Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: ADAMTS1 levels were increased in the serum of patients with myocardial fibrosis, in the hearts of mice after CFPMI and in cardiac fibroblasts stimulated with fibrotic factors. A . ADAMTS1 levels in clinical serum samples were evaluated via ELISA. N = 30. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. B . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. C . Survival rate of mice in each group. D . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. E and F . ADAMTS1 expression in mouse heart tissue was determined through Western blot and IHC. Human cardiac fibroblasts were treated with 10 ng/mL TGF-β1 or 0.1 μM Ang II for 12, 24, or 48 h to construct in vitro models. The cells were divided into four groups: the Control, TGF-β1/Ang II (12 h), TGF-β1/Ang II (24 h), and TGF-β1/Ang II (48 h) groups. G and H . Western blot detection of the expression of ADAMTS1, Collagen I and FN. I. The expression levels of ADAMTS1, Collagen I and FN were measured via qRT‒PCR. After the optimal time point (48 h) was determined, the mice were further divided into the control, TGF-β1 and Ang II groups. J . ADAMTS1 expression in cells was assessed by IF. Human cardiac fibroblasts were treated with 10 ng/mL TGF-β1 or 0.1 μM Ang II for 15, 30, 60, 90, and 120 min. K. qRT-PCR detection of the mRNA expression of ADAMTS1, Collagen I and FN. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Construct, Staining, Expressing, Western Blot, In Vitro, Control, Quantitative RT-PCR

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: SMAD2 regulated ADAMTS1 expression in human and mouse cardiac fibroblasts induced by TGF-β1. We used 10 ng/mL TGF-β1 to induce human and mouse cardiac fibroblasts for 15, 30, 60, 90, and 120 min. A and B . Western blot analysis of the expression of SMAD2, p-SMAD2 and ADAMTS1. Human and mouse cardiac fibroblasts were treated with 10 ng/mL TGF-β1 for 48 h, and the cells were divided into Control and TGF-β1 groups. C . Human and mouse cardiac fibroblasts were induced with 10 ng/mL TGF-β1 for 12 h, 24 h, 48 h, and 72 h. qRT‒PCR was performed to detect the mRNA levels of ADAMTS1, Collagen I, and FN. D . IF staining was conducted to determine the expression of p-SMAD2 and ADAMTS1 at 48 h. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. E . p-SMAD2 expression in mouse heart tissue was measured by IHC. F . ChIP verification of the interaction between SMAD2 and ADAMTS1. Then, we interfered with SMAD2 expression. The groups were the si-NC, si-SMAD2-1, and si-SMAD2-2 groups. G . SMAD2 expression was assessed via qRT‒PCR. After the best si-SMAD2 was selected, the cells were further divided into the si-NC, TGF-β1 (48 h), si-SMAD2, and TGF-β1 + si-SMAD2 groups. H . Western blot detection of ADAMTS1, Collagen I, and FN expression in cells. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Expressing, Western Blot, Control, Staining, Construct

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: Overexpression of ADAMTS1 enhanced the production of collagen fiber proteins in human and mouse cardiac fibroblasts induced by TGF-β1. We overexpressed ADAMTS1 and divided the cells into the oe-NC, TGF-β1 (48 h), oe-ADAMTS1, and TGF-β1 + oe-ADAMTS1 groups. A . Western blot detection of ADAMTS1, Collagen I, FN, and α-SMA expression. B-D . IF staining of Collagen I and FN expression. Additionally, we interfered with ADAMTS1; the cells were grouped as follows: si-NC, si-ADAMTS1-1, and si-ADAMTS1-2. E . ADAMTS1 expression was determined through qRT‒PCR. After the best si-ADAMTS1 was selected, the cells were further divided into the si-NC, TGF-β1 (48 h), si-ADAMTS1, and TGF-β1 + si-ADAMTS1 groups. F and G . Western blot detection of ADAMTS1, Collagen I, FN, and α-SMA expression. H and I . IF staining of Collagen I and FN expression. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Over Expression, Western Blot, Expressing, Staining

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: Knockdown of ADAMTS1 alleviated TGF-β1-induced fibrosis by downregulating HDAC6 protein expression. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. A . HDAC6 expression in mouse heart tissue was determined through IHC. After the best si-ADAMTS1 was selected, the cells were further divided into the si-NC and si-ADAMTS1 groups. B . ADAMTS1 and HDAC6 expression was assessed via qRT‒PCR. C . Western blot analysis of ADAMTS1 and HDAC6 expression. Furthermore, we overexpressed ADAMTS1 and divided the cells into the oe-NC, TGF-β1, oe-ADAMTS1, and TGF-β1 + oe-ADAMTS1 groups. D . Western blot detection of HDAC6 expression. After the si-ADAMTS1 group was selected, the cells were further divided into the si-NC, TGF-β1, si-ADAMTS1, and TGF-β1 + si-ADAMTS1 groups. E. Western blot detection of HDAC6 expression. Moreover, cells were treated with the HDAC6 inhibitor ACY1215 (5 μM) for 48 h and then divided into the si-NC, si-ADAMTS1, si-NC + ACY1215, and si-ADAMTS1 + ACY1215 groups. F. Western blot detection of TGF-β1, Collagen I, FN, and ADAMTS1 expression. Subsequently, experiments were performed in human and mouse cardiac fibroblasts with ADAMTS1 overexpression followed by treatment with the HDAC6 inhibitor ACY1215. G. Western blot analysis was conducted to detect the expression levels of ADAMTS1 and HDAC6. H. Western blot analysis was performed to determine the expression levels of TGF-β1, Collagen I, and FN. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Knockdown, Expressing, Construct, Western Blot, Over Expression

