Journal: iScience

Article Title: Combined HIF-1α blockade and CHIR99021 treatment reverses pulmonary fibrosis via modulation endothelial-to-mesenchymal transition

doi: 10.1016/j.isci.2025.114028

Figure Lengend Snippet: HIF-1α deletion and CHIR99021 synergistically reduce radiation-induced fibrosis and promote endothelial repair (A) Schematic representation of collagen irradiation and drug administration in WT and Col1a2-HIF1α KO mice. Col1a2 Cre-ER mutant mice carry a tamoxifen-inducible Cre recombinase. For Tomato expression and HIF1α deletion in fibroblasts, mice carrying a loxP site in the HIF1a gene and mice carrying the Tomato gene were crossed with Col1a2 Cre-ER mice. Tamoxifen was injected into mice to induce the expression of Tomato and deletion of HIF1α in fibroblasts. WT and Col1a2-HIF1α KO mice were irradiated in the left lung with 90 Gy using a 4 mm diameter field. Col1a2-Tomato mice were treated with tamoxifen (2 mg/day) once daily for 4 days starting 6 days post-irradiation and administered CHIR99021 (30 mg/kg) starting 4 days post-irradiation, with dosing continued every 2 days. Lung samples (n ≧ 5/group) were obtained 21 days post-irradiation from non-irradiated and irradiated mice. (B) Representative images of Hematoxylin & eosin staining, Masson trichrome staining, and Tomato immunofluorescence staining in non-irradiated or irradiated lung tissues from WT and Col1a2-HIF1α KO mice, with or without drug treatment (magnification, 200×). Scale bars = 40 μm. Scoring of fibrosis grade, quantification of collagen deposition, and Tomato + area are shown in the graph. (C) Immunohistochemistry staining of CD31 in the lung tissues of mice 21 days post-irradiation (magnification, 200×). Scale bars = 10 μm. Bar graphs quantify the CD31 area. (D) Immunofluorescence staining of CD31 (green), Tomato (red), and αSMA (white) in the lung tissues of mice 21 days post-irradiation (magnification, 400×). Scale bars = 10 μm. Scale bar of cropped images = 5 μm. Quantification of the Tomato + CD31 + αSMA + area and the Tomato + CD31 + αSMA − area. (E) Immunofluorescence staining of Tomato (red) and αSMA (green in the lung tissues of mice 21 days post-irradiation (magnification, 400×). Scale bars = 10 μm. Bar graphs quantify the αSMA and αSMA + Tomato + /αSMA + area. In the Ashcroft score graph (A) error bars indicate SD. In all other graphs, error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns: not significant (one-way ANOVA for multiple comparisons). The data shown are representative of repeated two independent experiments.

Article Snippet: Specific pathogen-free C57BL/6 Col1a2-CreERT2, HIF1α flox/flox and CAG-tdTomato mice were obtained from the Jackson Laboratory.

Techniques: Irradiation, Mutagenesis, Expressing, Injection, Staining, Immunofluorescence, Immunohistochemistry

Journal: iScience

Article Title: Combined HIF-1α blockade and CHIR99021 treatment reverses pulmonary fibrosis via modulation endothelial-to-mesenchymal transition

doi: 10.1016/j.isci.2025.114028

Figure Lengend Snippet: HIF-1α deletion and CHIR99021 synergistically regulate p -smad2/3, histone modifications, and pluripotent factor (A) Immunofluorescence staining of p -smad2/3 (green) and Tomato (red) in non-irradiated or irradiated lung tissues from WT and Col1a2-HIF1α KO mice 21 days post-irradiation (magnification, 400×). Scale bars = 20 μm. Scale bar of cropped images = 5 μm. Quantification of the p -smad2/3 + Tomato + /Tomato + area. (B) Immunofluorescence staining of histone modifications in lung tissues. Representative images of H3K27ac (green), H3K9ac (green), H3K9me3 (green), and Tomato (red) in the lung tissues of 21 days post-irradiation, treated with or without drug treatment (magnification, 400×) (right panel). Scale bars = 10 μm. Bar graphs quantify the H3K27ac, H3K9ac, and H3K9me3 area per nucleus area (left panel). (C) Immunofluorescence staining of OCT4 (green) and Tomato (red) in the lung tissues of mice 21 days post-irradiation (magnification, 400×). Scale bars = 10 μm. Scale bar of cropped images = 5 μm. Quantification of the OCT4 + Tomato + /Tomato + area. (D) Uniform Manifold Approximation and Projection (UMAP) plot of Single-Cell Transcriptomes. The plot was generated with single-cell RNA sequencing data using scanpy package. Each sample is represented by a distinct color: ‘WT, IR’ in blue and ‘Col1a2-HIF1α KO+ CHIR99021, IR’ in orange (left panel). Comparison of endothelial- and mesenchymal-related gene expression between two conditions: ‘WT, IR’ and ‘Col1a2-HIF1α KO + CHIR99021, IR’. The bar plot represents the ratio of positive cells expressing each gene. The y-axis lists genes associated with endothelium or mesenchyme, while the x-axis represents the ratio of positive cells between the two conditions (right panel). All graph error bars indicate SEM. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001; ns: not significant (one-way ANOVA for multiple comparisons).

