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Increased ID1 and ID3 expression in pulmonary fibrosis. (A) (Left) Representative immunofluorescence images of lung sections from healthy donors and IPF patients showing ID1/ID3 (green) and Fibronectin (red) co-staining. Nuclei were counterstained with DAPI (blue). Scale bar: 100 µm. (Right) Quantification of the data. n = 6 patients per group. (B) (Left) UMAP plots showing the distribution of COL1A1⁺ fibroblasts in healthy and IPF samples, along with PDGFRA and PDGFRB expression. (Right) Violin plots showing the expression levels of Col1a1, ID1, and ID3 in fibroblasts from healthy and IPF lungs. (C-D) ID1 and ID3 <t>mRNA</t> (C) and protein (D) levels determined by real time qPCR and Western blot analyses in lung fibroblasts isolated from healthy donors and patients with IPF (n = 6/group). (E) ID1 and ID3 mRNA levels in healthy human lung fibroblasts treated with TGF-β1 (5 ng/ml) for 48 hours. n = 6-7 experiments performed in triplicate. (F) ID1 and ID3 protein levels in healthy human lung fibroblasts treated with TGF-β1 (5 ng/ml) for 48 hours. n = 4. (G) ID1 and ID3 mRNA levels in lungs of mice with pulmonary fibrosis (n = 6-8 mice/group). (H) ID1 and ID3 protein levels in lung homogenates 10 and 21 days after PBS or BLM injection (n = 3 mice/group). (I) ID1 and ID3 mRNA levels in lung fibroblasts isolated from the lungs of PBS or BLM-treated mice (n = 4 mice/group). (J) ID1 and ID3 protein levels in lung fibroblasts from control and BLM-treated mice (n = 4 mice/group). * P < 0.05, ** P < 0.01, by t-test for all panels, except for (H), which was analyzed using one-way-ANOVA.
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Increased ID1 and ID3 expression in pulmonary fibrosis. (A) (Left) Representative immunofluorescence images of lung sections from healthy donors and IPF patients showing ID1/ID3 (green) and Fibronectin (red) co-staining. Nuclei were counterstained with DAPI (blue). Scale bar: 100 µm. (Right) Quantification of the data. n = 6 patients per group. (B) (Left) UMAP plots showing the distribution of COL1A1⁺ fibroblasts in healthy and IPF samples, along with PDGFRA and PDGFRB expression. (Right) Violin plots showing the expression levels of Col1a1, ID1, and ID3 in fibroblasts from healthy and IPF lungs. (C-D) ID1 and ID3 mRNA (C) and protein (D) levels determined by real time qPCR and Western blot analyses in lung fibroblasts isolated from healthy donors and patients with IPF (n = 6/group). (E) ID1 and ID3 mRNA levels in healthy human lung fibroblasts treated with TGF-β1 (5 ng/ml) for 48 hours. n = 6-7 experiments performed in triplicate. (F) ID1 and ID3 protein levels in healthy human lung fibroblasts treated with TGF-β1 (5 ng/ml) for 48 hours. n = 4. (G) ID1 and ID3 mRNA levels in lungs of mice with pulmonary fibrosis (n = 6-8 mice/group). (H) ID1 and ID3 protein levels in lung homogenates 10 and 21 days after PBS or BLM injection (n = 3 mice/group). (I) ID1 and ID3 mRNA levels in lung fibroblasts isolated from the lungs of PBS or BLM-treated mice (n = 4 mice/group). (J) ID1 and ID3 protein levels in lung fibroblasts from control and BLM-treated mice (n = 4 mice/group). * P < 0.05, ** P < 0.01, by t-test for all panels, except for (H), which was analyzed using one-way-ANOVA.

