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: The Journal of Clinical Investigation

Article Title: Age-associated callus senescent cells produce TGF- β 1 that inhibits fracture healing in aged mice

doi: 10.1172/JCI148073

Figure Lengend Snippet: Young and aged mice were sacrificed on 10 dpf. ( A ) Heatmap of the expression of SASP factors in callus as determined by qPCR. n = 3. ( B ) Expression of SASP factors in callus and nonfractured bone was assessed by qPCR. n = 4. Two-way ANOVA followed by Tukey’s post hoc test. ( C ) p16 + SCs isolated from young and aged callus. Expression of SASP factors examined by qPCR. * P < 0.05, by unpaired, 2-tailed Student’s t test. ( D ) p16 + , TGF-β1 + , and p16 + TGF-β1 + cells were identified by flow cytometry. ( E ) Fold changes in p16 + , TGF-β1 + , and p16 + TGF-β1 + cell percentages in callus and nonfractured tibiae of aged versus young mice. n = 4–6. * P < 0.05, for callus versus bone, by unpaired, 2-tailed Student’s t test. ( F ) Percentage of p16 + TGF-β1 + cells in callus and nonfractured tibiae. * P < 0.05; ^ P < 0.05; # P < 0.05, for aged versus young, by 2-way ANOVA followed by Tukey’s post hoc test. ( G ) Expression of TGF-β1 in callus tissues following D+Q treatment by as determined by Western blotting. ( H ) CaMPCs were treated for 2 days with CM from young or aged callus with or without TGF-β–neutralizing Ab or IgG. Cell growth and proliferation were assessed by methylene blue staining or a BrdU incorporation assay. n = 4 wells. * P < 0.05, for IgG versus anti–TGF-β Ab; # P < 0.05, for aged anti–TGF-β Ab versus young IgG, by 2-way ANOVA followed by Tukey’s post hoc test. ( I ) Concentration of active TGF-β1 in CM from young and aged callus cultures measured by ELISA. * P < 0.05, by unpaired, 2-tailed Student’s t test. ( J ) Callus pieces were harvested from young and aged mice and infected with Tgfb1 or scrambled (Ctl) shRNA lentivirus. CM was collected. The expression of Tgfb1 was measured by qPCR. CaMPCs were treated with the CM. Cell growth was assessed using a CCK8 kit. n = 3 wells. * P < 0.05, for control versus Tgfb1 shRNA, by 2-way ANOVA followed by Tukey’s post hoc test. The experiment was repeated once.

Article Snippet: The shRNA lentiviral particles targeting Tgfb1 (sc-37192-V) and a scrambled sequence (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Isolation, Flow Cytometry, Western Blot, Staining, BrdU Incorporation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, shRNA, Control

Journal: The Journal of Clinical Investigation

Article Title: Age-associated callus senescent cells produce TGF- β 1 that inhibits fracture healing in aged mice

doi: 10.1172/JCI148073

Figure Lengend Snippet: Young and aged mice underwent tibial fracture surgery. ( A ) The expression of Tgfb1 in fracture callus at indicated time points was measured by qPCR. n = 3. Relative mRNA expression is the fold-change versus young mice as 1. ( B ) The concentration of active TGF-β1 protein in fracture callus at indicated time points was measured by ELISA. n = 4. * P < 0.05, for aged versus young; # P < 0.05, for young versus young 0 dpf; ^ P < 0.05, for aged versus aged 0 dpf, by 2-way ANOVA followed by Tukey’s post hoc test ( A and B ). ( C ) Outline of the experimental design. Aged mice were given 2 μg in 10 μL TGF-β Ab, 1D11, or isotype IgG vehicle by intra-callus injection on 1, 3, 5, and 7 dpf and sacrificed on 10 dpf ( D – F and H – J ) or 28 dpf ( G ). n = 4–5. ( D ) Callus volume was measured by micro-CT. * P < 0.05, by unpaired, 2-tailed Student’s t test. ( E ) Representative images of ABH-stained sections showing more woven bone and callus areas in the anti–TGF-β Ab–treated mice. Scale bar: 1 mm. ( F ) Woven bone and cartilage areas were analyzed using Visiopharm software. ( G ) Bone stiffness, strength and toughness were assessed by biomechanical testing at 28 dpf. ( H ) The percentage and number of MPCs identified as CD45 – CD31 – CD105 + cells in fracture callus were determined by flow cytometry. ( I ) Representative paraffin sections of callus immunostained with anti-Ki67 Ab to detect proliferating cells (arrowheads). External callus is indicated by the dashed lines. Scale bars: 500 μm. Original magnification, ×4 (enlarged insets). ( J ) The percentage of Ki67 + cells was quantified by ImageJ. * P < 0.05, by unpaired, 2-tailed Student’s t test ( F , G , H , and J ).

Article Snippet: The shRNA lentiviral particles targeting Tgfb1 (sc-37192-V) and a scrambled sequence (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Injection, Micro-CT, Staining, Software, Flow Cytometry