Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cold Shock Proteins Mediate GN with Mesangioproliferation

doi: 10.1681/ASN.2015121367

Figure Lengend Snippet: Expression of DbpA in human kidney disease and cell lines. (A) Immunohistochemistry shows that DbpA expression is not detected in glomerular cells in minimal change (MC) GN, however DbpA expression is interspersed in tubulointerstitial cells (a–c). In contrast, DbpA protein was detected within the mesangial compartment of the glomeruli from patients with IgA nephritis and in cell infiltrates of the interstitium (d–f). A similar pattern of glomerular expression was observed in patients with lupus ISN/RPS class 4 G(A) GN with upregulated DbpA expression in tubular cells (g–i). The most profound tubular cell and interstitial upregulation of DbpA was seen with interstitial nephritis, where glomerular cells were immune negative (g–l). Images were made with a Leica DM6000 B Microscope (Leica Microsystems) using either a 100× (a, d, g, and j) or a 400× objective (b, c, e, f, h, i, k, and l). Scale bars, 200 μm in a, d, g, and j; 50 μm in b, c, e, f, h, i, k, and l. (B) Immunohistochemistry for DbpA and DbpB/YB-1 in sequential tissue slices from a patient with IgA nephritis reveals concordant upregulation of both proteins in mesangial compartment; at the same time, the staining pattern in the tubular cells and tubulointerstitial infiltrate markedly differs. (C) Western blot analysis of DbpA protein expression in cell lysates of human proximal tubular epithelial cells HK-2 and HKC-8, rMCs, human primary mesangial cells (hMCs), human embryonic kidney cells (HEK-293s), human podocytes, and human umbilical vein endothelial cells (HUVECs). Three major bands are shown: *55 kD; **50 kD; ***44 kD.

Article Snippet: Primary human mesangial cells (CC-2559; Lonza Group Ltd) were grown in a Clonetics MsGM Culture System (CC-3146; Lonza Group Ltd) containing mesangial cell basal medium supplemented with 5% FBS and GA-1000 at 37°C in humidified 5% CO 2 in air.

Techniques: Expressing, Immunohistochemistry, Microscopy, Staining, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cold Shock Proteins Mediate GN with Mesangioproliferation

doi: 10.1681/ASN.2015121367

Figure Lengend Snippet: Overexpression of DbpA in rMCs leads to increased cell proliferation. (A) Cellular morphology of rMCs after overexpression of DbpA isoforms DbpA_a and DbpA_b for 3 days. Both interventions result in increased cells proliferation. (B) Western blot analysis reveals the overexpression of both isoforms of DbpA (40 and 55 kD; asterisks in left panel) by pCDH lentiviral transduction, which is accompanied by upregulation of cell proliferation markers (e.g., PCNA and cyclin D1). *P<0.05 (n=4). (C) Immunofluorescence staining shows both cytoplasmic and nuclear DbpA expression, whereas overexpressed DbpA protein mainly localizes in a punctuate manner within the cytoplasm. Scale bar, 50 μm. (D) BrdU cell proliferation assay shows increased cell proliferation in DbpA_a and DbpA_b overexpressing mesangial cells compared with transfection of control vector. **P<0.01 (n=3). (E) Overexpression of DbpA in rMCs with stable knockdown of DbpA leads to much stronger induction of BrdU incorporation. **P<0.01 (n=3).

Article Snippet: Primary human mesangial cells (CC-2559; Lonza Group Ltd) were grown in a Clonetics MsGM Culture System (CC-3146; Lonza Group Ltd) containing mesangial cell basal medium supplemented with 5% FBS and GA-1000 at 37°C in humidified 5% CO 2 in air.

Techniques: Over Expression, Western Blot, Transduction, Immunofluorescence, Staining, Expressing, BrdU Cell Proliferation Assay, Transfection, Control, Plasmid Preparation, Knockdown, BrdU Incorporation Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cold Shock Proteins Mediate GN with Mesangioproliferation

doi: 10.1681/ASN.2015121367

Figure Lengend Snippet: DbpA protein expression is upregulated by PDGF-BB stimulation in mesangial cells in vitro. (A, upper panel) Western blot analysis of DbpA protein expression in PDGF-BB–challenged primary human mesangial cells (hMCs). *55 kD; **50 kD; ***44 kD. (A, lower panel) Quantification of band intensities reveals 2.5-fold induction of DbpA protein expression (44 and 55 kD) after 24 hour incubation with human PDGF-BB (50 ng/ml). *P<0.05 (n=3). (B, upper panel) Western blot analysis of DbpA protein expression in rMCs stimulated with rat PDGF-BB for 24 hours at increasing doses. A dose-dependent upregulation of DbpA is shown from 5 to 10 ng/ml PDGF-BB stimulation. No significant change of DbpA expression was observed after stimulation with 50–200 ng/ml PDGF-BB. *55 kD; **50 kD; ***44 kD. (B, lower panel) Two bands at 50 and 44 kD are seen, with the predominant band at 44 kD. ctrl, Control. **P<0.01 (n=3). (C) Real–time PCR analysis for DbpA transcripts in rMCs reveals a profound upregulation after 24 and 48 hours of PDGF-BB incubation. *P<0.05 (n=3). (D) Immunofluorescence staining for DbpA in rMCs. Compared with vehicle, DbpA expression increases after 24 hours of incubation with rPDGF-BB (50 ng/ml). DbpA protein accumulates mainly within the cytoplasm. Scale bar, 50 μm. (E) Changes of BrdU incorporation rates are determined for rMCs undergoing overexpression of DbpA_a and DbpA_b or knockdown of DbpA. Stimulation with rPDGF-BB (50 ng/ml) was performed for 24 hours. *P<0.05 (n=3).

