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. 2022 Jun 1;13:3059. doi: 10.1038/s41467-022-30618-8

Fig. 3. Extracellular DMP1 inhibits VEGF signalling in the OF in vivo and in endothelial cells in vitro.

Fig. 3

a Quantification of the centre to periphery ratio of phosphorylated VEGFR2 (pVEGFR2) and DMP1 amount at different ages. At 4 and 5 weeks n = 7, 5.5-6 weeks n = 6, 8 weeks n = 5, 12–15 weeks n = 4. Pooled data from three independent experiments. Mean ± SD. b Quantification of pVEGFR2 amount in the centre relative to periphery in control and Dmp1i∆OB OF. Two-tailed Wilcoxon matched-pairs signed-rank test. c, Quantification of the total central pVEGFR2 amount in control and Dmp1i∆OB group. Two-tailed unpaired t-test. b, c Pooled data from three independent experiments. Control n = 9, Dmp1i∆OB n = 11. Age: 6 weeks. Mean ± SD. d pVEGFR2 immunostaining showing no decrease of pVEGFR2 in the OF centre of Dmp1i∆OB femurs in contrast to control femurs at 6 weeks of age. Scale bar 25 µm. e Quantification of VEGFR2 intensity in EMCN+ endothelial cells in the OF of control and Dmp1i∆OB mice. Control n = 4, Dmp1i∆OB n = 4. Pooled data from two independent experiments. Two-tailed Mann–Whitney test. Median ± range. f Quantification of VEGFR3 intensity in the OF of control and Dmp1i∆OB mice. Control n = 6, Dmp1i∆OB n = 9. Pooled data from three independent experiments. Two-tailed Mann–Whitney test. Median ± range. g, h VEGFR2 and EMCN (g) and VEGFR3 and EMCN (h) immunostaining of the OF centre in 6-week-old control and Dmp1i∆OB mice. Scale bar 50 µm. i pVEGFR2 immunostaining of bEnd.3 cells, which were either treated with VEGF only (left) or pre-treated with DMP1 (40 nM) for 3 h before adding VEGF (50 ng/ml). Scale bar 25 µm. j Quantification of the pVEGFR2 intensity per cell of bEnd.3 cells, which were either treated with VEGF only or pre-treated with DMP1 before adding VEGF. Two-tailed Mann–Whitney test. Mean. k qPCR of c-fos expression, one of the target genes of VEGF signalling, in bEnd.3 cells, which were treated either with DMP1 only, or with VEGF only, or first with DMP1 and then with VEGF. Tukey’s multiple comparisons test. Mean + SD. i–k Representative plots of 2 independent experiments. a–c, e, f, j, k Source data are provided in Source Data file.