Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: HNF4A P1 cannot antagonize TGF-β1 induced dedifferentiation of HK-2 cells. A Representative Western blots and C , D relative quantitation of WB results of HNF4A P1, SNAI1, and VIM of HK-2 cells with or without HNF4A P1 overexpression. E Representative Western blots and F–H relative quantitation of WB results of HNF4A P1, SNAI1, and VIM of HK-2 cells with or without HNF4A knocking down. I Representative Western blots and J–L the relative quantitation of protein expression of HNF4A, SNAI1, and VIM of HK-2 cells treated with or without TGF-β1 at different concentration ( n = 3). M Immunofluorescence of HNF4A P1 isoform of HK-2 cells under normal condition or treated with TGF-β1 10 ng/ml, with or without HNF4A P1 overexpression. Bars, 10 μm. N–P Representative Western blots and the relative quantitation of protein expression of SNAI1 and VIM of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1 overexpression ( n = 3). Q , R The mRNA expression of SNAI1 and VIM in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1 overexpression ( n = 3). The protein expression was quantified by densitometry using ImageJ software. Data are presented as mean ± SD; t -test was used in B–D and F–H ; ANOVA test was used in J–L and O–R ; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: To induce fibroblast activation, following overnight starvation in serum-free medium, subconfluent NRK-49F fibroblasts were incubated with tubular cell-conditioned medium for 48 h. To inhibit SRC activation, HK-2 and TCMK-1 cells were incubated with 10 ng/ml TGF-β1 for 48 h in the presence of PP1 (5 μM, TargetMol, MA, China).

Techniques: Western Blot, Quantitation Assay, Over Expression, Expressing, Concentration Assay, Immunofluorescence, Software

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: HNF4A P2 can antagonize TGF-β1-induced dedifferentiation in HK-2 cells. A Western blot results of HNF4A P1 and P2 isoforms in HK-2 cells treated with TGF-β1 either individually or simultaneously exposed. B Immunofluorescence of VIM of HK-2 cells under normal condition or treated with TGF-β1 10 ng/ml, with or without HNF4A P2 overexpression. Bars, 10 μm. C–E Representative Western blots and the relative quantitation of protein expression of SNAI1 and VIM of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). F, G The mRNA expression of SNAI1 and VIM in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). H Flowchart for culturing NRK-49F cells using conditioned medium derived from HK2 cells. I, J Representative Western blots and the relative quantitation of protein expression of α-SMA of NRK-49F cells treated with different conditioned medium derived from HK2 cells ( n = 3). Data are presented as mean ± SD; ANOVA test was used; * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: To induce fibroblast activation, following overnight starvation in serum-free medium, subconfluent NRK-49F fibroblasts were incubated with tubular cell-conditioned medium for 48 h. To inhibit SRC activation, HK-2 and TCMK-1 cells were incubated with 10 ng/ml TGF-β1 for 48 h in the presence of PP1 (5 μM, TargetMol, MA, China).

Techniques: Western Blot, Immunofluorescence, Over Expression, Quantitation Assay, Expressing, Concentration Assay, Derivative Assay

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: HNF4A P2 antagonises TGF-β1 induced dedifferentiation through JAG1/NOTCH pathway. A Differentially expressed genes in HK-2 cells with HNF4A P2 overexpression versus without HNF4A P2 overexpression subjected to TGF-β1 10 ng/ml. B Western blots of JAG1 of HK-2 cells subjected to TGF-β1 10 ng/ml. C , D Representative Western blots and the relative quantitation of protein expression of JAG1 in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). E The mRNA expression of JAG1 in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). F qPCR analysis of ChIP assays with anti-Flag antibody on HK-2 cells treated with TGF-β1 and empty plasmid or HNF4A P2-Flag overexpression ( n = 3). G – J Representative Western blots and the relative quantitation of protein expression of JAG1 of kidney tissues from UIRI and UUO ( n = 6). K – N Representative Western blots and the relative quantitation of protein expression of JAG1 of kidney tissues from UIRI and UUO treated with or without HNF4A P1/P2 plasmids ( n = 6). O – R Representative Western blots and the relative quantitation of protein expression of NOTCH1, NOTCH2, and NOTCH3 of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). Data are presented as mean ± SD; t -test was used in panels H and J ; ANOVA test was used in other statistical test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: To induce fibroblast activation, following overnight starvation in serum-free medium, subconfluent NRK-49F fibroblasts were incubated with tubular cell-conditioned medium for 48 h. To inhibit SRC activation, HK-2 and TCMK-1 cells were incubated with 10 ng/ml TGF-β1 for 48 h in the presence of PP1 (5 μM, TargetMol, MA, China).