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: ADAMTS1 interacted with HDAC6 during fibrosis. A . Co-IP verification of the interaction of ADAMTS1 with HDAC6, with ADAMTS1 as the bait protein. In the experiment, we first captured the ADAMTS1 protein using a specific antibody and then used Co-IP technology to detect its interaction with HDAC6. B . Co-IP verification of the interaction of HDAC6 with ADAMTS1, with HDAC6 as the bait protein. In the experiment, we captured the HDAC6 protein using a specific antibody and then used Co-IP technology to detect its interaction with ADAMTS1. We used 10 ng/mL TGF-β1 to induce human and mouse cardiac fibroblasts for 48 h, and the cells were further divided into control and TGF-β1 groups. C . IF staining was performed to evaluate the colocalization of ADAMTS1 and HDAC6 in TGF-β1-treated human and mouse cardiac fibroblasts. D . Changes in the level of ubiquitinated HDAC6 protein in human and mouse cardiac fibroblasts transfected with oe-ADAMTS1/si-ADAMTS1 or oe-NC/si-NC in the presence of 10 μM MG132. E. In si-ADAMTS1-treated human and mouse cardiac fibroblasts, MG132 (10 μM) was added to detect the ubiquitination levels of ADAMTS1 protein. F. Human and mouse cardiac fibroblasts were treated with TGF-β1 and subjected to SMAD2 knockdown to determine the ubiquitination levels of ADAMTS1 protein. N = 3. *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Co-Immunoprecipitation Assay, Control, Staining, Transfection, Ubiquitin Proteomics, Knockdown

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: AAV-shRNA-ADAMTS1 treatment alleviated myocardial fibrosis and improved cardiac function after CFPMI. The mice were randomly divided into the Sham, CFPMI, and CFPMI + sh-ADAMTS1 groups. A . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. B . Survival rate of mice in each group. C . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. D . HDAC6 expression was determined through qRT‒PCR. E. Western blot analysis of the expression of TGF-β1, SMAD2, and p-SMAD2 in mouse heart tissue. F. Western blot detection of Collagen I, and FN expression. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: shRNA, Staining, Expressing, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: AAV-shRNA-HDAC6 transfection combined with ADAMTS1 inhibitor treatment alleviated myocardial fibrosis and improved cardiac function after CFPMI. The mice were randomly divided into the Sham, CFPMI, CFPMI + anti-ADAMTS1, CFPMI + sh-HDAC6, and CFPMI + anti-ADAMTS1 + sh-HDAC6 groups. A . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. B . Survival rate of mice in each group. C and D . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. E. HDAC6 expression was determined through qRT‒PCR. F. Western blot analysis of the expression of TGF-β1, SMAD2, and p-SMAD2 in mouse heart tissue. G. Western blot detection of Collagen I and FN expression. N = 3. ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: shRNA, Transfection, Staining, Expressing, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: TGF-β1/SMAD2 regulated ADAMTS1 by mediating CFPMI through HDAC6 ubiquitination

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Ubiquitin Proteomics

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: ( A ) Representative images of collagen invasion of spheroids composed of HPMCs or TGF-β1–stimulated HPMCs. Scale bars, 200 μm. ( B ) The bar graph showing TGF-β1–stimulated HPMCs showed a higher invasion ability into the collagen layer. ( C ) Images of migration or invasion cells using the Transwell assay. Scale bar, 200 μm. ( D ) Linear plots showing that TGF-β1–stimulated HPMCs had a higher migration and invasion ability compared with the control HPMCs. ( E ) Bar graph showing the concentration of TGF-β1 in the supernatant in EOC cells when inhibiting TGF-β1 with siRNA. ( F ) Representative images of spheroid collagen invasion. Scale bars, 100 μm. ( G ) Bar graphs showing that inhibition of TGF-β1 by siRNA in OV90 cells or the TGF-β1 receptor blocker in HPMCs reduced the invasion ability of ACMSs compared with the control. ( H and I ) Differences in the spheroids in ascites when mice were injected with OV90 (green) and HPMCs (red). The number of spheroids was significantly decreased when OV90 cells were treated with siRNA for TGF-β1 than those with si-control. Scale bars, 100 μm. ( J and K ) Representative images of metastases on the omentum and bar graph showing the metastasis area on the omentum. Scale bars, 1000 μm. ( L ) Bar graph showing the TGF-β1 concentration in the culture supernatant in sh-control– or TGF-β1–transduced OV90 cells. ( M and N ) Representative images and bar graph showing the omental metastatic area ( n = 8). Scale bars, 1000 μm. ( O and P ) Representative images of confocal imaging of the omental micrometastasis area and bar graph showing the invasion depth of mesothelial cells from the metastatic border. Scale bars, 100 μm. ( Q and R ) Representative images and bar graph showing that both the number and size of spheroids in ascites were significantly decreased in mice injected with sh-TGF-β1 no. 1 or 2 O90 cells compared with sh-control mice. Scale bars, 100 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: As none of these siRNAs achieved significant mRNA suppression in EOC cells, we used a commercially available siRNA mix (Santa Cruz Biotechnology, sc-270322) for subsequent experiments (no. 4).

Techniques: Migration, Transwell Assay, Control, Concentration Assay, Inhibition, Injection, Imaging

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