Journal: PLoS ONE

Article Title: Kidney Specific Protein-Positive Cells Derived from Embryonic Stem Cells Reproduce Tubular Structures In Vitro and Differentiate into Renal Tubular Cells

doi: 10.1371/journal.pone.0064843

Figure Lengend Snippet: (a) Mouse neonate kidney stained with our anti-KSP antibody. The left picture is a low magnification, and the right picture is a high magnification. A micron scale is shown at the lower right corner. (b) Mouse kidney stained with our anti-KSP antibody, anti-AQP1 antibody, anti-Megalin antibody, anti-E-cadherin antibody and anti-AQP2 antibody. Scale bar, 400 µm. (c) Mouse small intestine stained with anti-E-cadherin antibody and our anti-KSP antibody. Scale bar, 400 µm. (d) Mouse ES cells stained using our anti-KSP antibody and anti-E-cadherin antibody after 18 days of differentiation with Activin (10 ng/mL).

Article Snippet: The samples were incubated with our anti-KSP antibody conjugated with biotin, anti-human specific mitochondria antibody conjugated with biotin (Abcam), anti-Megalin antibody, anti-AQP2 (Alomone Labs) or anti-Podocalyxin (R&D) antibodies.

Techniques: Staining

Journal: PLoS ONE

Article Title: Kidney Specific Protein-Positive Cells Derived from Embryonic Stem Cells Reproduce Tubular Structures In Vitro and Differentiate into Renal Tubular Cells

doi: 10.1371/journal.pone.0064843

Figure Lengend Snippet: (a) PCR showing the expression of segment-specific genes of renal tubular cells. The samples were KSP-positive cells examined right after cell purification with flow cytometry, KSP-positive cells forming tubular structures co-cultured with NIH3T3-Wnt4, and NIH3T3-Wnt4 alone. The bands were quantified using ImageJ, and graphs normalized against GAPDH were shown. After sorting: a sample of cells collected right after cell sorting of KSP-positive cells. Tubular structures: a sample of KSP-positive cells that form tubular structures and feeder cells of NIH3T3-Wnt4. Feeder alone: a sample of feeder cells, NIH3T3-Wnt4. (b) Immunofluorescence showing Megalin, AQP2 and Podocalyxin in tubular structures formed by KSP-positive cells co-cultured with NIH3T3-Wnt4. A micron scale is shown at the lower right corner.

Article Snippet: The samples were incubated with our anti-KSP antibody conjugated with biotin, anti-human specific mitochondria antibody conjugated with biotin (Abcam), anti-Megalin antibody, anti-AQP2 (Alomone Labs) or anti-Podocalyxin (R&D) antibodies.

Techniques: Expressing, Purification, Flow Cytometry, Cell Culture, FACS, Immunofluorescence

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Reduced Notch Signaling Leads to Renal Cysts and Papillary Microadenomas

doi: 10.1681/ASN.2009090925

Figure Lengend Snippet: The mechanism driving epithelial stratification and cyst formation is not loss of apical-basal polarity or reduced TCF2/HNFβ1 expression. (A) Na+/K+-ATPase is expressed in the basolateral membrane of PT epithelia in P1 wild-type (RBP-J f/f) littermates. (B) RBP-J deficient PT epithelia lining the cysts retain basolateral expression of Na+/K+-ATPase, although some regions (marked by asterisks) show a reduction in Na+/K+-ATPase expression. Inset in B shows a magnified view of the cyst-lining epithelia in which the arrowhead points at a stratified epithelium that retains polarized expression of Na+/K+-ATPase. (C) Kidneys from P1 Rarb2-Cretg;RBP-Jf/f mice were stained for TCF2/HNF1β (red). Although TCF2/HNF1β is downregulated in some cyst-lining epithelia in Notch-deficient kidneys (box 1), TCF2/HNF1β was expressed in all normal diameter PTs (box 2) and in some cyst-lining epithelia (box 3) containing stratified epithelia (arrowhead in box 4). (D) E-cadherin expression is restricted to the basolateral membrane, with a strong punctate staining at the adherence junctions of PT cells that express megalin in the apical membrane of wild-type kidneys. The inset shows magnified PTs with an asterisk in the lumen of a PT composed of epithelia with properly polarized expression of E-cadherin and megalin. (E, F) In the 5-week-old Six2-GFP::Cretg;N2f/f kidneys, PT epithelia retain apical-basal polarity (asterisks), except for a few epithelia lining the cysts, which either lack apical megalin and have apical E-cadherin (arrow) or have colocalization of megalin and E-cadherin in apical surface (arrowhead). All scale bars are 100 μm.

Article Snippet: Additional primary antibodies and lectins utilized in the study include p21 (1:200; Santa Cruz SC-6246), neural cell adhesion molecule (1:200; Sigma), E-cadherin (1:400; BD Transduction Labs C20820), β-catenin (1:200, BD Transduction Labs), WT-1 (1:100; Santa Cruz Sc-192), CLCK (1:100; Alomone labs ACL-004), AQP2 (1:500; gift from Dr. Mark Knepper 53 ), Na + /K + -ATPase α-1 (1:500; Upstate 05-369), megalin (1:1000; gift from Marilyn Farquhar), Claudin 1 (1:100, Invitrogen), TCF2/HNF1β (1:400; Santa Cruz SC-22840 and gift from Dr. Marco Pontoglio), acetylated-tubulin (1:500; Sigma T6793), FITC-conjugated LTL (1:200; Vector Labs), Ki-67 (1:200; Novocastra NCL-Ki67p), and pS10H3 (1:500; Upstate 05 to 817).

Techniques: Expressing, Staining