Journal: Nature Communications

Article Title: Neuronal development is promoted by weakened intrinsic antioxidant defences due to epigenetic repression of Nrf2

doi: 10.1038/ncomms8066

Figure Lengend Snippet: ( a ) Impact of Nrf2 deficiency on vulnerability to H 2 O 2 treatment, in DIV10 astrocytes (>98% GFAP + astrocytes) or neurons (cortical cultures containing >98% NeuN + neurons, <0.02% GFAP + astrocytes). Cells were treated with H 2 O 2 , fixed after 24 h, DAPI stained and scored for survival/death based on nuclear morphology. * P <0.05, Student's t -test here and throughout unless otherwise stated (neurons: n =4, 700–2000 cells analysed per condition per genotype (PCPG); astrocytes wt n =5, 700–2,000 cells PCPG, ko n =7, 2,100–2,700 cells PCPG). Data are displayed as mean±s.e.m. throughout. ( b ) Nrf2 target genes Cat and Gclc expression analysed by qRT–PCR, normalized to 18s rRNA, expressed relative to the level in pure neuronal cultures. Mixed cultures: approximately 10% GFAP + astrocytes, 90% NeuN + neurons. * P <0.05, Cat n =6, Gclc n =7. ( c , d ) Impact of Nrf2 deficiency on Cat or Gclc mRNA expression in neurons ( c ) and astrocytes ( d ). * P <0.05 compared with wild type (WT); Cat n =4, Gclc n =5. ( e ) qRT–PCR analysis of Nrf2 target gene (Hmox1, Srxn1 and xCT) expression in response to tBHQ (10 μM, 8 h) treatment of neuronal or mixed cultures. * P <0.05 ( n =5–6). ( f ) Immunohistochemical analysis of Hmox1 expression in tBHQ-treated mixed cultures. * P <0.05 ( n =5). Upper: example pictures illustrating elevation of Hmox1 in astrocytes within tBHQ-treated mixed cultures. Scale bar, 25 μm. ( g – i ) The effect of Keap1 deficiency on Nrf2 target genes. * P <0.05 (compared with WT control (Con), n =8–15). ( j ) Nrf2 mRNA analysed in parallel cultures of the indicated types. * P <0.05, ( n =10). ( k ) Western blot analysis of Nrf2 expression in different cell types treated with tBHQ (TB; 10 μM) or MG132 (MG; 5 μM) for 16 h. ( l ) Images of mixed cultures treated as indicated and stained with an anti-Nrf2 antibody (red) and a neuronal marker (Milli-Mark). White arrows highlight lack of Nrf2 induction in neurons. Scale bar, 25 μm. ( m ) qRT–PCR analysis of Nrf2 and neuronal/non-neuronal marker gene expression in RNA isolated from FAC-sorted adult cortical NeuN + neurons, expressed relative to levels in NeuN − cells. * P <0.05 ( n =4). See . ( n ) Expression of NRF2 ( NEF2L2 ) and GRIN1 analysed in astrocytes and neurons derived from H9 hESCs. * P <0.05 ( n =3). NS, not significant.

Article Snippet: Employed primary antibodies include: rabbit anti-Hmox1 (1:1,000, Stressgen Biotechnologies), mouse anti-GFAP (1:400, Sigma), rabbit anti-GFP (1:750, Life Technologies Ltd), Milli-Mark neuronal antibody cocktail (Millipore, 1:500) and Anti-Nrf2 (Cell Signaling).

Techniques: Staining, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Western Blot, Marker, Isolation, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Oxygen Desaturation Is Associated With Fibrocyte Activation via Epidermal Growth Factor Receptor/Hypoxia-Inducible Factor-1α Axis in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2022.852713

Figure Lengend Snippet: Higher percentage of circulating fibrocytes with greater lung function decline in COPD desaturators (A) . NANT cells were isolated from peripheral blood of non-desaturators (n = 12) and desaturators (n = 12) and the percentage of collagen I + /CD45 + fibrocyte in freshly isolated NANT cells on day 0 was determined by flow cytometry. Horizontal lines represent the mean values for each group. (B–F) All participants performed 6-minute walking tests at the beginning and during the 5-year follow-up. The correlation between the percentage of fibrocytes and the extent of post-exercise reduction in SpO 2 at baseline (B) , annualized change in FEV 1 /FVC ratio (C) , FEV 1 (D) , FVC (E) and 6MWD (F) are shown. The differences between non-desaturators and desaturators were determined by Mann-Whitney test and the correlation was determined by Spearman’s rank correlation. **** P < 0.0001. COPD, chronic obstructive pulmonary disease; NANT cells, non-adherent non-T fraction of peripheral blood mononuclear cells; FEV 1 , forced expiratory volume in 1 second; FVC, forced vital capacity; %pred, percent of predicted value; Ex, exercise; SpO 2 , peripheral blood oxygen saturation; 6MWD, 6-minute walking distance.

