Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Age-Specific Effects on Rat Lung Glutathione and Antioxidant Enzymes after Inhaling Ultrafine Soot

doi: 10.1165/rcmb.2012-0108OC

Figure Lengend Snippet: TAQMAN GENE EXPRESSION ASSAY CATALOG NUMBERS

Article Snippet: GCLm , Rn00568900_m1 , Glutamate-cysteine ligase regulatory subunit , {"type":"entrez-nucleotide","attrs":{"text":"NM_017305.2","term_id":"51036644"}} NM_017305.2.

Techniques: Gene Expression

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Age-Specific Effects on Rat Lung Glutathione and Antioxidant Enzymes after Inhaling Ultrafine Soot

doi: 10.1165/rcmb.2012-0108OC

Figure Lengend Snippet: GCLc and GCLm gene expression. RT-PCR expression in airway and parenchyma compartments in neonates and adult rats exposed to PFPs. Basal GCLc was consistently expressed in higher abundance than GCLm, and its highest expression was seen in adult airways (A). After PFP exposure, a transient drop in neonatal airway GCLc expression was observed in PFP24 compared with PFP2 (B). No treatment effects were detected in adult animals (C). Data are plotted as means ± SEM (n = 5–7 rats per group, per compartment, per gene). P < 0.05 are denoted as follows: *significantly different from neonates in the same compartment, and †significantly different from airways in the same age. PFP2, PFP24, and PFP48 refer to PFP exposure for 4, 24, and 48 hours, respectively.

Article Snippet: GCLm , Rn00568900_m1 , Glutamate-cysteine ligase regulatory subunit , {"type":"entrez-nucleotide","attrs":{"text":"NM_017305.2","term_id":"51036644"}} NM_017305.2.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Age-Specific Effects on Rat Lung Glutathione and Antioxidant Enzymes after Inhaling Ultrafine Soot

doi: 10.1165/rcmb.2012-0108OC

Figure Lengend Snippet: GCLc and GCLm protein analysis through Western blotting and immunohistochemistry. Representative GCLc and GCLm Western blots with actin loading control (A). GCLc/m Western blots were quantified: while neonatal GCLc/m expression remained unchanged after exposure (B), a significant up-regulation in GCLc was detected in PFP2 adult rats compared against FA controls (C). Data are plotted as means ± SEM (n = 6 rats per group). P < 0.05 is denoted as follows: ‡significantly different from FA in the same age. GCL immunohistochemical images in neonatal (D–G) and adult (H–K) rats reared in FA (D and H) and exposed to PFP: PFP2 (E and I), PFP24 (F and J), and PFP48 (G and K). Intense GCL staining was observed in adult and neonatale PFP2. In contrast to neonates, staining in adult PFP48 was continually up-regulated. High magnification inserts highlight GCL-positive cells in treated groups. Scale bars for D–K (shown in K) are 50 μm.

Article Snippet: GCLm , Rn00568900_m1 , Glutamate-cysteine ligase regulatory subunit , {"type":"entrez-nucleotide","attrs":{"text":"NM_017305.2","term_id":"51036644"}} NM_017305.2.

Techniques: Western Blot, Immunohistochemistry, Control, Expressing, Immunohistochemical staining, Staining

Journal: eBioMedicine

Article Title: Dysregulated anti-oxidant signalling and compromised mitochondrial integrity negatively influence regulatory T cell function and viability in liver disease

doi: 10.1016/j.ebiom.2023.104778

Figure Lengend Snippet: The Nrf2/HO-1 redox pathway is dysregulated in Tregs from LC patients. a. Nrf2 abundance in freshly isolated CD4 + CD25 + Tregs of healthy controls (HC) and liver cirrhotic (LC) patients relative to the loading control TFIID measured by Western Blot. HC n = 6, LC n = 5; unpaired t test. Mean (SD). b. Nuclear Nrf2 translocation following 12-h prostaglandin J2 (PGJ2) stimulation in freshly isolated HC and LC Tregs measured by ImageStream. HC/LC n = 5. Two-way repeated measures ANOVA with Bonferroni's correction for multiple pairwise comparison tests. Mean (SD). c–d. Relative abundance of HO-1 ( c. ) and Bach1 ( d. ) in freshly isolated HC and LC Tregs relative to the loading control TFIID measured by Western Blot. HO1: HC n = 6, LC n = 5; Bach1: HC n = 7, LC n = 5; unpaired t-test following log10 transformation. Median (IQR). e. Relative gene expression of Nrf2 targets NQO1 , GCLM and SOD1 in freshly isolated HC and LC Tregs (normalised to a HC calibrator sample). HC n = 15, LC n = 12. Multiple unpaired t-tests with Bonferroni's adjustment following log10 transformation. Median (IQR). f. GSEA of oxygen radical response in HC and LC patients. Top enriched genes are listed to the right. n = 7. g–i. Phenotype and function of murine Nrf2 −/− Tregs. g. Expression (MFI) of phenotypic markers (FoxP3 left, CD39 middle, CTLA-4 right) in freshly isolated Tregs of WT and Nrf2 −/− mice. n = 3, multiple unpaired t-tests with Bonferroni's adjustment. Mean (SD). h. Proportion of Annexin V + CD4 + CD25 + Tregs from WT and Nrf2 −/− mice following 3-days of CD3/CD28 stimulation. n = 5, unpaired t-test. Mean (SD). i. Suppressive function of Nrf2 −/− Tregs at different Treg:Teff ratios. n = 7, Two-way repeated measures ANOVA with Bonferroni's correction for multiple pairwise comparison. Mean (SD). j–l. CRISPR/Cas9 mediated HO-1 knock-out in human Tregs. j. Representative flow cytometry plot (left) and summary (right) of CRISPR/Cas9-mediated human HO-1 knock-out Tregs. Control conditions are wild-type (WT) Tregs and negative control (NC) Tregs (i.e. electroporated with a CRISPR/Cas9 complex not targeting the human genome). WT n = 10, NC n = 11, KO n = 12. Ordinary one-way ANOVA with Bonferroni's correction for multiple pairwise comparison tests following log10 transformation. Median (IQR). k. Suppressive capacity of modified Tregs (KO) and its controls (WT, NC) to inhibit Teff proliferation at various Treg:Teff ratios. n = 3 (NC) n = 4 (WT, KO). Linear mixed effect model with Bonferroni's correction for multiple pairwise comparison. (SD). p-values indicate comparison between NC and KO. l. Percentage of viable WT, NC and HO-1 KO Tregs following 18-h incubation at different concentrations of Hydrogen Peroxide (H 2 O 2 ). WT/NC/KO n = 3. Two-way repeated measures ANOVA with Bonferroni's correction for multiple pairwise comparison (SD). p-values indicate comparison between NC and KO. Abbreviations: Healthy control (HC), liver cirrhosis (LC), Nuclear factor erythroid 2-related factor 2 (Nrf2). Heme-oxygenase 1 (HO-1). NAD(P)H quinone dehydrogenase 1 (NQO1). Glutamate-cysteine ligase modifier subunit (GCLM). Superoxide dismutase 1 (SOD1). Prostaglandin J2 (PGJ2). Gene Set enrichment analysis (GSEA), Wild-type (WT), negative control (NC), knock-out (KO).

Article Snippet: Total RNA was extracted using the Quick-RNA MiniPrep kit (R1054, ZymoResearch) according to the manufacturer's protocol and reverse transcription was performed using the Reverse transcription High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Cat. 4374966). qPCR reactions were run with TaqMan Universal MasterMix II, no UNG (Applied Biosystems, Cat. 4440040) and TaqMan Gene Expression Assay Probes (Applied Biosystems; ACTB Hs01060665_g1 (VIC), NQO1 Hs01045993_g1 (FAM), GCLM Hs00978072_m1 (FAM), SOD1 Hs00533490_m1 (FAM), GPX2 Hs01591589_m1 (FAM) GSTA2 Hs00747232_mH (FAM)).

Techniques: Isolation, Control, Western Blot, Translocation Assay, Comparison, Transformation Assay, Gene Expression, Expressing, CRISPR, Knock-Out, Flow Cytometry, Negative Control, Modification, Incubation