Journal: Advanced Science

Article Title: HSP90AB1‐Mediated Ubiquitin‐Proteasome Degradation of ITGBL1 Promotes Osteosarcoma Progression by Inhibiting Endoplasmic Reticulum Stress‐Induced Autophagy

doi: 10.1002/advs.202515651

Figure Lengend Snippet: ITGBL1 is downregulated in OS, and low expression of ITGBL1 is related to poor prognosis in OS patients. (A,B) The mRNA and protein expression levels of ITGBL1 in OS tissues and their normal bone tissues ( n = 18 pairs). (C,D) The mRNA and protein expression levels of ITGBL1 in the indicated cell lines. (E) The mRNA expression level of ITGBL1 in normal osteoblast and OS tissues. Data from GSE33382 . (F) The mRNA expression level of ITGBL1 in bone tissues and OS tissues. Data from GSE19276 . (G,H) The volcano map and heatmap show the differentially expressed genes between primary tumors of with or without lung metastasis (log FC > 2 and P.adj < 0.05). Data from the TARGET database. (I) The mRNA expression level of ITGBL1 in primary tumors of OS patients with or without lung metastasis. Data from the TARGET database. (J) IHC staining of ITGBL1 in primary tumor tissues of OS patients with ( n = 12) and without lung metastasis ( n = 6). Scale bar = 50 µm. (K) Kaplan–Meier analyses of the overall survival rate and disease‐free survival rate of OS patients with high and low expression of ITGBL1. All in vitro experiments were conducted three independent experiments. All data are presented as the mean ± SD, and differences between groups were assessed by Student's t ‐test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To detect the ubiquitination of endogenous ITGBL1, OS cells were treated with 20 μ m MG132 (MCE, USA) for 9 h to inhibit proteasome‐mediated degradation.

Techniques: Expressing, Immunohistochemistry, In Vitro

Journal: Advanced Science

Article Title: HSP90AB1‐Mediated Ubiquitin‐Proteasome Degradation of ITGBL1 Promotes Osteosarcoma Progression by Inhibiting Endoplasmic Reticulum Stress‐Induced Autophagy

doi: 10.1002/advs.202515651

Figure Lengend Snippet: Downregulation of ITGBL1 promotes OS progression and stemness, while inhibiting apoptosis of OS cells. (A) OS cell viability was measured by the CCK8 assay. (B) OS cell migration was measured by the wound healing assay. Scale bar = 100 µm. (C) OS cell migration and invasion were measured by the Transwell assay. Scale bar = 100 µm. (D) The stemness of OS cells was measured by sphere formation assay, and the spheres were stained with Calcein‐AM and imaged. Scale bar = 50 µm. (E) The expression of stemness‐related proteins was detected by immunofluorescence in spheres. Scale bar = 50 µm. (F) The apoptotic cells were detected in 143B and HOS cells by TUNEL assay. Scale bar = 100 µm. (G) Bioluminescence images and quantification of orthotopic OS models ( n = 5/group). (H,I) The tumor weight and growth in OS orthotopic models. (J) Typical X‐ray images of the tibia in the orthotopic OS model. (K) HE, IHC (ITGBL1, Ki67, OCT4, SOX2, and NANOG), and TUNEL assays of orthotopic xenograft tumors. Scale bar = 50 µm in images of HE and IHC; Scale bar = 100 µm in TUNEL images. (L) Bioluminescence images and quantification of OS lung metastasis models. (M) Representative images of Lung and lung tissue HE staining in OS lung metastasis models. Scale bar = 100 µm. All in vitro experiments were conducted in three independent experiments. All data are presented as the means ± SD, and differences between groups were assessed by Student's t ‐test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To detect the ubiquitination of endogenous ITGBL1, OS cells were treated with 20 μ m MG132 (MCE, USA) for 9 h to inhibit proteasome‐mediated degradation.