Article Snippet: Permeabilised NANT cells were incubated with FITC-conjugated rabbit anti-human CTGF antibodies (Abcam, Cambridge, UK), followed by staining with anti-COL I-PE (Merck Milli-Mark, CA, USA) and anti-CD45-PerCp polyclonal antibodies to evaluate the expression of CTGF in the fibrocytes.

Techniques: Isolation, Flow Cytometry, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Oxygen Desaturation Is Associated With Fibrocyte Activation via Epidermal Growth Factor Receptor/Hypoxia-Inducible Factor-1α Axis in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2022.852713

Figure Lengend Snippet: Identification of COL I + /CD34 + fibrocytes in the lung tissue of COPD desaturators (A–C) . The paraffin sections of lung tissue from COPD desaturators were stained for the CD34 surface marker ( green ), intracellular COL I ( red ), and nuclei ( blue ) (A) , and for hematoxylin and eosin staining (B) . The immunofluorescence staining and confocal microscopic analysis are described in Methods. (C) Shows a magnification of the merged image of (A) , and arrows indicate the double staining of COL I + /CD34 + fibrocytes with yellow enhancement in the merged image. Bar , 75 μm (A) and 25μm (C) . (D, E) The paraffin sections of airway tissue from COPD non-desaturators were stained for indicated immunofluorescence staining (D) and hematoxylin and eosin staining (E) , as described above. Bar , 75 μm (D) and 30 μm (E) . (F) The differences between the numbers of fibrocytes in specimens from non-desaturators and desaturators were determined by Mann-Whitney test. ** P < 0.01. COL I, collagen I; COPD, chronic obstructive pulmonary disease; In, interstitium; Ep, epithelium; En, endothelium; Al, alveoli.

Article Snippet: Permeabilised NANT cells were incubated with FITC-conjugated rabbit anti-human CTGF antibodies (Abcam, Cambridge, UK), followed by staining with anti-COL I-PE (Merck Milli-Mark, CA, USA) and anti-CD45-PerCp polyclonal antibodies to evaluate the expression of CTGF in the fibrocytes.

Techniques: Staining, Marker, Immunofluorescence, Double Staining, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Oxygen Desaturation Is Associated With Fibrocyte Activation via Epidermal Growth Factor Receptor/Hypoxia-Inducible Factor-1α Axis in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2022.852713

Figure Lengend Snippet: Higher CXCR4, CTGF, EGFR and HIF-1α expression with greater increased in number and myofibroblastic differentiation in fibrocytes from COPD desaturators. The number of Col I + /CD45 + fibrocytes (A) and differentiating α-SMA + fibrocytes (B) in NANT cells were determined by flow cytometry after 14 days in culture. NANT cells were isolated from COPD non-desaturators and desaturators. The proportion of CXCR4- (C) , CTGF- (D) , EGFR- (E) and HIF-1α- (F) expressing fibrocytes within freshly isolated NANT cells from desaturators, determined by flow cytometry, was higher compared to that from non-desaturators. Horizontal lines represent the median values for each group. The differences between disease groups were determined by Mann-Whitney test. * P < 0.05, ** P < 0.01, *** P < 0.001. CXCR4, CXC chemokine receptor 4; CTGF, connective tissue growth factor; EGFR, epidermal growth factor receptor; HIF-1α, hypoxia-inducible factor-1α; COPD, chronic obstructive pulmonary disease; NANT cells, non-adherent non-T fraction of peripheral blood mononuclear cells; α-SMA, α-smooth muscle actin.

Article Snippet: Permeabilised NANT cells were incubated with FITC-conjugated rabbit anti-human CTGF antibodies (Abcam, Cambridge, UK), followed by staining with anti-COL I-PE (Merck Milli-Mark, CA, USA) and anti-CD45-PerCp polyclonal antibodies to evaluate the expression of CTGF in the fibrocytes.

Techniques: Expressing, Flow Cytometry, Isolation, MANN-WHITNEY