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. 2012 Oct 29;109(46):18827–18832. doi: 10.1073/pnas.1210037109

Fig. 2.

Fig. 2.

1,25D signaling enhances stability and expression of MXD1. (A) Gradual rise in MXD1 mRNA levels in SCC25 cells treated with 100 nM 1,25D. (B) 1,25D treatment induces expression of MXD1 protein. (Upper) Western blots for MXD1 and actin from total cell extracts. (Lower) Western blots of nuclear and cytoplasmic extracts of cells treated with 1,25D as indicated. The band marked by an asterisk in the top and bottom Western blots is a nonspecific cytoplasmic protein. (Right) Quantification of the results of four independent analyses of MXD1 protein expression. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 as determined by one-way ANOVAs followed by Tukey’s post hoc test for multiple comparisons. (C) Effect of short-term (6 h) treatment with 1,25D on turnover of MXD1 protein cycloheximide-treated cells. A quantification of the results of three different experiments is shown. (D) MXD1 coimmunoprecipitates in a 1,25D-dependent manner with the VDR from extracts of SCC25 cells treated with vehicle or 1,25D as indicated. (E) Knockdown of FBW7 enhances expression of MXD1. (F) Ablation of FBW7 expression reduces MXD1 turnover in cycloheximide-treated cells (Scr; scrambled control siRNA). A quantification of four independent experiments is shown. Statistical analysis was performed by two-way ANOVA with Bonferroni correction for multiple comparisons; *P = 0.001. (G) Ablation of FBW7 abolishes the effect of 1,25D treatment on MXD1 turnover. A quantification of five independent experiments is shown.