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. 2024 Jan 26;12:RP89762. doi: 10.7554/eLife.89762

Figure 4. 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 green fluorescent protein (GFP) (Ad5-GFP, left) or Cre-expressing adenovirus (Ad5-cre, right) as determined by quantitative real-time PCR (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 three technical replicates. (C) Western blot detection of collagen a1(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 Ad5-GFP was 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 three technical replicates.

Figure 4—source data 1. Original gel images of Col11a1 fl/fl excision PCR assay in Figure 4A.
Figure 4—source data 2. Figure 4A and original gel images of Col11a1 fl/fl excision PCR assay with highlighted and labeled bands.
Figure 4—source data 3. Original western blot images (anti-COL11A1, anti-MMP3, anti-GAPDH) shown in Figure 4C.
Figure 4—source data 4. Figure 4C and original western blot images (anti-COL11A1, anti-MMP3, anti-GAPDH) with highlighted bands and labels.

Figure 4.

Figure 4—figure supplement 1. Relative expression of MMP3 compared to COL11A1 in human spinal tissues.

Figure 4—figure supplement 1.

Values are plotted from average TPM values for each gene.
Figure 4—figure supplement 2. Immunofluorescence microscopy of Rosa26+/-:ATC P0 spines, without doxycycline treatment (left) and after doxycycline treatment starting at embryonic stage 15.5 (E15.5) (right).

Figure 4—figure supplement 2.

Figure 4—figure supplement 3. PCR assays in DNA from costal cartilage.

Figure 4—figure supplement 3.

Top gel shows detection of ATC Cre transgene (top, 416 bp band) in Col11a1fl/fl:ATC mice (lanes 2,3,7) and Col11a1fl/fl mice (lanes 4,5,6) after doxycycline treatment. Lane 1 is positive control. Bottom gel shows Col11a1 fl/fl excision-specific band (321 bp) in Col11a1fl/fl:ATC mice (lanes 2,3,7) and Col11a1fl/fl mice (lanes 4,5,6) after doxycycline treatment.
Figure 4—figure supplement 3—source data 1. Original gel images of Col11a1 fl/fl excision PCR assay in Figure 4—figure supplement 3.
Figure 4—figure supplement 3—source data 2. Original gel images of Col11a1 fl/fl excision PCR assay in Figure 4—figure supplement 3 with highlighted and labeled bands.