Journal: Bone Research
Article Title: Inhibition of sympathetic tone via hypothalamic descending pathway propagates glucocorticoid-induced endothelial impairment and osteonecrosis of the femoral head
doi: 10.1038/s41413-024-00371-3
Figure Lengend Snippet: Fig. 6 Sympathetic nerves stimulates endothelial alternation of glycolysis. a Schematic graph of RNA-seq analysis of the sorted femoral head ECs from vehicle- or MPS-treated mice following Adrb2 agonist (clenbuterol) treatment every other day for 1 week. b Heatmap of RNA-seq data showed expression changes encoding glucose metabolism-related genes of the sorted femoral head ECs from vehicle- or MPS-treated mice following Adrb2 agonist (clenbuterol) treatment every other day for 1 week. c Quantitative RT-PCR analysis of Pfkfb3, Pfkl, Pfkp. Eno, and Hk1 genes expression in femoral head ECs from vehicle- or MPS-treated mice following Adrb2 agonist (clenbuterol) treatment every other day for 1 week. d Quantitative RT-PCR analysis of Pfkfb3 gene expression for ECs treated with vehicle, NE, E, NPY, and DA at the concentration of 0.1 μmol/L, respectively. e Quantitative RT-PCR analysis of Pfkfb3 gene expression for femoral head ECs treated with NE for 0–6 h respectively. f, g Representative images of WB and quantitative analysis of PFKFB3 expression for femoral head ECs treated with NE for 0–6 h respectively. h Quantitative RT-PCR analysis of Pfkfb3 gene expression for femoral head ECs treated with vehicle, NE, MPS, or MPS + NE. i, j Representative images of WB and quantitative analysis of PFKFB3 expression for femoral head ECs treated with vehicle, NE, MPS, or MPS + NE. k, l Representative tube formation images and quantification of total loops and total tube length of femoral head ECs under different treatments as indicated. Scale bar: 100 μm. m, n ECAR profile showing glycolytic function and quantification of glycolytic function parameters for femoral head ECs under different treatments as indicated. Vertical lines indicate the time of addition of glucose (10 mmol/L), oligomycin (1 μmol/L), and 2-DG (50 mmol/L). o−s Measurement of glucose uptake, extracellular and intracellular lactate levels, and intracellular G-6-P as well as pyruvate levels for femoral head ECs under different treatments as indicated. t Quantitative analysis of ELISA assay for cAMP in femoral head ECs in response to different treatments as indicated. u Quantitative analysis of PKA activity assay for cell homogenates of femoral head ECs receiving different treatments as indicated. v, w Representative images of WB and quantitative analysis of pCREB/CREB expression for femoral head ECs under different treatments as indicated. All data were presented as means ± SD, n = 6 per group; *P < 0.05. **P < 0.01. ***P < 0.001. Statistical significance was determined by two-tailed Student’s t-test (c). Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test (d, e, g, h, j, n−u, w). Statistical significance was determined by two-way ANOVA with Bonferroni post hoc test (l)
Article Snippet: ChIP-grade antibodies against CREB (1:50, 9198, Cell Signaling Technology), rabbit IgG (negative control), or histone H3 were used to immunoprecipitate the chromatin fragments, which were then incubated with ChIP-grade protein G magnetic beads at 4 °C for 2 h. The eluted DNA was purified and analyzed using qRT-PCR to evaluate the degree of enrichment of the region of the Pfkfb3 gene promoter after rinsing.
Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Gene Expression, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Two Tailed Test