Article Snippet: Primary human mesangial cells (CC-2559; Lonza Group Ltd) were grown in a Clonetics MsGM Culture System (CC-3146; Lonza Group Ltd) containing mesangial cell basal medium supplemented with 5% FBS and GA-1000 at 37°C in humidified 5% CO 2 in air.

Techniques: Expressing, In Vitro, Western Blot, Incubation, Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, BrdU Incorporation Assay, Over Expression, Knockdown

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Cold Shock Proteins Mediate GN with Mesangioproliferation

doi: 10.1681/ASN.2015121367

Figure Lengend Snippet: DbpA protein expression is induced in experimental models of anti-Thy1.1 and mice MsPGN. (A) DbpA protein was identified by immunohistochemistry in kidney tissue from healthy rats (a and b) and the time course of anti-Thy1.1 nephritis (c and d, 4 hours; e and f, day 4; g and h, day 7; and i and j, day 21). Whereas no DbpA was detected in the glomeruli of healthy rats, significant DbpA expression was observed within the cytoplasm of mesangial cells after the induction of anti-Thy1.1 nephritis on day 4, peaking at day 7 and returning to background after 3 weeks. Magnification, ×100 in a, c, e, g, and i; ×400 in b, d, f, h, and j. Scale bars, 100 μm in a, c, e, g, and i; 25 μm in b, d, f, h and j. (B) Western blot analysis reveals profound upregulation of DbpA protein (44 kD) expression that peaks at day 7 and subsequently, returns to basal levels. DbpA protein levels normalized to β-actin are shown. **P<0.01; ***44 kD. (C) DbpA protein expression by immunohistochemistry staining in kidney tissue from healthy mice (a and b) and after induction of MsPGN for 3 (c and d) and 6 (e and f) days. Quantification of the DbpA staining is shown for 50 visual fields. **P<0.01.

Article Snippet: Primary human mesangial cells (CC-2559; Lonza Group Ltd) were grown in a Clonetics MsGM Culture System (CC-3146; Lonza Group Ltd) containing mesangial cell basal medium supplemented with 5% FBS and GA-1000 at 37°C in humidified 5% CO 2 in air.

Techniques: Expressing, Immunohistochemistry, Western Blot, Staining

Journal: Methods in cell biology

Article Title: Analysis of primary cilia in renal tissue and cells

doi: 10.1016/bs.mcb.2019.04.008

Figure Lengend Snippet: Immortalized and primary renal epithelial cells.

Article Snippet: The balance between cilia assembly and disassembly regulates cilia length ( Mirvis, Stearns, & James Nelson, 2018 ; Spalluto, Wilson, & Hearn, 2013 ). table ft1 table-wrap mode="anchored" t5 caption a7 Cell line Description Source References M-1 Murine cortical collecting duct epithelial cells ATCC Stoos et al. (1991) IMCD-3 Murine inner medullary collecting duct epithelial cells ATCC Rauchman et al. (1993) LLC-PK1 Porcine proximal tubule epithelial cells ATCC Nielsen et al. (1998) MDCK Madin-Darby canine kidney epithelial cells ATCC Gaush et al. (1966) NHK/ADPKD Primary cortical epithelial cells from normal human kidney (NHK) or Autosomal Dominant PKD (ADPKD) KUMC Graham et al. (1977) and Reif et al. (2011) Open in a separate window Immortalized and primary renal epithelial cells. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Company Cilia structure References Acetylated-α-tubulin Sigma-Aldrich Axoneme Ishikawa et al. (2012) , Seixas et al. (2015) , Silva et al. (2018) , and Yu, Sharma, Skowronek, and Erdmann (2016) γ-tubulin Sigma-Aldrich Centrioles Breslow et al. (2013) Pericentrin Covance Centrioles Ishikawa et al. (2012) ARL13B Proteintech Ciliary membrane Seixas et al. (2015) INPP5E Proteintech Ciliary membrane Plotnikova et al. (2015) IFT52 Proteintech Axoneme Silva et al. (2018) IFT81 Proteintech Axoneme Silva et al. (2018) IFT88 Proteintech Axoneme Silva et al. (2018) IFT140 Proteintech Axoneme Silva et al. (2018) BBS2 Proteintech Axoneme Silva et al. (2018) BBS5 Proteintech Axoneme Silva et al. (2018) Open in a separate window Markers of primary cilia.

Techniques: