 -KO), Atg7 flox/flox ;Alb- Cre ; p62 S351A/S351A ( Atg7 -KO; p62 S351A ) and Atg7 flox/flox ;Alb- Cre ; p62 T352A/T352A ( Atg7 -KO; p62 T352A ) mice. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques: Immunohistofluorescence

Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A , B ) Immunoblot analysis. Livers from cont., p62 S351A , Atg7 -KO, and Atg7 -KO; p62 S351A mice ( A ) and from cont., p62 T352A , Atg7 -KO, and Atg7 -KO; p62 T352A mice ( B ) at 3 month-old were separated into nuclear and cytoplasmic fractions. The cytoplasmic fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Cont., in which ATG7 is efficiently expressed at similar level of the wild-type mice were used as control. Graph shows the results of quantitative densitometric analysis of ubiquitinated proteins, KEAP1, p62 and Ser351-phosphorylated p62 relative to the whole protein content estimated using Ponceau-S staining ( n = 3). Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. Asterisks indicate non-specific bands, as they did not increase in the Atg7 knockout background. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques: Western Blot, SDS Page, Control, Staining, Knock-Out

Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A , B ) Immunoblot analysis. Wild-type p62 and indicated p62 mutants were introduced into p62 -knockout ( A ) or p62 and FIP200 -double knockout ( B ) Huh-1 cells, and the cell lysates were subjected to immunoblot analysis with indicated antibodies. Bar graph shows the results of quantitative densitometric analysis of KEAP1 relative to the whole protein content estimated using Ponceau-S staining ( n = 3). Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. ( C ) Immunofluorescence microscopy. Huh-1 cells indicated in ( A ) were immunostained with the indicated antibodies. Scale bars, 10 μm (main panels). The graph shows the mean intensity of KEAP1 in p62 bodies comprising of wild-type p62 ( n = 2469), p62 S349E ( n = 1057), p62 S349A ( n = 2992), or p62 T350A ( n = 2033). Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. ( D ) Immunofluorescence microscopy. Huh-1 cells indicated in ( B ) were immunostained with the indicated antibodies. Scale bars, 10 μm (main panels). The graph shows the mean intensity of KEAP1 in p62 bodies comprising of wild-type p62 ( n = 2892), p62 S349E ( n = 2611), p62 S349A ( n = 5191), or p62 T350A ( n = 3522). Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques: Western Blot, Knock-Out, Double Knockout, Staining, Immunofluorescence, Microscopy

Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A , B ) Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) from cont. ( n = 10), p62 S351A ( n = 7), Atg7 -KO ( n = 13), and Atg7 -KO; p62 S351A ( n = 7) mice ( A ) and from cont. ( n = 3), p62 T352A ( n = 10), Atg7 -KO ( n = 5), and Atg7 -KO; p62 T352A ( n = 6) mice ( B ) were measured. IU/l, international units/liter. Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. ( C ) Mechanism of liver injury development induced by autophagy suppression: KEAP1 retention via p62 body. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques:

Journal: Journal of Physiological Investigation

Article Title: MiR-367-3p Alleviates Oxidative Stress Injury in the Ischemia/Reperfusion Injury Cell Model by Targeting Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4-mediated Keap1/Nrf2/ARE Pathway

doi: 10.4103/ejpi.ejpi-d-24-00103

Figure Lengend Snippet: Figure 5: The regulatory role of miR-367-3p/NOX4 axis on inflammation, apoptosis, and oxidative stress-related molecules in oxygen-glucose deprivation/ reoxygenation (OGD/R) injury model. SH-SY5Y cells were transfected with si-NC, si-NOX4, miR-NC, mimics, or mimics plus oe-NOX4, respectively, followed by exposure to OGD/R. (a) Western blot analysis was employed to detect the protein expression of iNOS and cleaved caspase-3, and ImageJ was utilized for quantification. (b) The interaction between Keap1 and Nrf2 was detected by means of a CO-IP assay. (c) Western blot analysis was used to determine the protein expression of nuclear Nrf2 and cytoplasmic Nrf2. (d) The protein expression of Keap1, heme oxygenase-1, and NQO1 was ascertained through western blot analysis. The gray values of nuclear Nrf2 protein normalized with Lamin B1 and other proteins normalized with GAPDH were quantified by ImageJ. Experiments were performed with three independent biological replicates. Data were presented as the mean ± standard deviation ***P < 0.001, compared with control; #P < 0.05, ###P < 0.001, compared with OGD/R + si-NC; &&&P < 0.001, compared with OGD/R + miR-NC; $$P < 0.01, $$$P < 0.001, compared with OGD/R + mimics. ODG/R: Oxygen-glucose deprivation/reoxygenation, IgG: Immunoglobulin G.

Article Snippet: Subsequently, the supernatants were incubated with antibodies against Keap1 (1:1000, #8047, Cell signaling) and Nrf2 (1:1000, #12721, Cell signaling) or negative control immunoglobulin G (IgG) and protein IgG beads at 4°C overnight while being rotated.

Techniques: Transfection, Western Blot, Expressing, Co-Immunoprecipitation Assay, Standard Deviation, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Formononetin promotes porcine oocytes maturation and improves embryonic development by reducing oxidative stress

doi: 10.3389/fcell.2024.1520429

Figure Lengend Snippet: Information about the primer sequences.

Article Snippet: And incubation with horseradish peroxidase-conjugated secondary antibodies at RT for 1 h. Primary antibodies were rabbit monoclonal antibodies against NRF2 (1:2000, #16396-1-AP, Proteintech), KEAP1 (1:2000, #10503-2-AP, Proteintech), and β-Actin (1:5000, #20536-1-AP, Proteintech).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Formononetin promotes porcine oocytes maturation and improves embryonic development by reducing oxidative stress

doi: 10.3389/fcell.2024.1520429

Figure Lengend Snippet: Effect of FMN on mitochondrial function in oocytes and early embryos. (A) The mitochondria distribution at MII stage were stained with Mito-Tracker Red; scale bar, 50 μm. (B) The relative abundance of mitochondria in oocyte were analyzed for control and FMN-treated group. (C) Relative mRNA levels of ATP5B, NRF2 and KEAP1 in oocytes. (D) 4-cell stage embryos stained with JC-1. Scale bar, 100 μm. (E) Relative levels of JC-1 Red/Green fluorescence intensity in embryos were analyzed for control and FMN-treated group. (F) Relative mRNA levels of TFAM and NRF1 in embryos. (G) ATP levels of 4-cell stage embryos in both control and FMN-treated groups. Data are presented as the mean ± standard deviation (SD). * p < 0.05,** p < 0.01, *** p < 0.001 vs. 0 μM FMN group.

Article Snippet: And incubation with horseradish peroxidase-conjugated secondary antibodies at RT for 1 h. Primary antibodies were rabbit monoclonal antibodies against NRF2 (1:2000, #16396-1-AP, Proteintech), KEAP1 (1:2000, #10503-2-AP, Proteintech), and β-Actin (1:5000, #20536-1-AP, Proteintech).

Techniques: Staining, Control, Fluorescence, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Formononetin promotes porcine oocytes maturation and improves embryonic development by reducing oxidative stress

doi: 10.3389/fcell.2024.1520429

Figure Lengend Snippet: Effect of FMN on the expression levels of the Nrf2/Keap1 signaling pathway-related protein and genes in porcine blastocysts. (A) Porcine blastocysts incubated with or without FMN were stained with NRF2 (green) and DAPI (blue), scale bar, 50 μm. (B) Relative levels of NRF2 fluorescence intensity in blastocysts were analyzed for the control and FMN-treated group. (C) Relative mRNA expression levels of genes related to Nrf2/Keap1 pathway, NRF2, KEAP1, NQO1, UCHL1 in blastocysts. (D) Western blot analysis of NRF2 and KEAP1 protein expressions in blastocysts in the control and FMN-treated groups. Data were presented as the mean ± standard deviation (SD). ** p < 0.01,*** p < 0.001 vs. 0 μM FMN group.

Article Snippet: And incubation with horseradish peroxidase-conjugated secondary antibodies at RT for 1 h. Primary antibodies were rabbit monoclonal antibodies against NRF2 (1:2000, #16396-1-AP, Proteintech), KEAP1 (1:2000, #10503-2-AP, Proteintech), and β-Actin (1:5000, #20536-1-AP, Proteintech).

Techniques: Expressing, Incubation, Staining, Fluorescence, Control, Western Blot, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Formononetin promotes porcine oocytes maturation and improves embryonic development by reducing oxidative stress

doi: 10.3389/fcell.2024.1520429

Figure Lengend Snippet: Summary of the effect of Formononetin (FMN) on porcine oocyte in vitro maturation and early embryo in vitro . This schematic view illustrates that during the IVM stage, FMN enhances oocyte maturation in vitro by up-regulating genes related to CCs expansion and antioxidants, reducing oxidative stress, and improving mitochondrial function. During the IVC stage, FMN promotes the nuclear expression of Nrf2 through the P62-Keap1-Nrf2 pathway, which exerts antioxidant function and enhances mitochondrial function. Then, FMN promotes cell proliferation, reduces apoptosis, and lowers autophagy levels.

Article Snippet: And incubation with horseradish peroxidase-conjugated secondary antibodies at RT for 1 h. Primary antibodies were rabbit monoclonal antibodies against NRF2 (1:2000, #16396-1-AP, Proteintech), KEAP1 (1:2000, #10503-2-AP, Proteintech), and β-Actin (1:5000, #20536-1-AP, Proteintech).

Techniques: In Vitro, Expressing

Journal: bioRxiv

Article Title: Optical imaging of treatment-naïve human NSCLC reveals changes associated with metastatic recurrence

doi: 10.1101/2024.10.14.618213

Figure Lengend Snippet: Stitched mosaic map for optical redox ratio of NSCLC sample (A), corresponding KEAP1 stain from serial slide (B) and resulting registration from both images (C). Quantification of the average mean ORR over each region of KEAP1 expression revealed a lower ORR for the medium KEAP1 expression regions compared to low (p<0.). Scale bar at 500 µm.

Article Snippet: Briefly, slides were fixed with paraformaldehyde, blocked with 10% goat serum, and exposed to rabbit primary antibody against KEAP1 (dilution 1: 200, Proteintech, Chicago, IL), followed by anti-rabbit secondary antibody (Alexa Fluor 568, Invitrogen, Carlsbad, CA).

Techniques: Staining, Expressing