serpin e1 pai 1 immunoassay  (R&D Systems)

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: WWP1 is highly upregulated in fibrotic human kidneys and correlates with a decline in renal function and disease progression. Renal disease datasets (ERCB Nephrotic Syndrome TubInt, ERCB Lupus TubInt) available from Nephroseq ( https://www.nephroseq.org (accessed on 5 June 2024)) ( A ) were analyzed for WWP1 mRNA levels in human diabetic, FSGS (focal segmental glomerulosclerosis), and lupus nephritis kidneys relative to healthy controls. Data in ( A ) are represented as the median. * p < 0.05, ** p < 0.01. Human renal disease specimens from ( A ) were further assessed for a correlation analysis between WWP1 expression and proteinuria ( B ) (r = 0.63, p = 0.0199) (ERCB Nephrotic Syndrome TubInt), or serum creatinine level ( C ) (r = 0.43, p = 0.0284) (ERCB Nephrotic Syndrome TubInt), or glomerular filtration rate (GFR) ( D , E ) (r = -0.79, p = 0.0349; r = -0.72, p = 0.0425) (ERCB Nephrotic Syndrome TubInt, ERCB Lupus TubInt). Lysates from human healthy and diabetic kidneys were immunoblotted for fibronectin, collagen 1, PAI-1, c-Myc, WWP1, and TRIM65 ( F ) proteins. A single cell RNA sequencing dataset (Accession No.: GSE183279 ) was analyzed for WWP1 transcript levels in the diseased kidneys relative to the reference (healthy controls) ( G ) represented as a dot plot, where the intensity of dot color (brown) dictates the WWP1 expression level, and dot size represents the percentage of cells expressing WWP1. UMAP analysis of the dataset (Accession No.: GSE183279 ) was performed to determine the compartment-specific expression of WWP1 in the kidney ( H ), where in the left panel, yellow-green represents epithelial cells, sea green represents endothelial cells, red represents immune cells, orange represents stroma cells, and a very small percentage of neuronal cells are represented by the blue color.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Biomarker Discovery, Expressing, Filtration, Single Cell, RNA Sequencing

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: PAI-1, WWP1, TRIM65, and c-Myc upregulation correlated with repression of the BMP-7/SMAD1/5 signaling axis in fibrotic UUO kidneys. Mice were subjected to unilateral ureteral obstruction (7 days) prior to the extraction of obstructed (UUO) and contralateral kidneys. Contralateral and UUO renal extracts were assessed for fibronectin ( A , B ), collagen 1 ( A , C ), PAI-1 ( A , D ) and c-Myc ( A , E ), WWP1 ( A , F ), and TRIM65 ( A , G ), BMP-7 ( A , H ), SMAD5 ( A , I ), and pSMAD1/5 ( A , J ) protein levels by immunoblot analysis. β-tubulin is serving as a loading control, and the expression of each indicated protein is normalized to tubulin level. Histograms ( B – J ) depict the expression (mean ± SD) comparisons for the indicated protein in the UUO and contralateral kidneys (reference) using a Student’s T -test for 5 animals per group shown as 1–5 in ( A ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Extraction, Western Blot, Control, Expressing

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Induction of PAI-1, WWP1, TRIM65, and c-Myc correlates with BMP-7/SMAD1/5 signaling axis deregulation in aristolochic acid (AA) induced fibrotic kidneys. Mice were administered either with a NaCl vehicle (control) or aristolochic acid (AA) sodium salt (5 mg/kg body weight dissolved in distilled water) via intraperitoneal injection, daily for 5 consecutive days, and termed as NaCl kidney and AAN kidney, respectively. Twenty-five days post-AA injections, mice in both groups were euthanized for kidney harvesting. Renal lysates from both groups were western blotted for fibronectin ( A , B ), collagen 1 ( A , C ), PAI-1 ( A , D ), c-Myc ( A , E ), WWP1 ( A , F ), TRIM65 ( A , G ), BMP-7 ( A , H ), SMAD5 ( A , I ), and pSMAD1/5 ( A , J ). Data are presented as the mean ± SD. The expression of each indicated protein is normalized to β-tubulin (loading control). Histograms ( B – J ) depict the expression comparisons for the indicated proteins in the AAN and NaCl (reference) kidneys using a Student’s T -test for 3 animals per group shown as 1–3 in ( A ). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Control, Injection, Western Blot, Expressing

