adrb2 sirna  (Santa Cruz Biotechnology)

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: Activation of Adrb2 prevents the onset of osteonecrosis with sympathetic denervation. a , b Representative immunofluorescence co-staining of CD31 (green), EMCN (red), and TH (white) and quantitative analysis for TH + sympathetic nerves and TH + H-type vessels in the femoral heads from vehicle-treated healthy control mice and MPS-induced ONFH mice. Scale bar: 50 μm. c Quantitative RT-PCR analysis of all adrenergic receptors from ECs in the femoral heads of wildtype mice. d , e Quantitative RT-PCR analysis for Adrb2 and Adra2a expression from H-type and L-type ECs in the femoral heads of wildtype mice. f , g FACS analysis of the femoral heads single-cell suspensions co-stained with CD31 and EMCN and quantitative analysis of CD31 hi EMCN hi cells in wildtype mice treated with vehicle, Adra2a agonist (clonidine), or Adrb2 agonist (clenbuterol). h , i Representative images of WB for Adrb2 expression and quantitative RT-PCR analysis for Adrb2 gene expression in the femoral heads from humans and mice. j Experimental design graph for validating the effects of Adrb2 activation on the femoral heads in MPS-treated mice after 6-OHDA-induced sympathetic denervation. Mice were harvested 6 weeks after the first injection of MPS. One down arrow (grey) represented one time vehicle or MPS treatment, and one down arrow (black) represented one time vehicle or 6-OHDA treatment. j − p MPS-treated mice were grouped as vehicle and 6-OHDA treatment and their littermates were treated with vehicle or clenbuterol. k, l μCT reconstruction images and quantitative analysis of Tb. BV/TV, Tb. Th, Tb. N and Tb. Sp of femoral heads. Scale bar: 1 mm. m H&E staining images of femoral heads. Scale bar: 200 μm. Black arrows indicate empty osteocytic lacunae. n Representative images of immunofluorescence staining of OCN (green) and co-staining of CD31 (green) with EMCN (red) ( o, p ) and quantitative analysis of the number of OCN + osteoblasts and CD31 + EMCN + cells in the femoral heads. Scale bar: 50 μm. 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 ( b, d, e, i ). Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test ( g ). Statistical significance was determined by two-way ANOVA with Bonferroni post hoc test ( l, o, p )

Article Snippet: Mouse Adrb2 siRNA (si- Adrb2 ) and negative control siRNA (si -Control ) were acquired from RiboBio Company (Guangzhou, China).

Techniques: Activation Assay, Immunofluorescence, Staining, Control, Quantitative RT-PCR, Expressing, Injection, Two Tailed Test

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: Knockout of Adrb2 abolishes the bone-protective effects of sympathetic outflow and accelerate ONFH in GC-treated mice. a Experimental design graph for investigating the effects of Adrb2 deletion on the femoral heads in MPS-treated mice receiving vehicle or RU486 treatment in PVN. Mice were harvested 6 weeks after the first injection of MPS. One down arrow (grey) represented one time treatment with vehicle or MPS. b, c Representative images of immunofluorescence staining of Adrb2 (red) and quantitative analysis of Adrb2 + cells in the femoral heads from Adrb2 WT and Adrb2 −/− mice. Scale bar: 200 μm. d Quantitative RT-PCR analysis for Adrb2 gene expression from ECs in the femoral heads of Adrb2 WT and Adrb2 −/− mice. e − j MPS-treated Adrb2 WT and Adrb2 −/− mice were grouped as vehicle or RU486 treatment in PVN. e , f μCT reconstruction images and quantitative analysis of Tb. BV/TV, Tb. Th, Tb. N and Tb. Sp of femoral heads. Scale bar: 1 mm. g H&E staining images of femoral heads. Scale bar: 200 μm. Black arrows indicate empty osteocytic lacunae. h Representative images of immunofluorescence staining of OCN (green) and co-staining of CD31 (green) with EMCN (red) ( i , j ) and quantitative analysis of the number of OCN + osteoblasts and CD31 + EMCN + cells in the femoral heads. Scale bar: 50 μm. 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, d ). Statistical significance was determined by two-way ANOVA with Bonferroni post hoc test ( f, i, j )

Article Snippet: Mouse Adrb2 siRNA (si- Adrb2 ) and negative control siRNA (si -Control ) were acquired from RiboBio Company (Guangzhou, China).

Techniques: Knock-Out, Injection, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Two Tailed Test

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: 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: Mouse Adrb2 siRNA (si- Adrb2 ) and negative control siRNA (si -Control ) were acquired from RiboBio Company (Guangzhou, China).

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Two Tailed Test

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: Sympathetic neurotransmitter NE-induced endothelial metabolic alternation and angiogenesis-osteogenesis coupling are mediated by Adrb2/cAMP/CREB/Pfkfb3 signaling. a − c Representative images of WB and quantitative analysis of PFKFB3 and pCREB/CREB expression for MPS-treated femoral head ECs under vehicle or NE treatment after knockdown of Adrb2 by si- Adrb2 or CREB inhibition by 666-15. d, e ECAR profile showed glycolytic function and quantification of glycolytic function parameters in MPS-treated 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). f − j 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. k, l Quantitative RT-PCR analysis of pro-angiogenic ( Vegfa, Vegfc, Tgfa, and Tgfb2 ) and pro-osteogenic ( Bmp2, Noggin, and Ptn ) genes expression for vehicle or 666-15-treated femoral head ECs with MPS and NE cotreatment following activation of cAMP by db-cAMP or Pfkfb3 overexpression by Ad- Pfkfb3 . m, n Representative tube formation images and quantification of total loops and total tube length for femoral head ECs under different treatments as indicated. Scale bar: 100 μm. o, p Representative images of ARS staining and quantification of the positively stained areas in BMSCs after EC-CM treatment. Scale bar: 50 μm. q, r ChIP-qRT-PCR analysis revealed CREB antibody (CREB Ab) immune-precipitate Pfkfb3 promoter domain enrichment relative to the input DNA in femoral head ECs under vehicle or NE treatment. Normal rabbit anti-IgG acted as a negative control. Histone H3 antibody (Histone H3 Ab) pulldown for Rpl30 gene enrichment acted as a positive control. 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-way ANOVA with Bonferroni post hoc test ( b, c, e − l, n, p ). Statistical significance was determined by two-tailed Student’s t -test ( r )

Article Snippet: Mouse Adrb2 siRNA (si- Adrb2 ) and negative control siRNA (si -Control ) were acquired from RiboBio Company (Guangzhou, China).

Techniques: Expressing, Knockdown, Inhibition, Quantitative RT-PCR, Activation Assay, Over Expression, Staining, Negative Control, Positive Control, Two Tailed Test

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: Overexpression of endothelial Pfkfb3 attenuates ONFH in GC-treated Adrb2 WT and Adrb2 −/− mice. a ECs were isolated from the femoral heads of Adrb2 WT and Adrb2 −/− mice following adeno-associated viral (AAV)- Control or AAV- Pfkfb3 transfection for 1 month. Schematic diagram showing the procedure of flow cytometry for apoptosis and tube formation assays. b , c Representative images and quantification of flow cytometry analysis of Annexin V/PI staining in femoral head ECs from different mice as indicated. d , e Representative tube formation images and quantification of total loops and total tube length of femoral head ECs from different mice as indicated. Scale bar: 100 μm. f Experimental design graph for exploring the effects of endothelial PFKFB3 overexpression on the femoral heads in MPS-treated Adrb2 WT and Adrb2 −/− mice. Mice were harvested 3 days after MPS daily injection or 6 weeks after the first injection of MPS. One down arrow (grey) represented one time vehicle or MPS treatment, and brown arrow represented AAV transfection. g − n MPS-treated Adrb2 WT and Adrb2 −/− mice were grouped as AAV- Control and AAV- Pfkfb3 transfection. g, h Representative immunofluorescence co-staining of EMCN (red) and c-Caspase-3 (white) and quantitative analysis for vessels expressing both EMCN and c-Caspase-3 in the femoral heads of 3-days MPS-treated Adrb2 WT and Adrb2 −/− mice and their littermates were received AAV- Control and AAV- Pfkfb3 transfection. Scale bar: 50 μm. i, j μCT reconstruction images and quantitative analysis of Tb. BV/TV, Tb. Th, Tb. N and Tb. Sp of femoral heads. Scale bar: 1 mm. k H&E staining images of femoral heads. Scale bar: 200 μm. Black arrows indicate empty osteocytic lacunae. l Representative images of immunofluorescence staining of OCN (green) and co-staining of CD31 (green) with EMCN (red) ( m, n ) and quantitative analysis of the number of OCN + osteoblasts and CD31 + EMCN + cells in the femoral heads. Scale bar: 50 μm. 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-way ANOVA with Bonferroni post hoc test

Article Snippet: Mouse Adrb2 siRNA (si- Adrb2 ) and negative control siRNA (si -Control ) were acquired from RiboBio Company (Guangzhou, China).

