normal human glomerular mesangial cells (nhmcs)  (Lonza)

Journal: Clinical and Experimental Nephrology

Article Title: Detecting and exploring kidney-derived extracellular vesicles in plasma

doi: 10.1007/s10157-024-02464-z

Figure Lengend Snippet: ITGA8 expression in mesangial cells in the biopsy specimen, NHMCs, and sEVs isolated from the NHMC supernatant. A Control immunohistochemistry images of human renal biopsy specimens without using primary antibody. Scale bars indicate 100 µm (left) and 20 µm (right). B The same specimen used in A stained with the anti-ITGA8 antibody. The arrows indicate positive staining in the glomerular mesangial area. Scale bars indicate 100 µm (left) and 20 µm (right). C Immunofluorescence images of HUVECs and NHMCs stained with DAPI (blue) and ITGA8 (green). D – G Immunoelectron microscopy images of sEVs capturing beads. Non-antibody-coated beads ( D ) and anti-CD63 (general sEV marker) antibody-coated beads with sEVs isolated from the supernatant of HUVECs ( E ) and NHMCs ( F , G ). Beads were then stained with either anti-ITGA8 antibody ( E , G ) or its isotype control antibody ( F ). The magnified images are shown on the right upper or the right side of the images. The arrowheads indicate small dots of positive staining for ITGA8. Scale bars indicate 200 nm. sEVs small extracellular vesicles, NHMCs normal human mesangial cells, HUVECs human umbilical vein endothelial cells, ITGA8 α8 integrin, Ab antibody

Article Snippet: Normal human mesangial cells (NHMCs; CC-2559) were obtained from Lonza (Basel, Switzerland) and cultured in a supplemented mesangial basal medium (CC-3147, CC-4146) [ ].

Techniques: Expressing, Isolation, Control, Immunohistochemistry, Staining, Immunofluorescence, Immuno-Electron Microscopy, Marker

Journal: Clinical and Experimental Nephrology

Article Title: Detecting and exploring kidney-derived extracellular vesicles in plasma

doi: 10.1007/s10157-024-02464-z

Figure Lengend Snippet: Detection of ITGA8-positive sEVs from human plasma samples. A Schematic diagram showing the ITGA8-positive sEVs detection system and an electron microscopy image of plasma-derived sEVs captured using anti-CD63 antibody-coated beads. Scale bar indicates 200 nm. B Western blotting analysis of a lysate from sEVs isolated from the plasma of the healthy control and a lysate from NHMC. C , D Representative images of sEVs signal detected using on-bead flow cytometry for CD9 ( C ) and ITGA8 ( D ). E , F Boxplots comparing MFI between anti-CD9 antibody-FITC and isotype control antibody-FITC ( E ) and between anti-ITGA8 antibody-Alexa flour and isotype control antibody-Alexa flour on anti-CD63 antibody-conjugated magnetic beads ( F ). sEVs small extracellular vesilces, HC healthy control, NHMC normal human mesangial cell, ITGA8 α8 integrin, MFI mean fluorescent intensity, CTRL control. * p < 0.05

Article Snippet: Normal human mesangial cells (NHMCs; CC-2559) were obtained from Lonza (Basel, Switzerland) and cultured in a supplemented mesangial basal medium (CC-3147, CC-4146) [ ].

Techniques: Clinical Proteomics, Electron Microscopy, Derivative Assay, Western Blot, Isolation, Control, Flow Cytometry, Magnetic Beads

Journal: Kidney Medicine

Article Title: Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor–Mediated AXL Phosphorylation

doi: 10.1016/j.xkme.2021.06.007

Figure Lengend Snippet: Overexpressed phospho-AXL in human glomeruli in immunoglobulin A nephropathy (IgAN) biopsy samples in relation to markers for podocytes and endothelial cells. Immunofluorescence staining of sections of remnant frozen kidney biopsy specimens from patients with IgAN revealed a mesangial pattern of phospho-AXL staining. Phospho-AXL (green) did not colocalize with markers for podocytes (A) synaptopodin (red) and (B) endothelial cells, Ulex europeus lectin (red), indicating that phospho-AXL is not expressed to any great extent in those cells. Phospho-AXL had stronger expression in kidney biopsy specimens from patients with IgAN than in biopsy specimens from patients with minimal change disease (MCD). Images were taken with a ×40 objective.

Article Snippet: Normal human mesangial cells were purchased from Lonza.

Techniques: Immunofluorescence, Staining, Expressing

Journal: Kidney Medicine

Article Title: Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor–Mediated AXL Phosphorylation

doi: 10.1016/j.xkme.2021.06.007

Figure Lengend Snippet: AXL activation by PDGF. (A) Kinomic studies revealed that multiple protein-tyrosine kinases were activated in primary normal human mesangial cells (NHMC) during a 15-minute stimulation with platelet-derived growth factor (PDGF)-AB (10 ng/mL). Protein-tyrosine kinases identified by kinomic profiling and analyzed with protein-tyrosine kinase UpKin, version 8.0 (BioNavigator), software are listed on the y-axis with the normalized kinase statistic scores shown on the x-axis. The bar color of protein-tyrosine kinases indicates activities induced by PDGF-AB, showing highest activities in red. This kinomic profiling showed that the kinase activity of AXL, a member of TAM family, was top ranked. (B) Western blot analysis of cell lysates using antibodies against the 3 members of the TAM family, TYRO3, AXL, MERTK, revealed that AXL was the major protein of TAM family expressed in NHMC. Molecular weights of the standard proteins in kDa are shown on the side. (C) Immunofluorescence staining for AXL in primary human mesangial cells. AXL was expressed in NHMC as well as in mesangial cells isolated from kidney-biopsy specimens from patients with immunoglobulin A nephropathy (IgAN). Three samples were used in each group; representative images are shown. Negative control is without primary antibody; only nuclei are stained (blue).

Article Snippet: Normal human mesangial cells were purchased from Lonza.

Techniques: Activation Assay, Derivative Assay, Software, Activity Assay, Western Blot, Immunofluorescence, Staining, Isolation, Negative Control

Journal: Kidney Medicine

Article Title: Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor–Mediated AXL Phosphorylation

doi: 10.1016/j.xkme.2021.06.007

Figure Lengend Snippet: Platelet-derived growth factor (PDGF) activation of AXL in primary human mesangial cells. Cell lysates prepared from normal human mesangial cells (NHMC) after a 15-minute stimulation with 10 ng/mL of PDGF-AB or mock-stimulation (Control) were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions followed by Western blotting with AXL- or phospho-AXL (P-AXL; Y779)-specific antibodies. (A) Typical example of a Western blot. Molecular weight of the standard protein (130 kDa) is shown on the side. These data were then evaluated by densitometry. (B) Individual data points and mean and standard deviation values calculated from 3 independent experiments ( P = 0.01). Black dots are the results of groups treated with PDGF-AB. Blue dots are the results of groups without treatment. AXL was phosphorylated after a 15-minute stimulation of NHMC with PDGF-AB.

Article Snippet: Normal human mesangial cells were purchased from Lonza.

Techniques: Derivative Assay, Activation Assay, Control, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Molecular Weight, Standard Deviation

Journal: Kidney Medicine

Article Title: Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor–Mediated AXL Phosphorylation

doi: 10.1016/j.xkme.2021.06.007

Figure Lengend Snippet: Platelet-derived growth factor (PDGF) activation of PDGF receptor (PDGFR)-β and cross-activation of AXL. Immunoprecipitation (IP) of cell lysates from normal human mesangial cells stimulated with PDGF-AB for 15 minutes. Antibodies specific for AXL or PDGFR-β were used for IP and the resultant material was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The blots were probed with antibodies specific for PDGFR-β, P-PDGFR-β, AXL, and P-AXL. PDGFR-β and AXL were co-precipitated by antibodies specific for AXL or PDGFR-β, suggesting that a fraction of each protein was associated with the other protein. Furthermore, PDGF-AB activated PDGFR-β and cross-activated AXL, as evidenced by the reactivity with antibodies specific for phosphorylated forms of AXL (Y779) and PDGFR-β (Y751). Abbreviations: AXL, immunoprecipitations with AXL-specific antibody; IB, immunoblotting; IP: PDGFR-β, immunoprecipitations with PDGFR-β–specific antibody. Example of 1 of 2 independent experiments with similar results is shown. Molecular weights of the standard proteins (130 and 250 kDa) are shown on the side.

