Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 1. TGF-β expression in osteosarcoma specimens and cell lines. (a) Immunohistochemical staining was used to analyze TGF-β expression in both osteosarcoma specimens and normal adjacent tissues. (b) Survival analysis was performed using the Kaplan-Meier method. Data are presented as mean ± SEM of three independent experiments. (c) TGF-β protein levels in osteosarcoma cell lines (*P < 0.05). (d) TGF-β mRNA expression in osteosarcoma cell lines (*P < 0.05).

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 2. Small interfering (si) RNA targeting of TGF-β (si-TGF-β) suppresses proliferation and promotes apoptosis in osteosarcoma. (a) RT-qPCR was employed to assess the knockdown efficiency of si-TGF-β, *P < 0.05 vs. the si-control group. (b) Western blotting was used to measure the knockdown efficiency of si-TGF-β, *P < 0.05 vs. the si-control group. (c)The MTT assay was used to assess the viability of U2OS and MG-63 cells transfected with si-control or si-TGF-β, *P < 0.05 vs. the si-control group. (d and e) The colony- forming ability of U2OS and MG-63 cells transfected with si-control or si-TGF -β, *P < 0.05 vs. the si-control group. (f and g) Flow cytometry analysis of the cell cycle phase distribution of U2OS and MG-63 cells transfected with si-control or si-TGF – β, *P < 0.05 vs. the si-control group. (h and i) The rate of apoptosis in U2OS and MG-63 cells transfected with si-TGF-β, *P < 0.05 vs. the si-control group. (j and k) TGF-β knockdown increases the expression of cleaved PARP, caspase-3, and BAX, *P < 0.05 vs. the si-control group.

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Quantitative RT-PCR, Knockdown, Control, Western Blot, MTT Assay, Transfection, Flow Cytometry, Expressing

Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 4. TGF-β enhances osteosarcoma cell stemness and increases the proportion of CD133+ cells. (a and b) Spheroid formation assay for U2OS and MG-63 cells transfected with si-TGF-β. Representative images (left panel) and statistical measurements (right panel). Representative micrographs of formed spheres were analyzed in cells treated with si-TGF-β or si-control. Scale bar, 100 μm. *P < 0.05 vs. the si-control group. (c and d) The percentage of CD133+ cells in U2OS- and MG-63-derived spheroids was analyzed by flow cytometry.*P < 0.05 vs. the si-control group. (e and f) Stem cell-associated protein expression was measured by western blot. *P < 0.05 vs. the si-control group.

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Tube Formation Assay, Transfection, Control, Derivative Assay, Flow Cytometry, Expressing, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 5. TGF-β regulates the PI3K/mTOR signaling pathway in osteosarcoma cells. (a) p-PI3K and p-Akt expression was measured by western blot, P < 0.05 vs. the si-control group. (b and c) The colony-forming ability of si-TGF-β-transfected U2OS and MG-63 cells with or without LY294002 treatment. P < 0.05 vs. the si-control group. (d and e) Transwell assays were used to assess the invasive ability of U2OS and MG-63 cells, P < 0.05 vs. the si-control group. (f and g) A wound-healing assay was performed to measure the migratory ability of U2OS and MG-63 cells, P < 0.05 vs. the si-control group. (h and i) Spheroid formation assay for U2OS/MG-63 cells transfected with si-TGF-β. Representative images (left panel) and statistical measurement (right panel). Representative micrographs of formed spheres were analyzed in cells treated with si-TGF-β or si-control with or without LY294002 treatment. Scale bar, 100 μm. *P < 0.05 vs. the si-control group. (j and k) The percentage of CD133+ cells in U2OS cell- and MG-63 cell-derived spheroids was analyzed by flow cytometry. *P < 0.05 vs. the si-control group.

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Expressing, Western Blot, Control, Transfection, Wound Healing Assay, Tube Formation Assay, Derivative Assay, Flow Cytometry

Journal: Cells

Article Title: The Warburg Trap: A Novel Therapeutic Approach for Targeting Osteosarcoma.

doi: 10.3390/cells13010061

Figure Lengend Snippet: Figure 2. Warburg Trap drugs significantly reduce the colony-forming capacity of OS cells. OS cell lines (n = 6) were seeded at low density in culture plates and treated with the WT drugs (Metformin (1 mM), Monensin (5 nM), and Fenofibrate (10 µM)) in an acidic culture medium (pH 6.5). After 24 h and 48 h of treatment, the culture medium was replaced by a normal cell culture medium with a physiological pH value of 7.4. Colonies that formed during a further seven days of culture were stained with haematoxylin and photographed before (A) the number of colonies and (B) the size of the colonies were quantified using the software ImageJ version 1.48v (* p < 0.01 compared to untreated controls). (C) Representative photographs of stained colonies obtained with cell line 143B analysed in duplicates. (D) Analysis of colony numbers that formed after pre-treatment of osteosarcoma cell lines (n = 6) with WT drugs and subsequent cultivation for 14 days in soft agar (* p < 0.01 compared to untreated controls).

Article Snippet: The following six commercially available human osteosarcoma cell lines were used in this study: HOS (American Tissue Culture Collection, Rockville, MD, USA), HOS 143B (Sigma-Aldrich, Taufkirchen, Germany), CAL-72 (DSMZ, Braunschweig, Germany), MNNG-HOS, SaOS-2, and U2OS (Cell Line Service GmbH, Eppelheim, Germany).

Techniques: Cell Culture, Staining, Software

Journal: Cells

Article Title: The Warburg Trap: A Novel Therapeutic Approach for Targeting Osteosarcoma.

doi: 10.3390/cells13010061

Figure Lengend Snippet: Figure 3. DDIT3-mediated downregulation of WNT target genes by WT drugs. Osteosarcoma cell lines (n = 3) were treated with WT drugs (Metformin (1 mM), Monensin (5 nM), and Fenofibrate (10 µM)) (MeMoFe) for the indicated time before the expression of DDIT3 and the WNT target genes AXIN2, CCND1 and LGR5 were analysed by real-time quantitative RT-PCR analysis. The expression of the reference gene RPS13 (ribosomal protein S13) was used for normalisation. Data are presented as percent of untreated 24 h controls (* p < 0.05 compared to untreated controls).

Article Snippet: The following six commercially available human osteosarcoma cell lines were used in this study: HOS (American Tissue Culture Collection, Rockville, MD, USA), HOS 143B (Sigma-Aldrich, Taufkirchen, Germany), CAL-72 (DSMZ, Braunschweig, Germany), MNNG-HOS, SaOS-2, and U2OS (Cell Line Service GmbH, Eppelheim, Germany).

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: A p53 Drug Response Signature Identifies Prognostic Genes in High-Risk Neuroblastoma

doi: 10.1371/journal.pone.0079843

Figure Lengend Snippet: (A) Consistent with our previously data , Western blotting showed an increase in p53 levels within 4 hours exposure to Nutlin-3a in p202, p218, and IMR32 cells, confirming that the p53 signaling pathway is active in our system. (B) Apoptosis induction was tested in five established neuroblastoma lines (JF, IMR32), early passage neuroblastoma tumor cultures (p202, p218, H), and four non-neuroblastoma human solid tumors including colorectal (HCT116), breast (MCF7) and osteosarcoma (SJSA-1). SJSA-1 line differs by the level of MDM2 expression, being amplified 25 fold. A p53 mutant neuroblastoma line (SJ3-12), which lacks part of the DNA binding domain, was used as control. Proliferating cells were treated with Nutlin-3a for 24 hours and TdT-positive fraction was measured by flow cytometry. (C) Pathways enriched upon Nutlin-3a treatment: p53 signaling (GSEA ID: M6370), DNA damage (GSEA ID: M8378), and chemotherapy response (bleo_human_lymph) genes. Vertical columns separate Nutlin-3a versus Nutlin-3b at 3, 8, and 16 hours, horizontal rows indicate genes.

Article Snippet: JF (ATCC), IMR32 (ATCC), LAN5 (LS Metelitsa, Houston TX), and LAN1 (ATCC) human NB lines were maintained in RPMI 1640; human colorectal cancer cell line, HCT 116 (ATCC), and human breast cancer cell line, MCF7 (ATCC), in McCoy’s 5A and DMEM plus 1% insulin respectively; human osteosarcoma line, SJSA-1 (ATCC), in RPMI 1640 medium with 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate; primary neuroblastoma lines (p202, p218, pH) (Texas Children’s Cancer Center, Houston, TX) and CHLA255 line (LS Metelitsa) in IMDM with 20% FBS and 0.1% ITS.

Techniques: Western Blot, Expressing, Amplification, Mutagenesis, Binding Assay, Control, Flow Cytometry