human osteosarcoma cell line mg  (Millipore)

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: Relative USP22 expression in osteosarcoma cells and tissues. (A) Relative mRNA levels of USP22 in 30 pairs of osteosarcoma (OS) and corresponding non‐tumour tissues, assessed by qRT‐PCR analysis. * p < 0.05. (B) Determination and quantification of USP22 protein levels in osteosarcoma (OS) and corresponding non‐tumour tissues by western blotting assay. GAPDH was used as a loading control. (C, D) Representative images (C) and quantification (D) of USP22 staining in 30 paired osteosarcoma (OS) and noncancer tissues. * p < 0.05. (E, F) mRNA and protein levels of USP22 in osteosarcoma (OS) cells (143B, HOS, MG‐63, U2‐OS) and the immortalised normal cells (hfoBI‐19) line.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: Effects of USP22 on osteosarcoma growth in vitro and in vivo. (A, B) the mRNA (A) and protein (B) levels of USP22 in 143B cells after transfection with shUSP22 or shNC (control). (C, D) CCK‐8 assay showing proliferation of OS cancer cells following overexpression (right) or knockdown (left) of USP22. * p < 0.05. (E, F) Representative images (left) and quantification (right) of EDU assays of osteosarcoma cells transfected with p‐USP22 or shUSP22. Scale bar, 50 μm. * p < 0.05. (G, H) Representative images of colony formation assays of osteosarcoma (OS) cells transfected with shUSP22 (G) or p‐USP22 (H). (I, J) The apoptosis rate of OS cells was detected by flow cytometry and was significantly increased in the shUSP22 cells but decreased in the p‐USP22 cells. (K–M) 143B/shUSP22 cells were subcutaneously injected into nude mice, and tumour volumes were measured on the indicated days; at the experimental endpoint, tumours were dissected, photographed and weighed ( n = 5, * p < 0.05).