Article Snippet: Normal human mesangial cells were purchased from Lonza.

Techniques: Derivative Assay, Activation Assay, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot

Journal: Kidney Medicine

Article Title: Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor–Mediated AXL Phosphorylation

doi: 10.1016/j.xkme.2021.06.007

Figure Lengend Snippet: AXL inhibitor R428 partial inhibition of cellular proliferation of mesangial cells induced by platelet-derived growth factor (PDGF)-AB. AXL inhibitor R428 reduced in a dose-dependent manner the cellular proliferation of normal human mesangial cells (NHMC) induced by PDGF-AB (10 ng/mL; black bars). R428 did not alter the cellular proliferation of control NHMC (open bars). Results are shown as individual data points and mean and standard deviation values calculated from 3 independent experiments with duplicates. Blue dots are results from groups treated with R428. Black dots are results from groups treated with R428 and PDGF-AB. Statistical differences were determined by 1-way analysis of variance test ( P = 0.002 for 0 vs 0.3 μmol/L of R428).

Article Snippet: Normal human mesangial cells were purchased from Lonza.

Techniques: Inhibition, Derivative Assay, Control, Standard Deviation

Journal: Kidney Medicine

Article Title: Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor–Mediated AXL Phosphorylation

doi: 10.1016/j.xkme.2021.06.007

Figure Lengend Snippet: AXL inhibitor R428 inhibition of platelet-derived growth factor (PDGF)-induced phosphorylation of AXL and PDGF receptor (PDGFR)-β and the downstream signaling to AKT1 and ERK1/2. Our previous experiment showed that R428, an AXL inhibitor, partially inhibited PDGF-induced cellular proliferation. Here, we assessed the capacity of R428 to block cellular signaling induced by PDGF. Normal human mesangial cells were preincubated with AXL inhibitor R428 (0.3 μmol/L) for 1.5 hour and then stimulated with PDGF-AB (10 ng/mL) for 15 minutes. The solvent for R428, dimethyl sulfoxide (DMSO), was used as an additional control. Cell lysates were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. R428 inhibited PDGF-induced phosphorylation of AXL as well as PDGFR-β. Downstream signaling to AKT1 and ERK1/2 was also inhibited by R428. White lines separate the noncontiguous parts of blots. Example of 1 of 2 independent experiments with similar results is shown. Molecular weights of the proteins are shown on the side (130, 180, 70, and 44/42kDa).

Article Snippet: Normal human mesangial cells were purchased from Lonza.

Techniques: Inhibition, Derivative Assay, Phospho-proteomics, Blocking Assay, Solvent, Control, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot

Journal: BMC Genomics

Article Title: Decoding the differentiation of mesenchymal stem cells into mesangial cells at the transcriptomic level

doi: 10.1186/s12864-020-06868-5

Figure Lengend Snippet: Additional file 7. Differentiated mesangial cells contraction video.

Article Snippet: Briefly, MSC were co-cultured with hydrogen peroxide-injured commercially available human mesangial cells (CloneticsTM Normal Human Mesangial Cells, Lonza, Cat# CC-2559) ( n = 3) in trans-well dishes separated by a membrane for 7 days.

Techniques:

Journal: BMC Genomics

Article Title: Decoding the differentiation of mesenchymal stem cells into mesangial cells at the transcriptomic level

doi: 10.1186/s12864-020-06868-5

Figure Lengend Snippet: a Biomarkers or key genes involved in the mesangial cell characteristic and functions were up-regulated during the differentiation process. The expression of these selected key genes in mesangial cells are also being reported in other research. b The connection between these selected ascending key genes and their upstream regulators/TFs. These co-expression TFs and key genes were supported with TRR database

Article Snippet: Briefly, MSC were co-cultured with hydrogen peroxide-injured commercially available human mesangial cells (CloneticsTM Normal Human Mesangial Cells, Lonza, Cat# CC-2559) ( n = 3) in trans-well dishes separated by a membrane for 7 days.

