Journal: Frontiers in Pediatrics

Article Title: Hydrogen regulates the aryl hydrocarbon receptor, improving bronchopulmonary dysplasia in neonatal rats and RLE-6TN cells exposed to hyperoxia

doi: 10.3389/fped.2025.1662922

Figure Lengend Snippet: H 2 ameliorates BPD induced by hyperoxia. (A) Hematoxylin–eosin staining was performed to assess the pathological conditions of the lung tissue in BPD rats, 20×, scale bar = 50 μm. (B) Radial alveolar count (RAC) analysis, AHR activation (phosphorylated form) is mainly enriched in the alveolar epithelial cell nuclei, while CPEB4 is highly expressed in the cytoplasm and perinuclear regions. (C) Quantification of the average surface area of a single alveolus (µm 2 ). (D) TUNEL staining was performed to detect apoptosis in the lung tissue. (E) Flow cytometry was employed to measure the expression of SP-A in BPD rats on day 14 from the Con, H 2 , BPD, and BPD + H 2 groups. (F) Flow cytometry was also used to evaluate the expression of SP-B on day 14 from the Con, H 2 , BPD, and BPD + H 2 groups. Data are presented as mean ± SD. ( n = 3).

Article Snippet: The cells were permeabilized with 0.5% Triton (9002-93-1, Solarbio, China) for 10 min and blocked with serum at 26 °C for 1 h. The cells were then incubated with CPEB4 antibody (1:250, 25342-1-AP, Proteintech, China) and AHR antibody (1:800, 67785-1-IG, Proteintech, China) at 4 °C overnight, followed with fluorescent secondary antibody, and incubated for 2 h at 26 °C.

Techniques: Staining, Activation Assay, TUNEL Assay, Flow Cytometry, Expressing

Journal: Frontiers in Pediatrics

Article Title: Hydrogen regulates the aryl hydrocarbon receptor, improving bronchopulmonary dysplasia in neonatal rats and RLE-6TN cells exposed to hyperoxia

doi: 10.3389/fped.2025.1662922

Figure Lengend Snippet: H 2 activates AHR and its downstream molecule CPEB4, thereby reducing endoplasmic reticulum stress. (A) Predicted downstream regulators of AHR are presented. (B) Immunohistochemical detection of AHR activation and CPEB4 expression in the lung tissue is shown, 60×, scale bar = 25 µm, AHR activation (phosphorylated form) is mainly enriched in the alveolar epithelial cell nuclei, while CPEB4 is highly expressed in the cytoplasm and perinuclear regions. (C–E) IOD quantitative analysis of the protein expression of p-AHR, AHR, and CPEB4. (F–H) Protein expression of p-AHR, AHR, CPEB4, p-IRE1α, IRE1α, and XBP1 was assessed by western blotting on days 3, 7, and 14, and the protein bands were quantified. Data are presented as mean ± SD. ( n = 3), * P < 0.05.

Article Snippet: The cells were permeabilized with 0.5% Triton (9002-93-1, Solarbio, China) for 10 min and blocked with serum at 26 °C for 1 h. The cells were then incubated with CPEB4 antibody (1:250, 25342-1-AP, Proteintech, China) and AHR antibody (1:800, 67785-1-IG, Proteintech, China) at 4 °C overnight, followed with fluorescent secondary antibody, and incubated for 2 h at 26 °C.

Techniques: Immunohistochemical staining, Activation Assay, Expressing, Western Blot

Journal: Frontiers in Pediatrics

Article Title: Hydrogen regulates the aryl hydrocarbon receptor, improving bronchopulmonary dysplasia in neonatal rats and RLE-6TN cells exposed to hyperoxia

doi: 10.3389/fped.2025.1662922

Figure Lengend Snippet: Effect of H 2 on RLE-6TN cells in a hyperoxic environment was investigated. (A) CCK-8 assay was performed to assess the viability of RLE-6TN cells. (B) Flow cytometry was performed to further evaluate the apoptosis of RLE-6TN cells. (C) qPCR was conducted to measure the mRNA levels of AHR and CPEB4 in RLE-6TN cells. (D) Western blotting was performed to analyze the expression of p-AHR, AHR, CPEB4, p-IRE1α, IRE1α, and XBP1 in RLE-6TN cells, followed by quantitative analysis of the protein bands. (E) Immunofluorescence microscopy was used to observe the nuclear translocation of AHR in RLE-6TN cells, 60×, scale bar = 25 µm. Data are presented as mean ± SD. ( n = 3), * P < 0.05.

