normal rat kidney fibroblasts  (ATCC)

Journal: Journal of Advanced Research

Article Title: Direct pharmacological targeting of Piezo1 by Paeoniflorin: a novel therapeutic approach for renal fibrosis

doi: 10.1016/j.jare.2025.07.015

Figure Lengend Snippet: PF inhibits matrix stiffness-induced acceleration of EndMT through Piezo1 activation (A–F) Matrix stiffness-dependent protein modulation in HUVECs. HUVECs were cultured on polyacrylamide hydrogels with soft (1.00 ± 0.31 kPa) or stiff (40.40 ± 2.39 kPa) for 48 h, with or without PF. (A) Western blot analysis of (B) Piezo1, (C) VE-Cadherin, (D) eNOS, (E) Vimentin, and (F) TGF-β1. Quantification normalized to GAPDH. (n = 3; *p < 0.05, **p < 0.01 vs. soft; #p < 0.05 vs. stiff without PF). (G-L) Piezo1 knockdown reverses stiffness-induced EndMT. HUVECs transfected with Piezo1 siRNA (50 nM, 24 h) or scramble siRNA (control) were cultured on stiffness hydrogels (40.40 ± 2.39 kPa) ± PF (400 μM). (G) Western blot analysis of (H) Piezo1, (I) VE-Cadherin, (J) eNOS, (K) Vimentin, and (L) TGF-β1. Quantification normalized to GAPDH (n = 3; *p < 0.05, **p < 0.01 vs. scramble siRNA control; ns vs. Piezo1 siRNA without PF).(M) Schematic of co-culture model. HUVECs and NRK-49F fibroblasts were co-cultured on stiffness-tunable hydrogels using a transwell system (0.4 μm pore size) for 5 days to assess paracrine signaling. (N) TGF-β1 levels in HUVEC supernatants from the co-culture system were quantified by ELISA. (n = 3; *p < 0.05 vs. softness; #p < 0.05 vs. stiffness without PF). (O) Fibrotic gene expression in NRK-49F cells. RT-qPCR analysis of Fibronectin , COL1A1 , Vimentin , and TGF-β1 mRNA levels. Data normalized to 18 s ( n = 3). (P-S) Fibrotic protein expression in NRK-49F cells. (P) Western blot analysis of (Q) Fibronectin, (R) COL1, (S) Vimentin, and (T) TGF-β1. Quantification normalized to GAPDH (n = 3). (U–V) Immunofluorescence of Fibronectin (red) in NRK-49F cells. Nuclei stained with DAPI (blue). (U) Representative images. (V) Quantification of fluorescence intensity using ImageJ (n = 3). Scale bar: 20 μm. Data presented as mean ± SEM. *p < 0.05, **p < 0.01 vs . softness co-culture; # p < 0.05 vs . stiffness co-culture without PF . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and rat kidney fibroblast cell line (NRK-49F) were purchased from ATCC (American Type Culture Collection, https://www.atcc.org ).

Techniques: Activation Assay, Cell Culture, Western Blot, Knockdown, Transfection, Control, Co-Culture Assay, Pore Size, Enzyme-linked Immunosorbent Assay, Gene Expression, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Advanced Research

Article Title: Direct pharmacological targeting of Piezo1 by Paeoniflorin: a novel therapeutic approach for renal fibrosis