Techniques: Over Expression, Western Blot, Quantitation Assay, Expressing, Concentration Assay, Plasmid Preparation

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: Overexpression of JAG1 antagonized the inhibitory effect of HNF4A P2 on the dedifferentiation of HK-2 cells. A – G Representative Western blots and the relative quantitation of protein expression of JAG1, NOTCH1, NOTCH2, NOTCH3, SNAI1, and VIM of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2/JAG1 overexpression ( n = 3). H, I The mRNA expression of SNAI1 and VIM in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2/JAG1 overexpression ( n = 3). J, K Representative Western blots and the relative quantitation of protein expression of α-SMA of NRK-49F cells treated with different conditioned medium derived from HK2 cells ( n = 3). Data are presented as mean ± SD; ANOVA test was used; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: To induce fibroblast activation, following overnight starvation in serum-free medium, subconfluent NRK-49F fibroblasts were incubated with tubular cell-conditioned medium for 48 h. To inhibit SRC activation, HK-2 and TCMK-1 cells were incubated with 10 ng/ml TGF-β1 for 48 h in the presence of PP1 (5 μM, TargetMol, MA, China).

Techniques: Over Expression, Western Blot, Quantitation Assay, Expressing, Concentration Assay, Derivative Assay

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: p-SRC selectively inhibits the function of HNF4A P1 instead of HNF4A P2. A Western blots of p-SRC/SRC in HK-2 cells treated with different concentration of TGF-β1. B , C Representative Western blots and the relative quantitation of protein expression of p-SRC of kidney tissues from UIRI and UUO ( n = 6). D , E Representative Western blots and the relative quantitation of protein expression of p-SRC in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1/P2 overexpression ( n = 3). F Phosphorylation of HNF4A P1/P2 protein in HK-2 cells treated with different concentration of TGF-β1 with or without HNF4A P1/P2 plasmids were determined via IP and Western blot assays ( n = 3). G – M Representative Western blots and the relative quantitation of protein expression of SNAI1, VIM, JAG1, NOTCH1, NOTCH2, and NOTCH3 of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1 overexpression and PP1 ( n = 3). N , O The mRNA level of SNAI1 and VIM in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1 overexpression and PP1. P , Q Representative Western blots and the relative quantitation of protein expression of α-SMA of NRK-49F cells treated with different conditioned medium derived from HK2 cells ( n = 3). R qPCR analysis of ChIP assays with anti-HNF4A P1 antibody on HK-2 cells from different groups and relative quantitation ( n = 3). Data are presented as mean ± SD; ANOVA test was used; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: To induce fibroblast activation, following overnight starvation in serum-free medium, subconfluent NRK-49F fibroblasts were incubated with tubular cell-conditioned medium for 48 h. To inhibit SRC activation, HK-2 and TCMK-1 cells were incubated with 10 ng/ml TGF-β1 for 48 h in the presence of PP1 (5 μM, TargetMol, MA, China).

Techniques: Western Blot, Concentration Assay, Quantitation Assay, Expressing, Over Expression, Phospho-proteomics, Derivative Assay

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: Schematic illustration of different roles of HNF4A P1/2 isoforms in TGF-β1-induced PTC dedifferentiation. Under basal conditions, overexpression of HNF4A-P1 and HNF4A-P2 leads to their occupancy at the JAG1 promoter. When tubular cells are in dedifferentiated state, TGF-β1 induced SRC activation and phosphorylated HNF4A P1 isoform and thereby inhibited binding between P1 isoform and the JAG1 promoter region, while HNF4A P2 isoform could bind at the JAG1 promoter region in the presence of TGF-β1 without being phosphorylated by p-SRC

Article Snippet: To induce fibroblast activation, following overnight starvation in serum-free medium, subconfluent NRK-49F fibroblasts were incubated with tubular cell-conditioned medium for 48 h. To inhibit SRC activation, HK-2 and TCMK-1 cells were incubated with 10 ng/ml TGF-β1 for 48 h in the presence of PP1 (5 μM, TargetMol, MA, China).