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: In Vitro, In Vivo, Transfection, Control, CCK-8 Assay, Over Expression, Knockdown, Flow Cytometry, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: USP22 promotes aerobic glycolysis in osteosarcoma cells. (A, B) ATP levels, Cellular G6P levels, glucose consumption and lactate production in 143B/shUSP22 cells (A) or U2OS/p‐USP22 cells (B). Three independent experiments were performed. * p < 0.05 versus control. (C, E) ECAR data showing the glycolytic rate and capacity in USP22‐silenced (C) or USP22‐overexpressing (E) osteosarcoma cells. Glucose (10 mM), the oxidative phosphorylation inhibitor oligomycin (1.0 μM) and the glycolytic inhibitor 2‐deoxyglucose (2‐DG, 50 mM) were sequentially injected into each well at the indicated time points. All measurements were normalised to the cell number calculated using crystal violet assay at the end of the experiment. * p < 0.05 versus control. (D, F) OCR results showing the basal respiration and maximum respiration in 143B/shUSP22 cells (D) or U2OS/p‐USP22 cells (F). Oligomycin (1.0 μM), the mitochondrial uncoupler carbonyl cyanide p‐trifluoromethoxy phenylhydrazone (FCCP, 1.0 μM) and the mitochondrial complex I inhibitor rotenone plus the mitochondrial complex III inhibitor antimycin A (Rote/AA, 0.5 μM) were sequentially injected. All measurements were normalised to the cell number calculated using crystal violet assay at the end of the experiment. * p < 0.05 versus control.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Control, Injection, Crystal Violet Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: Stable knockdown of USP22 decreased HK2 expression in osteosarcoma cells. (A, B) Western blotting and qRT‐PCR analyses of HK2 expression levels in 143B cells stably transfected with shNC or shUSP22 plasmid. * p < 0.05. (C, D) Western blotting and qRT‐PCR analyses of HK2 expression levels in U2OS cells stably transfected with control vector or p‐USP22 plasmid. * p < 0.05. (E, F) Determination of HK2 protein levels in osteosarcoma tissues ( n = 30) and paired non‐tumour tissues ( n = 30) by western blotting. GAPDH was used as a loading control. (G) Representative images of HK2 staining in 30 paired osteosarcoma (OS) and noncancer tissues. (H) Determination of HK2 mRNA levels in osteosarcoma tissues ( n = 30) and paired non‐tumour tissues ( n = 30) by qRT‐PCR. (I) Scatter plots of USP22 and HK2 mRNA expression in osteosarcoma. (J) Quantification of HK2 protein levels in osteosarcoma (OS) and corresponding non‐tumour tissues by western blotting assay. (K) Scatter plots of USP22 and HK2 protein expression in osteosarcoma.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Knockdown, Expressing, Western Blot, Quantitative RT-PCR, Stable Transfection, Transfection, Plasmid Preparation, Control, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: Tumour‐suppressive effects of USP22 silencing in osteosarcoma cells partially reversed by HK2 overexpression. (A) Western blotting of USP22 or HK2 in 143B cells stably transfected with shUSP22 in the presence or absence of p‐HK2. (B) CCK‐8 assays showing proliferation capacity of 143B cells stably transfected with shUSP22 in the presence or absence of p‐HK2. * p < 0.05. (C) Representative images of EdU assays of 143B cells stably transfected with shUSP22 in the presence or absence of p‐HK2. (D) ATP levels, Cellular G6P levels, glucose consumption and lactate production in 143B cells stably transfected with shUSP22 in the presence or absence of p‐HK2. * p < 0.05. (E) ECAR of USP22‐silenced 143B cells with and without HK2 overexpression. * p < 0.05. (F) OCR values of USP22 silenced 143B cells with and without HK2 overexpression. * p < 0.05.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Over Expression, Western Blot, Stable Transfection, Transfection, CCK-8 Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: USP22 regulates HK2 expression through β‐catenin in osteosarcoma. (A) Co‐immunoprecipitation (Co‐IP) showing that endogenous USP22 and HK2 were not directly bound. (B) Protein and mRNA levels of β‐catenin assessed by western blotting and qRT‐PCR in osteosarcoma cells transfected with shUSP22 or shNC. (C) Protein and mRNA levels of β‐catenin assessed by western blotting and qRT‐PCR in osteosarcoma cells transfected with p‐USP22 or control vector. (D, E) The total and nuclear protein levels of β‐catenin were assessed by western blotting in USP22‐silencing 143B cells (D) or USP22‐overexpression U2OS cells (E). GAPDH and Histone 3 were used as a loading control, respectively. (F) The protein levels of β‐catenin and HK2 were assessed by western blotting in USP22‐overexpression U2OS cells following treatment with shβ‐catenin. (G) Quantification for CCK‐8 assays of USP22‐overexpression U2OS cells transfected with shβ‐catenin. * p < 0.05. (H, I) Quantification (H) and representative images (I) for EDU assays of USP22‐overexpression U2OS cells transfected with shβ‐catenin. * p < 0.05. (J) ECAR of USP22‐overexpression U2OS cells transfected with shβ‐catenin. (K) OCR values of USP22‐overexpression U2OS cells transfected with shβ‐catenin.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Over Expression, CCK-8 Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: USP22 stabilises β‐catenin by regulating the ubiquitination of β‐catenin in osteosarcoma cells. (A) Co‐immunoprecipitation (Co‐IP) showing direct binding of endogenous USP22 and β‐catenin in osteosarcoma cells. (B, C) Osteosarcoma cells were treated with MG132 (15 μmol/L) for the indicated times, and levels of β‐catenin were determined. (D, E) Representative results of β‐catenin protein level in USP22‐silencing cells. The cells were treated with cycloheximide (CHX, 100 mg/mL) for indicated time points were subjected to western blot analysis. (F, G) OS cells transduced with shUSP22 (F) or p‐USP22 (G) were treated with 10 μM MG132. Cells were collected at 6 h and immunoblotted with the antibodies indicated. (H, I) Lysates from OS cells transduced with shUSP22 (H) or p‐USP22 (I) were immunoprecipitated with the anti‐Ub and immunoblotted with the anti‐β‐catenin. Cells were treated with MG132 for 6 h before collection. (J) Proposed model by which ubiquitin‐specific protease USP22 promotes osteosarcoma growth and aerobic glycolysis by upregulating HK2 via stabilisation of β‐catenin.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Transduction

Journal: International Journal of Molecular Sciences

Article Title: Assessing Cytotoxicity, Proteolytic Stability, and Selectivity of Antimicrobial Peptides: Implications for Orthopedic Applications

doi: 10.3390/ijms252413241

Figure Lengend Snippet: The bar graphs show the effects of KSL on the cellular metabolism of ( a ) MG63 cells, ( b ) L929 cells, and ( c ) hMSCs in the absence and presence of FBS. Bioluminescence readings in relative luminescence units (RLUs) were normalized to the mean of the reference control set to 100%. Bars in the graph represent the mean ± S.D. of three independent experiments ( N = 9), except for the assay performed with MG63 and hMSCs without FBS (four independent experiments, N = 12). The statistically significant difference with respect to the Reference control: * p < 0.05; ** p < 0.01; *** p < 0.001. Detailed information on the statistical analysis performed and exact p -values can be found in .

Article Snippet: The cytotoxicity of the test AMPs was assayed on three different mammalian cell lines, namely, the human osteosarcoma cell line MG63 (ATCC, Rockville, MD, USA); the murine immortalized fibroblast line L929 (ATCC, Rockville, MD, USA), and bone marrow-derived, human mesenchymal cells (hMSCs, BioWhittaker Inc., Walkersville, MD, USA).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Assessing Cytotoxicity, Proteolytic Stability, and Selectivity of Antimicrobial Peptides: Implications for Orthopedic Applications

doi: 10.3390/ijms252413241

Figure Lengend Snippet: The bar graphs show the effects of KSL-W on the cellular metabolism of ( a ) MG63 cells, ( b ) L929 cells, and ( c ) hMSCs in the absence and presence of FBS. Bioluminescence readings in relative luminescence units (RLUs) were normalized to the mean of the reference control set to 100%. Bars in the graph represent the mean ± S.D. of three independent experiments ( N = 9), except for the assay performed with MG63 and hMSCs without FBS (four independent experiments, N = 12). The statistically significant difference with respect to the Reference control: * p < 0.05; ** p < 0.01; *** p < 0.001. Detailed information on the statistical analysis performed and exact p -values can be found in .

Article Snippet: The cytotoxicity of the test AMPs was assayed on three different mammalian cell lines, namely, the human osteosarcoma cell line MG63 (ATCC, Rockville, MD, USA); the murine immortalized fibroblast line L929 (ATCC, Rockville, MD, USA), and bone marrow-derived, human mesenchymal cells (hMSCs, BioWhittaker Inc., Walkersville, MD, USA).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Assessing Cytotoxicity, Proteolytic Stability, and Selectivity of Antimicrobial Peptides: Implications for Orthopedic Applications

doi: 10.3390/ijms252413241

Figure Lengend Snippet: The bar graph reports the calculated IC 50 values of Dadapin-1 when tested on MG63 cells, L929 cells, and hMSCs, in the presence (blue bars) and absence of FBS (red bars). Plotted values for MG63 cells refer to our previously published work . Conversely, the values for L929 cells and hMSCs are newly introduced by the present investigation. It may be noticed that Dadapin-1 exhibits no or very low cytotoxicity. For both MG63 cells and hMSCs, the level of cytotoxicity increased in the absence of serum.

Article Snippet: The cytotoxicity of the test AMPs was assayed on three different mammalian cell lines, namely, the human osteosarcoma cell line MG63 (ATCC, Rockville, MD, USA); the murine immortalized fibroblast line L929 (ATCC, Rockville, MD, USA), and bone marrow-derived, human mesenchymal cells (hMSCs, BioWhittaker Inc., Walkersville, MD, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Assessing Cytotoxicity, Proteolytic Stability, and Selectivity of Antimicrobial Peptides: Implications for Orthopedic Applications

doi: 10.3390/ijms252413241

Figure Lengend Snippet: The bar graph presents the calculated IC 50 values of ( a ) KSL and ( b ) KSL-W when tested on MG63 cells, L929 cells, and hMSCs, in the presence (blue bars) and absence of FBS (red bars). Confidence intervals for IC 50 values can be found in .

Article Snippet: The cytotoxicity of the test AMPs was assayed on three different mammalian cell lines, namely, the human osteosarcoma cell line MG63 (ATCC, Rockville, MD, USA); the murine immortalized fibroblast line L929 (ATCC, Rockville, MD, USA), and bone marrow-derived, human mesenchymal cells (hMSCs, BioWhittaker Inc., Walkersville, MD, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Assessing Cytotoxicity, Proteolytic Stability, and Selectivity of Antimicrobial Peptides: Implications for Orthopedic Applications

doi: 10.3390/ijms252413241

Figure Lengend Snippet: Representative images of MG63 cells treated for 24 h with the indicated substances. The nuclei staining (Hoechst 333432) is shown in blue, and the actin cytoskeleton staining (Phalloidin-FITC) is shown in green. Bottom scale bars: 50 µm.

Article Snippet: The cytotoxicity of the test AMPs was assayed on three different mammalian cell lines, namely, the human osteosarcoma cell line MG63 (ATCC, Rockville, MD, USA); the murine immortalized fibroblast line L929 (ATCC, Rockville, MD, USA), and bone marrow-derived, human mesenchymal cells (hMSCs, BioWhittaker Inc., Walkersville, MD, USA).

Techniques: Staining