A WT Wnt6 smact merge VK/A KO ctrl IgG WT KO Wnt6 smact DAPI SUPPLEMENTAL FIGURE I: Wnt6 expression in MGP-deficient aortae. Immunostaining for Wnt6 and smooth muscle actin (smact) in aortae from 7 day old (A) or 4.5 week old () wild-type (WT) or MGPdeficient (KO) mice [nuclei counterstained with DAPI (blue)]. In A, adjacent sections were incubated with control IgG. And in, adjacent sections were stained for deposition of calcified (black) and glycosaminoglycan-rich (blue) matrices by von Kossa (VK) and Alcian blue (A) stains, respectively. Scale bar in A = mm. Scale bar in = 5 mm.
b-catenin luciferase activity (fold) A pwnt6: - + Wnt6 GAPDH 8 6 4 Medium Cell lysate pwnt6-medium: - + SUPPLEMENTAL FIGURE II: Active Wnt6 is secreted by transfected Cos7 cells. A, Western blot for Wnt6 protein in equal volumes of conditioned medium collected from mock-transfected Cos7 cells or Cos7 cells transfected with Wnt6 plasmid (pwnt6). Roughly equal numbers of cells were used in this experiment as evident from Western blot for GAPDH in total cell lysates., Activity of the TCF/LEF-responsive luciferase reporter dependent on canonical b-catenin signaling in cells treated with mock- or pwnt6-conditioned medium (N=3)., p<.5.
Smad-dependent luciferase activity 5 4 3 MP: + + Noggin: + - LDN9389: - - - + SUPPLEMENTAL FIGURE III: Efficiency of MP inhibitors. Noggin ( ng/ml) or LDN9389 (5 nmol/l) effects on MP- induced activity of Smad-dependent luciferase reporter in A cells treated with recombinant MP (5 ng/ml) for 48 hours (N=4)., p<.
SUPPLEMENTAL FIGURE IV: Adapted representation of Ingenuity Pathway Analysis (IPA). RNA deep sequencing data revealing the interactions between 33 (gray symbols) out of 6 signature transcripts and molecules of the Mechanistic IPA network. The presence of known direct and indirect relationships between signature genes and members of the network are indicated with solid and dashed lines, respectively. For clarity, relationships between non-signature molecules were omitted.
A psmad smactin psmad/smact/dapi phase WT KO psmad/5/dapi WT-d3 KO-d3 vertebra limb/bone SUPPLEMENTAL FIGURE V: Smad activation in MGP-null aortic tissue. A, Immunostaining for phosphorylated Smads (psmad) implicates activation of TGFb signaling in aortae from 4.5 week old (d3) MGP-null (KO) but not in wild-type (WT) mice [nuclei counterstained with DAPI (blue)]. Phase images of tissue morphology (right panels) show cartilaginous metaplasia in KO aorta in contrast to typical aortic morphology in WT tissue with characteristic elastic lamina. Scale bar = mm, Lack of immunostaining for phosphorylated Smads and 5 (psmad/5) suggests no activity of the MP signaling in aortae from 4.5 week old (d3) wild-type (WT) and MGP-null (KO) mice [nuclei counterstained with DAPI (blue)]. Cartilaginous metaplasia is outlined with white dashed oval. Control sections of neonatal mice showed positive stain for psmad/5 in both developing vertebrae and limb bones. Scale bar = mm in aortae. Scale bar = 5 mm in neonatal tissues.
8 6 4 GAG deposition (fold) DMEM DMEM-S TGFb: - + - - TGFb: - - + - TGFb3: - - - + SUPPLEMENTAL FIGURE VI: TGFb-induced chondrogenic transformation in WT VSMCs. Equal pro-chondrogenic activities of the TGFb isoforms ( ng/ml) on A micromasses cultured for 8 days is shown by similar levels of glycosaminoglycan (GAG) synthesis detected with Alcian blue stain. Graph shows quantitation of extracted Alcian blue dye normalized to crystal violet stain for cell density (N=4)., p<..
Wnt6 mrna (fold) Wnt6 protein (fold) A..8.6.4. TGFb: - + Wnt6 GAPDH..8.6.4. TGFb: - + TGFb: - - + + Wnt6: - + - + SUPPLEMENTAL FIGURE VII: TGFb-induced repression of Wnt6 in VSMCs and effects on chondrogenesis. A, Expression of Wnt6 is reduced by TGFb3 ( ng/ml, 7 hours) in rat A VSMCs. Real-time PCR analysis for expression of Wnt6 mrna (left) and Western blot for protein levels (right) (N=3)., p<.., Representative images of A VSMCs micromasses treated with TGFb3 and Wnt6 and stained with Alcian blue dye to detect GAG.
osteogenic gene expression (fold) relative cell number (% of untreated) LDH release in medium (AU) SMC markers (fold) SMC markers (fold) A..8.6.4. Cnn MHC sma smact Wnt6 shscr: + - shwnt6: - + + - - + + - + - - + - + + - - + C D.4..8.6.4. SP OPN TGFb: + + Wnt6: - - + - - + 3 5 5 5 TGFb: + Wnt6: - + SUPPLEMENTAL FIGURE VIII: Effects of Wnt6 on gene expression in wild type VSMC. A-, Wnt6 supports expression of smooth muscle contractile markers. Down-regulation of Wnt6 by sirna results in down-regulation of contractile markers (A). In contrast, exogenous Wnt6 induces contractile markers expression inhibited by TGFb ().Gene expression analyzed by real-time PCR and compared to expression in non-transformed VSMC after normalization to ribosomal protein L9 expression (N=3). C, Expression of osteogenic genes in 8 days old rat A VSMCs micromasses in the presence or absence of exogenous Wnt6 (N=4). D-E, Exogenous Wnt6 did not affect either total cell number (detected by DNA staining with Crystal violet (D) or cytotoxicity in 4 day old chondrogenic micromasses (detected by release of lactate dehydrogenase into the medium) (E) compared to micromasses cultured in plain DMEM (N=4). NS not significant..5.5.5 NS calponin MHC sma smact E TGFb: + + + + Wnt6: - - + - - + - - + - - + 3.5.5.5 NS TGFb: + Wnt6: - - +
Notch gene expression (fold) Notch gene expression (fold) A..8.6.4. Dll Notch Jag Hey Hes DAPT: - + - + - + - + - +.5 Notch Dll Jag Jag.5 TGFb: - + - + - + - + SUPPLEMENTAL FIGURE IX. TGFb3 inhibits Notch signaling in wild type VSMC. Real-time PCR analysis of the expression of Notch genes in A VSMCs treated with either Notch inhibitor DAPT (A, N=3) or exogenous TGFb ( ng/ml) (, N=3)., p<.5;, p<.;, p<..