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Sustained epithelial PAI-1 expression promotes maladaptive repair (tubular dysfunction). Schematic for the generation of PAI-1-overexpressing cells ( A ). Lysates of CMV-Con and CMV-PAI-1 cells were immunoblotted for PAI-1 ( B , C ), fibronectin ( B , D ), collagen 1 ( B , E ), CTGF ( B , F ), osteopontin ( B , G ), E-cadherin ( B , H ), vimentin ( B , I ), snail ( B , J ), pSMAD3 ( B , K ), p53 ( B , L ), p21 ( B , M ), pHistone H3 ( B , N ), and β-tubulin (loading control) ( B ). Histograms ( C – N ) depict the expression (mean ± SD) differences of PAI-1 ( C ), fibronectin ( D ), collagen 1 ( E ), CTGF ( F ), osteopontin ( G ), E-cadherin ( H ), vimentin ( I ), snail ( J ), pSMAD3 ( K ), p53 ( L ), p21 ( M ), and pHistone H3 ( N ) in the CMV-Con (reference) and CMV-PAI-1 cell populations in three independent experiments ( n = 3) in triplicate. A Student’s T -test was used for statistical comparisons. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Expressing, Control

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: PAI-1-mediated WWP1 upregulation is causatively linked to tubular dysfunction. Western blot analysis of CMV-Con (reference) and CMV-PAI-1 cell lysates for WWP1 expression ( A ); the histogram in ( B ) represents the expression (mean ± SD) differences of WWP1 between groups for three independent studies ( n = 3) in triplicate. *** p < 0.001. WWP1 expressions in CMV-Con and CMV-PAI-1 transgenic cell monolayers are confirmed in ( C ) immunofluorescence imaging, showing WWP1 (in magenta) and nuclear staining with Hoechst (in blue) (40× magnification and scale bars = 100 μm in ( C ), n = 3). CMV-PAI-1 cultures were stably infected with either control shRNA (reference) or WWP1 shRNA lentiviral particles and double transgenic lysates were subjected to western blot assessment for WWP1 ( D , E ), fibronectin ( D , F ), collagen 1 ( D , G ), CTGF ( D , H ), osteopontin ( D , I ), pSMAD3 ( D , J ), p53 ( D , K ), p21 ( D , L ), pHistone H3 ( D , M ), snail ( D , N ), TRIM65 ( D , O ), c-Myc ( D ), and PAI-1 ( D ) expressions. Histograms ( E – O ) depict the expression comparisons of the indicated markers ( n = 3) between the groups. CMV-PAI-1 + Control shRNA and CMV-PAI-1 + WWP1 shRNA cultures were seeded equally, allowed to grow for 5 days, and subjected to Crystal Violet staining to assess the differences in cell counts ( P ). Histogram ( Q ) shows the quantification of cell number per field (three fields per plate) (scale bar = 400 μm, 10× magnification) from ( P ) for three independent studies ( n = 3). Data are presented as the mean ± SD, and a Student’s T -test was utilized for statistical comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Western Blot, Expressing, Transgenic Assay, Immunofluorescence, Imaging, Staining, Stable Transfection, Infection, Control, shRNA

