normal rat kidney fibroblasts  (ATCC)

Journal: Renal Failure

Article Title: Ubiquitin-specific protease 11 facilitates the activation and proliferation of renal interstitial fibroblasts through epidermal growth factor receptor signaling pathways

doi: 10.1080/0886022X.2026.2666452

Figure Lengend Snippet: USP11 is highly upregulated in UA-stimulated RIF. In vitro , we stimulated NRK49F with UA in dose-dependent manner (0, 200, 400, 800 μM) and in time-dependent manner (0, 12, 24, 36 hours). Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (A, C). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (B, D). Data were expressed as mean± SD ( n = 4 for each group). N.S.: no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Rat renal interstitial fibroblast (NRK49F) cells were obtained from ATCC (Manassas, VA).

Techniques: In Vitro, Western Blot

Journal: Renal Failure

Article Title: Ubiquitin-specific protease 11 facilitates the activation and proliferation of renal interstitial fibroblasts through epidermal growth factor receptor signaling pathways

doi: 10.1080/0886022X.2026.2666452

Figure Lengend Snippet: USP11 contributes to RIF activation in the UA-stimulated NRK49F. NRK49F cells were seeded to 70–80% confluence in the antibiotic-free medium and grown followed by transfection with USP11 siRNA or USP11-pcDNA 3.0 plasmid. After transfection, the medium was changed to DMEM with F12 containing 0.5% FBS for starvation and then cells were incubated with or without uric acid (800 μM) for an additional 36 hours before being harvested for analysis. Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (A, F, H). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (B, G, I). Representative images and quantitative analysis of immunofluorescence staining for Fibronectin and Vimentin with DAPI nuclear counterstaining in each group (C). Then NRK49F cells were starved for 24 h with DMEM containing 0.5% FBS before they were exposed to uric acid in the presence or absence of MTX (1, 5, and 10 μM). Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (D). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (E). Data were expressed as mean± SD ( n = 4 for each group). N.S.: no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bars = 100μm.

Article Snippet: Rat renal interstitial fibroblast (NRK49F) cells were obtained from ATCC (Manassas, VA).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Incubation, Western Blot, Immunofluorescence, Staining

Journal: Renal Failure

Article Title: Ubiquitin-specific protease 11 facilitates the activation and proliferation of renal interstitial fibroblasts through epidermal growth factor receptor signaling pathways

doi: 10.1080/0886022X.2026.2666452

Figure Lengend Snippet: USP11 directly interacts with EGFR and stabilizes its protein level. Immunofluorescence co-staining of USP11 and EGFR in the UA-stimulated NRK49F (A). Co-IP assay in NRK49F cells. Flag-tagged USP11 and Myc-tagged EGFR were immunoprecipitated using an anti-Flag/Myc antibody, and the presence of Myc-tagged EGFR and Flag-tagged USP11 in the precipitates was detected by immunoblotting (B, C). Western blot analysis of USP11 and EGFR protein expressions in each group were conducted, with normalization to GAPDH (D). Quantitative analysis of EGFR protein levels in each group (E). Data were expressed as mean± SD ( n = 4 for each group). N.S.: no significant difference, *** p < 0.001. Scale bars = 50μm.

Article Snippet: Rat renal interstitial fibroblast (NRK49F) cells were obtained from ATCC (Manassas, VA).

Techniques: Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot

Journal: Renal Failure

Article Title: Ubiquitin-specific protease 11 facilitates the activation and proliferation of renal interstitial fibroblasts through epidermal growth factor receptor signaling pathways

doi: 10.1080/0886022X.2026.2666452

Figure Lengend Snippet: USP11 activates EGFR signaling pathway in the UA-stimulated NRK49F. Western blot analysis of p-EGFR and EGFR protein expressions in each group were conducted, with normalization to GAPDH (A, C, E). Quantitative analysis of p-EGFR and EGFR protein levels in each group (B, D, F). Data were expressed as mean ± SD (n = 4 for each group). N.S.: no significant difference. *** p < 0.001, **** p < 0.0001.

Article Snippet: Rat renal interstitial fibroblast (NRK49F) cells were obtained from ATCC (Manassas, VA).

Techniques: Western Blot

Journal: Renal Failure

Article Title: Ubiquitin-specific protease 11 facilitates the activation and proliferation of renal interstitial fibroblasts through epidermal growth factor receptor signaling pathways

doi: 10.1080/0886022X.2026.2666452

Figure Lengend Snippet: USP11 is involved in the activation of EGFR signaling pathway in the UA-stimulated NRK49F. To fully demonstrate the relationship between USP11 and EGFR signaling, we stimulated starved NRK49F cells with EGF (5 ng/ml) in the presence or absence of USP11 siRNA or treated with gefitinib (1 nM and 5 nM), a highly selective EGFR inhibitor in the presence of USP11 pcDNA 3.0 plasmid for an additional 36 hours. Western blot analysis of p-EGFR and EGFR protein expressions in each group were conducted, with normalization to GAPDH (A, E). Quantitative analysis of p-EGFR and EGFR protein levels in each group (B, F). Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (C, G). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (D, H). Data were expressed as mean ± SD ( n = 4 for each group). N.S.: no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Rat renal interstitial fibroblast (NRK49F) cells were obtained from ATCC (Manassas, VA).

Techniques: Activation Assay, Plasmid Preparation, Western Blot

Journal: Renal Failure

Article Title: Ubiquitin-specific protease 11 facilitates the activation and proliferation of renal interstitial fibroblasts through epidermal growth factor receptor signaling pathways

doi: 10.1080/0886022X.2026.2666452

Figure Lengend Snippet: Inhibition of USP11 attenuates the proliferation and migration in the UA-stimulated NRK49F. Photomicrographs of migrating cells in wound healing assay were taken at 0h and 36h (A). The migratory rate was calculated as (A-B)/A*100%, where A and B reflect the width of the wound at 0h and 36h respectively (B). The CCK-8 proliferation kit was used according to the manufacturer’s instructions and the final optical density values were read at 450 nm (C). Representative images and quantitative analysis of immunofluorescence staining for Ki67 with DAPI nuclear counterstaining in each group (D, E). Western blot analysis of PCNA and Cyclin E protein expressions in each group were conducted, with normalization to GAPDH (F, H). Quantitative analysis of PCNA and Cyclin E protein levels in each group (G, I). Data were expressed as mean± SD ( n = 4 for each group). N.S., no significant difference. * p < 0.05, *** p < 0.001, **** p < 0.0001. Scale bars = 500μm (A) and 50μm (D).

Article Snippet: Rat renal interstitial fibroblast (NRK49F) cells were obtained from ATCC (Manassas, VA).

Techniques: Inhibition, Migration, Wound Healing Assay, CCK-8 Assay, Immunofluorescence, Staining, Western Blot

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: Redox Report : Communications in Free Radical Research

Article Title: Ligustroflavone alleviates chronic kidney disease by inhibiting ferroptosis through the GSK3β/NRF2 signaling pathway

doi: 10.1080/13510002.2026.2636421

Figure Lengend Snippet: Supernatant from ligustroflavone-pretreated MPTCs inhibits NRK-49F activation. (A)EdU assay of NRK-49F proliferation. Semi-quantitative ImageJ analysis shown below (scale bar, 20 μm). (B) Immunofluorescence staining of α-SMA expression in NRK-49F cells. Semi-quantitative ImageJ analysis shown below. (scale bar, 20 μm). Data are presented as mean ± SD (n = 6). **** P < 0·0001, ** P < 0·01 ns: no statistical difference (one-way ANOVA).

Article Snippet: MPTCs (CRL-3361, ATCC, USA) and NRK-49F fibroblasts (CRL-1570, ATCC, USA) were obtained from the American Type Culture Collection (ATCC, Virginia, USA).

Techniques: Activation Assay, EdU Assay, Immunofluorescence, Staining, Expressing