Fig. 5

IGF-1/PI3K signaling is activated in the resting zone. a Schematic diagram for transcriptome analysis using laser microdissection and RNA sequencing. P30 tibial and femoral growth plates in Axin2Cre^ERT2;R26R^TdTomato mice (pulsed on P23–25) were subjected to laser microdissection. b KEGG pathways enriched by genes significantly upregulated in the resting zone compared to the proliferative zone. The red arrow indicates the PI3-Akt signaling pathway. Logarithmic P values of significance are indicated on the x-axis. c Representative image of immunohistochemistry for p-Akt in the proximal tibial growth plate at P28. The right three panels show magnified views of each zone of the growth plate. d CPM values of Igf-1 in the resting and proliferative zone samples. RZ (n = 4), PZ (n = 4). **P = 0.004 1. e Representative image for in situ hybridization of Igf-1 in the proximal tibial growth plate at P28. The right panel shows a magnified view of the resting zone of the growth plate. f, g Representative image of immunohistochemistry for p-Akt in the resting zone of the proximal-tibial growth plate at P28 after receiving either vehicle (dimethyl sulfoxide/corn oil, 9:1) (f) or IGF-1 receptor tyrosine kinase inhibitor (PPP, 20 μg·g⁻¹ body weight) (g) 30 min before euthanasia. h Percentage of p-Akt⁺ cells in the top 50 μm. Vehicle group (n = 6), PPP group (n = 6). *P = 0.043 5. p-AKT phosphorylation of the protein kinase Akt, SOC secondary ossification, RZ resting zone, PZ proliferative zone, HZ hypertrophic zone, CPM count per million, PPP picropodophyllin. The white dashed lines demarcate the growth plate from the surrounding tissues. Scale bars: 200 μm (c [left-most], e [left]), 100 µm (e [right]), 20 µm (c [right three], f, g). All data are presented as the mean ± SD. Statistical significance was determined by unpaired two-tailed t test