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: E3 ligase TRIM65 upregulation by PAI-1 is necessary for tubular dysfunction. Analysis of a single cell RNA sequencing dataset (Accession No.: GSE183279 ) for TRIM65 mRNA levels in human CKD patients relative to reference kidneys (healthy controls) represented as a dot plot ( A ), where the intensity of dot color (red) dictates TRIM65 expression level, and dot size represents the percentage of cells expressing a TRIM65 gene. Immunoblot comparisons of TRIM65 protein levels between CMV-Con (reference) and CMV-PAI-1 transgenic cells lysates ( B ). The histogram in ( C ) represents the expression (mean ± SD) differences of TRIM65 protein between the groups for three independent studies ( n = 3) in triplicate. ** p < 0.01. CMV-PAI-1 cells were infected with either Control shRNA (reference) or TRIM65 shRNA lentiviral particles followed by stable selection. The double transgenic cell lysates were assessed by western blotting for TRIM65 ( D , E ), fibronectin ( D , F ), collagen 1 ( D , G ), CTGF ( D , H ), osteopontin ( D , I ), pSMAD3 ( D , J ), p53 ( D , K ), p21 ( D , L ), c-Myc ( D ), WWP1 ( D ), and PAI-1 ( D ) levels. Histograms in ( E – L ) depict the expression differences of the indicated markers for three independent experiments ( n = 3). Data are represented as the mean ± SD, and a Student’s T -test was used for statistical comparisons between groups. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Single Cell, RNA Sequencing, Expressing, Western Blot, Transgenic Assay, Infection, Control, shRNA, Selection

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: c-Myc silencing in PAI-1 stable transductants attenuates PAI-1-induced fibrotic reprogramming and WWP1 and TRIM65 induction. Assessment of Nephroseq ( https://www.nephroseq.org (accessed on 5 June 2024)) renal disease datasets for c-Myc transcript levels during human CKD (Nakagawa CKD Kidney) and diabetic nephropathy (Woroniecka Diabetes TubInt, Ju CKD TubInt) progression ( A ). Data in ( A ) are represented as the median. * p < 0.05, **** p < 0.0001. CMV-Con (reference) and CMV-PAI-1 transgenic culture lysates were subjected to immunoblotting for c-Myc protein ( B ). The histogram in ( C ) represents the differences in c-Myc expression (mean ± SD) for three independent studies ( n = 3) in triplicate. Immunofluorescence of CMV-Con and CMV-PAI-1 transgenic populations with c-Myc specific antibodies (red color) followed by Hoechst counterstaining (blue color) ( D ) (40× magnification and scale bars = 100 μm in ( D ), n = 3). CMV-PAI-1 cells were infected with either Control shRNA (reference) or c-Myc shRNA lentiviral particles followed by stable selection. CMV-PAI-1 + Control shRNA (reference) and CMV-PAI-1 + c-Myc shRNA double transgenic lysates were western blotted for c-Myc ( E , F ), fibronectin ( E , G ), collagen 1 ( E , H ), CTGF ( E , I ), osteopontin ( E , J ), pSMAD3 ( E , K ), p53 ( E , L ), p21 ( E , M ), pHistone H3 ( E , N ), snail ( E , O ), WWP1 ( E , P ), TRIM65 ( E , Q ), and PAI-1 ( E ) expressions. Histograms ( F – Q ) depict expression (mean ± SD) differences of the indicated markers ( n = 3). A Student’s T -test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Transgenic Assay, Western Blot, Expressing, Immunofluorescence, Infection, Control, shRNA, Selection

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Tubular PAI-1 upregulation impairs the BMP-7/SMAD1/5 signaling network, and rescue of BMP-7 expression mitigates PAI-1-induced fibrogenesis and c-Myc, WWP1, and TRIM65 upregulation. CMV-Con and CMV-PAI-1 cell lysates were subjected to western blot analysis for the expression of BMP-7, SMAD5, and pSMAD1/5 ( A ). Histograms ( B – D ) depict the expression comparisons of BMP-7 ( B ), SMAD5 ( C ), and pSMAD1/5 ( D ) between CMV-Con (reference) and CMV-PAI-1 cell populations in three independent experiments ( n = 3) in triplicate. ** p < 0.01, *** p < 0.001, **** p < 0.0001. PAI-1-overexpressing cells were infected with either CMV-Control (reference) or CMV-BMP-7 expressing lentiviral particles prior to stable selection. CMV-BMP-7 constructs have a GFP tag at their C terminal end. Double transgenic cell lysates were extracted and immunoblotted for GFP ( E ), pSMAD1/5 ( E , F ), pSMAD3 ( E , G ), fibronectin ( E , H ), collagen 1 ( E , I ), CTGF ( E , J ), osteopontin ( E , K ), p53 ( E , L ), p21 ( E , M ), pHistone H3 ( E , N ), snail ( E , O ), c-Myc ( E , P ), WWP1 ( E , Q ), TRIM65 ( E , R ), PAI-1 ( E ), and β-tubulin ( E ) levels. Histograms ( F – R ) depict expression differences of the indicated markers between CMV-PAI-1 + CMV-Control Vector (reference) and CMV-PAI-1 + CMV-BMP-7 Vector cells for three independent experiments ( n = 3). All data are presented as the mean ± SD, and a Student’s T -test was used for statistical comparison between the indicated groups. *** p < 0.001, **** p < 0.0001.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Expressing, Western Blot, Infection, Control, Selection, Construct, Transgenic Assay, Plasmid Preparation, Comparison

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Restoration of SMAD5 expression attenuates PAI-1-driven fibrogenic responses as well as c-Myc, WWP1, and TRIM65 induction. CMV-PAI-1 cells were stably infected with either CMV-Control (reference) or CMV-SMAD5 lentiviral particles followed by stable selection. CMV-SMAD5 constructs are linked to a GFP Tag at their C terminal end. Immunoblot analysis of double transgenic lysates for GFP ( A ), pSMAD1/5 ( A , B ), pSMAD3 ( A , C ), fibronectin ( A , D ), collagen 1 ( A , E ), CTGF ( A , F ), osteopontin ( A , G ), p53 ( A , H ), p21 ( A , I ), pHistone H3 ( A , J ), snail ( A , K ), c-Myc ( A , L ), WWP1 ( A , M ), TRIM65 ( A , N ), PAI-1 ( A ), and β-tubulin ( A ) proteins. Histograms ( B – N ) depict expression differences for indicated proteins between CMV-PAI-1 + CMV-Control Vector (reference) and CMV-PAI-1 + CMV-SMAD5 Vector lysates in three independent experiments ( n = 3). Data are presented as the mean ± SD, and a Student’s T -test was utilized for statistical comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Expressing, Stable Transfection, Infection, Control, Selection, Construct, Western Blot, Transgenic Assay, Plasmid Preparation

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Model of WWP1 and TRIM65 involvement in renal tubular dysfunction and fibrosis (created with BioRender: https://www.biorender.com/ ). PAI-1 strongly induces the expression of the E3 ubiquitin ligases WWP1 and TRIM65, which are causatively linked to maladaptive tubular repair. Sustained renal epithelial PAI-1 induction represses BMP-7 expression, leading to tubular dysfunction. PAI-1-mediated suppression of the anti-fibrotic BMP-7–SMAD5 signaling axis triggers upregulation of the transcription factor c-Myc, which in turn drives WWP1 and TRIM65 induction, subsequent SMAD3 and p53 activation, and renal maladaptive repair.

Article Snippet: To generate stable double-transgenic epithelial cell lines for BMP-7 and SMAD5 expression rescue experiments, semiconfluent PAI-1 stable transductants were reinfected with CMV-BMP-7-GFP (LPP-A0309-Lv122) and CMV-SMAD5-GFP (LPP-I0510-Lv122) lentiviral particles, respectively, or CMV-Control vector lentiviral particles (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) for 1–2 days in complete medium with Polybrene at 5 μg/mL before stable selection.