Techniques: Over Expression, Isolation, Control, Transfection, Flow Cytometry, Staining, Injection, Immunofluorescence, Expressing

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: Activation of Adrb2 prevents the onset of osteonecrosis with sympathetic denervation. a , b Representative immunofluorescence co-staining of CD31 (green), EMCN (red), and TH (white) and quantitative analysis for TH + sympathetic nerves and TH + H-type vessels in the femoral heads from vehicle-treated healthy control mice and MPS-induced ONFH mice. Scale bar: 50 μm. c Quantitative RT-PCR analysis of all adrenergic receptors from ECs in the femoral heads of wildtype mice. d , e Quantitative RT-PCR analysis for Adrb2 and Adra2a expression from H-type and L-type ECs in the femoral heads of wildtype mice. f , g FACS analysis of the femoral heads single-cell suspensions co-stained with CD31 and EMCN and quantitative analysis of CD31 hi EMCN hi cells in wildtype mice treated with vehicle, Adra2a agonist (clonidine), or Adrb2 agonist (clenbuterol). h , i Representative images of WB for Adrb2 expression and quantitative RT-PCR analysis for Adrb2 gene expression in the femoral heads from humans and mice. j Experimental design graph for validating the effects of Adrb2 activation on the femoral heads in MPS-treated mice after 6-OHDA-induced sympathetic denervation. Mice were harvested 6 weeks after the first injection of MPS. One down arrow (grey) represented one time vehicle or MPS treatment, and one down arrow (black) represented one time vehicle or 6-OHDA treatment. j − p MPS-treated mice were grouped as vehicle and 6-OHDA treatment and their littermates were treated with vehicle or clenbuterol. k, l μCT reconstruction images and quantitative analysis of Tb. BV/TV, Tb. Th, Tb. N and Tb. Sp of femoral heads. Scale bar: 1 mm. m H&E staining images of femoral heads. Scale bar: 200 μm. Black arrows indicate empty osteocytic lacunae. n Representative images of immunofluorescence staining of OCN (green) and co-staining of CD31 (green) with EMCN (red) ( o, p ) and quantitative analysis of the number of OCN + osteoblasts and CD31 + EMCN + cells in the femoral heads. Scale bar: 50 μm. 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 ( b, d, e, i ). Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test ( g ). Statistical significance was determined by two-way ANOVA with Bonferroni post hoc test ( l, o, p )

Article Snippet: In brief, femoral head ECs were seeded in 6-well plates, cultured for 24 h, and then transfected with Adrb2 siRNA (100 nmol/L) or negative control siRNA using the RiboFECT CP Transfection Kit (C10511, RiboBio) according to the manufacturer’s protocols.

Techniques: Activation Assay, Immunofluorescence, Staining, Control, Quantitative RT-PCR, Expressing, Gene Expression, Injection, Two Tailed Test

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: Knockout of Adrb2 abolishes the bone-protective effects of sympathetic outflow and accelerate ONFH in GC-treated mice. a Experimental design graph for investigating the effects of Adrb2 deletion on the femoral heads in MPS-treated mice receiving vehicle or RU486 treatment in PVN. Mice were harvested 6 weeks after the first injection of MPS. One down arrow (grey) represented one time treatment with vehicle or MPS. b, c Representative images of immunofluorescence staining of Adrb2 (red) and quantitative analysis of Adrb2 + cells in the femoral heads from Adrb2 WT and Adrb2 −/− mice. Scale bar: 200 μm. d Quantitative RT-PCR analysis for Adrb2 gene expression from ECs in the femoral heads of Adrb2 WT and Adrb2 −/− mice. e − j MPS-treated Adrb2 WT and Adrb2 −/− mice were grouped as vehicle or RU486 treatment in PVN. e , f μCT reconstruction images and quantitative analysis of Tb. BV/TV, Tb. Th, Tb. N and Tb. Sp of femoral heads. Scale bar: 1 mm. g H&E staining images of femoral heads. Scale bar: 200 μm. Black arrows indicate empty osteocytic lacunae. h Representative images of immunofluorescence staining of OCN (green) and co-staining of CD31 (green) with EMCN (red) ( i , j ) and quantitative analysis of the number of OCN + osteoblasts and CD31 + EMCN + cells in the femoral heads. Scale bar: 50 μm. 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, d ). Statistical significance was determined by two-way ANOVA with Bonferroni post hoc test ( f, i, j )

Article Snippet: In brief, femoral head ECs were seeded in 6-well plates, cultured for 24 h, and then transfected with Adrb2 siRNA (100 nmol/L) or negative control siRNA using the RiboFECT CP Transfection Kit (C10511, RiboBio) according to the manufacturer’s protocols.