Techniques: Over Expression, Activation Assay, Binding Assay

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: HNF4A P1 cannot antagonize TGF-β1 induced dedifferentiation of HK-2 cells. A Representative Western blots and C , D relative quantitation of WB results of HNF4A P1, SNAI1, and VIM of HK-2 cells with or without HNF4A P1 overexpression. E Representative Western blots and F–H relative quantitation of WB results of HNF4A P1, SNAI1, and VIM of HK-2 cells with or without HNF4A knocking down. I Representative Western blots and J–L the relative quantitation of protein expression of HNF4A, SNAI1, and VIM of HK-2 cells treated with or without TGF-β1 at different concentration ( n = 3). M Immunofluorescence of HNF4A P1 isoform of HK-2 cells under normal condition or treated with TGF-β1 10 ng/ml, with or without HNF4A P1 overexpression. Bars, 10 μm. N–P Representative Western blots and the relative quantitation of protein expression of SNAI1 and VIM of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1 overexpression ( n = 3). Q , R The mRNA expression of SNAI1 and VIM in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1 overexpression ( n = 3). The protein expression was quantified by densitometry using ImageJ software. Data are presented as mean ± SD; t -test was used in B–D and F–H ; ANOVA test was used in J–L and O–R ; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: TCMK-1 was incubated in serum-free culture medium with 10 ng/ml recombinant mouse TGF-β1 (TargetMol, Shanghai, China).

Techniques: Western Blot, Quantitation Assay, Over Expression, Expressing, Concentration Assay, Immunofluorescence, Software

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: HNF4A P2 can antagonize TGF-β1-induced dedifferentiation in HK-2 cells. A Western blot results of HNF4A P1 and P2 isoforms in HK-2 cells treated with TGF-β1 either individually or simultaneously exposed. B Immunofluorescence of VIM of HK-2 cells under normal condition or treated with TGF-β1 10 ng/ml, with or without HNF4A P2 overexpression. Bars, 10 μm. C–E Representative Western blots and the relative quantitation of protein expression of SNAI1 and VIM of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). F, G The mRNA expression of SNAI1 and VIM in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). H Flowchart for culturing NRK-49F cells using conditioned medium derived from HK2 cells. I, J Representative Western blots and the relative quantitation of protein expression of α-SMA of NRK-49F cells treated with different conditioned medium derived from HK2 cells ( n = 3). Data are presented as mean ± SD; ANOVA test was used; * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: TCMK-1 was incubated in serum-free culture medium with 10 ng/ml recombinant mouse TGF-β1 (TargetMol, Shanghai, China).

Techniques: Western Blot, Immunofluorescence, Over Expression, Quantitation Assay, Expressing, Concentration Assay, Derivative Assay

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: HNF4A P2 antagonises TGF-β1 induced dedifferentiation through JAG1/NOTCH pathway. A Differentially expressed genes in HK-2 cells with HNF4A P2 overexpression versus without HNF4A P2 overexpression subjected to TGF-β1 10 ng/ml. B Western blots of JAG1 of HK-2 cells subjected to TGF-β1 10 ng/ml. C , D Representative Western blots and the relative quantitation of protein expression of JAG1 in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). E The mRNA expression of JAG1 in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). F qPCR analysis of ChIP assays with anti-Flag antibody on HK-2 cells treated with TGF-β1 and empty plasmid or HNF4A P2-Flag overexpression ( n = 3). G – J Representative Western blots and the relative quantitation of protein expression of JAG1 of kidney tissues from UIRI and UUO ( n = 6). K – N Representative Western blots and the relative quantitation of protein expression of JAG1 of kidney tissues from UIRI and UUO treated with or without HNF4A P1/P2 plasmids ( n = 6). O – R Representative Western blots and the relative quantitation of protein expression of NOTCH1, NOTCH2, and NOTCH3 of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2 overexpression ( n = 3). Data are presented as mean ± SD; t -test was used in panels H and J ; ANOVA test was used in other statistical test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: TCMK-1 was incubated in serum-free culture medium with 10 ng/ml recombinant mouse TGF-β1 (TargetMol, Shanghai, China).