Techniques: Expressing, Ubiquitin Proteomics, Activation Assay

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: WWP1 is highly upregulated in fibrotic human kidneys and correlates with a decline in renal function and disease progression. Renal disease datasets (ERCB Nephrotic Syndrome TubInt, ERCB Lupus TubInt) available from Nephroseq ( https://www.nephroseq.org (accessed on 5 June 2024)) ( A ) were analyzed for WWP1 mRNA levels in human diabetic, FSGS (focal segmental glomerulosclerosis), and lupus nephritis kidneys relative to healthy controls. Data in ( A ) are represented as the median. * p < 0.05, ** p < 0.01. Human renal disease specimens from ( A ) were further assessed for a correlation analysis between WWP1 expression and proteinuria ( B ) (r = 0.63, p = 0.0199) (ERCB Nephrotic Syndrome TubInt), or serum creatinine level ( C ) (r = 0.43, p = 0.0284) (ERCB Nephrotic Syndrome TubInt), or glomerular filtration rate (GFR) ( D , E ) (r = -0.79, p = 0.0349; r = -0.72, p = 0.0425) (ERCB Nephrotic Syndrome TubInt, ERCB Lupus TubInt). Lysates from human healthy and diabetic kidneys were immunoblotted for fibronectin, collagen 1, PAI-1, c-Myc, WWP1, and TRIM65 ( F ) proteins. A single cell RNA sequencing dataset (Accession No.: GSE183279 ) was analyzed for WWP1 transcript levels in the diseased kidneys relative to the reference (healthy controls) ( G ) represented as a dot plot, where the intensity of dot color (brown) dictates the WWP1 expression level, and dot size represents the percentage of cells expressing WWP1. UMAP analysis of the dataset (Accession No.: GSE183279 ) was performed to determine the compartment-specific expression of WWP1 in the kidney ( H ), where in the left panel, yellow-green represents epithelial cells, sea green represents endothelial cells, red represents immune cells, orange represents stroma cells, and a very small percentage of neuronal cells are represented by the blue color.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Biomarker Discovery, Expressing, Filtration, Single Cell, RNA Sequencing

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: PAI-1, WWP1, TRIM65, and c-Myc upregulation correlated with repression of the BMP-7/SMAD1/5 signaling axis in fibrotic UUO kidneys. Mice were subjected to unilateral ureteral obstruction (7 days) prior to the extraction of obstructed (UUO) and contralateral kidneys. Contralateral and UUO renal extracts were assessed for fibronectin ( A , B ), collagen 1 ( A , C ), PAI-1 ( A , D ) and c-Myc ( A , E ), WWP1 ( A , F ), and TRIM65 ( A , G ), BMP-7 ( A , H ), SMAD5 ( A , I ), and pSMAD1/5 ( A , J ) protein levels by immunoblot analysis. β-tubulin is serving as a loading control, and the expression of each indicated protein is normalized to tubulin level. Histograms ( B – J ) depict the expression (mean ± SD) comparisons for the indicated protein in the UUO and contralateral kidneys (reference) using a Student’s T -test for 5 animals per group shown as 1–5 in ( A ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Extraction, Western Blot, Control, Expressing

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Induction of PAI-1, WWP1, TRIM65, and c-Myc correlates with BMP-7/SMAD1/5 signaling axis deregulation in aristolochic acid (AA) induced fibrotic kidneys. Mice were administered either with a NaCl vehicle (control) or aristolochic acid (AA) sodium salt (5 mg/kg body weight dissolved in distilled water) via intraperitoneal injection, daily for 5 consecutive days, and termed as NaCl kidney and AAN kidney, respectively. Twenty-five days post-AA injections, mice in both groups were euthanized for kidney harvesting. Renal lysates from both groups were western blotted for fibronectin ( A , B ), collagen 1 ( A , C ), PAI-1 ( A , D ), c-Myc ( A , E ), WWP1 ( A , F ), TRIM65 ( A , G ), BMP-7 ( A , H ), SMAD5 ( A , I ), and pSMAD1/5 ( A , J ). Data are presented as the mean ± SD. The expression of each indicated protein is normalized to β-tubulin (loading control). Histograms ( B – J ) depict the expression comparisons for the indicated proteins in the AAN and NaCl (reference) kidneys using a Student’s T -test for 3 animals per group shown as 1–3 in ( A ). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Control, Injection, Western Blot, Expressing