Techniques: Expressing

Journal: BMC Genomics

Article Title: Decoding the differentiation of mesenchymal stem cells into mesangial cells at the transcriptomic level

doi: 10.1186/s12864-020-06868-5

Figure Lengend Snippet: Immunohistochemistry showing glomerular expression patterns of the selected monotonic ascending pattern target genes with DE ≤ 4. These genes were expressed in human mesangial cells in glomeruli. These images were collected from the Human Protein Atlas ( www.proteinatlas.org ) after cropping the glomeruli from the original full images

Article Snippet: Briefly, MSC were co-cultured with hydrogen peroxide-injured commercially available human mesangial cells (CloneticsTM Normal Human Mesangial Cells, Lonza, Cat# CC-2559) ( n = 3) in trans-well dishes separated by a membrane for 7 days.

Techniques: Immunohistochemistry, Expressing

Journal: BMC Genomics

Article Title: Decoding the differentiation of mesenchymal stem cells into mesangial cells at the transcriptomic level

doi: 10.1186/s12864-020-06868-5

Figure Lengend Snippet: Selected analysis of functional pathways for each level. Three developmental-stage transitions can be observed. Stage 1: differentiation preparation stage (orange); pathways related to cell proliferation have been enriched. Stage 2: differentiation initiation stage (red); pathways related to regulating or driving differentiation have been enriched. Stage 3: maturation stage (green); pathways related to mesangial cell function and characteristics have been enriched

Article Snippet: Briefly, MSC were co-cultured with hydrogen peroxide-injured commercially available human mesangial cells (CloneticsTM Normal Human Mesangial Cells, Lonza, Cat# CC-2559) ( n = 3) in trans-well dishes separated by a membrane for 7 days.

Techniques: Functional Assay, Cell Function Assay

Journal: BMC Genomics

Article Title: Decoding the differentiation of mesenchymal stem cells into mesangial cells at the transcriptomic level

doi: 10.1186/s12864-020-06868-5

Figure Lengend Snippet: For purposes of validating one of the KEGG functional pathway: Vascular smooth muscle contraction (hsa04270), has been enriched in differentiated cells by bioinformatics analysis. MSC co-cultured with injured mesangial cells were treated with AngII; cell contraction was observed as indicated by white arrows. Pure MSC (Control) did not show any contraction after treated with AngII

Article Snippet: Briefly, MSC were co-cultured with hydrogen peroxide-injured commercially available human mesangial cells (CloneticsTM Normal Human Mesangial Cells, Lonza, Cat# CC-2559) ( n = 3) in trans-well dishes separated by a membrane for 7 days.

Techniques: Functional Assay, Cell Culture, Control

Journal: BMC Genomics

Article Title: Decoding the differentiation of mesenchymal stem cells into mesangial cells at the transcriptomic level

doi: 10.1186/s12864-020-06868-5

Figure Lengend Snippet: Experimental design of the study. a MSC were co-cultured with injured mesangial cells. On indicated days, co-cultured MSC were harvested and RNA was extracted. cDNA was synthesised and subsequently underwent RNA-seq. b After RNA-seq, raw data was processed by initial read processing and normalizations before the data were further analyzed simultaneously using two methods. c Key genes related to MSC or mesangial cells with ascending or descending monotonic patterns over the differentiation process were identified with MFSelector. d Co-expression patterns between TF and construction TF co-expression network were determined by TO-GCN. Once the data from both methods was generated, both sets of data were analysed synergistically ( e ). The data obtained from both methods were used to correlate the key genes at specific time points to the TO-GCN at different levels. This helps to elucidate the network interaction between TF-TF and TF-key genes at each level of TO-GCN. Additionally, this builds our understanding of the overall gene expression during the MSC differentiation into mesangial cells

Article Snippet: Briefly, MSC were co-cultured with hydrogen peroxide-injured commercially available human mesangial cells (CloneticsTM Normal Human Mesangial Cells, Lonza, Cat# CC-2559) ( n = 3) in trans-well dishes separated by a membrane for 7 days.

Techniques: Cell Culture, RNA Sequencing, Expressing, Generated, Gene Expression