Techniques: CCK-8 Assay, Migration, Wound Healing Assay, Transwell Assay, Tube Formation Assay, Staining, Expressing, Immunofluorescence, TUNEL Assay, In Vitro

Journal: Advanced Science

Article Title: HSP90AB1‐Mediated Ubiquitin‐Proteasome Degradation of ITGBL1 Promotes Osteosarcoma Progression by Inhibiting Endoplasmic Reticulum Stress‐Induced Autophagy

doi: 10.1002/advs.202515651

Figure Lengend Snippet: Overexpression of ITGBL1 inhibits OS progression. (A) OS cell viability was measured by the CCK8 assay. (B) OS cell migration was measured by the wound healing assay. Scale bar = 100 µm. (C) OS cell migration and invasion were measured by the transwell assay. (D) OS cell stemness was measured by sphere formation assay. Scale bar = 50 µm. (E) The expression of stemness‐related proteins was detected by immunofluorescence in OS spheres. Scale bar = 50 µm. (F) Apoptotic cells in OS cells were detected by the TUNEL assay. Scale bar = 100 µm. (G) Bioluminescence images and quantification of orthotopic OS model ( n = 5). (H,I) The tumor weight and growth in the orthotopic OS model. (J) Typical X‐ray image of tibia in the orthotopic OS model. (K) HE, IHC (ITGBL1, Ki67, OCT4, SOX2, and NANOG), and TUNEL staining of orthotopic xenograft tumors. Scale bar = 50 µm in images of HE and IHC, Scale bar = 100 µm in TUNEL images. (L) Bioluminescence images and quantification of OS lung metastatic models. (M) Representative images of Lung and lung tissue HE staining in OS lung metastasis models. Scale bar = 100 µm. All in vitro experiments were conducted three independent experiments. All data are presented as the means ± SD, and differences between groups were assessed by Student's t ‐test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To detect the ubiquitination of endogenous ITGBL1, OS cells were treated with 20 μ m MG132 (MCE, USA) for 9 h to inhibit proteasome‐mediated degradation.

Techniques: Over Expression, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Assay, Tube Formation Assay, Expressing, Immunofluorescence, TUNEL Assay, Staining, In Vitro

Journal: Advanced Science

Article Title: HSP90AB1‐Mediated Ubiquitin‐Proteasome Degradation of ITGBL1 Promotes Osteosarcoma Progression by Inhibiting Endoplasmic Reticulum Stress‐Induced Autophagy

doi: 10.1002/advs.202515651

Figure Lengend Snippet: ITGBL1 activates ER stress and autophagy in OS cells. (A) Differentially expressed genes between ITGBL1 overexpression and the vector group with log 2 FC ≥ 1 and P.adj < 0.05 were presented with a volcano map and a heatmap. (B,C) KEGG and GO pathway enrichment analysis of DEGs. (D) GSEA shows that ITGBL1 expression levels are positively correlated with the response to endoplasmic reticulum (ER) stress and endoplasmic reticulum unfolded protein response. (E) The morphology and thickness changes of ER in OS cells were examined using transmission electron microscopy (TEM), and the ER was indicated by a red arrow. Scale bar = 500 nm. (F) The expression level of BIP mRNA was detected by RT‐qPCR. (G,H) The expression of ER stress‐related proteins including PERK, IRE1α, BIP, and CHOP were detected by Western blot. (I) ITGBL1‐GFP or Vector‐GFP and pDsRed‐ER plasmids were transfected into 143B and HOS cell lines for confocal microscopic analysis. Scale bar = 10 µm. (J) Autolysosomes in 143B cells were detected using TEM, and the autolysosomes were marked in red arrow. Scale bar = 500 nm. (K) The expression of autophagy‐related proteins was detected by Western blot. (L) LC3B‐GFP‐mCherry plasmids were transfected into OS cells that overexpress ITGBL1 or vector for confocal microscopic analysis to examine the expression of GFP and mCherry. Scale bar = 10 µm. All data are presented as the means ± SD, and differences between groups were assessed by Student's t ‐test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To detect the ubiquitination of endogenous ITGBL1, OS cells were treated with 20 μ m MG132 (MCE, USA) for 9 h to inhibit proteasome‐mediated degradation.