Article Snippet: Specific pathogen-free C57BL/6 Col1a2-CreERT2, HIF1α flox/flox and CAG-tdTomato mice were obtained from the Jackson Laboratory.

Techniques: Immunofluorescence, Staining, Irradiation, Generated, RNA Sequencing, Comparison, Gene Expression, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: SRSF7 promotes pulmonary fibrosis through regulating PKM alternative splicing in lung fibroblasts

doi: 10.1016/j.apsb.2025.04.017

Figure Lengend Snippet: Increased expression of SRSF7 in lung fibroblasts during pulmonary fibrosis. (A) Heatmap of mRNA expression levels of serine/arginine-rich splicing factors in lung samples from GSE166036 , GSE213001 and GSE83717 . The color bars represent the range of log 2 FC values for each gene, with higher log 2 FC values shown in red and lower values in blue. ∗ P < 0.05, ∗∗ P < 0.01. (B) Single-cell RNA sequencing of annotated human lung fibrosis populations generated from Chan-Zuckerberg CELL by GENE Discover online database depicting relative expression of SRSF gene. Circle color denotes mean gene expression within each fibroblast subtype while circle size represents the proportion of each cell population expressing the indicated gene. (C) The Pearson’s correlation analysis between the expression of COL1A1 and SRSF7 in the advanced IPF samples from GSE134692 dataset. The X -axis shows the FPKM-normalized expression values of the SRSF7 gene. The Y -axis shows the expression level of COL1A1. Pearson correlation ( r ) and P -value ( P ) are shown. (D) The IHC staining of SRSF7 in non-IPF and IPF patients. Non-IPF: n = 6, IPF: n = 8. (E) The protein expression of SRSF7 in lung tissues. Non-IPF: n = 5, IPF: n = 7. (F) The protein expression of SRSF7 in BLM treated mice. Saline: n = 3, BLM: n = 6. (G) In GSE159354 , marker genes were identified using the rank_genes_groups function in Scanpy, with the wilcoxon test applied. Genes with a log 2 FC ≥ 4 and P < 0.05 were considered markers and were marked with an asterisk. (H) Dual immunofluorescence in TGF- β 1 treated MRC-5 cells. n = 3, scale bar = 10 μm. (I) Dual immunofluorescence of S100A4 (red) and SRSF7 (green) in frozen lung sections. n = 3, scale bar = 20 μm.

Article Snippet: Srsf7 fl/fl and Col1a2 -CreERT2 were purchased from Cyagen (Cyagen Biosciences) and wild-type C57BL/6 mice (22–25 g) were purchased from Liaoning Changsheng Biotechnology (Benxi, China).

Techniques: Expressing, RNA Sequencing, Generated, Gene Expression, Immunohistochemistry, Saline, Marker, Immunofluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: SRSF7 promotes pulmonary fibrosis through regulating PKM alternative splicing in lung fibroblasts

doi: 10.1016/j.apsb.2025.04.017

Figure Lengend Snippet: Overexpression of SRSF7 led to pulmonary function decline. (A) micro-CT was used to observe the fibrosis of mouse lung; n = 5. (B) FVC, IC, FRC, Cdyn, FEF 50% and Flow-volume loop were detected in mice treated with AAV- NC and AAV- Srsf7 ; n = 5. (C) Masson staining was used to evaluate the content of collagen in lung tissues; n = 4, scale bar = 500 μm. (D) The alveoli structure detected by H&E staining and the degree of interstitial lung fibrosis were determined by using a predetermined numerical scale of 0–8, based on the Ashcroft scoring method; n = 5, scale bar = 500 μm. (E, F) Collagen I and α -SMA were stained by IHC ( n = 3, scale bar = 500 μm) and immunofluorescence staining ( n = 4, scale bar = 20 μm). (G) The mRNA expression of Fn1, Col1α1, Col3α1 and Acta2 in mice treated with AAV- NC and AAV- Srsf7 ; n = 5. (H) The protein expression of FN1, α -SMA and SRSF7 in the lung fibroblasts of mice. (I) The mRNA expression of fibrosis markers in mice treated with AAV- NC and AAV- Srsf7 ; n = 4. Data are presented as mean ± SEM; ns, no significance.