Techniques: Knock-Out, Injection, Immunofluorescence, Staining, Quantitative RT-PCR, Gene Expression, Two Tailed Test

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: 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: In brief, femoral head ECs were seeded in 6-well plates, cultured for 24 h, and then transfected with Adrb2 siRNA (100 nmol/L) or negative control siRNA using the RiboFECT CP Transfection Kit (C10511, RiboBio) according to the manufacturer’s protocols.

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Gene Expression, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Two Tailed Test

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: Sympathetic neurotransmitter NE-induced endothelial metabolic alternation and angiogenesis-osteogenesis coupling are mediated by Adrb2/cAMP/CREB/Pfkfb3 signaling. a − c Representative images of WB and quantitative analysis of PFKFB3 and pCREB/CREB expression for MPS-treated femoral head ECs under vehicle or NE treatment after knockdown of Adrb2 by si- Adrb2 or CREB inhibition by 666-15. d, e ECAR profile showed glycolytic function and quantification of glycolytic function parameters in MPS-treated 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). f − j 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. k, l Quantitative RT-PCR analysis of pro-angiogenic ( Vegfa, Vegfc, Tgfa, and Tgfb2 ) and pro-osteogenic ( Bmp2, Noggin, and Ptn ) genes expression for vehicle or 666-15-treated femoral head ECs with MPS and NE cotreatment following activation of cAMP by db-cAMP or Pfkfb3 overexpression by Ad- Pfkfb3 . m, n Representative tube formation images and quantification of total loops and total tube length for femoral head ECs under different treatments as indicated. Scale bar: 100 μm. o, p Representative images of ARS staining and quantification of the positively stained areas in BMSCs after EC-CM treatment. Scale bar: 50 μm. q, r ChIP-qRT-PCR analysis revealed CREB antibody (CREB Ab) immune-precipitate Pfkfb3 promoter domain enrichment relative to the input DNA in femoral head ECs under vehicle or NE treatment. Normal rabbit anti-IgG acted as a negative control. Histone H3 antibody (Histone H3 Ab) pulldown for Rpl30 gene enrichment acted as a positive control. 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-way ANOVA with Bonferroni post hoc test ( b, c, e − l, n, p ). Statistical significance was determined by two-tailed Student’s t -test ( r )

Article Snippet: In brief, femoral head ECs were seeded in 6-well plates, cultured for 24 h, and then transfected with Adrb2 siRNA (100 nmol/L) or negative control siRNA using the RiboFECT CP Transfection Kit (C10511, RiboBio) according to the manufacturer’s protocols.

Techniques: Expressing, Knockdown, Inhibition, Quantitative RT-PCR, Activation Assay, Over Expression, Staining, Negative Control, Positive Control, Two Tailed Test

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: Overexpression of endothelial Pfkfb3 attenuates ONFH in GC-treated Adrb2 WT and Adrb2 −/− mice. a ECs were isolated from the femoral heads of Adrb2 WT and Adrb2 −/− mice following adeno-associated viral (AAV)- Control or AAV- Pfkfb3 transfection for 1 month. Schematic diagram showing the procedure of flow cytometry for apoptosis and tube formation assays. b , c Representative images and quantification of flow cytometry analysis of Annexin V/PI staining in femoral head ECs from different mice as indicated. d , e Representative tube formation images and quantification of total loops and total tube length of femoral head ECs from different mice as indicated. Scale bar: 100 μm. f Experimental design graph for exploring the effects of endothelial PFKFB3 overexpression on the femoral heads in MPS-treated Adrb2 WT and Adrb2 −/− mice. Mice were harvested 3 days after MPS daily injection or 6 weeks after the first injection of MPS. One down arrow (grey) represented one time vehicle or MPS treatment, and brown arrow represented AAV transfection. g − n MPS-treated Adrb2 WT and Adrb2 −/− mice were grouped as AAV- Control and AAV- Pfkfb3 transfection. g, h Representative immunofluorescence co-staining of EMCN (red) and c-Caspase-3 (white) and quantitative analysis for vessels expressing both EMCN and c-Caspase-3 in the femoral heads of 3-days MPS-treated Adrb2 WT and Adrb2 −/− mice and their littermates were received AAV- Control and AAV- Pfkfb3 transfection. Scale bar: 50 μm. i, j μCT reconstruction images and quantitative analysis of Tb. BV/TV, Tb. Th, Tb. N and Tb. Sp of femoral heads. Scale bar: 1 mm. k H&E staining images of femoral heads. Scale bar: 200 μm. Black arrows indicate empty osteocytic lacunae. l Representative images of immunofluorescence staining of OCN (green) and co-staining of CD31 (green) with EMCN (red) ( m, n ) and quantitative analysis of the number of OCN + osteoblasts and CD31 + EMCN + cells in the femoral heads. Scale bar: 50 μm. 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-way ANOVA with Bonferroni post hoc test

