Figure 6: Impaired functional interaction between a pathological missense Foxc1 mutant and Gli2.
From: The transcription factor Foxc1 is necessary for Ihh–Gli2-regulated endochondral ossification
(a) 293FT cells were transfected with empty vector, Flag-tagged WT Foxc1 (Flag–Foxc1) or Flag-tagged-F112S mutant Foxc1 (Flag-F112S), and cell lysates were immunoblotted with anti-Flag (upper panel) and anti-β-actin (lower panel) antibodies. (b) Effect of F112S mutant on PTHrP. PTHrP mRNA expression was determined by RT–qPCR. Data are shown as fold activation normalized to control (mean±s.d., n=3). **P<0.01 (versus Foxc1); one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test. (c) Effect of the F112S mutation on Foxc1 transcriptional activity on the PTHrP promoter. Data are expressed in relative luciferase units (mean±s.d., n=3). **P<0.01 (versus Foxc1); one-way ANOVA followed by the Tukey–Kramer test. (d) Inhibitory effect of F112S on PTHrP mRNA expression induced by Ihh. Data are shown as the mean±s.d. (n=3). **P<0.01(versus Ihh); one-way ANOVA followed by the Tukey–Kramer test. (e) Impaired synergistic effect between F112S and Gli2 on PTHrP mRNA expression. Primary chondrocytes were infected with the indicated adenoviruses and PTHrP mRNA expression was analysed by RT–qPCR analysis. Data are shown as the mean±s.d. (n=3). **P<0.01, *P<0.05; one-way ANOVA followed by the Tukey–Kramer test. (f) DNA pull-down assays using the Foxc1-binding element from the PTHrP promoter. Samples precipitated with the biotin-labelled Foxc1-BE oligonucleotide (upper panel) and total cell lysates (lower panel) were immunoblotted with an anti-Flag antibody. (g) F112S failed to interact with Gli2. Lysates of the 293FT cells transfected as indicated were immunoprecipitated with an anti-Myc antibody and immunoblotted with an anti-Flag antibody (top). The cell lysates were immunoblotted with an anti-Myc (middle) or anti-Flag (bottom) antibody. (h) Nuclear localization of F112S and Gli2. HEK293 cells transfected with RFP-tagged F112S (RFP-F112S) and Venus-tagged Gli2 (Venus-Gli2). Confocal cross sections of xy (top left), yz (right) and xz (bottom) were visualized under a confocal microscope. Scale bar, 5 μm.