doi: 10.1016/j.jare.2025.07.015

Figure Lengend Snippet: PF inhibits EndMT through the Piezo1-mediated HIF-1α signaling pathway (A–C) Renal HIF-1α expression analysis. (A) Representative RT-qPCR analysis of HIF-1α mRNA levels in kidney tissues. Data normalized to 18 s . (B-C) Western blot and quantification of HIF-1α protein expression in renal tissues. Data normalized to GAPDH (n = 5–6) . (D–H) Effects of PF or HIF-1α inhibitor BAY 87-2243 (10 μM, 24 h) on endothelial markers in HUVECs cultured with or without Piezo1 activation by Yoda1 (5 μM, 12 h). (D) Western blot analysis of (E) Piezo1, (F) HIF-1α, (G) VE-Cadherin, and (H) eNOS. Quantification normalized to GAPDH. Quantification showing Yoda1-induced Piezo1 upregulation and HIF-1α/VE-Cadherin/eNOS downregulation, reversed by PF or BAY 87-2243 (n = 3). (I-K) PF or BAY 87-2243 inhibits Yoda1-induced EndMT in HUVECs. (I) Western blot analysis of (J) Vimentin and (K) TGF-β1. Yoda1 increased Vimentin and TGF-β1, suppressed by PF or BAY 87-2243 (n = 3). (L) Schematic of HUVEC-NRK-49F co-culture. HUVECs pre-treated with/without Yoda1 (5 μM, 6 h) were co-cultured with NRK-49F fibroblasts for 48 h. (M) TGF-β1 levels in HUVEC supernatants from the co-culture system were quantified by ELISA. (N) RT-qPCR analysis of Fn1 , COL1A1 , and Vimentin mRNA in NRK-49F cells co-cultured with Yoda1-treated HUVECs. PF attenuated Yoda1-induced fibrotic marker expression (n = 3). (O-S) Western blot validation of (P) Fibronectin, (Q) COL1, (R) Vimentin, and (S) TGF-β1 in NRK-49F cells. PF reduced Yoda1-induced protein expression (n = 3). Data presented as mean ± SEM. *p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTL/control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CRF/Yoda1.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and rat kidney fibroblast cell line (NRK-49F) were purchased from ATCC (American Type Culture Collection, https://www.atcc.org ).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Cell Culture, Activation Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Marker, Biomarker Discovery, Control

Journal: Cell Death Discovery

Article Title: Inhibition of epigenetic regulator UHRF1 attenuates renal fibrosis and retains transcription factor Krüppel-like factor 15 expression

doi: 10.1038/s41420-025-02549-y

Figure Lengend Snippet: A Masson staining (×200) and immunochemistry staining (×200) for α-SMA and Fibronectin on days 7 in UUO mice and on days 14 in UIR mice. Scale bar: 100 μm. B The bar graph summarizes the relative fibrotic area or α-SMA-positive and Fibronectin positive areas in each group. C , D Relative renal KLF15 protein expression in UUO mice or in UIR mice, analyzed by western blots. E , F Immunofluorescence double-labeling with antibodies to UHRF1 (Red) and PDFGRα + β (green) or AQP1 (green) (×200). The white arrow represents UHRF1 + fibroblasts (top panel) and UHRF1 + proximal tubular epithelial cells (bottom panel). Scale bar: 50 μm. n = 6 mice per group. Data were shown as mean ± SEM. Data were analyzed using unpaired Student’s t test. * P < 0.05, ** P < 0.01.

Article Snippet: Rat normal renal fibroblast (NRK-49F cells, ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

Techniques: Staining, Expressing, Western Blot, Immunofluorescence, Labeling

Journal: Cell Death Discovery

Article Title: Inhibition of epigenetic regulator UHRF1 attenuates renal fibrosis and retains transcription factor Krüppel-like factor 15 expression

doi: 10.1038/s41420-025-02549-y

Figure Lengend Snippet: Col1a2-Cre + /UHRF1 flox/flox mice (fibroblast-specific depletion of UHRF1) and their littermate controls received Sham or UUO operation for 7 days. A Generation of mice with fibroblast-specific depletion of UHRF1 using Cre-LoxP recombination system. B Genotyping of conditional knockout (cKO) mice (Col1a2-Cre + /UHRF1 flox/flox mice). C Illustration of experimental design to induce fibroblast-specific depletion of UHRF1 in UUO mice. D Relative renal UHRF1 protein expression analyzed by Western blots. E Immunofluorescence double-labeling with antibodies to UHRF1 (Red) and PDFGRα + β (green) (×200). The white arrow represents UHRF1 + fibroblasts. Scale bar: 50 μm. Masson staining ( F , H ) and immunochemistry staining for α-SMA and Fibronectin ( G , I ) in kidney sections (×200). Scale bar: 100 μm. ( J , K ) Western blots show the renal expression of α-SMA and Fibronectin. n = 6 mice per group. Data were shown as mean ± SEM. Data were analyzed using One-way ANOVA followed by Tukey. * P < 0.05, ** P < 0.01.

