Journal: Nature Communications

Article Title: Genome-wide RNAi screening identifies TMIGD3 isoform1 as a suppressor of NF-κB and osteosarcoma progression

doi: 10.1038/ncomms13561

Figure Lengend Snippet: ( a ) Subcutaneous tumour formation assays using SJSA-1 cells (5 × 10 5 ) downregulated for non-silencing shRNA (Control), A3AR ( A2a ) or TMIGD3 ( T6U ). Tumours were measured twice a week until day 20 after injections. Error bars: means±s.d. ( n =5 mice per group). ** P <0.01; two-way analysis of variance (ANOVA). ( b ) Primary tumours and metastases following orthotopic injections of SJSA-1 (top, n =5 mice per group) and Saos2 (bottom, n =5 mice per group) cells with or without TMIGD3 knockdown ( T6U ). Cells (1 × 10 5 ) were injected into femurs of NOD-scid IL2Rγ null (NSG) mice. Mice were killed ∼2 months (SJSA-1) or 5 months (Saos2) later, when thigh diameter became ∼2 cm in mice injected with TMIGD3 knockdown cells. Graphs showing primary tumour weight and number of metastatic nodules in the lungs (SJSA-1) or livers (Saos2). Representative images of primary tumours and metastases (arrows) below the graphs. Error bars: means±s.d. * P <0.05 and ** P <0.01; Student's t -test (two-tailed). ( c ) Tail vein injection assays using SJSA-1 cells (5 × 10 4 ) infected with non-silencing (control: GFP+, green), T6U (GFP+, green) or T3 (DsRed+, red) shRNAs. Mice (Control : n =7, T6U : n =7, T3: n =4) were killed 6 weeks later. Representative pictures of lungs (top) and images from a fluorescence dissecting stereo-microscope (bottom). Scale bar, 5 mm. ( d ) Primary tumours and metastases (arrows) following orthotopic injections of SJSA-1 cells (1 × 10 5 ) with or without A3AR knockdown. Mice were monitored for ∼2 months. Graphs showing primary tumour weight and number of metastatic nodules in the lungs. Representative images of the primary tumours and metastatic nodules below the graphs. Error bars: means±s.d. ( n =5 mice per group). * P <0.05 and ** P <0.01; Student's t -test (two-tailed). ( e ) Immunohistochemistry (IHC) for TMIGD3 (left) and A3AR (right) in human OS and normal tissues. IHC analyses were performed independently in OS tissues ( n =54 primary tissues and 17 metastases), as well as normal lung ( n =10) and bone ( n =10) tissues. Scoring was done on a scale of 0–6. Representative images of IHC are below the graphs. The horizontal lines in the plots represent the median. ** P <0.01; one-way ANOVA. Scale bar, 50 μm.

Article Snippet: All the human OS cell lines including SJSA-1, U2OS, KHOS/NP, MG63 and Saos2 were purchased from ATCC and maintained in DMEM medium or Roswell Park Memorial Institute medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: shRNA, Control, Knockdown, Injection, Two Tailed Test, Infection, Fluorescence, Microscopy, Immunohistochemistry

Journal: Clinical Cancer Research

Article Title: The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 Upregulation in Osteosarcoma Preclinical Models

doi: 10.1158/1078-0432.ccr-12-2293

Figure Lengend Snippet: Figure 1. Effects of sorafenib and everolimus on the mTOR pathway in preclinical models of osteosarcoma both in vitro and in vivo. A, quantification of Western blot analysis bands obtained after 5 hours of treatment of KHOS, MNNG-HOS, and U-2 OS with sorafenib (5 mmol/L) compared with the vehicle-treated control. B, immunuhistochemistry analysis of P-S6 and P-mTOR Ser2481 as functional readouts of mTORC1 and mTORC2 activity, respectively, in vehicle- and sorafenib-treated MNNG-HOS xenografts, , P < 0.05. C, top, Western blot analysis for the activation status of mTORC1 (P-mTOR Ser2448, P-p70 S6K Ser387, P-4E BP1 Ser65), and mTORC2 (P-mTOR Ser2481, P- Rictor Thr1135, P-AKT Ser247) after a 5 hours exposure of KHOS cells to sorafenib (5 mmol/L), everolimus (10 nmol/L), and their combination. Bottom, analysis of the mTORC2 complex under the same conditions: mTOR and Rictor staining following Rictor immunoprecipitation.

Article Snippet: Cell cultures, drugs, and reagents Human osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2 OS, SJSA-1, and SAOS-2) were purchased from the American Type Culture Collection and were cultured in RPMI-1640 (Invitrogen) augmented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin.

