Journal: Bioscience Reports

Article Title: Knockdown of the oncogene lncRNA NEAT1 restores the availability of miR-34c and improves the sensitivity to cisplatin in osteosarcoma

doi: 10.1042/BSR20180375

Figure Lengend Snippet: ( A ) The expression of NEAT1 in OS cell lines MG63, 143B, HOS, and Saos2 cells and one human osteoblastic cell line hFOB1.19 cells were determined by Q-PCR. ( B ) The NEAT1 was knocked down in MG63 and HOS cell lines and ( C , D ) the cell vitalities ( E , F ) apoptosis and ( G , H ) cell cycle of two cell lines were analyzed by CCK-8 and flow cytometry. ( I ) The expressions of BCL-2, caspase-3, BAX, cyclin D1, and CDK were analyzed by western blot in MG63 and HOS cell lines. * P <0.05, ** P <0.01, *** P <0.001, data represent the means ± S.D.

Article Snippet: The proteins were separated by SDS/PAGE and transferred on to nitrocellulose membrane (Bio-Rad, Hercules, CA) which were blocked in 5% BSA-contained TBST buffer (TBS containing 0.1% Tween-20) for 1 h at room temperature, and subsequently the membrane was incubated with anti-Bcl-2, BAX, caspase-3, and cyclin D1, CDK2, and GAPDH antibodies overnight at 4°C.

Techniques: Expressing, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Bioscience Reports

Article Title: Knockdown of the oncogene lncRNA NEAT1 restores the availability of miR-34c and improves the sensitivity to cisplatin in osteosarcoma

doi: 10.1042/BSR20180375

Figure Lengend Snippet: ( A ) The expressions of miR-34c in OS tissues were analyzed by Q-PCR and ( B ) its correlation with NEAT1 was determined. ( C ) The expression of miR-34c in NEAT1 knockdown cells was assessed. After the transfection of siRNA-NEAT1 with/without miR-34c inhibitor, ( D , E ) the cell vitalities, ( F ) apoptosis and ( G , H ) cell cycle were determined by CCK-8 and flow cytometry in MG63 and HOS cell lines. ( I ) The expressions of BCL-2, caspase-3, BAX, cyclin D1, and CDK were analyzed by western blot in MG63 and HOS cell lines. * P <0.05, ** P <0.01, *** P <0.001, data represent the means ± S.D.

Article Snippet: The proteins were separated by SDS/PAGE and transferred on to nitrocellulose membrane (Bio-Rad, Hercules, CA) which were blocked in 5% BSA-contained TBST buffer (TBS containing 0.1% Tween-20) for 1 h at room temperature, and subsequently the membrane was incubated with anti-Bcl-2, BAX, caspase-3, and cyclin D1, CDK2, and GAPDH antibodies overnight at 4°C.

Techniques: Expressing, Transfection, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Bioscience Reports

Article Title: Knockdown of the oncogene lncRNA NEAT1 restores the availability of miR-34c and improves the sensitivity to cisplatin in osteosarcoma

doi: 10.1042/BSR20180375

Figure Lengend Snippet: The 2 × 10 6 conditional MG63 cells were transfected with lentivirus vector of siRNA-NEAT1 or negative control and were subcutaneously injected in rear flank of nude mice (six per group). ( A , B ) The mean tumor size (mm 3 ) and weight was analyzed. ( C ) The expressions of NEAT1 and miR-34c in tumor tissues were determined by Q-PCR. ( D , E ) The expressions of Ki-67, BCL-2, caspase-3, BAX, cyclin D1, and CDK were analyzed by IHC and western blot. * P <0.05, ** P <0.01, *** P <0.001, data represent the means ± S.D.

Article Snippet: The proteins were separated by SDS/PAGE and transferred on to nitrocellulose membrane (Bio-Rad, Hercules, CA) which were blocked in 5% BSA-contained TBST buffer (TBS containing 0.1% Tween-20) for 1 h at room temperature, and subsequently the membrane was incubated with anti-Bcl-2, BAX, caspase-3, and cyclin D1, CDK2, and GAPDH antibodies overnight at 4°C.

Techniques: Transfection, Plasmid Preparation, Negative Control, Injection, Western Blot

Journal:

Article Title: mTOR Activator Rheb Is Frequently Overexpressed In Human Carcinomas And Is Critical And Sufficient For Skin Epithelial Carcinogenesis

doi: 10.1158/0008-5472.CAN-09-3467

Figure Lengend Snippet: (A) STAT3 pathway activation in wild-type back skin, transgenic papilloma, and transgenic papillomas following RAD001 treatment for either 20 hours or 4 weeks. (B) Focal epidermal STAT3 activation (yellow arrow) with concomitant stromal inflammatory cell infiltration in hyperplastic transgenic back skin. (C) Induction of cyclin D1 expression in hyperplasia (upper panel) to nascent papilloma transition (lower panel). (D) STAT3 activation in wild-type and transgenic keratinocytes cultured for 16 hours without or with serum. Serum-starved transgenic and nontransgenic keratinocytes stimulated with 10ng/ml IL-6 for the indicated intervals alone or pretreated with rapamycin 4h before IL-6 stimulation. Bars (A, C): 100μm.

Article Snippet: Additional antibodies used were: rabbit anti-loricrin, anti-keratin 8, and anti-keratin 10 (1:500, 1:5000, and 1:50 respectively, Covance, Princeton, NJ), anti-cyclin D1 (1:100, Abcam), rat anti-CD45, anti-meca32, anti-CD4, anti-CD8a, anti-Gr-1 (1:50, 1:20, 1:20, 1:20, 1:20 respectively, BD Biosciences, San Jose, CA), anti-FoxP3 (1:200, eBioscience, San Diego, CA), anti-F4/80 (Serotec, Raleigh, NC) antibodies, and biotinylated mouse antibody against keratin 14 (1:1000, Lab Vision, Fremont, CA).

Techniques: Activation Assay, Transgenic Assay, Expressing, Cell Culture