Figures and data
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.
Study cohorts. USA (TX): Texas cohort, USA (MO)
Missouri cohort, SW-D: Swedish-Danish cohort, JP: Japanese cohort, HK: Hong Kong cohort, 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) Detection of collagen type XI alpha chain 1 in P0.5 mouse spine. Immunohistochemistry (IHC) shown at top, with immunofluorescence (IF) staining below. “-ab” refers to negative controls lacking primary antibody (shown at left). Results are representative of N≥3 technical replicates in whole spines.
c) Detection of collagen α1(XI) in P28 mouse spine. 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. Results are representative of N≥3 technical replicates in whole spines.
Assessing Pax1 regulation of Col11a1.
a) IF staining of P28 IVD from thoracic regions of Pax1-/- (bottom and wildtype littermate (middle, top) mice using PAX1-(green) and collagen α1(XI)-specific (red) antibodies and DAPI nuclear counterstain. Antibody-negative controls are shown at top as (-ab). Results are representative of N≥3 technical replicates in whole spines.
b) Heatmap of differentially expressed genes (P-value<0.0001) in E12.5 tails of WT and Pax1-/- mice
c) Gene ontology (GO) analysis of differentially expressed genes in E12.5 tail WT and Pax1-/- null mice
d-g) Gene expression levels dissected from E12.5 mouse tail from wild type (WT) and Pax1-/- (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 costal chondrocytes.
b) Gene expression levels from Col11a1fl/fl cultured costal chondrocytes transduced with GFP (Ad5-GFP, left) or Cre-expressing adenovirus (Ad5-cre, right) as determined by qRT-PCR. Values represent the ratio of each gene expression to that of GAPDH, and values are mean ± standard deviation. The expression value of control Ad5-GFP results was arbitrarily set at 1.0. Statistical differences were determined using a two-sided paired t test (P*<0.05). Results shown for N≥3 biologic replicates, each including 3 technical replicates.
c) Western blot detection of collagen α1(XI), MMP3, and GAPDH loading control in cultured costal chondrocytes after Ad5-GFP or Ad5-cre transduction. Results are representative of N= 4 biologic replicates. Protein size ladder is shown in lane 1. Quantification of bands detected by Western blotting, where scramble results were set to 1.0, is shown at right. Statistical differences were determined using a two-sided paired t test (P*<0.05).
d) Gene expression levels from dissected Col11a1fl/fl ATC costal cartilage, analyzed as described in a). Results shown for N=3 biologic replicates, each including 3 technical replicates.
Col11a1P1335L regulation of Mmp3 expression in lentiviral transduced mouse GPCs.
a) qRT-PCR of human COL11A1 and endogenous mouse Mmp3 in SV40 immortalized mouse costal chondrocytes transduced with the lentiviral vector only (lanes 1,2), human WT COL11A1 (lane 3), or COL11A1P1335L. Values represent the ratio of each gene expression to that of GAPDH, and values are mean ± standard deviation. Significant quantitative changes (P ≤0.05) relative to vector-only transfected cells as measured by unpaired t-tests are shown by *. Results shown for N=4 biologic replicates, each including 3 technical replicates.
b) Western blot corresponding to experiments shown in (a) using HA antibody to detect epitope-tagged human collagen α1(XI), COL11A1 antibody to detect mouse and human collagen α1(XI), MMP3 antibody to detect endogenous mouse MMP3, and GAPDH. Values are mean after normalization to GAPDH, ± standard deviation. Significant differences (P ≤0.05) relative to vector-only, Ad5-negative transfected cells as measured by unpaired t-tests are shown by *.
Effects of estrogen receptor beta on Col11a1-Mmp3 signaling axis.
a) Representative Western blot (of N=4 biologic replicates) of cultured costal chondrocytes after scramble or Col11a1-specific siRNA knockdown. Protein size ladder is shown in lane 1. Quantification of bands detected by Western blotting are shown at right, where scramble results were set to 1.0. Values are mean after normalization to GAPDH, ± standard deviation.
b) Gene expression levels of Col11a1 (left), Mmp3 (middle), and Esr2 (right) mRNA in cultured costal chondrocytes showing fold change relative to the scramble control. dKD = double Col11a1-Esr2-specific siRNA knockdowns. Each value represents the ratio of each gene expression to that of GAPDH, and values are mean ± standard deviation. Results are representative of N≥3 biologic replicates, each including 3 technical replicates.
c) Gene expression levels from rat CEP cells, as described in (b).
Cartoon depiction of a collagen XI-mediated signaling axis in chondrocytes. 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.