Techniques: In Vitro, In Vivo, Western Blot, Control, Functional Assay, Activity Assay, Activation Assay, Staining, Immunoprecipitation

Journal: Clinical Cancer Research

Article Title: The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 Upregulation in Osteosarcoma Preclinical Models

doi: 10.1158/1078-0432.ccr-12-2293

Figure Lengend Snippet: Figure 2. Combined sorafenib- and everolimus-induced apoptosis mediated by AMPK activation and ROS production in osteosarcoma cells. A, Western blot analysis for the phosphorylation status of ERK1/2, LKB1 (negative regulatory site Ser428), and AMPK (activation site Thr172) after a 5-hour exposure of KHOS cells to sorafenib 2.5 mmol/L, everolimus 10 nmol/L, and their combination. Total ERK, AMPK, and LKB1 are shown. B, percentage of early and late apoptotic cells (annexin V–positive cells) and ROS-containing cells (carboxy-H2-DCFDA–positive cells) after 24 hours and

Article Snippet: Cell cultures, drugs, and reagents Human osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2 OS, SJSA-1, and SAOS-2) were purchased from the American Type Culture Collection and were cultured in RPMI-1640 (Invitrogen) augmented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin.

Techniques: Activation Assay, Western Blot, Phospho-proteomics

Journal: Clinical Cancer Research

Article Title: The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 Upregulation in Osteosarcoma Preclinical Models

doi: 10.1158/1078-0432.ccr-12-2293

Figure Lengend Snippet: Figure 3. Sorafenib and everolimus in combination potentiates the antiproliferative and proapoptotic effects of the single drugs in osteosarcoma cells. A, dose- dependence curve of viability inhibition effect calculated after 72 hours treatment of U-2 OS cells with scalar doses of sorafenib, everolimus, and their combination. B, colony growth assay. After colony establishment, KHOS, MNNG-HOS, and U-2 OS cells were cultured for 10 days in the presence of 2.5 mmol/L sorafenib, 10 nmol/L everolimus, and their combination. They were then fixed and stained with crystal violet. C, average surface occupied by cell colonies, Y error bars ¼ SD; , P 0.05 versus control and single agents. D, cell-cycle analysis. Flow cytometry evaluation of DNA content (PI staining) of KHOS cells after 48 hours of treatment with 2.5 mmol/L sorafenib, 10 nmol/L everolimus, and their combination. E, Western blot analysis evaluation of PARP cleavage in KHOS cells after 24 hours of treatment with 2.5 mmol/L sorafenib, 10 nmol/L everolimus, and their combination. Vinculin staining is visualized as a loading control.

Article Snippet: Cell cultures, drugs, and reagents Human osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2 OS, SJSA-1, and SAOS-2) were purchased from the American Type Culture Collection and were cultured in RPMI-1640 (Invitrogen) augmented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin.

Techniques: Inhibition, Growth Assay, Cell Culture, Staining, Control, Cell Cycle Assay, Flow Cytometry, Western Blot

Journal: Clinical Cancer Research

Article Title: The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 Upregulation in Osteosarcoma Preclinical Models

doi: 10.1158/1078-0432.ccr-12-2293

Figure Lengend Snippet: Figure 4. The combination of sorafenib and everolimus increases the antiproliferative, proapoptotic, and antimetastatic effects of the single drugs in mouse models of human osteosarcoma. A, percentage of PCNA-positive (proliferating) cells and (B) TUNEL-positive (apoptotic) cells in MNNG-HOS xenograft sections from treatments: vehicle (control), sorafenib (5 mg/kg/d), everolimus (1 mg/kg/d), combination, and 10-fold-diluted combination.

Article Snippet: Cell cultures, drugs, and reagents Human osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2 OS, SJSA-1, and SAOS-2) were purchased from the American Type Culture Collection and were cultured in RPMI-1640 (Invitrogen) augmented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin.

Techniques: TUNEL Assay, Control

Journal: Cancer Management and Research

Article Title: A Novel Predictive Model Associated with Osteosarcoma Metastasis

doi: 10.2147/CMAR.S332387

Figure Lengend Snippet: The qPCR result of seven lncRNAs in five OS highly metastasis and weakly metastasis cell lines. ( A ) AL512422.1. ( B ) AL008718.3. ( C ) AC006033.2. ( D ) AL357507.1. ( E ) AL360182.2. ( F ) C5orf66-AS1. ( G ) CEBPA-DT. *<0.05, **<0.01, ***<0.001, ****<0.0001.

Article Snippet: Highly metastasis human OS cell lines (MNNG) and weakly metastasis human OS cell lines (U2S2, SAOS-2, SJSA-1, and HOS) were purchased from iCell (Shanghai, China).

Techniques: