Journal: bioRxiv

Article Title: Temporal Coordination of the Transcription Factor Response to H 2 O 2 stress

doi: 10.1101/2023.03.07.531593

Figure Lengend Snippet: (A) Uniform manifold approximation and projection (UMAP) plot of cells treated with PBS, 50μM and 75μM of H 2 O 2 after unsupervised clustering (n ≥ 10,000). Colors for cells based on the six clusters obtained. (B) UMAP of the same cells in A but colored based on sample. (C) UMAPs of cells from A colored by deviation scores for FOXO1 (left), RelA (middle) and HSF1 motifs (right) (D) UMAPs of cells from A showing deviation scores for p53 (left), Jun(middle) and NRF2 motifs (right) (E) Density colored scatter plots of log nuclear p53 (x-axis) and nuclear fraction of RelA (y-axis) at indicated levels of H 2 O 2 treatment for 5 hours (F) Percentage of cells activating both RelA and p53 (Both), RelA only, p53 only or neither for all concentrations of H 2 O 2 (G) Density colored scatter plots of log nuclear p53 (x-axis) and nuclear fraction of NFAT1 (y-axis) at indicated levels of H 2 O 2 treatment for 5 hours (H) Percentage of cells activating both NFAT1 and p53 (Both), NFAT1 only, p53 only or neither for all concentrations of H 2 O 2 . (I) Density colored scatter plots of log nuclear NRF2 (x-axis) and nuclear fraction of FOXO1(y-axis) at indicated levels of H 2 O 2 treatment for 5 hours (J) Percentage of cells activating both FOXO1 and NRF2 (Both), FOXO1 only, NRF2 only or neither for all concentrations of H 2 O 2 . (K) Density colored scatter plots of log nuclear JUN (x-axis) and nuclear fraction of FOXO1 (y-axis) at indicated levels of H 2 O 2 treatment for 5 hours (L) Percentage of cells activating both FOXO1 and JUN (Both), FOXO1 only, JUN only or neither for all concentrations of H 2 O 2 .

Article Snippet: Primary antibodies used: Anti-FOXO1 (C29H4) from Cell Signaling Cat# 2880S (1:500), Anti-p53 (DO-1) from Santa Cruz Cat# sc-126 (1:500), Anti-Sulfiredoxin from Santa Cruz Cat# sc-166786 (1:100), Recombinant Anti-Peroxiredoxin 1/PAG antibody [EPR5433] (ab109498) (1:1000), NRF2 (D1Z9C) XP Rabbit mAb #12721 (1:500), NFAT1 (D43B1) XP Rabbit mAb #5861 (1:300), NF-kB p65 (D14E12) XP Rabbit mAb #8242 (1:400), Anti-HSF1 antibody 10H8 from Stress Marq Biosciences (1:200), c-Fos (9F6) Rabbit mAb # 2250 (1:1000), and c-Jun (60A8) Rabbit mAb #9165 (1:400) from Cell Signaling.

Techniques:

Journal: bioRxiv

Article Title: Temporal Coordination of the Transcription Factor Response to H 2 O 2 stress

doi: 10.1101/2023.03.07.531593

Figure Lengend Snippet: (A) GSEA of p53 target genes in J14 + H 2 O 2 samples as compared to SRXN1-OE + H 2 O 2 samples (B) GSEA of NRF2 target genes in J14 + H 2 O 2 samples as compared to SRXN1-OE + H 2 O 2 samples. (C) Wald statistic and (D) log 2 fold changes of p53 target genes in H 2 O 2 treated cells vs. PBS controls, H 2 O 2 + J14 treated cells vs J14 treated controls and H 2 O 2 treated SRXN1-OE cells vs. SRXN1-OE PBS controls. (E) Wald statistic and (F) log 2 fold changes of NRF2 target genes in H 2 O 2 treated cells vs. PBS controls, H 2 O 2 + J14 treated cells vs J14 treated controls and H 2 O 2 treated SRXN1-OE cells vs. SRXN1-OE PBS controls. H 2 O 2 concentration is 50μM, J14 20μM.

Article Snippet: Primary antibodies used: Anti-FOXO1 (C29H4) from Cell Signaling Cat# 2880S (1:500), Anti-p53 (DO-1) from Santa Cruz Cat# sc-126 (1:500), Anti-Sulfiredoxin from Santa Cruz Cat# sc-166786 (1:100), Recombinant Anti-Peroxiredoxin 1/PAG antibody [EPR5433] (ab109498) (1:1000), NRF2 (D1Z9C) XP Rabbit mAb #12721 (1:500), NFAT1 (D43B1) XP Rabbit mAb #5861 (1:300), NF-kB p65 (D14E12) XP Rabbit mAb #8242 (1:400), Anti-HSF1 antibody 10H8 from Stress Marq Biosciences (1:200), c-Fos (9F6) Rabbit mAb # 2250 (1:1000), and c-Jun (60A8) Rabbit mAb #9165 (1:400) from Cell Signaling.

Techniques: Concentration Assay

Journal: Biomedicines

Article Title: Cadmium-Induced Kidney Injury in Mice Is Counteracted by a Flavonoid-Rich Extract of Bergamot Juice, Alone or in Association with Curcumin and Resveratrol, via the Enhancement of Different Defense Mechanisms

doi: 10.3390/biomedicines9121797

Figure Lengend Snippet: Oligonucleotide primers used for the quantitative Real-time PCR analyses.

Article Snippet: Primary antibodies IL-1β (1:250, Santa Cruz Biotechnology, Dallas, TX, USA) and Nrf2 (1:150, St. John’s Laboratory, London, UK) were incubated overnight at 4 °C in a moisturized chamber.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Biomedicines

Article Title: Cadmium-Induced Kidney Injury in Mice Is Counteracted by a Flavonoid-Rich Extract of Bergamot Juice, Alone or in Association with Curcumin and Resveratrol, via the Enhancement of Different Defense Mechanisms

doi: 10.3390/biomedicines9121797

Figure Lengend Snippet: Real-time PCR analysis of Nrf2 ( A ), Nqo1 ( B ) and Hmox1 ( C ). *** p < 0.001 vs. control mice; §§§ p < 0.001 vs. CdCl 2 -treated mice.

Article Snippet: Primary antibodies IL-1β (1:250, Santa Cruz Biotechnology, Dallas, TX, USA) and Nrf2 (1:150, St. John’s Laboratory, London, UK) were incubated overnight at 4 °C in a moisturized chamber.

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: Biomedicines

Article Title: Cadmium-Induced Kidney Injury in Mice Is Counteracted by a Flavonoid-Rich Extract of Bergamot Juice, Alone or in Association with Curcumin and Resveratrol, via the Enhancement of Different Defense Mechanisms

doi: 10.3390/biomedicines9121797

Figure Lengend Snippet: Immunohistochemical localization of Nrf2 in the kidneys. ( A ) In all control groups, Nrf2 immunoreactivity is particularly strong in the tubular wall (arrow). ( B ) In CdCl 2 plus vehicle-treated mice, no Nrf2 immunoreactivity is present. ( C – E ) In mice treated with CdCl 2 plus both doses of Cur and with CdCl 2 plus Re, Nrf2 shows a moderate positivity in some tubules (arrow). ( F ) In mice challenged with CdCl 2 plus BJe at the lower dose, Nrf2 immunoreactivity was lower (arrow) when compared to Cur and Re. ( G – I ) Mice treated with CdCl 2 plus BJe at 40 mg/kg and with CdCl 2 plus both associations: Nrf2 immunoreactivity is high (arrow), similar to controls. ( J ) Morphometric results for Nrf2 expression. Data are expressed in optical units/unit area (OU/UA) (from 0 = black to 255 = white). * p < 0.05 vs. control; § p < 0.05 vs. CdCl 2 plus vehicle. Scale bar: 50 µm.

Article Snippet: Primary antibodies IL-1β (1:250, Santa Cruz Biotechnology, Dallas, TX, USA) and Nrf2 (1:150, St. John’s Laboratory, London, UK) were incubated overnight at 4 °C in a moisturized chamber.

Techniques: Immunohistochemical staining, Control, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Protective effects of Rosavin on bleomycin-induced pulmonary fibrosis via suppressing fibrotic and inflammatory signaling pathways in mice.

doi: 10.1016/j.biopha.2019.108870

Figure Lengend Snippet: Fig. 8. The effect of Rosavin on the expression of TGF-β1, α-SMA, Nrf2 and NF-κB p65 of BIM-induced PF in mice. Values are the mean ± SD (n = 3); nsP > 0.05, ##P < 0.01 vs. control group. *P < 0.05, **P < 0.01 vs. Bleomycin control group.

Article Snippet: Rabbit anti-TGF-β1 antibody (21898-1-AP) was purchased from Proteintech (Wuhan, China), and mouse anti-α-SMA antibody (BM0002), rabbit anti-NF-Kb p65 antibody, rabbit anti-Nrf2 antibody were all obtained from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Expressing, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Distinct Modes of Balancing Glomerular Cell Proteostasis in Mucolipidosis Type II and III Prevent Proteinuria

doi: 10.1681/ASN.2019090960

Figure Lengend Snippet: Glomerular cells of MLII but not of MLIII mice activate the integrated stress response (ISR) to alleviate proteostatic stress. (A) Immunoblot for the mTORC1 target S6 and its phosphorylated form p-S6 in MLII and MLIII mice demonstrates downregulation of total S6 in MLII and MLIII glomeruli, whereas the ratio of p-S6/S6 is not altered. Graphs show densitometric quantification normalized to β-actin of the same membrane, the relative levels to respective wild-type littermate controls are shown (mean±SEM, Mann–Whitney U test, n≥7, *P≤0.05). (B) High-resolution confocal microscopy for the transcription factor (TF) TFEB, which translocates to the nucleus to initiate the transcription of lysosomal genes in the setting of decreased mTORC1 activity. White arrows point toward podocyte (p) and endothelial cells (ec) with nuclear TFEB signal. (C) Activation of the ISR as a pathway to downregulate protein translation was evaluated by qPCR in MLIII and MLII glomeruli. Graph shows the relative gene of interest (GOI) expression to respective wild-type (WT) littermate controls (dashed line); internal controls used were 18S and glyceraldehyde-3-phosphate dehydrogenase (mean±SEM, Mann–Whitney U test, n≥5, *P≤0.05). (D) Immunoblot for the expression and activity (phosphorylation) levels of eIF2α as the core event of the ISR leading to a decrease in global protein synthesis and to the induction of selected genes such as the transcription factor ATF4 to promote cellular recovery in isolated glomeruli of MLIII and MLII mice. Graphs represent densitometric quantification normalized to β-actin of the same membrane, relative levels to respective wild-type littermate controls are shown (mean±SEM, Mann–Whitney U test, n≥7, *P≤0.05). High-resolution confocal microscopy exhibits expression of (E) activated p-eIF2α and its downstream target the transcription factor ATF4, (F) the activated ER stress sensor p-PERK, and (G) the transcription factors NRF1 and activated p-NRF2, which induce the transcription of proteasome genes. Podocyte slit membrane was visualized by staining for nephrin (red), DNA was counterstained with Hoechst (blue). Note the enhanced signal for p-PERK in MLII endothelial cells, for NRF1 and to a lesser extent for p-NRF2 in MLIII podocytes, of p-eIF2α in MLII podocytes and endothelial cells, and the marked ATF4 nuclear signal in MLII glomerular cells (white arrows). Rel., relative.

Article Snippet: Primary antibodies used for the study were rat anti–UCH-L1 (immunofluorescence [IF] microscopy 1:50; Western blot [WB] 1:250, self-made clone U104 17 ), guinea pig anti-nephrin (IF 1:100; Acris), mouse anti-ubiquitin (WB 1:250; Millipore), rabbit anti-ubiquitin (IF 1:300; Novus), rabbit anti–K48-polyubiquitin (WB 1:1000; Abcam), rabbit anti- β 5 (WB 1:1000; X. Wang, University of South Dakota), rabbit anti-LMP7 (WB 1:5000; E.K.), rabbit anti-Limp2 (WB 1:1000, IF 1:1000; P. Saftig, University of Kiel, Kiel, Germany), rabbit anti-Lamp2 (WB 1:1000, IF 1:400; Sigma-Aldrich), rabbit anti-LC3B (WB 1:1000, IF 1:50; Sigma-Aldrich), mouse anti– β -actin (WB 1:10,000; Sigma-Aldrich), goat anti–kidney injury molecule 1 (IF 1:1000; R&D Systems), rabbit anti–smooth muscle actin (IF 1:400; Abcam), rabbit anti–cleaved caspase 3 (IF 1:100; Cell Signaling), rhodamine wheat germ agglutinin (IF 1:400; Vector), rabbit anti-pS6 (IF 1:100, WB 1:1000; Cell Signaling), rabbit anti-S6 (WB 1:1000), rabbit anti–p-PERK (IF 1:100; Invitrogen), rabbit anti-ATF4 (WB 1:1000, IF 1:100; Bioss), rabbit anti-NRF1 (WB 1:1000, IF 1:100; Cell Signaling), rabbit anti–p(S40)-NRF2 (WB 1:1000, IF 1:50; Bioss), rabbit anti–eukaryotic translation initiation factor 2 α (anti-eIF2 α ; WB 1:1000), rabbit anti–p-eIF2 α (WB 1:1000, IF 1:50; Cell Signaling), and guinea pig anti-p62 (IF 1:100; Progen).

