Journal: Cancers

Article Title: Ciliary Neurotrophic Factor Modulates Multiple Downstream Signaling Pathways in Prostate Cancer Inhibiting Cell Invasiveness

doi: 10.3390/cancers14235917

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: pAb Rabbit anti-human GLUT4 (#PA5-23052) , // , 1:500 , // , Thermo Fisher Scientific, Waltham, MA, USA.

Techniques:

Journal: Nature Communications

Article Title: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

doi: 10.1038/s41467-024-52965-4

Figure Lengend Snippet: a , b Osteogenic activity of MC3T3-E1 osteoblastic cells with 11β-HSD1 overexpression (MC3T3-HSD1 cells) and transfected control cells (MC3T3-GFP cells). a Alkaline phosphatase (ALP) and alizarin red staining. b The mRNA expression of RUNX family transcription factor 2 ( Runx2 ) and bone gamma-carboxyglutamate protein ( Bglap ). c , d Combination analysis of RNA sequencing (RNA-seq) and assays for transposase-accessible chromatin sequencing (ATAC-seq) in MC3T3-GFP cells and MC3T3-HSD1 cells. c Intersection of differential genes from RNA-seq and ATAC-seq. d Biological processes associated with Egr2. e The mRNA and protein expression of Egr2 in MC3T3-GFP cells and MC3T3-HSD1 cells. f The mRNA expression of Egr2 during osteogenic differentiation. g , h The osteogenic activity of MC3T3-HSD1 cells with Egr2 overexpressing. g Alizarin red staining. h Bglap mRNA expression. i The glucose uptake test of MC3T3-GFP cells and MC3T3-HSD1 cells. j , k The mRNA expression of key glucose transporter proteins and components of insulin-dependent glucose uptake pathway in MC3T3-GFP cells and MC3T3-HSD1 cells. j Glucose transporter type 1 (Glut1) , 3 (Glut3) and 4 (Glut4) . k: Insulin receptor (Ir), Insulin receptor substrate 1 (Irs1), Phosphatidylinositol-3-kinase catalytic subunit regulatory subunit 1 (Pik3r), alpha (Pik3ca) and subunit beta (Pik3cb) . l , m The glucose uptake test and mRNA expression of Glut4 and Pik3cb in MC3T3-HSD1 cells with Egr2 overexpressing. l Glucose uptake test. m Glut4 and Pik3cb . n Dual-luciferase reporter analysis and ChIP quantitative polymerase chain reaction (ChIP-qPCR) analysis of Ege2 target interactions for Pik3cb and Glut4 gene promotors. n Pik3cb and Glut4 gene promotors. o ChIP-qPCR analysis. p Pik3cb and Glut4 gene promotors with different mutations. Note: Data were presented as mean value ± SEM for ( a , b , e – m , p ). n = 3 biologically independent samples for RNA-seq, ATAC-seq, western blot analysis, glucose uptake test and osteogenic staining; n = 6 biologically independent samples for RT-qPCR analysis. Statistical significance was calculated using two-tailed Student’s t-test ( e ), one-way ANOVA followed by Tukey’s post-hoc test ( n , p ), and two-way ANOVA followed by a two-stage step-up method by Benjamini, Krieger and Yekutieli ( a , b , f – m ) to adjust for multiple comparisons. All tests were two-sided.

Article Snippet: Following blocking with 5% nonfat milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies (1:500) against Actin (4967, Cell Signaling, USA), p110β (ab151549, abcam, UK), AKT (4691 T, Cell Signaling, USA), phosphorylated AKT (4060 T, pAKT, Cell Signaling, USA), Glut4 (21619, SAB, USA), 11β-HSD1 (AF3397, R&D system, USA), Egr2 (EPR4004, Abcam, UK), H6PDH (EPR12338, Abcam, UK).

Techniques: Activity Assay, Over Expression, Transfection, Control, Staining, Expressing, RNA Sequencing, Sequencing, Luciferase, Real-time Polymerase Chain Reaction, ChIP-qPCR, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