Article Snippet: Srsf7 fl/fl and Col1a2 -CreERT2 were purchased from Cyagen (Cyagen Biosciences) and wild-type C57BL/6 mice (22–25 g) were purchased from Liaoning Changsheng Biotechnology (Benxi, China).

Techniques: Over Expression, Micro-CT, Staining, Immunofluorescence, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: SRSF7 promotes pulmonary fibrosis through regulating PKM alternative splicing in lung fibroblasts

doi: 10.1016/j.apsb.2025.04.017

Figure Lengend Snippet: SRSF7 promotes fibroblast activation and fibrogenesis. (A) The mRNA expression of fibrosis related genes after MRC-5 cells transfected with SRSF7 ; n = 6. (B) Western blot demonstrated increased expression level of Fn1 in SRSF7 transfected MRC-5 cells; n = 3. (C) Relative quantification of the collagen contraction area after transfected with SRSF7 ; n = 3. (D, E) EdU assay (scale bar = 50 μm, n = 4) and wound healing experiment (scale bar = 200 μm, n = 4) indicated that transfected with SRSF7 promoted the proliferation and migration of MRC-5. (F) Overexpression of SRSF7 promoted the fibroblast-myofibroblast transformation of MRC-5 cells; n = 3, scale bar = 10 μm. (G) qRT-PCR analysis demonstrating the relative expression of FN1 , COL1A1 , COL3A1 and ACTA2 in TGF- β 1-induced MRC-5 cells transfected with or without si- SRSF7 ; n = 6. (H) Western blot showed that the si- SRSF7 abrogated the upregulation of fibrotic proteins in MRC-5 cells induced by TGF- β 1; n = 4. (I, J) EdU (scale bar = 50 μm, n = 4) and wound healing assays (scale bar = 200 μm, n = 3) indicated that si- SRSF7 inhibited the proliferation and migration of MRC-5 cells. Data are presented as mean ± SEM.

Article Snippet: Srsf7 fl/fl and Col1a2 -CreERT2 were purchased from Cyagen (Cyagen Biosciences) and wild-type C57BL/6 mice (22–25 g) were purchased from Liaoning Changsheng Biotechnology (Benxi, China).

Techniques: Activation Assay, Expressing, Transfection, Western Blot, Quantitative Proteomics, EdU Assay, Migration, Over Expression, Transformation Assay, Quantitative RT-PCR

Journal: Acta Pharmaceutica Sinica. B

Article Title: SRSF7 promotes pulmonary fibrosis through regulating PKM alternative splicing in lung fibroblasts

doi: 10.1016/j.apsb.2025.04.017

Figure Lengend Snippet: SRSF7 is able to bind to and regulate the alternative splicing of PKM. (A) Heatmap illustrating differentially expressed genes. Red represents up-regulated, and blue represents down-regulated. In the heatmap, red indicates high gene expression, while blue represents low gene expression. The color bars display the scale of values for each gene, ranging from −2 to 2 as shown on the right. (B) GO enrichment analysis regulated by overexpression of SRSF7 . (C) GSEA showed that U2 SnRNP and spliceosome related genes were significantly enriched after overexpression of SRSF7 . (D) The differentially alternative splicing genes regulated by SRSF7 were mainly enriched in exon skipped events. (E) Prediction of SRSF7 regulated alternative splicing genes overlap in MRC-5 and IPF patients. (F) SRSF7 enhanced the alternative splicing of PKM exon 2; n = 4. (G) Silencing SRSF7 can reduce the skipping of PKM exon 2 induced by TGF- β 1; n = 4. (H) Lung fibroblasts were isolated from mice to detect the alternative splicing events of PKM . (I) Minigene reporters of PKM were introduced into SRSF7-overexpression cells; n = 6. (J) Deficiency of the SRSF7 function domain on alternative splicing of PKM exon 2. (K) RIP experiments were performed to detect the binding relationship between SRSF7 and PKM ; n = 3. (L) The effect of SRSF7 on the ability of binding with snRNAs was investigated by the RIP experiment; n = 3. Data are presented as mean ± SEM.

Article Snippet: Srsf7 fl/fl and Col1a2 -CreERT2 were purchased from Cyagen (Cyagen Biosciences) and wild-type C57BL/6 mice (22–25 g) were purchased from Liaoning Changsheng Biotechnology (Benxi, China).