Techniques: Over Expression, Western Blot, Quantitation Assay, Expressing, Concentration Assay, Plasmid Preparation

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: Overexpression of JAG1 antagonized the inhibitory effect of HNF4A P2 on the dedifferentiation of HK-2 cells. A – G Representative Western blots and the relative quantitation of protein expression of JAG1, NOTCH1, NOTCH2, NOTCH3, SNAI1, and VIM of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2/JAG1 overexpression ( n = 3). H, I The mRNA expression of SNAI1 and VIM in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P2/JAG1 overexpression ( n = 3). J, K Representative Western blots and the relative quantitation of protein expression of α-SMA of NRK-49F cells treated with different conditioned medium derived from HK2 cells ( n = 3). Data are presented as mean ± SD; ANOVA test was used; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: TCMK-1 was incubated in serum-free culture medium with 10 ng/ml recombinant mouse TGF-β1 (TargetMol, Shanghai, China).

Techniques: Over Expression, Western Blot, Quantitation Assay, Expressing, Concentration Assay, Derivative Assay

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: p-SRC selectively inhibits the function of HNF4A P1 instead of HNF4A P2. A Western blots of p-SRC/SRC in HK-2 cells treated with different concentration of TGF-β1. B , C Representative Western blots and the relative quantitation of protein expression of p-SRC of kidney tissues from UIRI and UUO ( n = 6). D , E Representative Western blots and the relative quantitation of protein expression of p-SRC in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1/P2 overexpression ( n = 3). F Phosphorylation of HNF4A P1/P2 protein in HK-2 cells treated with different concentration of TGF-β1 with or without HNF4A P1/P2 plasmids were determined via IP and Western blot assays ( n = 3). G – M Representative Western blots and the relative quantitation of protein expression of SNAI1, VIM, JAG1, NOTCH1, NOTCH2, and NOTCH3 of HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1 overexpression and PP1 ( n = 3). N , O The mRNA level of SNAI1 and VIM in HK-2 cells treated with different concentration of TGF-β1, with or without HNF4A P1 overexpression and PP1. P , Q Representative Western blots and the relative quantitation of protein expression of α-SMA of NRK-49F cells treated with different conditioned medium derived from HK2 cells ( n = 3). R qPCR analysis of ChIP assays with anti-HNF4A P1 antibody on HK-2 cells from different groups and relative quantitation ( n = 3). Data are presented as mean ± SD; ANOVA test was used; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: TCMK-1 was incubated in serum-free culture medium with 10 ng/ml recombinant mouse TGF-β1 (TargetMol, Shanghai, China).

Techniques: Western Blot, Concentration Assay, Quantitation Assay, Expressing, Over Expression, Phospho-proteomics, Derivative Assay

Journal: Cellular & Molecular Biology Letters

Article Title: HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling

doi: 10.1186/s11658-025-00845-0

Figure Lengend Snippet: Schematic illustration of different roles of HNF4A P1/2 isoforms in TGF-β1-induced PTC dedifferentiation. Under basal conditions, overexpression of HNF4A-P1 and HNF4A-P2 leads to their occupancy at the JAG1 promoter. When tubular cells are in dedifferentiated state, TGF-β1 induced SRC activation and phosphorylated HNF4A P1 isoform and thereby inhibited binding between P1 isoform and the JAG1 promoter region, while HNF4A P2 isoform could bind at the JAG1 promoter region in the presence of TGF-β1 without being phosphorylated by p-SRC

Article Snippet: TCMK-1 was incubated in serum-free culture medium with 10 ng/ml recombinant mouse TGF-β1 (TargetMol, Shanghai, China).

Techniques: Over Expression, Activation Assay, Binding Assay

Journal: Genes & Diseases

Article Title: Autophagy-induced NR2F1 activation promotes the apoptosis of lens epithelial cells and facilitates cataract-associated fibrosis through targeting STAT3

doi: 10.1016/j.gendis.2025.101549

Figure Lengend Snippet: TGF-β1 mediates fibrosis of SRA01/04 cells. (A) Light field images of SRA01/04 cells treated with PBS or TGF-β1. (B, C) The protein expression and quantification of fibrosis-related markers FN1, VIM, and α-SMA in SRA01/04 cells with or without TGF-β1. n = 3 per group; mean ± standard deviation; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; unpaired student's t -test. (D – F) Immunofluorescence of FN1, VIM, and α-SMA in the two groups mentioned above. Scale bar, 100 μm. TGF-β1, transforming growth factor-β1; PBS, phosphate-buffered saline; FN1, fibronectin 1; α-SMA, α-smooth muscle actin; VIM, vimentin.

Article Snippet: For TGF-β1 treatment, cells were seeded in 6-well plates and treated with 10 ng/mL of TGF-β1 (PeproTech, Suzhou, China) for 48 h. The autophagy inhibitor chloroquine (T8689, TargetMol, USA) was applied at concentrations of 5, 10, 20, 40, and 80 μM.

Techniques: Expressing, Standard Deviation, Immunofluorescence, Saline

Journal: Genes & Diseases

Article Title: Autophagy-induced NR2F1 activation promotes the apoptosis of lens epithelial cells and facilitates cataract-associated fibrosis through targeting STAT3

doi: 10.1016/j.gendis.2025.101549

Figure Lengend Snippet: TGF-β1 mediated autophagy dysfunction resulting in an increased protein level of NR2F1. (A) The mRNA profile of NR2F1 in lens epithelial cells and fiber cells using single-cell data. (B) The mRNA level of NR2F1 in SRA01/04 cells with PBS or TGF-β1. n = 3 per group; mean ± standard deviation; ∗ p < 0.05; unpaired student's t -test. (C, D) The protein expression and quantification of NR2F1 in TGF-β1-induced SRA01/04 cells. n = 3 per group; mean ± standard deviation; ∗ p < 0.05; unpaired student's t -test. (E) Subcellular location of NR2F1 in PBS- or TGF-β1-treated SRA01/04 cells. Scale bar, 50 μm. (F, G) The protein level and quantification of NR2F1 in SRA01/04 cells stimulated with the autophagy inhibitor chloroquine at concentrations of 5, 10, 20, 40, and 80 μM, respectively. n = 3 per group; mean ± standard deviation; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA. (H) Co-localization of LC3B and NR2F1 along with P62 and NR2F1 in SRA01/04 cells with TGF-β1. Scale bar, 50 μm. TGF-β1, transforming growth factor-β1; NR2F1, nuclear receptor subfamily 2 group F member 1; PBS, phosphate-buffered saline; LC3B, microtubule-associated protein 1 light-chain 3B.

Article Snippet: For TGF-β1 treatment, cells were seeded in 6-well plates and treated with 10 ng/mL of TGF-β1 (PeproTech, Suzhou, China) for 48 h. The autophagy inhibitor chloroquine (T8689, TargetMol, USA) was applied at concentrations of 5, 10, 20, 40, and 80 μM.

Techniques: Single Cell, Standard Deviation, Expressing, Saline

Journal: Genes & Diseases

Article Title: Autophagy-induced NR2F1 activation promotes the apoptosis of lens epithelial cells and facilitates cataract-associated fibrosis through targeting STAT3

doi: 10.1016/j.gendis.2025.101549

Figure Lengend Snippet: Knockdown of NR2F1 significantly attenuates fibrosis both in vivo and in vitro . (A) Adeno-associated adenovirus (AAV) was administered into the anterior chamber of mouse eyes. (B) Representative pictures of the ASC modeling group versus the negative control group under a slit lamp following AAV injection. (C) Hematoxylin and eosin staining in the two groups mentioned above. Scale bar, 100 μm. (D) Masson staining in the two groups. Scale bar, 100 μm. (E) The immunofluorescence intensity of α-SMA in the lens sections of AAV-NC and AAV-NR2F1 ASC mice. Scale bar, 50 μm. (F, G) The protein level and quantification of NR2F1 in SRA01/04 cells transfected with Sh-NC, Sh-NR2F1-1, Sh-NR2F1-2, or Sh-NR2F1-3 lentivirus. n = 3 per group; mean ± standard deviation; ∗∗∗ p < 0.001; one-way ANOVA. (H–K) The protein expression and quantitative graphs of FN1, VIM, and α-SMA in TGF-β1-induced SRA01/04 cells with Sh-NC or Sh-NR2F1. n = 3 per group; mean ± standard deviation; ∗ p < 0.05, ∗∗ p < 0.01; unpaired student's t -test. (L – N) Immunofluorescence images of FN1, VIM, and α-SMA in TGF-β1-mediated SRA01/04 cells with Sh-NC or Sh-NR2F1. Scale bar, 100 μm. NR2F1, nuclear receptor subfamily 2 group F member 1; ASC, anterior subcapsular cataract; FN1, fibronectin 1; α-SMA, α-smooth muscle actin; VIM, vimentin; TGF-β1, transforming growth factor-β1.

Article Snippet: For TGF-β1 treatment, cells were seeded in 6-well plates and treated with 10 ng/mL of TGF-β1 (PeproTech, Suzhou, China) for 48 h. The autophagy inhibitor chloroquine (T8689, TargetMol, USA) was applied at concentrations of 5, 10, 20, 40, and 80 μM.

Techniques: Knockdown, In Vivo, In Vitro, Negative Control, Injection, Staining, Immunofluorescence, Transfection, Standard Deviation, Expressing

Journal: Genes & Diseases

Article Title: Autophagy-induced NR2F1 activation promotes the apoptosis of lens epithelial cells and facilitates cataract-associated fibrosis through targeting STAT3

doi: 10.1016/j.gendis.2025.101549

Figure Lengend Snippet: NR2F1 inhibition suppresses epithelial cell apoptosis and migration. (A) The TUNEL staining in lens sections of AAV-NC and AAV-NR2F1 mice with ASC. Scale bar: 50 μm. (B – D) The protein levels and quantitative charts of BAX and CASP3 in the two groups mentioned above. n = 3 per group; mean ± standard deviation; ∗ p < 0.05, ∗∗ p < 0.01; unpaired student's t -test. (E, F) The TUNEL staining in the TGF-β1-mediated group with Sh-NR2F1 compared with that with Sh-NC. n = 3 per group; mean ± standard deviation; ∗ p < 0.05; unpaired student's t -test; scale bar, 200 μm. (G – I) The protein expression and quantification of BAX and CASP3 in TGF-β1-induced SRA01/04 cells with or without Sh-NR2F1. n = 3 per group; mean ± standard deviation; ∗ p < 0.05; unpaired student's t -test. (J, K) The transwell assay in the two groups mentioned above. n = 4 per group; mean ± standard deviation; ∗∗ p < 0.01; unpaired student's t -test. (I–K) The protein expression and quantification of BAX and CASP3 in TGF-β1-induced SRA01/04 cells with or without Sh-NR2F1. n = 3 per group; mean ± standard deviation; ∗ p < 0.05; unpaired student's t -test. NR2F1, nuclear receptor subfamily 2 group F member 1; AAV, adeno-associated adenovirus; ASC, anterior subcapsular cataract; BAX, Bcl-2-associated X; CASP3, caspase 3; TGF-β1, transforming growth factor-β1.

Article Snippet: For TGF-β1 treatment, cells were seeded in 6-well plates and treated with 10 ng/mL of TGF-β1 (PeproTech, Suzhou, China) for 48 h. The autophagy inhibitor chloroquine (T8689, TargetMol, USA) was applied at concentrations of 5, 10, 20, 40, and 80 μM.

Techniques: Inhibition, Migration, TUNEL Assay, Staining, Standard Deviation, Expressing, Transwell Assay

Journal: Genes & Diseases

Article Title: Autophagy-induced NR2F1 activation promotes the apoptosis of lens epithelial cells and facilitates cataract-associated fibrosis through targeting STAT3

doi: 10.1016/j.gendis.2025.101549

Figure Lengend Snippet: NR2F1 directly binds to STAT3 and regulates the expression of p-STAT3. (A, B) The protein levels and quantification of JAK1, p-STAT3, SMAD2, and MYD88 in TGF-β1-treated SRA01/04 cells with or without Sh-NR2F1. n = 3 per group; mean ± standard deviation; ns, >0.05; ∗∗ p < 0.01; unpaired student's t -test. (C, D) The protein level and quantitative chart of p-STAT3 in AAV-NC or AAV-NR2F1 ASC mice. n = 3 per group; mean ± standard deviation; ∗ p < 0.01; unpaired student's t -test. (E) The motif of NR2F1 predicted by the JASPAR website. (F) A dual-luciferase vector. (G) Mutant and wild-type STAT3 plasmids consequences. (H) The dual-luciferase assay for NR2F1 and STAT3 promoter. n = 3 per group; mean ± standard deviation; ns, >0.05; ∗ p < 0.05; unpaired student's t -test. (I, J) The protein level of p-STAT3 in TGF-β1-induced SRA01/04 cells following NR2F1 agonist treatment at 0.5 or 1 μM. n = 3 per group; mean ± standard deviation; ns > 0.05; ∗ p < 0.05; one-way ANOVA. (K) Structure of p-STAT3 specific inhibitor NSC 74859. (L, M) The protein expression and quantification of p-STAT3, STAT3, JAK1, FN1, VIM, and α-SMA in TGF-β1-induced SRA01/04 cells treated with the specific P-STAT3 inhibitor. n = 3 per group; mean ± standard deviation; ns, >0.05, ∗ p < 0.05, ∗∗ p < 0.01; one-way ANOVA. (N, O) The protein expression of the apoptosis-related markers BAX and CASP3 in TGF-β1-induced SRA01/04 cells treated with NSC 74859. n = 3 per group; mean ± standard deviation; ∗ p < 0.05; one-way ANOVA. NR2F1, nuclear receptor subfamily 2 group F member 1; STAT3, signal transducer and activator of transcription 3; p-STAT3, phosphorylated STAT3; ASC, anterior subcapsular cataract; FN1, fibronectin 1; α-SMA, α-smooth muscle actin; VIM, vimentin; BAX, Bcl-2-associated X; CASP3, caspase 3; TGF-β1, transforming growth factor-β1; JAK1, Janus kinase 1; SMAD2, SMAD family member 2; AAV, adeno-associated adenovirus.

Article Snippet: For TGF-β1 treatment, cells were seeded in 6-well plates and treated with 10 ng/mL of TGF-β1 (PeproTech, Suzhou, China) for 48 h. The autophagy inhibitor chloroquine (T8689, TargetMol, USA) was applied at concentrations of 5, 10, 20, 40, and 80 μM.

Techniques: Expressing, Standard Deviation, Luciferase, Plasmid Preparation, Mutagenesis

Journal: Genes & Diseases

Article Title: Autophagy-induced NR2F1 activation promotes the apoptosis of lens epithelial cells and facilitates cataract-associated fibrosis through targeting STAT3

doi: 10.1016/j.gendis.2025.101549

Figure Lengend Snippet: The regulatory mechanism of NR2F1 in TGF-β1-induced SRA01/04 cells. NR2F1, nuclear receptor subfamily 2 group F member 1; TGF-β1, transforming growth factor-β1.

Article Snippet: For TGF-β1 treatment, cells were seeded in 6-well plates and treated with 10 ng/mL of TGF-β1 (PeproTech, Suzhou, China) for 48 h. The autophagy inhibitor chloroquine (T8689, TargetMol, USA) was applied at concentrations of 5, 10, 20, 40, and 80 μM.

Techniques:

Journal: NPJ Microgravity

Article Title: BMP9 attenuates microgravity-related disuse osteoporosis by modulating TGFβ and BMP signaling

doi: 10.1038/s41526-025-00510-y

Figure Lengend Snippet: A Micro-CT reconstruction image. B Micro-CT analysis ( n = 6). C Morphology of distal femoral bone tissue, H.E. staining, TRAP staining, AB-PAS staining and silver nitrate staining. GP Growth Plate. D Cortical bone and bone marrow TGFβ1 immunohistochemistry, p-Smad2/3 immunohistochemistry, and p-Smad1/5/8 immunohistochemistry. Scale bar: Black/Red 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Bone marrow stromal cells were extracted using the previously mentioned method , infected with Adv-BMP9 or control virus, and then treated with osteogenic induction medium (OM) containing TGFβ1 at concentrations of 1, 5, and 10 ng/mL (Topscience, China) to assess its impact on osteogenic function.

Techniques: Micro-CT, Staining, Immunohistochemistry

Journal: NPJ Microgravity

Article Title: BMP9 attenuates microgravity-related disuse osteoporosis by modulating TGFβ and BMP signaling

doi: 10.1038/s41526-025-00510-y

Figure Lengend Snippet: A Western blot analysis of phosphorylated Smads in primary osteocytes stimulated with a high dose of TGFβ1 for 3 days. B Expression of BMP-pSmad1/5/8 downstream target genes in primary osteocytes treated with varying concentrations of TGFβ1 for 3 days. C Intracellular pH changes in response to different concentrations of TGFβ1. D Expression of osteolytic factors in primary osteocytes induced by different concentrations of TGFβ1. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Bone marrow stromal cells were extracted using the previously mentioned method , infected with Adv-BMP9 or control virus, and then treated with osteogenic induction medium (OM) containing TGFβ1 at concentrations of 1, 5, and 10 ng/mL (Topscience, China) to assess its impact on osteogenic function.

Techniques: Western Blot, Expressing

Journal: NPJ Microgravity

Article Title: BMP9 attenuates microgravity-related disuse osteoporosis by modulating TGFβ and BMP signaling

doi: 10.1038/s41526-025-00510-y

Figure Lengend Snippet: A Alkaline phosphatase staining and Alizarin Red S staining. B Alkaline phosphatase enzyme activity. C Quantification of mineralization. D Osteogenic gene expression ( n = 3). E Phosphorylation of Smads following high-dose TGFβ1 treatment in vitro, as detected by western blot. F Alteration of BMP-pSmad1/5/8 signaling target genes in marrow stromal cells by high-dose TGFβ1 in vitro ( n = 3). CM Control culture medium, OM=Osteogenic induction medium. ns no significance * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Bone marrow stromal cells were extracted using the previously mentioned method , infected with Adv-BMP9 or control virus, and then treated with osteogenic induction medium (OM) containing TGFβ1 at concentrations of 1, 5, and 10 ng/mL (Topscience, China) to assess its impact on osteogenic function.

Techniques: Staining, Activity Assay, Gene Expression, Phospho-proteomics, In Vitro, Western Blot, Control

Journal: NPJ Microgravity

Article Title: BMP9 attenuates microgravity-related disuse osteoporosis by modulating TGFβ and BMP signaling

doi: 10.1038/s41526-025-00510-y

Figure Lengend Snippet: A Alkaline phosphatase staining. B Quantification of alkaline phosphatase enzyme activity. C Osteogenic gene expression following infection with Adv-BMP9 or control virus in the presence of 10 ng/ml TGFβ1 ( n = 3). D Phosphorylation of Smads in marrow stromal cells following infection with Adv-BMP9 or control virus in the presence of 10 ng/ml TGFβ1, as detected by western blot. E BMP-pSmad1/5/8 signaling target gene expression in marrow stromal cells following infection with Adv-BMP9 or control virus in the presence of 10 ng/ml TGFβ1 ( n = 3). ns no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Bone marrow stromal cells were extracted using the previously mentioned method , infected with Adv-BMP9 or control virus, and then treated with osteogenic induction medium (OM) containing TGFβ1 at concentrations of 1, 5, and 10 ng/mL (Topscience, China) to assess its impact on osteogenic function.

Techniques: Staining, Activity Assay, Gene Expression, Infection, Control, Virus, Phospho-proteomics, Western Blot, Targeted Gene Expression

Journal: NPJ Microgravity

Article Title: BMP9 attenuates microgravity-related disuse osteoporosis by modulating TGFβ and BMP signaling

doi: 10.1038/s41526-025-00510-y

Figure Lengend Snippet: A Phosphorylation of Smads in primary osteocytes following infection with Adv-BMP9 or control virus in the presence of 10 ng/ml TGFβ1, as detected by western blot. B BMP-pSmad1/5/8 signaling target gene expression in primary osteocytes following infection with Adv-BMP9 or control virus in the presence of 10 ng/ml TGFβ1 ( n = 3). C Intracellular pH changes in primary osteocytes following infection with Adv-BMP9 or control virus in the presence of 10 ng/ml TGFβ1, normalized to control group. D Expression of osteoclastic genes in primary osteocytes following infection with Adv-BMP9 or control virus in the presence of 10 ng/ml TGFβ1 ( n = 3). ns no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Bone marrow stromal cells were extracted using the previously mentioned method , infected with Adv-BMP9 or control virus, and then treated with osteogenic induction medium (OM) containing TGFβ1 at concentrations of 1, 5, and 10 ng/mL (Topscience, China) to assess its impact on osteogenic function.

Techniques: Phospho-proteomics, Infection, Control, Virus, Western Blot, Targeted Gene Expression, Expressing