doi: 10.1038/s41467-024-52965-4

Figure Lengend Snippet: a The mRNA expression of glucocorticoid target gene Glucocorticoid-induced leucine zipper ( Gilz ) in MC3T3-GFP cells and MC3T3-HSD1 cells after treating with 11β-HSD1 inhibitor (AZD8329). b , c The mRNA and protein expression of early growth response 2 ( Egr2 ) in MC3T3-GFP cells and MC3T3-HSD1 cells after treatment of AZD8329. b The mRNA expression. c The protein expression. d The mRNA expression of Egr2 in MC3T3-GFP cells and MC3T3-HSD1 cells after treating with AZD8329 during osteogenic differentiation. e – h The osteogenic activity of MC3T3-GFP cells and MC3T3-HSD1 cells after treatment of AZD8329. e Alizarin red staining. f Bone gamma-carboxyglutamate protein (Bglap) mRNA expression. g Runt-related transcription factor 2 (Runx2) mRNA expression. h Osterix mRNA expression. i – j The glucose uptake test and mRNA expression levels of Glucose transporter type 4 (Glut4) and Phosphatidylinositol-3-kinase catalytic subunit beta (Pik3cb) in MC3T3-GFP cells and MC3T3-HSD1 cells after treatment of AZD8329. g: Glucose uptake test. h Glut4 and Pik3cb mRNA expression. Note: Data were presented as mean value ± SEM for ( a , b , d – j ). n = 3 biologically independent samples for osteogenic staining, western blot analysis and LC-MS/MS analysis, n = 6 biologically independent samples for RT-qPCR analysis. Statistical significance was calculated using one-way ANOVA followed by Tukey’s post-hoc test ( a – c ), and two-way ANOVA followed by a two-stage step-up method by Benjamini, Krieger and Yekutieli ( d – j ) to adjust for multiple comparisons. All tests were two-sided.

Article Snippet: Following blocking with 5% nonfat milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies (1:500) against Actin (4967, Cell Signaling, USA), p110β (ab151549, abcam, UK), AKT (4691 T, Cell Signaling, USA), phosphorylated AKT (4060 T, pAKT, Cell Signaling, USA), Glut4 (21619, SAB, USA), 11β-HSD1 (AF3397, R&D system, USA), Egr2 (EPR4004, Abcam, UK), H6PDH (EPR12338, Abcam, UK).

Techniques: Expressing, Activity Assay, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

doi: 10.1038/s41467-024-52965-4

Figure Lengend Snippet: a Design of the bone-targeted 11β-HSD1 inhibitor, (DSS) 6 -AZD8329. b , c Organ distribution and skeletal distribution of (DSS) 6 -AZD8329 ( n = 3) and (RKK) 6 -AZD8329 ( n = 3). b The organ distribution. c The skeletal distribution, scale bar=20 μm. d Experimental design of wild-type mice treated with (DSS) 6 -AZD8329 and (RKK) 6 -AZD8329. e – g The micro-CT analysis and bone histometric analysis. e The trabecular bone and cortical bone microstructure. f The percentage change of trabecular bone volume/total volume (Tb. BV/TV) and trabecular bone density (Tb. v. BMD). g The percentage change of cortical bone density (Ct. v. BMD). h The calcein double labeling, scale bar=20 μm. i The percentage change of mineral apposition rate (MAR), bone formation rate per bone surface (BFR/BS) and the number of osteoblasts per bone surface (N.Ob/BS). j – k Weight gain and energy intake during HFD. * P < 0.05, ** P < 0.01, *** P < 0.001 when other groups vs. HFD+vehicle group. j Weight gain. k Energy intake. l Weights and representative photographs of gonadal white adipose tissues (gWAT). m – o Glucose handling tests ( n = 8 for two vehicle-treated groups, n = 12 for two drug-treated groups). * P < 0.05, ** P < 0.01, *** P < 0.001 when other groups vs. HFD + vehicle group. m Fasting blood glucose. n: Insulin tolerance test (ITT). o Oral glucose tolerance test (oGTT). p Glucose uptake tests ( n = 8 for two vehicle-treated groups, n = 12 for two drug-treated groups). Left: Representative images of glucose uptake. Right: Quantitative analysis of glucose uptake into liver, WAT, muscle and bone. q Skeletal mRNA expression of Hsd11b1 , Gilz , Egr2 , Pik3cb and Glut4 . Note: Data were presented as mean value ± SEM for ( f , g , i – q ). Chow+vehicle ( n = 8), mice with Chow feeding and start administration of vehicle since week 8; HFD+vehicle ( n = 8), HFD + (RKK) 6 -AZD8329 ( n = 13), HFD + (DSS) 6 -AZD8329 ( n = 13), mice with HFD feeding and start administration of vehicle, (RKK) 6 -AZD8329, or (DSS) 6 -AZD8329, respectively, since week 8. All samples are biologically independent samples. Statistical significance was calculated using one-way ANOVA followed by Tukey’s post-hoc test ( f – g , i , l , m – q ) and two-way ANOVA followed by Sidak’s multiple comparisons test ( j – k , n – o ). All tests were two-sided.

Article Snippet: Following blocking with 5% nonfat milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies (1:500) against Actin (4967, Cell Signaling, USA), p110β (ab151549, abcam, UK), AKT (4691 T, Cell Signaling, USA), phosphorylated AKT (4060 T, pAKT, Cell Signaling, USA), Glut4 (21619, SAB, USA), 11β-HSD1 (AF3397, R&D system, USA), Egr2 (EPR4004, Abcam, UK), H6PDH (EPR12338, Abcam, UK).

Techniques: Micro-CT, Labeling, Expressing