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Sustained epithelial PAI-1 expression promotes maladaptive repair (tubular dysfunction). Schematic for the generation of PAI-1-overexpressing cells ( A ). Lysates of CMV-Con and CMV-PAI-1 cells were immunoblotted for PAI-1 ( B , C ), fibronectin ( B , D ), collagen 1 ( B , E ), CTGF ( B , F ), osteopontin ( B , G ), E-cadherin ( B , H ), vimentin ( B , I ), snail ( B , J ), pSMAD3 ( B , K ), p53 ( B , L ), p21 ( B , M ), pHistone H3 ( B , N ), and β-tubulin (loading control) ( B ). Histograms ( C – N ) depict the expression (mean ± SD) differences of PAI-1 ( C ), fibronectin ( D ), collagen 1 ( E ), CTGF ( F ), osteopontin ( G ), E-cadherin ( H ), vimentin ( I ), snail ( J ), pSMAD3 ( K ), p53 ( L ), p21 ( M ), and pHistone H3 ( N ) in the CMV-Con (reference) and CMV-PAI-1 cell populations in three independent experiments ( n = 3) in triplicate. A Student’s T -test was used for statistical comparisons. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Control

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: PAI-1-mediated WWP1 upregulation is causatively linked to tubular dysfunction. Western blot analysis of CMV-Con (reference) and CMV-PAI-1 cell lysates for WWP1 expression ( A ); the histogram in ( B ) represents the expression (mean ± SD) differences of WWP1 between groups for three independent studies ( n = 3) in triplicate. *** p < 0.001. WWP1 expressions in CMV-Con and CMV-PAI-1 transgenic cell monolayers are confirmed in ( C ) immunofluorescence imaging, showing WWP1 (in magenta) and nuclear staining with Hoechst (in blue) (40× magnification and scale bars = 100 μm in ( C ), n = 3). CMV-PAI-1 cultures were stably infected with either control shRNA (reference) or WWP1 shRNA lentiviral particles and double transgenic lysates were subjected to western blot assessment for WWP1 ( D , E ), fibronectin ( D , F ), collagen 1 ( D , G ), CTGF ( D , H ), osteopontin ( D , I ), pSMAD3 ( D , J ), p53 ( D , K ), p21 ( D , L ), pHistone H3 ( D , M ), snail ( D , N ), TRIM65 ( D , O ), c-Myc ( D ), and PAI-1 ( D ) expressions. Histograms ( E – O ) depict the expression comparisons of the indicated markers ( n = 3) between the groups. CMV-PAI-1 + Control shRNA and CMV-PAI-1 + WWP1 shRNA cultures were seeded equally, allowed to grow for 5 days, and subjected to Crystal Violet staining to assess the differences in cell counts ( P ). Histogram ( Q ) shows the quantification of cell number per field (three fields per plate) (scale bar = 400 μm, 10× magnification) from ( P ) for three independent studies ( n = 3). Data are presented as the mean ± SD, and a Student’s T -test was utilized for statistical comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot, Expressing, Transgenic Assay, Immunofluorescence, Imaging, Staining, Stable Transfection, Infection, Control, shRNA