Journal: Theranostics

Article Title: Simultaneous inhibition of ID1 and ID3 mitigates fibroblast activation via cell cycle and MEK/ERK pathways in pulmonary fibrosis

doi: 10.7150/thno.127118

Figure Lengend Snippet: Increased ID1 and ID3 expression in pulmonary fibrosis. (A) (Left) Representative immunofluorescence images of lung sections from healthy donors and IPF patients showing ID1/ID3 (green) and Fibronectin (red) co-staining. Nuclei were counterstained with DAPI (blue). Scale bar: 100 µm. (Right) Quantification of the data. n = 6 patients per group. (B) (Left) UMAP plots showing the distribution of COL1A1⁺ fibroblasts in healthy and IPF samples, along with PDGFRA and PDGFRB expression. (Right) Violin plots showing the expression levels of Col1a1, ID1, and ID3 in fibroblasts from healthy and IPF lungs. (C-D) ID1 and ID3 mRNA (C) and protein (D) levels determined by real time qPCR and Western blot analyses in lung fibroblasts isolated from healthy donors and patients with IPF (n = 6/group). (E) ID1 and ID3 mRNA levels in healthy human lung fibroblasts treated with TGF-β1 (5 ng/ml) for 48 hours. n = 6-7 experiments performed in triplicate. (F) ID1 and ID3 protein levels in healthy human lung fibroblasts treated with TGF-β1 (5 ng/ml) for 48 hours. n = 4. (G) ID1 and ID3 mRNA levels in lungs of mice with pulmonary fibrosis (n = 6-8 mice/group). (H) ID1 and ID3 protein levels in lung homogenates 10 and 21 days after PBS or BLM injection (n = 3 mice/group). (I) ID1 and ID3 mRNA levels in lung fibroblasts isolated from the lungs of PBS or BLM-treated mice (n = 4 mice/group). (J) ID1 and ID3 protein levels in lung fibroblasts from control and BLM-treated mice (n = 4 mice/group). * P < 0.05, ** P < 0.01, by t-test for all panels, except for (H), which was analyzed using one-way-ANOVA.

Article Snippet: RNA integrity analysis, library preparation, sequencing, and raw read count generation were performed commercially by Novogene (Sacremento, CA, USA) using their Human mRNA Sequencing pipeline.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Isolation, Injection, Control

Knockdown of ID1/ID3 reduces human lung fibroblast proliferation, migration, and differentiation into myofibroblasts. (A) ID1 and ID3 mRNA levels in healthy human lung fibroblasts transfected with siRNA-Ctrl, siRNA-ID1 or siRNA-ID3 (5nM each) for 48 hours. n = 4-5 experiments performed in triplicate. (B) (Left) Representative immunoblots showing ID1, ID3 and GAPDH protein levels in healthy human lung fibroblasts transfected with siRNA-Ctrl, siRNA-ID1 or siRNA-ID3 (5nM each) for 48 hours. (Right) Quantification of the data. n = 4. (C) Proliferation of healthy and IPF-diseased human lung fibroblasts in the presence of the indicated treatments. n = 6 experiments performed in triplicate. (D-E) Migration of healthy (D) and IPF-derived (E) human lung fibroblasts in the presence of the indicated treatments. n = 4-6 experiments performed in duplicate. Scale bar: 100 µm. (F) qPCR assessment of Acta2 and Col1a1 mRNA levels in healthy HLF treated with siRNA-Ctrl or siRNA-ID1ID3 in the absence or presence of TGF-β1 (5 ng/ml). n = 3 experiments performed in triplicate. (G) (Left) Representative immunoblots showing Collagen1, α-SMA and GAPDH protein levels in healthy human lung fibroblasts transfected with siRNA-Ctrl or siRNA-ID1ID3 in the absence or presence of TGF-β1 (5 ng/ml). (Right) Quantification of the data. n = 4. (H) qPCR assessment of Acta2 and Col1a1 mRNA levels in IPF-derived HLF treated with siRNA-Ctrl or siRNA-ID1ID3 in the absence or presence of TGF-β1 (5 ng/ml). n = 3 experiments performed in triplicate. (G) (Left) Representative immunoblots showing Collagen1, α-SMA and GAPDH protein levels in IPF-derived HLFs transfected with siRNA-Ctrl or siRNA-ID1ID3 in the absence or presence of TGF-β1 (5 ng/ml). (Right) Quantification of the data. n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA (A and B) or two-way ANOVA (C-I).

