Journal: The Journal of Biological Chemistry

Article Title: Disruption of the integrin-linked kinase (ILK) pseudokinase domain affects kidney development in mice

doi: 10.1016/j.jbc.2021.100361

Figure Lengend Snippet: ILK mutants differentially regulate kidney collecting duct cell functions. A , ILK WT and mutants were transfected into ILK-null (KO) kidney CD cells, and clones were selected for equal expression of the mutants. B–E , WT and mutant CD cells were placed in 3D collagen I/Matrigel gels for 7 days in the presence of 5% FBS, stained with rhodamine-phalloidin, and visualized by confocal microscopy. The arrows depict lumens present within the tubules. Scale bar 50 μm. F , the number of branches of at least 50 tubules per genotype was quantified and the average branch number ± SD is shown. Differences between WT and the E359K, PBS, and K220M mutants (∗) were significant ( p < 0.01). G , CD cells were allowed to adhere to collagen I (Coll 1) or Matrigel for 1 h. Data are mean ± SD of three experiments in triplicate. ∗ p < 0.05 (between wild type and K220M mutant and ∗∗ p < 0.01 (between WT and E359K or PBS mutants). H , CD cells were plated on transwells coated with Coll 1 or Matrigel and migration, measured as cells per high-powered field (hpf), was evaluated after 4 h. Data are mean ± SD of three experiments in triplicate. (∗) and (∗∗) are as in G . I , CD cells were plated on Coll I or Matrigel. After 24 h, the cells were treated with [ 3 H] thymidine and incubated for a further 24 h after which thymidine incorporation (in cpm) was determined. Data are mean ± SD of three experiments in triplicate. ∗∗ p < 0.01 between WT and E359K or PBS mutants. J–M , CD cells were plated on Matrigel in serum-free medium for 4 h after which they were fixed and stained with rhodamine phalloidin and anti-paxillin antibody. Scale bar 5 μm. N , the area of at least 50 individual cells was measured as μm 2 . The mean cell area ± SD of the different mutant cells is shown. ∗∗ p < 0.01 between WT and E359K or PBS mutants (N).

Article Snippet: In total, 1.5 × 10 3 CD cells were placed in 3D gels comprising rat tail collagen I and Matrigel (Becton Dickinson) and DMEM containing 20 mM HEPES (pH 7.2) as previously described ( ).

Techniques: Transfection, Clone Assay, Expressing, Mutagenesis, Staining, Confocal Microscopy, Migration, Incubation

Journal: Journal of Virology

Article Title: Jaagsiekte Sheep Retrovirus Transformation in Madin-Darby Canine Kidney Epithelial Cell Three-Dimensional Culture ▿

doi: 10.1128/JVI.02323-09

Figure Lengend Snippet: Effect of JSRV Env on acinus structure and polarization. (A) LXSN- and LXSNenvHA-transduced MDCK cells were grown in 3-D culture and counterstained with DAPI (nuclei; blue) and phalloidin (actin; red) at the indicated time points. Shown are confocal cross sections through the center of the structures. (B) Acini were analyzed for hollowness at days 5 and 20, and the results are represented as percentages of total acini analyzed (≥100) that were hollow. The experiment was performed in duplicate, and the means (± standard errors) from 3 independent experiments are shown. Statistical significance was determined by Student's t test. *, P = 0.001; **, P = 0.00007. (C) Acini were immunostained with anti-GM130 (green) and DAPI (blue) at days 10 and 20 of culture and then analyzed by confocal microscopy. Higher-magnification images of the indicated areas (boxes) are shown in the insets. (D) Transduced MDCK cells were seeded in collagen (bottom) or Matrigel (top), immunostained with anti-GM130 (green), phalloidin (red), and DAPI (blue) at days 1, 2, and 5, and examined by confocal microscopy. Magnification, ×400; bars, 50 μm.

Article Snippet: Specifically, single-cell suspensions were resuspended in a collagen-Matrigel (BD Bioscience) mixture (80:20) at 6 × 10 3 cells/ml, and 2 × 10 4 cells/well were seeded into 4-well chamber slides.

Techniques: Confocal Microscopy