File S1 - <i>NPPB</i> and <i>ACAN</i>, Two Novel SHOX2 Transcription Targets Implicated in Skeletal Development

File includes Figures S1–S4 and Tables S1–S5. Fig. S1: Specificity of the different antibodies employed. A) Immunohistochemical controls performed in 38-wk fetal growth plates and adult normal colon sections: PBS - primary antibody replaced by PBS, Isotype - rabbit polyclonal IgG isotype control antibody, SHOX2 – SHOX2 antibody incubated with sections from adult normal colon where this protein is not expected to be expressed. Note the negative staining for the majority of the cells. Images performed at 20× magnification. B) Immunoblots showing the specificity of the SHOX2 antibody. Nuclear extracts of HEK293 cells overexpressing SHOX, SHOX2, SOX5, SOX6 and SOX9 were separated on SDS polyacrylamide gels and probed with anti-SHOX2. Anti-GAPDH was used as loading control. Fig. S2: SHOX2 cooperates with SOX5 and SOX9 to activate the Acan enhancer. Luciferase reporter activity of U2OS cells transfected with a reporter plasmid containing the Acan enhancer, renilla luciferase control plasmid and different combinations of SHOX, SHOX2 WT, SHOX2(p.L155V), SHOX2(p.Q234X), SOX5 and SOX9 expression plasmid as indicated. Fold-increase values were obtained by normalizing the relative luciferase units of each sample with the relative luciferase units of the sample transfected only with the reporter plasmid. All values represent the mean and standard deviation of three independent samples, with each sample assayed in triplicate. Significant p-values <0.001 obtained comparing different independent samples are indicated with two asterisks. Fig. S3: Expression of SHOX2 in different stages of the human fetal growth plate. Inmunohistochemistry performed with anti-SHOX2 antibody in normal fetal growth plates of 18, 27, 32 and 38-weeks. SHOX2 is expressed in the reserve (R), proliferative (P) and hypertrophic (H) chondrocytes. Images performed at 10× magnification. Fig. S4: SHOX2 is coexpressed with SHOX, SOX5, SOX6 and SOX9 in the 38-week human fetal growth plate. Immunohistochemistry performed on 38-weeks human fetal tibia growth plates using antibodies against SHOX, SHOX2, SOX5, SOX6 and SOX9. Specific staining can be observed in the reserve (R), proliferative (P) and hypertrophic (H) zones of the growth plate for all the analysed proteins. Images performed at 20× magnification. Table S1: Oligonucleotide sequences for the amplification of the SHOX2a cDNA and the cloning of the SHOX2 constructs. The incorporated enzymes sites are indicated in small letters. SHOX2a PCR A antisense oligonucleotide and SHOX2a PCR B sense oligonucleotide flank the sequence to be eliminated. Table S2: Oligonucleotide sequences for the generation of various SHOX2 fragments and SHOX2 missense mutants. The mutated site is indicated by a small letter. Table S3: Oligonucleotide sequences for the mutation screening of the coding exons and intron/exon boundaries of SHOX2. Table S4: Oligonucleotide sequences, PCR conditions and amplicon sizes of the SHOX2 microsatellite markers. Microsatellites are listed in order from telomere to centromere. Table S5: SHOX2 self-designed MLPA. Chromosomal and SHOX2 location, probe lengths and ligation site sequences are indicated for the SHOX2 and three control fragments. (PDF)