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. 2018 Jun 26;16(6):e2005086. doi: 10.1371/journal.pbio.2005086

Fig 4. Compensatory proliferation is bone-intrinsic and takes place when chondrocyte density is lower than normal.

Fig 4

(A) Summary of the ex vivo tibial culture experiment. The boxed regions correspond to the epiphyses shown in panel B. (B) Immunohistochemistry for the indicated molecules. (C) EdU quantification on distal PZ sections obtained from E15.5 ePit-Col-p21 tibiae cultured for 2 d. p-Value for 2-tailed paired t test comparing left and right proliferative ratios of p21− chondrocytes is shown (n = 4). The distal cartilage was quantified because the proximal one (bulkier) shows proliferation only in the periphery. (D) % of p21+ or p21− chondrocytes that have EdU+ nuclei in the PZ in the left and right proximal humerus of E17.5 ePit-p21 (Control, n = 3) and ePit-Col-p21 (Exp, n = 3) embryos. p21− cells from Control and Exp mice were compared by 2-way ANOVA with Side and Genotype as variables (p-values below graphs). (E) Correlation analysis between the extent of EdU incorporation in p21− cells and the amount of p21+ nuclei in left and right PZ of ePit-Col-p21 (n = 5 in vivo and n = 4 ex vivo) and Pit-tTA-p21 tibial cartilage (n = 4) at E17.5. The inflection point revealed by unconstrained segmental regression was rounded up and used as a dividing threshold for the 2 correlation analyses (colour-coded). Pearson correlation coefficients and 2-tailed p-values are shown. (F) Comparison of chondrocyte density in the PZ of left and right ePit-p21 and ePit-Col-p21 proximal tibial cartilage at E15.5 (n = 4 and n = 3), E17.5 (n = 5 and n = 5) and P0 (n = 4 and 7) and analysed by 2-way ANOVA for Genotype and Side (p-values shown in the embedded tables). When p < 0.05 for these variables, colour-coded p-values for Sidak’s post hoc tests are shown. (G) EdU incorporation in p21− chondrocytes of left and right PZ from E17.5 ePit-p21 and ePit-Col-p21 embryos (n = 5 each), plotted against cell density in the PZ. Note the sharp change in proliferation beyond 9,000 cells/mm2. For (C–G), see also S3 Data. Dox, doxycycline; E, embryonic day; EdU, 5-ethynyl-2’-deoxyuridine; Exp, Experimental; P, postnatal day; PZ, proliferative zone.