Journal: Theranostics

Article Title: Simultaneous inhibition of ID1 and ID3 mitigates fibroblast activation via cell cycle and MEK/ERK pathways in pulmonary fibrosis

doi: 10.7150/thno.127118

Figure Lengend Snippet: Knockdown of ID1/ID3 reduces human lung fibroblast proliferation, migration, and differentiation into myofibroblasts. (A) ID1 and ID3 mRNA levels in healthy human lung fibroblasts transfected with siRNA-Ctrl, siRNA-ID1 or siRNA-ID3 (5nM each) for 48 hours. n = 4-5 experiments performed in triplicate. (B) (Left) Representative immunoblots showing ID1, ID3 and GAPDH protein levels in healthy human lung fibroblasts transfected with siRNA-Ctrl, siRNA-ID1 or siRNA-ID3 (5nM each) for 48 hours. (Right) Quantification of the data. n = 4. (C) Proliferation of healthy and IPF-diseased human lung fibroblasts in the presence of the indicated treatments. n = 6 experiments performed in triplicate. (D-E) Migration of healthy (D) and IPF-derived (E) human lung fibroblasts in the presence of the indicated treatments. n = 4-6 experiments performed in duplicate. Scale bar: 100 µm. (F) qPCR assessment of Acta2 and Col1a1 mRNA levels in healthy HLF treated with siRNA-Ctrl or siRNA-ID1ID3 in the absence or presence of TGF-β1 (5 ng/ml). n = 3 experiments performed in triplicate. (G) (Left) Representative immunoblots showing Collagen1, α-SMA and GAPDH protein levels in healthy human lung fibroblasts transfected with siRNA-Ctrl or siRNA-ID1ID3 in the absence or presence of TGF-β1 (5 ng/ml). (Right) Quantification of the data. n = 4. (H) qPCR assessment of Acta2 and Col1a1 mRNA levels in IPF-derived HLF treated with siRNA-Ctrl or siRNA-ID1ID3 in the absence or presence of TGF-β1 (5 ng/ml). n = 3 experiments performed in triplicate. (G) (Left) Representative immunoblots showing Collagen1, α-SMA and GAPDH protein levels in IPF-derived HLFs transfected with siRNA-Ctrl or siRNA-ID1ID3 in the absence or presence of TGF-β1 (5 ng/ml). (Right) Quantification of the data. n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA (A and B) or two-way ANOVA (C-I).

Article Snippet: RNA integrity analysis, library preparation, sequencing, and raw read count generation were performed commercially by Novogene (Sacremento, CA, USA) using their Human mRNA Sequencing pipeline.

Techniques: Knockdown, Migration, Transfection, Western Blot, Derivative Assay

Genetic deletion of ID1 and ID3 reduces bleomycin-induced lung fibrosis. (A) Design of the study. (B) PCR analysis of ID1 and ID3 mRNA levels in lungs of the indicated groups. n=6-7 mice/group. (C) Lung function parameters, including inspiratory capacity, compliance and single frequency elastance. n=7 mice/group. (D) Lung protein expression of Collagen1 and α-SMA in lungs from the indicated groups. n=4-6 mice/group. (E) (Left) Representative images from Fast Green/Sirius-Red-stained lungs of the indicated groups. n=7 mice/group. Scale bars: 200 μm. (Middle) Quantitative analysis of fibrosis. (Right) Ashcroft scores representing the extent of fibrosis. n= 6-7 mice/group. (F) Hydroxyproline content in lungs from the indicated groups. n = 7 mice/group. * P < 0.05; ** P < 0.01. *** P < 0.001 by two-way ANOVA for all panels, except for the Ashcroft score, which was analyzed using the Kruskal-Wallis test followed by Dunn's multiple-comparison test.

Journal: Theranostics

Article Title: Simultaneous inhibition of ID1 and ID3 mitigates fibroblast activation via cell cycle and MEK/ERK pathways in pulmonary fibrosis

doi: 10.7150/thno.127118

Figure Lengend Snippet: Genetic deletion of ID1 and ID3 reduces bleomycin-induced lung fibrosis. (A) Design of the study. (B) PCR analysis of ID1 and ID3 mRNA levels in lungs of the indicated groups. n=6-7 mice/group. (C) Lung function parameters, including inspiratory capacity, compliance and single frequency elastance. n=7 mice/group. (D) Lung protein expression of Collagen1 and α-SMA in lungs from the indicated groups. n=4-6 mice/group. (E) (Left) Representative images from Fast Green/Sirius-Red-stained lungs of the indicated groups. n=7 mice/group. Scale bars: 200 μm. (Middle) Quantitative analysis of fibrosis. (Right) Ashcroft scores representing the extent of fibrosis. n= 6-7 mice/group. (F) Hydroxyproline content in lungs from the indicated groups. n = 7 mice/group. * P < 0.05; ** P < 0.01. *** P < 0.001 by two-way ANOVA for all panels, except for the Ashcroft score, which was analyzed using the Kruskal-Wallis test followed by Dunn's multiple-comparison test.

Article Snippet: RNA integrity analysis, library preparation, sequencing, and raw read count generation were performed commercially by Novogene (Sacremento, CA, USA) using their Human mRNA Sequencing pipeline.

Techniques: Expressing, Staining, Comparison

Pharmacological inhibition of ID1/ID3 reduces lung fibroblast proliferation, migration, and differentiation into myofibroblast. (A-B) (Left) Representative immunoblots showing ID1, ID3 and GAPDH protein levels in healthy (A) and IPF-derived (B) human lung fibroblasts treated with 0.1%FBS or 5%FBS in the presence or absence of an ID1/ID3 pharmacological inhibitor (AGX51, 20µM). (Right) Quantification of the data. n = 4. (C-D) Proliferation of healthy (C) and IPF-derived (D) human lung fibroblasts in the presence or absence of AGX51 (20µM). n = 5-6 experiments performed in triplicate. (E-F) Migration of healthy (E) and IPF-derived (F) human lung fibroblasts in the presence or absence of AGX51 (20µM). n = 5-6 experiments performed in triplicate. Scale bar: 100 µm. (G-H) qPCR assessment of Acta2, Col1a1 and Ctgf mRNA levels 48h after healthy (G) and IPF-derived (H) human lung fibroblasts treatment with TGF-β1 (5 ng/ml) and AGX51 (20 µM). n = 4-5 experiments performed in triplicate. * P < 0.05; ** P < 0.01. *** P < 0.001 by two-way ANOVA.

Journal: Theranostics

Article Title: Simultaneous inhibition of ID1 and ID3 mitigates fibroblast activation via cell cycle and MEK/ERK pathways in pulmonary fibrosis

doi: 10.7150/thno.127118

Figure Lengend Snippet: Pharmacological inhibition of ID1/ID3 reduces lung fibroblast proliferation, migration, and differentiation into myofibroblast. (A-B) (Left) Representative immunoblots showing ID1, ID3 and GAPDH protein levels in healthy (A) and IPF-derived (B) human lung fibroblasts treated with 0.1%FBS or 5%FBS in the presence or absence of an ID1/ID3 pharmacological inhibitor (AGX51, 20µM). (Right) Quantification of the data. n = 4. (C-D) Proliferation of healthy (C) and IPF-derived (D) human lung fibroblasts in the presence or absence of AGX51 (20µM). n = 5-6 experiments performed in triplicate. (E-F) Migration of healthy (E) and IPF-derived (F) human lung fibroblasts in the presence or absence of AGX51 (20µM). n = 5-6 experiments performed in triplicate. Scale bar: 100 µm. (G-H) qPCR assessment of Acta2, Col1a1 and Ctgf mRNA levels 48h after healthy (G) and IPF-derived (H) human lung fibroblasts treatment with TGF-β1 (5 ng/ml) and AGX51 (20 µM). n = 4-5 experiments performed in triplicate. * P < 0.05; ** P < 0.01. *** P < 0.001 by two-way ANOVA.