Techniques: Over Expression, Plasmid Preparation, Expressing, Transmission Assay, Electron Microscopy, Quantitative RT-PCR, Western Blot, Transfection

Journal: Advanced Science

Article Title: HSP90AB1‐Mediated Ubiquitin‐Proteasome Degradation of ITGBL1 Promotes Osteosarcoma Progression by Inhibiting Endoplasmic Reticulum Stress‐Induced Autophagy

doi: 10.1002/advs.202515651

Figure Lengend Snippet: ITGBL1 suppresses OS progression through ER stress. (A) The expression of ER stress‐related proteins was detected by Western blot after overexpressing ITGBL1 or/and administration of ER stress inhibitor 4‐PBA (1 m m ) in 143B and HOS. (B) ER was detected by TEM in 143B after overexpressing ITGBL1 with or without 4‐PBA (1 m m for 24 h). Scale bar = 500 nm. (C) The expression of p62 and LC3B was detected by Western blot after overexpressing ITGBL1 or/and administration 4‐PBA in 143B and HOS. (D) LC3B‐GFP‐mCherry plasmids were transfected into OS cells that overexpress ITGBL1 or administered 4‐PBA for confocal microscopic analysis to examine the expression of GFP and mCherry. Scale bar = 10 µm. (E) Schematic diagram for orthotopic OS model and OS lung metastasis model experiments. (F) Bioluminescence images and quantification of orthotopic OS model (n = 5). (G,H) The tumor weight and growth in orthotopic OS models. (I) Typical X‐ray image of tibia in orthotopic OS models. (J) HE, IHC (ITGBL1, Ki67, OCT4, SOX2, and NANOG), and TUNEL staining of orthotopic xenograft tumors. Scale bar = 50 µm in HE and IHC images, Scale bar = 100 µm in TUNEL images. (K) Bioluminescence images and quantification of OS lung metastasis models. (L) Representative images of lung and lung tissue HE staining in OS lung metastasis models. Scale bar = 100 µm. All data are presented as the means ± SD, and differences between groups were assessed by Student's t ‐test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To detect the ubiquitination of endogenous ITGBL1, OS cells were treated with 20 μ m MG132 (MCE, USA) for 9 h to inhibit proteasome‐mediated degradation.

Techniques: Expressing, Western Blot, Transfection, TUNEL Assay, Staining

Journal: Advanced Science

Article Title: HSP90AB1‐Mediated Ubiquitin‐Proteasome Degradation of ITGBL1 Promotes Osteosarcoma Progression by Inhibiting Endoplasmic Reticulum Stress‐Induced Autophagy

doi: 10.1002/advs.202515651

Figure Lengend Snippet: HSP90AB1 directly interacts with ITGBL1 in OS cells. (A) Schematic diagram for Co‐IP/MS analysis in OS cells. (B) Silver staining of proteins that isolated from OS cells by IgG or FLAG antibodies. (C) Fifteen proteins were screened out among the intersection between 143B and HOS anti‐FLAG groups, while ruled out proteins in the anti‐IgG group. (D) Co‐IP analysis was performed with anti‐ITGBL1 or anti‐HSP90AB1 antibodies in 143B and HOS cells. (E) 293T cells were transfected with plasmid that expressing HA‐HSP90AB1 or FLAG‐ITGBL1, and then cell lysates were subjected to Co‐IP assay with anti‐HA or anti‐FLAG antibodies. (F) The subcellular localization of HSP90AB1 and ITGBL1 in 143B and HOS cells was detected by IF staining. (G) The binding sites between HSP90AB1 and ITGBL1 were predicted by Molecular Docking. (H) Schematic representation of HA‐HSP90AB1 truncations was shown (upper panel). 293T cells were co‐transfected with FLAG‐ITGBL1 and the indicated HA‐HSP90AB1 truncations. The cells were lysed for immunoprecipitation using anti‐HA antibody, and anti‐FLAG antibody was used to detect the protein binding (lower panel). (I) A schematic representation of ITGBL1 truncations was shown (upper panel). The truncations of FLAG‐ITGBL1 and full‐length HA‐HSP90AB1 were co‐transfected in 293T cells, followed with Co‐IP analysis.