Article Snippet: The cells were permeabilized with 0.5% Triton (9002-93-1, Solarbio, China) for 10 min and blocked with serum at 26 °C for 1 h. The cells were then incubated with CPEB4 antibody (1:250, 25342-1-AP, Proteintech, China) and AHR antibody (1:800, 67785-1-IG, Proteintech, China) at 4 °C overnight, followed with fluorescent secondary antibody, and incubated for 2 h at 26 °C.

Techniques: CCK-8 Assay, Flow Cytometry, Western Blot, Expressing, Immunofluorescence, Microscopy, Translocation Assay

Journal: Frontiers in Pediatrics

Article Title: Hydrogen regulates the aryl hydrocarbon receptor, improving bronchopulmonary dysplasia in neonatal rats and RLE-6TN cells exposed to hyperoxia

doi: 10.3389/fped.2025.1662922

Figure Lengend Snippet: Effect of AHR on RLE-6TN cells in a hyperoxic environment was investigated. (A) Cell viability of RLE-6TN cells. (B) Apoptosis of RLE-6TN cells. (C) mRNA expression levels of AHR in RLE-6TN cells. (D) Efficiency of AHR overexpression and knockdown in RLE-6TN cells was verified using western blotting. (E) Efficiency of AHR overexpression and knockdown in RLE-6TN cells was verified using qPCR. (F) CCK-8 method was employed to assess cell viability in RLE-6TN cells following AHR overexpression and knockdown. (G) ChIP-qPCR was utilized to examine the binding of AHR to the CPEB4 promoter ( n = 9 biological replicates from three independent experiments). Data are presented as mean ± SD, ( n = 3) for A-F, * P < 0.05.

Article Snippet: The cells were permeabilized with 0.5% Triton (9002-93-1, Solarbio, China) for 10 min and blocked with serum at 26 °C for 1 h. The cells were then incubated with CPEB4 antibody (1:250, 25342-1-AP, Proteintech, China) and AHR antibody (1:800, 67785-1-IG, Proteintech, China) at 4 °C overnight, followed with fluorescent secondary antibody, and incubated for 2 h at 26 °C.

Techniques: Expressing, Over Expression, Knockdown, Western Blot, CCK-8 Assay, ChIP-qPCR, Binding Assay

Journal: Frontiers in Pediatrics

Article Title: Hydrogen regulates the aryl hydrocarbon receptor, improving bronchopulmonary dysplasia in neonatal rats and RLE-6TN cells exposed to hyperoxia

doi: 10.3389/fped.2025.1662922

Figure Lengend Snippet: H 2 ameliorates BPD induced by hyperoxia. (A) Hematoxylin–eosin staining was performed to assess the pathological conditions of the lung tissue in BPD rats, 20×, scale bar = 50 μm. (B) Radial alveolar count (RAC) analysis, AHR activation (phosphorylated form) is mainly enriched in the alveolar epithelial cell nuclei, while CPEB4 is highly expressed in the cytoplasm and perinuclear regions. (C) Quantification of the average surface area of a single alveolus (µm 2 ). (D) TUNEL staining was performed to detect apoptosis in the lung tissue. (E) Flow cytometry was employed to measure the expression of SP-A in BPD rats on day 14 from the Con, H 2 , BPD, and BPD + H 2 groups. (F) Flow cytometry was also used to evaluate the expression of SP-B on day 14 from the Con, H 2 , BPD, and BPD + H 2 groups. Data are presented as mean ± SD. ( n = 3).

Article Snippet: The antibodies utilized in this study were as follows: p-AHR (1:1000, PA5-104880, Thermo Fisher, USA), AHR (1:800, 67785-1-IG, Proteintech, China), CPEB4 (1:250, 25342-1-AP, Proteintech, China), Goat Anti-Rabbit IgG H&L (HRP) (1:1000,GB23303, Servicebio, 1:200), and Goat Anti-Mouse IgG/HRP (1:1000, GB23301, Servicebio, 1:200).

Techniques: Staining, Activation Assay, TUNEL Assay, Flow Cytometry, Expressing

Journal: Frontiers in Pediatrics

Article Title: Hydrogen regulates the aryl hydrocarbon receptor, improving bronchopulmonary dysplasia in neonatal rats and RLE-6TN cells exposed to hyperoxia

doi: 10.3389/fped.2025.1662922

Figure Lengend Snippet: H 2 activates AHR and its downstream molecule CPEB4, thereby reducing endoplasmic reticulum stress. (A) Predicted downstream regulators of AHR are presented. (B) Immunohistochemical detection of AHR activation and CPEB4 expression in the lung tissue is shown, 60×, scale bar = 25 µm, AHR activation (phosphorylated form) is mainly enriched in the alveolar epithelial cell nuclei, while CPEB4 is highly expressed in the cytoplasm and perinuclear regions. (C–E) IOD quantitative analysis of the protein expression of p-AHR, AHR, and CPEB4. (F–H) Protein expression of p-AHR, AHR, CPEB4, p-IRE1α, IRE1α, and XBP1 was assessed by western blotting on days 3, 7, and 14, and the protein bands were quantified. Data are presented as mean ± SD. ( n = 3), * P < 0.05.

Article Snippet: The antibodies utilized in this study were as follows: p-AHR (1:1000, PA5-104880, Thermo Fisher, USA), AHR (1:800, 67785-1-IG, Proteintech, China), CPEB4 (1:250, 25342-1-AP, Proteintech, China), Goat Anti-Rabbit IgG H&L (HRP) (1:1000,GB23303, Servicebio, 1:200), and Goat Anti-Mouse IgG/HRP (1:1000, GB23301, Servicebio, 1:200).

Techniques: Immunohistochemical staining, Activation Assay, Expressing, Western Blot

Journal: Frontiers in Pediatrics

Article Title: Hydrogen regulates the aryl hydrocarbon receptor, improving bronchopulmonary dysplasia in neonatal rats and RLE-6TN cells exposed to hyperoxia

doi: 10.3389/fped.2025.1662922

Figure Lengend Snippet: Effect of H 2 on RLE-6TN cells in a hyperoxic environment was investigated. (A) CCK-8 assay was performed to assess the viability of RLE-6TN cells. (B) Flow cytometry was performed to further evaluate the apoptosis of RLE-6TN cells. (C) qPCR was conducted to measure the mRNA levels of AHR and CPEB4 in RLE-6TN cells. (D) Western blotting was performed to analyze the expression of p-AHR, AHR, CPEB4, p-IRE1α, IRE1α, and XBP1 in RLE-6TN cells, followed by quantitative analysis of the protein bands. (E) Immunofluorescence microscopy was used to observe the nuclear translocation of AHR in RLE-6TN cells, 60×, scale bar = 25 µm. Data are presented as mean ± SD. ( n = 3), * P < 0.05.

Article Snippet: The antibodies utilized in this study were as follows: p-AHR (1:1000, PA5-104880, Thermo Fisher, USA), AHR (1:800, 67785-1-IG, Proteintech, China), CPEB4 (1:250, 25342-1-AP, Proteintech, China), Goat Anti-Rabbit IgG H&L (HRP) (1:1000,GB23303, Servicebio, 1:200), and Goat Anti-Mouse IgG/HRP (1:1000, GB23301, Servicebio, 1:200).

Techniques: CCK-8 Assay, Flow Cytometry, Western Blot, Expressing, Immunofluorescence, Microscopy, Translocation Assay

Journal: Frontiers in Pediatrics

Article Title: Hydrogen regulates the aryl hydrocarbon receptor, improving bronchopulmonary dysplasia in neonatal rats and RLE-6TN cells exposed to hyperoxia

doi: 10.3389/fped.2025.1662922

Figure Lengend Snippet: Effect of AHR on RLE-6TN cells in a hyperoxic environment was investigated. (A) Cell viability of RLE-6TN cells. (B) Apoptosis of RLE-6TN cells. (C) mRNA expression levels of AHR in RLE-6TN cells. (D) Efficiency of AHR overexpression and knockdown in RLE-6TN cells was verified using western blotting. (E) Efficiency of AHR overexpression and knockdown in RLE-6TN cells was verified using qPCR. (F) CCK-8 method was employed to assess cell viability in RLE-6TN cells following AHR overexpression and knockdown. (G) ChIP-qPCR was utilized to examine the binding of AHR to the CPEB4 promoter ( n = 9 biological replicates from three independent experiments). Data are presented as mean ± SD, ( n = 3) for A-F, * P < 0.05.

Article Snippet: The antibodies utilized in this study were as follows: p-AHR (1:1000, PA5-104880, Thermo Fisher, USA), AHR (1:800, 67785-1-IG, Proteintech, China), CPEB4 (1:250, 25342-1-AP, Proteintech, China), Goat Anti-Rabbit IgG H&L (HRP) (1:1000,GB23303, Servicebio, 1:200), and Goat Anti-Mouse IgG/HRP (1:1000, GB23301, Servicebio, 1:200).

Techniques: Expressing, Over Expression, Knockdown, Western Blot, CCK-8 Assay, ChIP-qPCR, Binding Assay

Journal: eLife

Article Title: Inhibition of Cpeb3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs and enhances object location memory

doi: 10.7554/eLife.90116

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-CPEB4 (rabbit polyclonal) , Proteintech , Cat# 25342-1-AP , 1:1000.

Techniques: Saline, Western Blot, Stripping, Bicinchoninic Acid Protein Assay, Lysis, SYBR Green Assay, Reverse Transcription, Software