Article Snippet: RNA integrity analysis, library preparation, sequencing, and raw read count generation were performed commercially by Novogene (Sacremento, CA, USA) using their Human mRNA Sequencing pipeline.

Techniques: Inhibition, Migration, Western Blot, Derivative Assay

Pharmacological inhibition of ID1/ID3 confers protection against pulmonary fibrosis in mice. (A) Design of the study. (B) (Left) Representative immunoblots showing ID1, ID3 and GAPDH protein levels in lung homogenates from PBS- or BLM-challenged mice treated or not with AGX51. (Right) Quantification of the data. n = 4 mice/group. (C) Lung function parameters, including inspiratory capacity, compliance, and single frequency elastance. n=5 mice/group. (D) PCR analysis of Col1a1, Col3a1 and Fn1 mRNA levels in lungs of the indicated groups. n=5-6 mice/group. (E) Lung protein expression of Collagen-I and Collagen-III in lungs from the indicated groups. n=5-6 mice/group. (F) (Left) Representative images from Fast Green/Sirius-Red-stained lungs of the indicated groups. n=5-9 mice/group. Scale bars: 200μm. (Middle) Quantitative analysis of fibrosis. (Right) Ashcroft scores representing the extent of fibrosis. n = 5-7 mice/group. (G) Hydroxyproline content in lungs from the indicated groups. n = 5-7 mice/group. * P < 0.05; ** P < 0.01. *** P < 0.001 by one-way ANOVA (B and E), two-way ANOVA (C, D, F, and G), and by Kruskal-Wallis test followed by Dunn's multiple-comparison test for the Ashcroft score.

Journal: Theranostics

Article Title: Simultaneous inhibition of ID1 and ID3 mitigates fibroblast activation via cell cycle and MEK/ERK pathways in pulmonary fibrosis

doi: 10.7150/thno.127118

Figure Lengend Snippet: Pharmacological inhibition of ID1/ID3 confers protection against pulmonary fibrosis in mice. (A) Design of the study. (B) (Left) Representative immunoblots showing ID1, ID3 and GAPDH protein levels in lung homogenates from PBS- or BLM-challenged mice treated or not with AGX51. (Right) Quantification of the data. n = 4 mice/group. (C) Lung function parameters, including inspiratory capacity, compliance, and single frequency elastance. n=5 mice/group. (D) PCR analysis of Col1a1, Col3a1 and Fn1 mRNA levels in lungs of the indicated groups. n=5-6 mice/group. (E) Lung protein expression of Collagen-I and Collagen-III in lungs from the indicated groups. n=5-6 mice/group. (F) (Left) Representative images from Fast Green/Sirius-Red-stained lungs of the indicated groups. n=5-9 mice/group. Scale bars: 200μm. (Middle) Quantitative analysis of fibrosis. (Right) Ashcroft scores representing the extent of fibrosis. n = 5-7 mice/group. (G) Hydroxyproline content in lungs from the indicated groups. n = 5-7 mice/group. * P < 0.05; ** P < 0.01. *** P < 0.001 by one-way ANOVA (B and E), two-way ANOVA (C, D, F, and G), and by Kruskal-Wallis test followed by Dunn's multiple-comparison test for the Ashcroft score.

Article Snippet: RNA integrity analysis, library preparation, sequencing, and raw read count generation were performed commercially by Novogene (Sacremento, CA, USA) using their Human mRNA Sequencing pipeline.

Techniques: Inhibition, Western Blot, Expressing, Staining, Comparison

Lung-specific inhibition of ID1 and ID3 attenuates bleomycin-induced lung fibrosis. (A) Schematic representation of the study design. (B) qPCR analysis of ID1 and ID3 mRNA levels in lungs of the indicated groups. n = 4 mice/group. (C) Lung function parameters, including inspiratory capacity, static compliance, and single-frequency elastance. n = 5-8 mice/group. (D) Relative mRNA expression of Col1a1, Col3a1, and Fn1 in lungs from the indicated groups. n=5 mice/group. (E) Hydroxyproline content in lungs from the indicated groups. n=5-6 mice/group. (F) (Left) Representative Fast Green/Sirius-Red-stained lung sections of the indicated groups. n=5-mice/group. Scale bar: 200μm. (Middle) Quantitative analysis of fibrosis. (Right) Ashcroft scores reflecting the extent of fibrosis. n = 5 mice/group. * P < 0.05; ** P < 0.01. *** P < 0.001 by two-way ANOVA for all panels, except for the Ashcroft score, which was analyzed using the Kruskal-Wallis test followed by Dunn's multiple-comparison test.

Journal: Theranostics

Article Title: Simultaneous inhibition of ID1 and ID3 mitigates fibroblast activation via cell cycle and MEK/ERK pathways in pulmonary fibrosis

doi: 10.7150/thno.127118

Figure Lengend Snippet: Lung-specific inhibition of ID1 and ID3 attenuates bleomycin-induced lung fibrosis. (A) Schematic representation of the study design. (B) qPCR analysis of ID1 and ID3 mRNA levels in lungs of the indicated groups. n = 4 mice/group. (C) Lung function parameters, including inspiratory capacity, static compliance, and single-frequency elastance. n = 5-8 mice/group. (D) Relative mRNA expression of Col1a1, Col3a1, and Fn1 in lungs from the indicated groups. n=5 mice/group. (E) Hydroxyproline content in lungs from the indicated groups. n=5-6 mice/group. (F) (Left) Representative Fast Green/Sirius-Red-stained lung sections of the indicated groups. n=5-mice/group. Scale bar: 200μm. (Middle) Quantitative analysis of fibrosis. (Right) Ashcroft scores reflecting the extent of fibrosis. n = 5 mice/group. * P < 0.05; ** P < 0.01. *** P < 0.001 by two-way ANOVA for all panels, except for the Ashcroft score, which was analyzed using the Kruskal-Wallis test followed by Dunn's multiple-comparison test.

Article Snippet: RNA integrity analysis, library preparation, sequencing, and raw read count generation were performed commercially by Novogene (Sacremento, CA, USA) using their Human mRNA Sequencing pipeline.

Techniques: Inhibition, Expressing, Staining, Comparison

ID1/ID3 act through modulation of cell cycle-associated genes and activation of the MEK signaling pathway. (A) Heat map of differentially expressed cell cycle-related genes from the Gene Ontology Biological Process pathway 'cell cycle G2/M phase transition' (GO:0044839) in PBS-treated versus AGX51-treated HLFs cultured in the presence of 0.1% FBS or 5% FBS. (B) PCR analysis of Ccna2, Ccnb2 and Cdk1 mRNA levels in HLF treated with PBS or AGX51 in the presence of Serum (5% FBS). n = 3-4 per group. (C) Pulmonary Ccna2, Ccnb2 and Cdk1 mRNA levels in lungs from the indicated groups. n = 5-6 per group. (D) (Left) Representative immunoblots showing CCNA2, CCNB2 and CDK1 protein levels in HLFs treated with 5%FBS in the presence or absence of AGX51. (Right) Quantification of protein expression. n = 4-6 per group. (E) (Left) Representative immunoblots showing CCNA2, CCNB2 and CDK1 protein levels in lung homogenates from the indicated groups. (Right) Quantification of protein expression. n = 4-6 per group. (F) Proliferation of human lung fibroblasts under the indicated treatment conditions. n = 5 experiments performed in triplicate. (G) Representative immunoblots showing phosphorylated MEK1 (p-MEK1) and total MEK1 protein levels in HLF treated with 5%FBS in the presence or absence of AGX51. (Right) Quantification of the data. n = 4 per group. (H) Representative immunoblots showing phosphorylated MEK1 and total MEK1 protein levels in HLF treated with Adenovirus control (Ad-Ctrl) or Adenoviruses ID1+ID3 (Ad-ID1/ID3). (Right) Quantification of the data. n = 3-4 per group. (I) qPCR analysis of Acta2, Col1a1, and Ctgf mRNA levels 48h after HLF treatment with Ad-ID1/ID3 or Ad-Ctrl in the presence or absence of the MEK1 inhibitor Selumetinib (10 µM). n = 4 experiments performed in duplicate. (J) qPCR analysis of Acta2, Col1a1, and Ctgf mRNA levels 48h after HLF treatment with Ad-MEK1 or Ad-Ctrl in the presence or absence of AGX51 (20 µM). n = 4 experiments performed in duplicate. * P < 0.05; ** P < 0.01. *** P < 0.001 by one-way ANOVA for all panel, expect for (H) which was analyzed using a t-test.

Journal: Theranostics

Article Title: Simultaneous inhibition of ID1 and ID3 mitigates fibroblast activation via cell cycle and MEK/ERK pathways in pulmonary fibrosis

doi: 10.7150/thno.127118

Figure Lengend Snippet: ID1/ID3 act through modulation of cell cycle-associated genes and activation of the MEK signaling pathway. (A) Heat map of differentially expressed cell cycle-related genes from the Gene Ontology Biological Process pathway 'cell cycle G2/M phase transition' (GO:0044839) in PBS-treated versus AGX51-treated HLFs cultured in the presence of 0.1% FBS or 5% FBS. (B) PCR analysis of Ccna2, Ccnb2 and Cdk1 mRNA levels in HLF treated with PBS or AGX51 in the presence of Serum (5% FBS). n = 3-4 per group. (C) Pulmonary Ccna2, Ccnb2 and Cdk1 mRNA levels in lungs from the indicated groups. n = 5-6 per group. (D) (Left) Representative immunoblots showing CCNA2, CCNB2 and CDK1 protein levels in HLFs treated with 5%FBS in the presence or absence of AGX51. (Right) Quantification of protein expression. n = 4-6 per group. (E) (Left) Representative immunoblots showing CCNA2, CCNB2 and CDK1 protein levels in lung homogenates from the indicated groups. (Right) Quantification of protein expression. n = 4-6 per group. (F) Proliferation of human lung fibroblasts under the indicated treatment conditions. n = 5 experiments performed in triplicate. (G) Representative immunoblots showing phosphorylated MEK1 (p-MEK1) and total MEK1 protein levels in HLF treated with 5%FBS in the presence or absence of AGX51. (Right) Quantification of the data. n = 4 per group. (H) Representative immunoblots showing phosphorylated MEK1 and total MEK1 protein levels in HLF treated with Adenovirus control (Ad-Ctrl) or Adenoviruses ID1+ID3 (Ad-ID1/ID3). (Right) Quantification of the data. n = 3-4 per group. (I) qPCR analysis of Acta2, Col1a1, and Ctgf mRNA levels 48h after HLF treatment with Ad-ID1/ID3 or Ad-Ctrl in the presence or absence of the MEK1 inhibitor Selumetinib (10 µM). n = 4 experiments performed in duplicate. (J) qPCR analysis of Acta2, Col1a1, and Ctgf mRNA levels 48h after HLF treatment with Ad-MEK1 or Ad-Ctrl in the presence or absence of AGX51 (20 µM). n = 4 experiments performed in duplicate. * P < 0.05; ** P < 0.01. *** P < 0.001 by one-way ANOVA for all panel, expect for (H) which was analyzed using a t-test.

Article Snippet: RNA integrity analysis, library preparation, sequencing, and raw read count generation were performed commercially by Novogene (Sacremento, CA, USA) using their Human mRNA Sequencing pipeline.

Techniques: Activation Assay, Sublimation, Cell Culture, Western Blot, Expressing, Control