Techniques: Western Blot, MANN-WHITNEY, Confocal Microscopy, Activity Assay, Activation Assay, Expressing, Isolation, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Distinct Modes of Balancing Glomerular Cell Proteostasis in Mucolipidosis Type II and III Prevent Proteinuria

doi: 10.1681/ASN.2019090960

Figure Lengend Snippet: Proposed compensatory mechanisms activated in MLII and MLIII glomerular cells to maintain proteostasis. (A) The Golgi-resident GlcNAc-1-phosphotransferase conjugates M6P residues to lysosomal enzymes, which then results in proper sorting of lysosomal enzymes to lysosomes after binding to M6P receptors. (B) In MLIII γ, the γ subunit of GlcNAc-1-phosphotransferase is mutated, resulting in a partial missorting of lysosomal enzymes outside the cell and to mild lysosomal dysfunction. In MLIII γ glomerular cell proteostasis (the balance between protein synthesis and degradation) is maintained (1) by upregulation of the ubiquitin proteasome system (UPS) by Nrf1/Nrf2 activation, and (2) by suppression of mTORC1 signaling, resulting in decreased protein translation through the S6Kinase-S6 pathway. (C) In MLII α/β, the α and β subunits of the GlcNAc-1-phosphotransferase are mutated, resulting in a severe missorting of lysosomal enzymes outside the cell and to severe lysosomal dysfunction. In MLII α/β glomerular cells, proteostasis is mainly maintained by suppression of protein translation by (1) suppression of mTORC1 signaling, and (2) activation of the integrated stress response (ISR) by the endoplasmic reticulum (ER) stress sensors and by cytosolic stress receptors. UPS transcripts and proteins are downregulated as a result of the “generally” suppressed protein translation. Ub, ubiquitin.

Article Snippet: Primary antibodies used for the study were rat anti–UCH-L1 (immunofluorescence [IF] microscopy 1:50; Western blot [WB] 1:250, self-made clone U104 17 ), guinea pig anti-nephrin (IF 1:100; Acris), mouse anti-ubiquitin (WB 1:250; Millipore), rabbit anti-ubiquitin (IF 1:300; Novus), rabbit anti–K48-polyubiquitin (WB 1:1000; Abcam), rabbit anti- β 5 (WB 1:1000; X. Wang, University of South Dakota), rabbit anti-LMP7 (WB 1:5000; E.K.), rabbit anti-Limp2 (WB 1:1000, IF 1:1000; P. Saftig, University of Kiel, Kiel, Germany), rabbit anti-Lamp2 (WB 1:1000, IF 1:400; Sigma-Aldrich), rabbit anti-LC3B (WB 1:1000, IF 1:50; Sigma-Aldrich), mouse anti– β -actin (WB 1:10,000; Sigma-Aldrich), goat anti–kidney injury molecule 1 (IF 1:1000; R&D Systems), rabbit anti–smooth muscle actin (IF 1:400; Abcam), rabbit anti–cleaved caspase 3 (IF 1:100; Cell Signaling), rhodamine wheat germ agglutinin (IF 1:400; Vector), rabbit anti-pS6 (IF 1:100, WB 1:1000; Cell Signaling), rabbit anti-S6 (WB 1:1000), rabbit anti–p-PERK (IF 1:100; Invitrogen), rabbit anti-ATF4 (WB 1:1000, IF 1:100; Bioss), rabbit anti-NRF1 (WB 1:1000, IF 1:100; Cell Signaling), rabbit anti–p(S40)-NRF2 (WB 1:1000, IF 1:50; Bioss), rabbit anti–eukaryotic translation initiation factor 2 α (anti-eIF2 α ; WB 1:1000), rabbit anti–p-eIF2 α (WB 1:1000, IF 1:50; Cell Signaling), and guinea pig anti-p62 (IF 1:100; Progen).

Techniques: Binding Assay, Activation Assay