Matrisome-wide association study.

a. Manhattan plot showing −log10 P-values (y axis) versus chromosomal position (x axis) for the 2,008 common coding variants tested in the discovery study USA (TX). The horizontal line represents the threshold for significance level (P-value<2.5×10-5) after Bonferroni multiple testing correction. b. Tests of association for SNPs rs3753841 and rs1042704 in discovery and independent replication cohorts. RAF reference allele frequency; OR odds ratio; CI-confidence interval.

Participating studies.

NHW: Non-Hispanic White. EAS: East Asian.

Col11a1 and Mmp14 expression in spine.

a) A heatmap of transcript per million (TPM) values of COL11A1, MMP14, and other published genes associated with AIS. The average TPM value of matrisome genes is represented as MATRISOME.

b) Localization of collagen type XI alpha chain 1 in P0.5 mouse IVD. Immunohistochemistry (IHC) and immunofluorescence (IF) staining in IVD at day P0.5. Negative antibody controls in IVD are shown at left; antibody-positive IHC and IF are shown at right.

c) Localization of collagen type XI alpha chain 1 in P28 mouse IVD. Negative antibody IHC control shown at left; antibody positive IHC shown at right. Enlarged, rotated view of white boxed area shows a biphasic staining pattern. CEP – cartilage endplate; GP – growth plate.

Assessing Pax1 regulation of Col11a1.

a) IF staining of P28 IVD using Pax1 (green) and Col11a1 specific (red) antibodies with DAPI nuclear counterstain. Arrow denotes cells at the CEP-condensing bone interface of IVD that show positive staining for both Pax1 and Col11a1.

b) Gene ontology (GO) analysis of differentially expressed genes in E12.5 tail WT and Pax1-null mice.

c) Heatmap of differentially expressed genes (P-value<0.0001) in E12.5 tails of WT and Pax1-null mice

d-g) Gene expression levels dissected from E12.5 mouse tail from wild type (WT) and Pax1-null (KO) mice as determined by qRT-PCR. Each value represents the ratio of each gene expression to that of β-actin, and values are mean ± standard deviation. The expression value of WT female group was arbitrarily set at 1.0. Each dot represents one embryo and statistical differences were determined using a two-sided unpaired t-test (P*<0.05, P**<0.01, P***<0.001).

Col11a1 regulation of Mmp3 expression in cartilage.

a) PCR assay of Col11a1 excision in Col11a1fl/fl cultured rib cartilage cells.

b) Gene expression levels from Col11a1fl/fl cultured rib cartilage cells transduced with GFP (Ad5-GFP, left) or Cre expressing adenovirus (Ad5-cre, right) as determined by qRT PCR. Each value represents the ratio of each gene expression to that of GAPDH, and values are mean ± standard deviation. The expression value of control Ad5-GFP results (N≥ 4 experiments) was arbitrarily set at 1.0. Statistical differences were determined using a two-sided unpaired t test (P*<0.05).

c) Representative Western blot of cultured cartilage cells after Ad5-GFP or Ad5-cre transduction. Protein size ladder is shown in lane 1. Quantification of bands detected by Western blotting, where scramble results were set to 1.0 from N=4 independent experiments is shown at right.

d) Gene expression levels from dissected Col11a1fl/fl ATC rib cartilage, analyzed as described in a). Results shown are from N=3 mice.

Col11a1P1335L regulation of Mmp3 expression in lentiviral transduced mouse GPCs.

a) qRT-PCR of human COL11A1 and endogenous mouse Mmp3. “SV40” refers to immortalized mouse chondrocytes transduced with the lentiviral vector only. Significant fold changes (P ≤0.5) relative to vector-only transfected cells are shown by *.

b) Western blot corresponding to experiments shown in (a) using antibodies to detect Col11a1, HA epitope tag representing human Col11a1 only, endogenous Mmp3, and GAPDH. Quantification at right shows Mmp3 expression in each experiment relative to GAPDH.

Effects of estrogen receptor beta on Col11a1 Mmp3 signaling axis.

a) Representative Western blot of cultured cartilage cells after scramble or Col11a1-specific siRNA knockdown. Protein size ladder is shown in lane 1. Quantification of bands detected by Western blotting, where scramble results were set to 1.0 from N=4 independent experiments is shown at right.

b) Gene expression levels of Col11a1 (left), Mmp3 (middle), and Esr2 (right) mRNA in cultured rib cartilage cells showing fold change relative to the scramble control. ddk = double Col11a1-Esr2-specific siRNA knockdowns. Results are representative of n≥3 experiments.

c) Gene expression levels from rat CEP cells, as described in (b).

Cartoon depiction of a collagen XI-mediated signaling axis in cartilage. Collagen XI is held in the pericellular space by integrins and DDR2. COL11A1, under the regulation of ESR2 and PAX1, signals through unknown mechanisms and inhibits MMP3 transcription.