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: E3 ligase TRIM65 upregulation by PAI-1 is necessary for tubular dysfunction. Analysis of a single cell RNA sequencing dataset (Accession No.: GSE183279 ) for TRIM65 mRNA levels in human CKD patients relative to reference kidneys (healthy controls) represented as a dot plot ( A ), where the intensity of dot color (red) dictates TRIM65 expression level, and dot size represents the percentage of cells expressing a TRIM65 gene. Immunoblot comparisons of TRIM65 protein levels between CMV-Con (reference) and CMV-PAI-1 transgenic cells lysates ( B ). The histogram in ( C ) represents the expression (mean ± SD) differences of TRIM65 protein between the groups for three independent studies ( n = 3) in triplicate. ** p < 0.01. CMV-PAI-1 cells were infected with either Control shRNA (reference) or TRIM65 shRNA lentiviral particles followed by stable selection. The double transgenic cell lysates were assessed by western blotting for TRIM65 ( D , E ), fibronectin ( D , F ), collagen 1 ( D , G ), CTGF ( D , H ), osteopontin ( D , I ), pSMAD3 ( D , J ), p53 ( D , K ), p21 ( D , L ), c-Myc ( D ), WWP1 ( D ), and PAI-1 ( D ) levels. Histograms in ( E – L ) depict the expression differences of the indicated markers for three independent experiments ( n = 3). Data are represented as the mean ± SD, and a Student’s T -test was used for statistical comparisons between groups. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Single Cell, RNA Sequencing, Expressing, Western Blot, Transgenic Assay, Infection, Control, shRNA, Selection

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: c-Myc silencing in PAI-1 stable transductants attenuates PAI-1-induced fibrotic reprogramming and WWP1 and TRIM65 induction. Assessment of Nephroseq ( https://www.nephroseq.org (accessed on 5 June 2024)) renal disease datasets for c-Myc transcript levels during human CKD (Nakagawa CKD Kidney) and diabetic nephropathy (Woroniecka Diabetes TubInt, Ju CKD TubInt) progression ( A ). Data in ( A ) are represented as the median. * p < 0.05, **** p < 0.0001. CMV-Con (reference) and CMV-PAI-1 transgenic culture lysates were subjected to immunoblotting for c-Myc protein ( B ). The histogram in ( C ) represents the differences in c-Myc expression (mean ± SD) for three independent studies ( n = 3) in triplicate. Immunofluorescence of CMV-Con and CMV-PAI-1 transgenic populations with c-Myc specific antibodies (red color) followed by Hoechst counterstaining (blue color) ( D ) (40× magnification and scale bars = 100 μm in ( D ), n = 3). CMV-PAI-1 cells were infected with either Control shRNA (reference) or c-Myc shRNA lentiviral particles followed by stable selection. CMV-PAI-1 + Control shRNA (reference) and CMV-PAI-1 + c-Myc shRNA double transgenic lysates were western blotted for c-Myc ( E , F ), fibronectin ( E , G ), collagen 1 ( E , H ), CTGF ( E , I ), osteopontin ( E , J ), pSMAD3 ( E , K ), p53 ( E , L ), p21 ( E , M ), pHistone H3 ( E , N ), snail ( E , O ), WWP1 ( E , P ), TRIM65 ( E , Q ), and PAI-1 ( E ) expressions. Histograms ( F – Q ) depict expression (mean ± SD) differences of the indicated markers ( n = 3). A Student’s T -test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Transgenic Assay, Western Blot, Expressing, Immunofluorescence, Infection, Control, shRNA, Selection

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Tubular PAI-1 upregulation impairs the BMP-7/SMAD1/5 signaling network, and rescue of BMP-7 expression mitigates PAI-1-induced fibrogenesis and c-Myc, WWP1, and TRIM65 upregulation. CMV-Con and CMV-PAI-1 cell lysates were subjected to western blot analysis for the expression of BMP-7, SMAD5, and pSMAD1/5 ( A ). Histograms ( B – D ) depict the expression comparisons of BMP-7 ( B ), SMAD5 ( C ), and pSMAD1/5 ( D ) between CMV-Con (reference) and CMV-PAI-1 cell populations in three independent experiments ( n = 3) in triplicate. ** p < 0.01, *** p < 0.001, **** p < 0.0001. PAI-1-overexpressing cells were infected with either CMV-Control (reference) or CMV-BMP-7 expressing lentiviral particles prior to stable selection. CMV-BMP-7 constructs have a GFP tag at their C terminal end. Double transgenic cell lysates were extracted and immunoblotted for GFP ( E ), pSMAD1/5 ( E , F ), pSMAD3 ( E , G ), fibronectin ( E , H ), collagen 1 ( E , I ), CTGF ( E , J ), osteopontin ( E , K ), p53 ( E , L ), p21 ( E , M ), pHistone H3 ( E , N ), snail ( E , O ), c-Myc ( E , P ), WWP1 ( E , Q ), TRIM65 ( E , R ), PAI-1 ( E ), and β-tubulin ( E ) levels. Histograms ( F – R ) depict expression differences of the indicated markers between CMV-PAI-1 + CMV-Control Vector (reference) and CMV-PAI-1 + CMV-BMP-7 Vector cells for three independent experiments ( n = 3). All data are presented as the mean ± SD, and a Student’s T -test was used for statistical comparison between the indicated groups. *** p < 0.001, **** p < 0.0001.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Western Blot, Infection, Control, Selection, Construct, Transgenic Assay, Plasmid Preparation, Comparison