Article Snippet: To detect the ubiquitination of endogenous ITGBL1, OS cells were treated with 20 μ m MG132 (MCE, USA) for 9 h to inhibit proteasome‐mediated degradation.

Techniques: Co-Immunoprecipitation Assay, Silver Staining, Isolation, Transfection, Plasmid Preparation, Expressing, Staining, Binding Assay, Immunoprecipitation, Protein Binding

Journal: Advanced Science

Article Title: HSP90AB1‐Mediated Ubiquitin‐Proteasome Degradation of ITGBL1 Promotes Osteosarcoma Progression by Inhibiting Endoplasmic Reticulum Stress‐Induced Autophagy

doi: 10.1002/advs.202515651

Figure Lengend Snippet: HSP90AB1 stimulates ITGBL1 degradation through K63‐linked ubiquitination. (A,B) The expression of indicated proteins was detected in 143B and HOS cells by Western blot after cells were transfected with the indicated plasmids. (C) The expression of ITGBL1 was detected in OS cells by WB after transfecting different doses of HA‐HSP90AB1. (D) The mRNA expression of ITGBL1 was measured by RT‐qPCR in OS cells after transfection with the indicated plasmids. (E) 143B, HOS, and 293T cells were transfected with HSP90AB1 expressing plasmids, and treated with or without 20 µ m MG132 for 9 h, and then assessed ITGBL1 expression. (F,G) OS cells transfected with HSP90AB1 expressing plasmids or vector were treated with 100 µg/mL CHX, and detected the expression levels of indicated proteins by WB at the indicated time points. Then ITGBL1 protein abundance was quantified by the Image J software. (H,I) The ubiquitination of ITGBL1 was analyzed in 143B cells that transfected with the indicated plasmids or treated with 17‐AAG. (J,K) 293T cells were transfected with the indicated constructs or treated with HSP90 inhibitor 17‐AAG. After 48 h of transfection, cells were treated with 20 µ m MG132 for 9 h. Lysates were subjected to IP assays, followed by immunoblotting analysis. Anti‐HIS antibody was used to bind HIS‐tagged Ub to indicate ubiquitination. (L) Immunoprecipitation assay was used to analyze the ubiquitination of ITGBL1 in 293T cells transfected with HIS‐Ub (WT, K6, K11, K27, K29, K33, K48, or K63) together with FLAG‐ITGBL1, HA‐HSP90AB1. (M) The ubiquitination of ITGBL1 was analyzed in 143B cells that transfected with the indicated plasmids.

Article Snippet: To detect the ubiquitination of endogenous ITGBL1, OS cells were treated with 20 μ m MG132 (MCE, USA) for 9 h to inhibit proteasome‐mediated degradation.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Plasmid Preparation, Quantitative Proteomics, Software, Construct, Immunoprecipitation

Journal: Advanced Science

Article Title: HSP90AB1‐Mediated Ubiquitin‐Proteasome Degradation of ITGBL1 Promotes Osteosarcoma Progression by Inhibiting Endoplasmic Reticulum Stress‐Induced Autophagy

doi: 10.1002/advs.202515651

Figure Lengend Snippet: The expression of HSP90AB1 in OS and correlation among ITGBL1 and BIP, HSP90AB1. (A) The protein expression level of HSP90AB1 in OS tissues and their normal bone tissues ( n = 18 pairs). (B) The protein expression level of HSP90AB1 in the indicated cells. (C) Kaplan–Meier analyses of the overall survival rate and disease‐free survival rate of OS patients with high and low expression levels of HSP90AB1. (D–F) IF staining of BIP, HSP90AB1, and ITGBL1 with OS tissue microarrays and the relationship among ITGBL1 and BIP, HSP90AB1. Scale bar = 20 µm. (G) Kaplan–Meier analyses of the overall survival rate of OS patients with high or low expression levels of ITGBL1 and HSP90AB1.

Article Snippet: To detect the ubiquitination of endogenous ITGBL1, OS cells were treated with 20 μ m MG132 (MCE, USA) for 9 h to inhibit proteasome‐mediated degradation.

Techniques: Expressing, Staining

Journal: Advanced Science

Article Title: HSP90AB1‐Mediated Ubiquitin‐Proteasome Degradation of ITGBL1 Promotes Osteosarcoma Progression by Inhibiting Endoplasmic Reticulum Stress‐Induced Autophagy

doi: 10.1002/advs.202515651

Figure Lengend Snippet: Ivermectin suppresses OS progression by blocking the interaction between HSP90AB1 and ITGBL1. (A,B) Virtual Screening revealed small molecules that targeting the interaction surface between HSP90AB1 and ITGBL1. (C) A diagram showing the structure of ivermectin and the potential binding surface at the HSP90AB1‐ ITGBL1 interaction. (D) Indicated proteins were detected by Western blot in the indicated OS cells that treated with the indicated concentrations of ivermectin for 24 h. (E) Indicated OS cells were treated with DMSO or 20 µ m ivermectin for 24 h, then performed Co‐IP using the indicated antibodies. (F) Schematic diagram for an ivermectin treatment animal experiment. (G) Co‐IP analysis was performed using orthotopic xenograft tumors that treated with PBS or Ivermectin. (H) Bioluminescence images and quantification of orthotopic OS model ( n = 5). (I,J) The tumor weight and growth in orthotopic OS models. (K) Typical X‐ray image of tibia in the orthotopic OS model. (L) HE, IHC (ITGBL1, Ki67, OCT4, SOX2, and NANOG), and TUNEL staining of orthotopic xenograft tumors. Scale bar = 50 µm in HE and IHC images; Scale bar = 100 µm in TUNEL images. (M) Representative images of lung and lung tissue HE staining in OS lung metastasis models. Scale bar = 100 µm. (N) Schematic diagram of HSP90AB1/ITGBL1 axis promoting OS progression (By Figdraw). All data are presented as the means ± SD, and differences between groups were assessed by Student's t‐ test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To detect the ubiquitination of endogenous ITGBL1, OS cells were treated with 20 μ m MG132 (MCE, USA) for 9 h to inhibit proteasome‐mediated degradation.

Techniques: Blocking Assay, Binding Assay, Western Blot, Co-Immunoprecipitation Assay, TUNEL Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Scanning electron microscopy (SEM) of the 3D cell–collagen scaffold construct. Representative SEM images: ( A ) the 3D osteoblast–collagen scaffold construct (non-loading), ( B ) the 3D osteoblast–collagen scaffold construct (after 24 h mechanical loading), ( C ) the 3D osteoblast–collagen scaffold construct (after 48 h non-loading), and ( D ) the 3D osteoblast–collagen scaffold construct (after 48 h mechanical loading). ( E ) Representative SEM images of a 3D chondrocyte–collagen scaffold construct (non-loading), ( F ) the 3D chondrocyte–collagen scaffold construct (after 24 h mechanical loading), ( G ) the 3D chondrocyte–collagen scaffold construct (48 h non-loading), and ( H ) the 3D chondrocyte–collagen scaffold construct (after 48 h mechanical loading) (each subgroup/time point, n = 12).

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Electron Microscopy, Construct

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Effects of mechanical loading on osteoblast activity. Cohen’s d between the control and the mechanical loading group is shown in the above Gardner–Altman estimation plot. Both groups are plotted on the left axes; the mean difference is plotted on a floating axes on the right as a bootstrap sampling distribution. The mean difference is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. The effect sizes and CIs are reported above as effect size (CI width lower bound; upper bound). ( A ) ALP: ( a ) control group versus mechanical loading (+)-24 h incubation group, ( b ) control group versus mechanical loading (+)-48 h incubation group. ( B ) Osteocalcin: ( a ) control group versus mechanical loading (+)-24 h incubation group, ( b ) control group versus mechanical loading (+)-48 h incubation group).

