Axin2 controls bone remodeling through the {beta}-catenin-BMP signaling pathway in adult mice -- Yan et al. 122 (19): 3566 Data Supplement - JCS051904 Supplementary Material -- Journal of Cell Science

Axin2 controls bone remodeling through the β-catenin–BMP signaling pathway in adult mice
J Cell Sci Yan et al. 122: 3566

JCS051904 Supplementary Material

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Supplemental Figure S1 -
Fig. S1. No significant changes in bone mass and bone mechanical property were observed in Col1-Axin2 transgenic mice. Three dimensional bone structure was analyzed in 3-month-old female Col1-Axin2 transgenic mice (n=8) and WT littermate controls (n=7) by micro-CT. No significant changes in bone volume fraction (BV/TV, %), bone mineral density (BMC/TV), structural model index (SMI), trabecular number (Tb.N.), trabecular thickness (Tb.Th.) and trabecular separation (Tb.Sp.) were found in Col1-Axin2 transgenic mice compared to WT littermate controls (A-F). Results from bone mechanical testing demonstrated that maximal stiffness, maximal force and yield force were not significantly altered in Col1-Axin2 transgenic mice compared to WT littermates (G-I).
Supplemental Figure S2 -
Fig. S2. Axin2 KO mice are partially resistant to ovariectomy-induced bone loss. Ovariectomy (Ovx) surgery and sham operation were performed in 6-month-old female Axin2 KO mice and WT controls. 4 weeks after Ovx, mice were sacrificed and bone volume (BV/TV, %) was measured by micro-CT. 48% bone loss was found in WT mice 4 weeks after Ovx compared to sham-operated mice (Sham: 8.1±0.9% versus Ovx: 4.2±0.7%). By contrast, 36% bone loss was found in Axin2 KO mice 4 weeks after Ovx compared to sham-operated Axin2 KO mice (Sham: 12.6±1.5% versus Ovx: 8.1±0.8%). *P<0.05, unpaired Student’s t-test, n=10.
Supplemental Figure S3 -
Fig. S3. Smad1 dephosphorylation by PPM1A cannot be altered by overexpression of Axins. HEK 293T cells were transfected with plasmids as indicated. Both Myc-Smad1 and endogenous Smad1 can be phosphylated by HA-ALK3 expression and such Smad1 phosphorylation can be reversed by PPM1A expression. Overexpression of Myc-Axin1 or Myc-Axin2 had no significant effects on phosphorylation or dephosphorylation of Smad1. Neither Axin1 nor Axin2 had any effect on PPM1A-induced Smad1 dephosphorylation.
Supplemental Figure S4 -
Fig. S4. Loss of Axin2 does not affect GSK3 phosphorylation of Smad1. 2T3 cells were transfected with non-targeting or Axin2 siRNA oligonulcleotides. At 48 hours post-transfection cell lysates were prepared and subjected to western blot analysis with the corresponding antibodies. Transfection of Axin2 siRNA had no significant effect on Smad1 phosphorylation at its linker domain GSK3 phosphorylation site.