Article Snippet: In brief, femoral head ECs were seeded in 6-well plates, cultured for 24 h, and then transfected with Adrb2 siRNA (100 nmol/L) or negative control siRNA using the RiboFECT CP Transfection Kit (C10511, RiboBio) according to the manufacturer’s protocols.

Techniques: Over Expression, Isolation, Control, Transfection, Flow Cytometry, Staining, Injection, Immunofluorescence, Expressing

Journal: Pflugers Archiv

Article Title: Increased β-adrenergic stimulation augments vascular smooth muscle cell calcification via PKA/CREB signalling

doi: 10.1007/s00424-021-02621-3

Figure Lengend Snippet: Knockdown or blockade of the β2-adrenergic receptor blunts the pro-calcific effects of isoproterenol in HAoSMCs. a Scatter dot plots and arithmetic means ± SEM ( n = 4; arbitrary units, a.u.) of β-adrenergic receptors ADRB1 , ADRB2 , and ADRB3 relative mRNA expression in HAoSMCs. b Scatter dot plots and arithmetic means ± SEM ( n = 6; a.u.) of ADRB2 relative mRNA expression in HAoSMCs following transfection with negative control (Negsi) or ADRB2 (ADRB2si) siRNA and treatment for 24 h with control or 1 μM isoproterenol (ISO). *( p < 0.05) statistically significant vs Negsi-transfected HAoSMCs. c , d Scatter dot plots and arithmetic means ± SEM ( n = 6; a.u.) of CBFA1 ( c ) and ALPL ( d ) relative mRNA expression in HAoSMCs following transfection with negative control (Negsi) or ADRB2 (ADRB2si) siRNA and treatment for 24 h with control or 1 μM isoproterenol (ISO). *( p < 0.05), ***( p < 0.001) statistically significant vs Negsi-transfected HAoSMCs; †( p < 0.05), †††( p < 0.001) statistically significant vs Negsi-transfected ISO-treated HAoSMCs. e , f Scatter dot plots and arithmetic means ± SEM (n = 10; a.u.) of CBFA1 ( e ) and ALPL ( f ) relative mRNA expression in HAoSMCs following treatment for 24 h with control (CTR) or 1 μM isoproterenol (ISO) without and with 1 μM β2-adrenergic receptor antagonist ICI 118,551 (ICI). g Scatter dot plots and arithmetic means ± SEM ( n = 8; a.u.) of normalized calcium content in HAoSMCs following treatment for 11 days with control (CTR) or 1 μM isoproterenol (ISO) and calcification medium (Calc.; 10 mM β-glycerophosphate + 1.5 mM CaCl 2 ) without and with 1 μM β2-adrenergic receptor antagonist ICI 118,551 (ICI). *( p < 0.05), **( p < 0.01), ***( p < 0.001) statistically significant vs control HAoSMCs; ††( p < 0.01), statistically significant vs ISO-/Calc. + ISO-treated HAoSMCs

Article Snippet: Where indicated, HAoSMCs were transfected with 10 nM PRKACA siRNA (ID:s11066), 10 nM CREB1 siRNA (ID:s3490), 10 nM ADRB2 siRNA (ID:s1122), or 10 nM negative control siRNA (ID:4390843) by using siPORT amine transfection agent (all from Fisher Scientific) [ ].

Techniques: Knockdown, Expressing, Transfection, Negative Control, Control