Figure 1.
FOXC1 induces MSX2 expression.
(A).U2OS cells were transiently transfected with Xpress-tagged FOXC1 expression vectors. Levels of exogenous FOXC1 protein were determined by immunoblotting with anti-Xpress antibody. (B) MSX2 mRNA levels were measured by qRT-PCR in FOXC1-over expressing cells. Error bars represent the standard error of the mean. *, p value <0.0001.
Figure 2.
FOXC1 binds to the Msx2 promoter in vivo.
(A) Sequence analysis of upstream regulatory elements reveals the presence of a FOXC1 binding motif, indicated in bold, (TAAAT/CAAT) located in a conserved motif near the predicted Msx2 transcription start site of the mouse, rat and human genes. Small arrows correspond to the position of ChIP primers located in the promoter region or the coding region of mouse Msx2. Nucleotide sequences of the Electrophoretic mobility shift assay (EMSA) probes for wild type (WT) and mutated (MUT) FOXC1 binding sites are indicated. (B) Chromatin immunoprecipitation assays confirm the binding of FOXC1 to the Msx2 promoter in vivo. Quantitative PCR (qPCR) was conducted on ChiP products isolated from 10T1/2 cells using antibodies recognizing FOXC1 or normal immunoglobulins (IgG). Primers were designed amplify regions in the promoter flanking the putative FOXC1 binding site or exon 2 of the mouse Msx2 gene. Amplification signals are presented a percentage compared to input chromatin fraction. (C) EMSAs demonstrate FOXC1 binding to DNA elements in the Msx2 promoter. Extracts from U2OS cells or cells transfected with FOXC1 were incubated with IR700-labeled oligonucleotides correspond to the WT or MUT FOXC1 binding sites. FOXC1-DNA complexes are indicated by the arrow.
Figure 3.
FOXC1 activates the MSX2 promoter.
(A) Luciferase (luc) reporter vectors consisting of the human (h) or mouse (m) MSX2 promoter which contained the conserved FOXC1 binding element were created. Transfection of 10T1/2 cells with FOXC1 expression vectors lead to a robust activation of both human and mouse MSX2 promoters. (B) The putative FOXC1 binding sites was mutated in the mouse Msx2-luc reporter. Wild type and mutated Msx2-luciferase vectors were co-transfected with empty pcDNA4 (vector) or Xpress tagged-FOXC1. Error bars correspond the standard error of the mean.
Figure 4.
FOXC1 shRNA expression reduces MSX2 mRNA levels.
(A) MB231 cells were transduced with lentiviral particles containing EGFP or FOXC1 shRNA. Pooled puromycin resistant colonies were expanded and FOXC1 expression analyzed semiquatitative rt-PCR. (B). qRT-PCR data indicating reduced expression of MSX2 mRNA in MB231 cells when FOXC1 levels are reduced by RNA interference.
Figure 5.
FOXC1 overexpression induced ectopic osteoblast transdifferentiation of C2C12 cells.
C2C12 myoblasts were transduced with retroviruses containing pBABE (empty vector control) or pBABE-FOXC1. Cells were grown to 95% confluence and stained for alkaline phosphatase activity after 4 days.
Figure 6.
Increased expression of osteogenic marker genes in FOXC1-expressing C2C12 cells.
Levels of Msx2, Runx2, Sp7 (Osterix), Alkaline Phosphatase (Alp) and Osteocalcin (Ocn) mRNA levels were determined by qRT-PCR from C2C12 cells transduced with empty pBABE or FOXC1 retroviral particles. * p<0.05; ns, not significant.