Journal: JCI Insight
Article Title: Tubular β -catenin and FoxO3 interactions protect in chronic kidney disease
doi: 10.1172/jci.insight.135454
Figure Lengend Snippet: (A) PT cells were treated with varying doses of Wnt3a either in control conditions (PT medium as described in Methods) or with oxidative stress (H2O2 100 μM in serum-free DMEM/F12) for 16 hours, and Axin2 transcripts were measured by qPCR and normalized to Gapdh. (B) PT cells stably transfected with a Topflash reporter construct (see Methods) were treated with various doses of Wnt3a in either control or oxidative stress medium (H2O2 100 μM in serum-free DMEM/F12). A luminometer measured the TCF/LEF-dependent activity (Steady Glo), which was normalized to cell number by using Cell Titer assay. For both A and B, Holm-Šídák multiple-comparisons test was used. (C) Cells treated with AA for 5 days showed increased oxidative stress reflected by increased nitrotyrosine on immunoblots. PT cells were treated with AA (30 μM), Wnt3a (10 ng/mL) was added during the last 48 hours, and nuclei were isolated (see Methods) and immunoblotted for FoxO1, FoxO3, or histone H3 for loading (D), and the results from 3 separate experiments were quantified (E). (F) PT cells were treated ± Wnt3a (10 ng/mL) and oxidative stress (H2O2 100 μM) for 16 hours, and then nuclei were isolated and coimmunoprecipitation was performed. Nuclear isolates had either β-catenin pull-down or IgG control, then were immunoblotted with FoxO3, and histone H3 was used for loading control of nuclear input. The nuclear input was also immunoblotted with α-tubulin, to assess for nuclear purity, and β-catenin. Levels of FoxO3 from the coimmunoprecipitation, normalized to histone H3 (nuclear input), were quantified from 3 separate experiments (G), as were nuclear β-catenin levels (H). Student’s t test was used for statistical analyses in G and H, and ANOVA was used for multiple comparisons in E with *P < 0.05 and **P < 0.01.
Article Snippet: Wnt/β-catenin reporter or Topflash ( Tcf/Lef:H2B-GFP ) mice harboring H2B-EGFP fusion protein expression under the control of TCF/LEF1 response element ( 26 ) were obtained from The Jackson Laboratory.
Techniques: Control, Stable Transfection, Transfection, Construct, Activity Assay, Titer Assay, Western Blot, Isolation
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