Techniques: Alternative Splicing, Gene Expression, Over Expression, Isolation, Binding Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: SRSF7 promotes pulmonary fibrosis through regulating PKM alternative splicing in lung fibroblasts

doi: 10.1016/j.apsb.2025.04.017

Figure Lengend Snippet: Silencing SRSF7 alleviates fibrogenesis by inhibiting the expression of PKM ∆ E2 isoform. (A) The mRNA expression of FN1 , COL1A1 , COL3A1 and ACTA2 ; n = 4. (B) Relative quantification of the collagen contraction area in MRC-5 cells; n = 3. (C) Immunofluorescence showed the fibroblasts transformation in MRC-5; n = 3, scale bar = 20 μm. (D, E) EdU (scale bar = 50 μm, n = 3) and wound healing assays (scale bar = 200 μm, n = 3) indicated that overexpression of PKM ∆ E2 promoted the cell proliferation and migration reduced by si SRSF7 . (F, G) Real-time ECAR and OCR were recorded in the MRC-5 transfected with si SRSF7 and PKM ∆ E2 after treatment with TGF- β 1. (H) The lactic acid content of MRC-5 cells, n = 6. Data are presented as mean ± SEM.

Article Snippet: Srsf7 fl/fl and Col1a2 -CreERT2 were purchased from Cyagen (Cyagen Biosciences) and wild-type C57BL/6 mice (22–25 g) were purchased from Liaoning Changsheng Biotechnology (Benxi, China).

Techniques: Expressing, Quantitative Proteomics, Immunofluorescence, Transformation Assay, Over Expression, Migration, Transfection

Journal: Acta Pharmaceutica Sinica. B

Article Title: SRSF7 promotes pulmonary fibrosis through regulating PKM alternative splicing in lung fibroblasts

doi: 10.1016/j.apsb.2025.04.017

Figure Lengend Snippet: Srsf7 deficiency attenuates BLM-induced experimental lung fibrosis in mice. (A) Diagram of the animal experimental model of Srsf7 -cKO. (B) Representative micro-CT images showed the lung fibrosis of Srsf7 -cKO mice treated with bleomycin for 21 days; n = 4. (C, D) FRC, FVC, IC, FEF50%, Cdyn and F–V Loop were detected in Srsf7 fl/fl or Srsf7 -cKO mice treated with saline or bleomycin for 21 days; n = 6. (E) Masson and (F) H&E staining were used to evaluate the content of collagen in lung tissues; n = 6, scale bar = 500 μm. (G) The hydroxyproline content detection in lung tissues was confirmed by hydroxyproline detection kit; n = 6. (H) qRT-PCR was used to determine the mRNA level of Srsf7 , Fn1 , Acta2 , Col1a1 and Col3a1 ; n = 6. (I, J) IHC ( n = 3, scale bar = 500 μm) and (K, L) immunofluorescence staining ( n = 4, scale bar = 20 μm) were used to evaluate the expression of Collagen Ⅰ and α -SMA in lung tissues. Data are presented as mean ± SEM.

Article Snippet: Srsf7 fl/fl and Col1a2 -CreERT2 were purchased from Cyagen (Cyagen Biosciences) and wild-type C57BL/6 mice (22–25 g) were purchased from Liaoning Changsheng Biotechnology (Benxi, China).

Techniques: Micro-CT, Saline, Staining, Quantitative RT-PCR, Immunofluorescence, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: SRSF7 promotes pulmonary fibrosis through regulating PKM alternative splicing in lung fibroblasts

doi: 10.1016/j.apsb.2025.04.017

Figure Lengend Snippet: Lomitapide inhibits SRSF7 expression to improve lung fibrosis and lung function in mice. (A) Chemical screen to systematically identify inhibitors of SRSF7 and prediction of Drug–Protein Interaction Networks from the Integration of SRSF7 Sequences and Lomitapide Chemical Structures (right panel). (B) Experimental protocol of lomitapide in BLM treated mice. (C) Representative micro-CT images showed the lung fibrosis of lomitapide treated mice; n = 4. (D) FVC, Cdyn and F–V Loop were detected in mice treated with lomitapide or pirfenidone; n = 6. (E) Masson and (F) H&E staining were used to evaluate the content of collagen in lung tissues; n = 5, scale bar = 500 μm. (G) The hyroxyproline content detection in lung tissues was confirmed by hydroxyproline detection kit; n = 6. (H) qRT-PCR was used to determine the mRNA level of Fn1 , Acta2 , Col1a1 and Col3a1 in lomitapide or pirfenidone treated mice; n = 6. (I, J) IHC ( n = 4, scale bar = 500 μm) and (K, L) immunofluorescence staining ( n = 3, scale bar = 20 μm) were used to evaluate the expression of Collagen Ⅰ and α -SMA in lung tissues. Data are presented as mean ± SEM.

Article Snippet: Srsf7 fl/fl and Col1a2 -CreERT2 were purchased from Cyagen (Cyagen Biosciences) and wild-type C57BL/6 mice (22–25 g) were purchased from Liaoning Changsheng Biotechnology (Benxi, China).

Techniques: Expressing, Micro-CT, Staining, Quantitative RT-PCR, Immunofluorescence