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Restoration of SMAD5 expression attenuates PAI-1-driven fibrogenic responses as well as c-Myc, WWP1, and TRIM65 induction. CMV-PAI-1 cells were stably infected with either CMV-Control (reference) or CMV-SMAD5 lentiviral particles followed by stable selection. CMV-SMAD5 constructs are linked to a GFP Tag at their C terminal end. Immunoblot analysis of double transgenic lysates for GFP ( A ), pSMAD1/5 ( A , B ), pSMAD3 ( A , C ), fibronectin ( A , D ), collagen 1 ( A , E ), CTGF ( A , F ), osteopontin ( A , G ), p53 ( A , H ), p21 ( A , I ), pHistone H3 ( A , J ), snail ( A , K ), c-Myc ( A , L ), WWP1 ( A , M ), TRIM65 ( A , N ), PAI-1 ( A ), and β-tubulin ( A ) proteins. Histograms ( B – N ) depict expression differences for indicated proteins between CMV-PAI-1 + CMV-Control Vector (reference) and CMV-PAI-1 + CMV-SMAD5 Vector lysates in three independent experiments ( n = 3). Data are presented as the mean ± SD, and a Student’s T -test was utilized for statistical comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Stable Transfection, Infection, Control, Selection, Construct, Western Blot, Transgenic Assay, Plasmid Preparation

Journal: Biomolecules

Article Title: Involvement of c-Myc/WWP1/TRIM65 Axis in Renal Fibrosis

doi: 10.3390/biom16030373

Figure Lengend Snippet: Model of WWP1 and TRIM65 involvement in renal tubular dysfunction and fibrosis (created with BioRender: https://www.biorender.com/ ). PAI-1 strongly induces the expression of the E3 ubiquitin ligases WWP1 and TRIM65, which are causatively linked to maladaptive tubular repair. Sustained renal epithelial PAI-1 induction represses BMP-7 expression, leading to tubular dysfunction. PAI-1-mediated suppression of the anti-fibrotic BMP-7–SMAD5 signaling axis triggers upregulation of the transcription factor c-Myc, which in turn drives WWP1 and TRIM65 induction, subsequent SMAD3 and p53 activation, and renal maladaptive repair.

Article Snippet: In order to generate PAI-1 stable transductants, semiconfluent HK-2 cultures were infected with lentiviruses carrying a cytomegalovirus (CMV) promoter–driven PAI-1 cDNA construct (termed CMV-PAI-1) (LPP-F0606-Lv105) or an empty vector (termed CMV-Con) (LPP-NEG-Lv105) (GeneCopoeia, Rockville, MD, USA) in the presence of Polybrene at 5 μg/mL (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% FBS/DMEM for 24 h. After a 24 h recovery, the cells were subjected to stable selection in complete medium (1X DMEM + GlutaMAX-I; 5% FBS; 5 units/mL penicillin + 5 μg/mL streptomycin) containing 5 μg/mL puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Ubiquitin Proteomics, Activation Assay