A novel feedback control mechanism of Wnt/β-catenin pathway.
(a) Upstream sequence of mouse wnt1 gene. Putative TRE is shaded. (b) Construct map of the luciferase reporter constructs used in luciferase activity assays. Constructs contain various portions of mouse wnt1 gene upstream sequence, as indicated. The putative TRE is denoted by a shaded box. (c) MC-3T3 cells were transfected with the reporter constructs pWNT, pWNTm and the control construct pGL3-Basic (pGL). The cells were then treated with either 20 mM LiCl or NaCl. Bar graph shows the results (mean ± SEM) on luciferase activities for transfection with control plasmid (pGL), pWNT or pWNTm, with 3 experiments in each group. (d) Primary chondrocytes were treated with 20 mM LiCl or NaCl for 24 hr and then ChIP assays were performed. No template: PCR amplification without DNA sample; 1% input: samples representing total input chromatin (1%) for each experiment; negative IgG: immunoprecipitated with negative IgG instead of β-catenin antibody. (e) Primary chondrocytes cultured on the stiff or soft ECM for 24 hr were subjected to quantitative ChIP assays. ChIP results were from 3 independent experiments and the bar graphs showing the combined results of 3 experiments on the stiff matrix as percentages (mean ± SEM) of the corresponding results on the soft matrix. *P < 0.05, **P < 0.01, n.s. stands for not statistically significant.