Article Snippet: Rat normal renal fibroblast (NRK-49F cells, ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

Techniques: Knock-Out, Expressing, Western Blot, Immunofluorescence, Labeling, Staining

Journal: Cell Death Discovery

Article Title: Inhibition of epigenetic regulator UHRF1 attenuates renal fibrosis and retains transcription factor Krüppel-like factor 15 expression

doi: 10.1038/s41420-025-02549-y

Figure Lengend Snippet: Col1a2-Cre + /UHRF1 flox/flox mice (fibroblast-specific depletion of UHRF1) and their littermate controls received Sham or UIR operation for 14 days. A Illustration of experimental design to induce fibroblast-specific depletion of UHRF1 in UIR mice. B Immunofluorescence double-labeling with antibodies to UHRF1 (Red) and PDFGRα + β (green) (×200). The white arrow represents UHRF1 + fibroblasts. Scale bar: 50 μm. C , D Masson staining and immunochemistry staining for α-SMA and Fibronectin in kidney sections (×200). Scale bar: 100 μm. n = 6 mice per group. Data were shown as mean ± SEM. Data were analyzed using One-way ANOVA followed by Tukey. * P < 0.05, ** P < 0.01.

Article Snippet: Rat normal renal fibroblast (NRK-49F cells, ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

Techniques: Immunofluorescence, Labeling, Staining

Journal: Cell Death Discovery

Article Title: Inhibition of epigenetic regulator UHRF1 attenuates renal fibrosis and retains transcription factor Krüppel-like factor 15 expression

doi: 10.1038/s41420-025-02549-y

Figure Lengend Snippet: A MeDIP-qPCR analysis shows the level of KLF15 promotor methylation. NRK-49F cells were transfected with UHRF1 siRNA or control siRNA before treatment of 10 ng/ml TGF-β1 for 48 h. n = 3 samples per group. B , C Col1a2-Cre + /UHRF1 flox/flox mice (fibroblast-specific depletion of UHRF1) and their littermate controls received Sham or UUO operation for 7 days. B Renal KLF15 promotor methylation levels were determined by MeDIP-qPCR. n = 4 mice per group. C Renal KLF15 protein levels were determined by Western blots. n = 6 mice per group. Data were shown as mean ± SEM. Data were analyzed using unpaired Student’s t test ( B ) or One-way ANOVA followed by Tukey ( A , C ). * P < 0.05, ** P < 0.01.

Article Snippet: Rat normal renal fibroblast (NRK-49F cells, ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

Techniques: Methylated DNA Immunoprecipitation, Methylation, Transfection, Control, Western Blot

Journal: Cell Death Discovery

Article Title: Inhibition of epigenetic regulator UHRF1 attenuates renal fibrosis and retains transcription factor Krüppel-like factor 15 expression

doi: 10.1038/s41420-025-02549-y

Figure Lengend Snippet: A Western blots showed the DNMT1 expression in NRK-49F cells transfected with UHRF1 siRNA or control siRNA with or without TGF-β1 treatment. B DNMT1 enzyme activity of NRK-49F cells transfected with UHRF1 siRNA or control siRNA with or without TGF-β1 treatment. C Co-IP analysis showed anti-UHRF1 antibody pulled down endogenous DNMT1. D PLA assay showed the interaction between UHRF1 and DNMT1 in NRK-49F cells with or without TGF-β1 treatment, presented by red fluorenscent signals (×400). Scale bar: 20 μm. E MeDIP-qPCR analysis showed inhibition of the interaction between UHRF1 and DNMT1 with NSC232003 prevented TGF-β1-induced KLF15 hypermethylation in NRK-49F cells. F Western blots demonstrated blocking effect of NSC232003 on TGF-β1-induced fibroblasts activation in NRK-49F cells. G NRK-49F cells infected with CRISPR-Cas9 lentivirus targeting KLF15 or scramble lentivirus were treated with TGF-β1 and NSC232003. Fibroblasts activation was assessed by immunoblots of α-SMA and Fibronectin. n = 3–6 samples per group. Data were shown as mean ± SEM. Data were analyzed using unpaired Student’s t test ( D , G ) or One-way ANOVA followed by Tukey ( A , B , E , F ). * P < 0.05, ** P < 0.01.

Article Snippet: Rat normal renal fibroblast (NRK-49F cells, ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

Techniques: Western Blot, Expressing, Transfection, Control, Activity Assay, Co-Immunoprecipitation Assay, Methylated DNA Immunoprecipitation, Inhibition, Blocking Assay, Activation Assay, Infection, CRISPR