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Activity Assay, Control, Sampling, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Effects of mechanical loading on chondrocyte activity. Effects of mechanical loading on osteoblast activity. Cohen’s d between the control and mechanical loading group is shown in the above Gardner–Altman estimation plot. Both groups are plotted on the left axes; the mean difference is plotted on a floating axes on the right as a bootstrap sampling distribution. The mean difference is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. The effect sizes and CIs are reported above as effect size (CI width lower bound; upper bound). ( A ) Proteoglycan: ( a ) control group versus mechanical loading (+)-24 h incubation group, ( b ) control group versus mechanical loading (+)-48 h incubation group. ( B ) Type II collagen: ( a ) control group versus mechanical loading (+)-24 h incubation group, ( b ) control group versus mechanical loading (+)-48 h incubation group).

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Activity Assay, Control, Sampling, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Expressions of Glut1, SIRT1, and Runx2 in osteoblasts. ( A ) The expression of Glut1 in non-loaded osteoblasts, 24 h loaded osteoblasts, and 48 h loaded osteoblasts. ( B ) The expression relative to β-actin. ( C ) The expression of SIRT1 in non-loaded osteoblasts, 24 h loaded osteoblasts, and 48 h loaded osteoblasts. ( D ) The expression relative to β-actin. ( E ) The expression of Runx2 in non-loaded osteoblasts, 24 h loaded osteoblasts, and 48 h loaded osteoblasts. ( F ) The expression relative to the level of β-actin.

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Effects of the Glut1 inhibitor on Glut-1, SIRT1, and Runx2 in osteoblasts. ( A ) The expression of Glut1 in mechanically loaded or non-loaded osteoblasts in the presence or absence of a Glut1 inhibitor (Western blot). ( a ) The culture medium only, ( b ) the control group (DMSO solution + culture medium), ( c ) the Glut1 inhibitor (WZB117)-treated group (10.0 μM WZB117 in DMSO solution + culture medium), and ( d ) the expression relative to β-actin. ( B ) The expression of SIRT1 in mechanically loaded or non-loaded osteoblasts in the presence or absence of a Glut1 inhibitor (Western blot). ( a ) The culture medium only, ( b ) the control group (DMSO solution + culture medium), ( c ) the Glut1 inhibitor (WZB117)-treated group (10.0 μM WZB117 in DMSO solution + culture medium), and ( d ) the expression relative to β-actin. ( C ) The expression of Runx2 in mechanically loaded or non-loaded osteoblasts in the presence or absence of a Glut1 inhibitor (Western blot). ( a ) The culture medium only, ( b ) the control group (DMSO solution + culture medium), ( c ) the Glut1 inhibitor (WZB117)-treated group (10.0 μM WZB117 in DMSO solution + culture medium), and ( d ) the expression relative to β-actin. * p < 0.05.

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Summary of our current study. ( A ) Involvement of osteocytes in osteoblast differentiation. Mechanical loading suppresses the expression of osteocytic sclerostin following activation of Wnt/beta-catenin signaling in osteocytes and osteoblasts, resulting in osteoblast differentiation and bone formation activity. Cellular interaction of osteoblasts with osteocytes, via the sclerostin–Wnt/beta-catenin signaling pathways, plays an important part in physiologic mechanical stress-mediated bone metabolism. ( B ) Mechanical loading may directly induce osteoblast differentiation and bone formation activity, without the molecular mechanism of osteocyte-to-osteoblast interaction. The mechanical stress-induced expression of Glut1 and resultant uptake of glucose may suppress the level of the energy sensor SIRT1 in osteoblast energy metabolism. Since SIRT1 is recognized to negatively regulate Runx2 activity, the mechanical stress-induced suppression of SIRT1 results in increased activity of the osteogenic transduction factor Runx2 in osteoblasts, leading to osteoblast differentiation and bone formation.

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Expressing, Activation Assay, Activity Assay, Protein-Protein interactions, Transduction

Journal: Advanced Healthcare Materials

Article Title: Ascorbic Acid Modulates Collagen Properties in Glucocorticoid‐Induced Osteoporotic Bone: Insights into Chemical, Mechanical, and Biological Regulation

doi: 10.1002/adhm.202502606

Figure Lengend Snippet: AA enhances COL1A1 biological activity and restores osteoblast function and COL1A1 regulation in the presence of Psl. A) ALP staining (red) of osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with (+AA), and in the Psl presence (+Psl) or absence (–Psl); Scale bar: 300 µm. B) Quantification of Mean Intensity of ALP (A.U.). C) Alzarin Red (AR) activity assay of osteo‐spheroids after 7 d with or without additional AA. Scale bar, 400 µm. D) Graph showing quantification of Mean Intensity of AR (A.U.). E) OsteoImaging mineralization assay quantifications of Mean Intensity (A.U.) of osteo‐spheroids incubated with or without additional AA for 7 d. F) Gene expression analysis of osteogenic markers ( BGLAP, DMP1, DLX3, RUNX2 ) of osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with (+AA), and in the Psl presence (+Psl) or absence (–Psl); Heatmap demonstrates the ∆CT averages. G) Gene expression analysis of COL1A1‐related genes ( PLOD1, PLOD3, DLX3, P3H1, P3H2, P3H3, LOX, SVCT2, COL1A2, COL22A1, IBSP, P4HA2, P4HA3, IFITM5 ) in osteo‐spheroids embedded in COL1A1 matrices, cultured for 7 d with or without ascorbic acid (AA), and in the presence (+Psl) or absence (–Psl) of prednisolone (Psl); Heatmap demonstrates the ∆CT averages H) Fold change of top collagen‐related putative metabolites elevated in the presence of endogenous ascorbic acid (+AA) within the 3D matrix of osteo‐spheroids after 7 d of Psl (+Psl) treatment, based on untargeted metabolomics analysis; fold change threshold > 2 with significance; FDR p‐value < 0.05. The data are expressed as mean ± SD; N = 3, n = 3; significant differences: * * p‐value < 0.01; *** p‐value < 0.005 ; **** p‐value < 0.0001.

Article Snippet: Human osteoblasts (HOBs) (PromoCell) were cultured in osteoblast growth medium (GM) (PromoCell) and differentiated in osteoblast mineralization medium (MM) (PromoCell).

Techniques: Activity Assay, Staining, Cell Culture, Mineralization Assay, Incubation, Gene Expression

Journal: Pancreas

Article Title: Stromal Fibrosis and Expression of Matricellular Proteins Correlate With Histological Grade of Intraductal Papillary Mucinous Neoplasm of the Pancreas

doi: 10.1097/mpa.0000000000000617

Figure Lengend Snippet: FIGURE 2. Area of positive staining revealed by immunohistochemistry in IPMN. Positive staining of the periductal stroma with anti–α-SMA (A), antiperiostin (B), and antigalectin-1 (C) at a magnification of 200. Positive staining of the acinar area with anti–α-SMA (D), antiperiostin (E), and antigalectin-1 (F) at a magnification of 100.

Article Snippet: The primary antibodies and dilutions employed were the following: anti–α-SMA (M0851; DAKO Japan, Tokyo, Japan, 1:50), antiperiostin (RD181045050; BioVendor, Czech Republic, 1:4000), and antigalectin-1 (sc-166618; Santa Cruz Biotechnology, Dallas, 1:40).

Techniques: Staining, Immunohistochemistry

Journal: Journal of biomedical materials research. Part A

Article Title: Electrophoretic deposition of MgO nanoparticles imparts antibacterial properties to poly-L-lactic acid for orthopedic applications.

doi: 10.1002/jbm.a.36174

Figure Lengend Snippet: Figure 12: The osteoblast cell density (cells/cm2) after 3 days of culture was directly proportional to the

Article Snippet: Osteoblasts were cultured in phenol-free osteoblast basal medium with osteoblast supplemental mix (PromoCell) and 1% penicillin-streptomycin (Sigma Aldrich).

Techniques: