Journal: Cell reports

Article Title: Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK

doi: 10.1016/j.celrep.2019.09.070

Figure Lengend Snippet:

Article Snippet: Hepa 1–6 , ATCC , CRL-1830.

Techniques: Virus, Plasmid Preparation, Recombinant, Lysis, Saline, Modification, Isolation, Cell Culture, Membrane, Activity Assay, DNA Extraction, Glucose Assay, Bioassay, cDNA Synthesis, SYBR Green Assay, Western Blot, Microarray, Software

Journal: Scientific Reports

Article Title: Elevated β-catenin pathway as a novel target for patients with resistance to EGF receptor targeting drugs

doi: 10.1038/srep13076

Figure Lengend Snippet: ( a ) Parental PC9 cells and PC9M2 cells were grown for 72 h in RPMI1640 with or without gefitinib at the indicated concentrations. Cell viability was measured using the MTT assay. The data are represented as mean ± SD, N = 4. ** P < 0.01 and *** P < 0.001. ( b ) DNA sequencing analysis to check for the presence of a secondary mutation in the EGFR in PC9M2 cells. ( c ) Western blotting of c ell lysates from parental PC9, from the M line of PC9 gefitinib-resistant clones including PC9M2 and from HEK293T cells, probed with anti-MET and anti-actin (loading control) antibodies. Pro-MET, precursor of MET. These cropped blots used in this figure were run under the same experimental conditions. ( d ) Western blotting of lysates of PC9 and PC9M2 cells, which were prepared from cells in the steady state, using anti-EGFR, anti-HER2, anti-HER3 and actin (loading control) antibodies. Experiments were performed at least three times and representative results were presented.

Article Snippet: Gefitinib, MM-2206 and ICG-001 were purchased from Selleck chemicals (Houston, TX, USA).

Techniques: MTT Assay, DNA Sequencing, Mutagenesis, Western Blot, Clone Assay, Control

Journal: Scientific Reports

Article Title: Elevated β-catenin pathway as a novel target for patients with resistance to EGF receptor targeting drugs

doi: 10.1038/srep13076

Figure Lengend Snippet: ( a ) Time course of mRNA sampling for DNA microarray analysis. PC9 and PC9M2 cells were starved for 48 h, then treated with or without gefitinib and cultured in the presence or absence of EGF. mRNA was extracted at the indicated times. ( b ) The mRNA expressions levels of the Wnt/β-catenin pathway related genes FZD5 , 7 and 9 and LEF of the cells in ( a ) were plotted using DNA microarray data. None/GFT; −/+ gefitinib respectively. ( c , d ) Western blotting of lysates of PC9 (P) and PC9M2 (M2) cells that were treated with gefitinib (GFT) at 1 μM for the indicated times ( c ) and at 0.5 μM for 1 hr ( d ). Treatment with DMSO is a control. Blots were probed with phospho-GSK3α, GSKα, β-actin (loading control), phospho-Akt and Akt antibodies. ( e ) Western blotting of β-catenin in the nucleus and cytosol/membrane fraction of PC9 (PC) or PC9M2 (M2) cells treated with gefitinib (GFT) at 1 μM or control DMSO for 1 hr. ( f ) The expression levels of Axin2 mRNA in PC9 and PC9M2 cells at the steady state, as measured using quantitative real-time (qRT)-PCR. The data are represented as mean ± SD, N = 4. *** P < 0.001 ( P = 4.89 e–6 ). ( g ) Western blotting of lysates of PC9 and PC9M2 cells that were treated with MM2206 at 10 μM for 5 hr. Blots were probed with phospho-Akt, Akt, phosphor-GSK3α, GSK3α, β-catenin and actin (loading control). ( c – e and g ) Experiments were perform e d at least three times and representative results were presented. These blots used in this figure were run under the same experimental conditions. Different pieces of the same protein blots or same samples with same conditions of electrophoresis and electrotransfer were used and presented.

Article Snippet: Gefitinib, MM-2206 and ICG-001 were purchased from Selleck chemicals (Houston, TX, USA).

Techniques: Sampling, Microarray, Cell Culture, Western Blot, Control, Membrane, Expressing, Quantitative RT-PCR, Electrophoresis, Electrotransfer

Journal: Scientific Reports

Article Title: Elevated β-catenin pathway as a novel target for patients with resistance to EGF receptor targeting drugs

doi: 10.1038/srep13076

Figure Lengend Snippet: ( a ) PC9M2 cells were transfected with β-catenin siRNA or control siRNA and these cells, or control PC9 cells, were treated with the indicated concentration of gefitinib for 72 h. Cell viability was determined using the MTT assay (N = 3). ( b ) PC9M2 cells were treated with the indicated concentration of ICG-001, or with control DMSO, in the presence or absence of gefitinib for 72 h. Cell viability was determined using the MTT assay (N = 3). The experiments were performed three times and the representative results were presented. The data are represented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.01.

Article Snippet: Gefitinib, MM-2206 and ICG-001 were purchased from Selleck chemicals (Houston, TX, USA).

Techniques: Transfection, Control, Concentration Assay, MTT Assay

Journal: Scientific Reports

Article Title: Elevated β-catenin pathway as a novel target for patients with resistance to EGF receptor targeting drugs

doi: 10.1038/srep13076

Figure Lengend Snippet: PC9 cells (upper graph) or PC9M2 cells (lower graph) were injected into the flanks of SCID mice. Oral administration of gefitinib (0 or 10 mg/kg/day) was started at 6 days after injection. Data were represented as mean ± SD (N = 6). ** P < 0.01.

Article Snippet: Gefitinib, MM-2206 and ICG-001 were purchased from Selleck chemicals (Houston, TX, USA).

Techniques: Injection

Journal: Scientific Reports

Article Title: Elevated β-catenin pathway as a novel target for patients with resistance to EGF receptor targeting drugs

doi: 10.1038/srep13076

Figure Lengend Snippet: ( a ) Parental PC9 or gefitinib resistant PC9M2 cells were injected into the flanks of SCID mice. The tumors derived from each cell type were harvested 21 days after cell injection. HE staining and immunohistochemical analysis of β-catenin expression and control IgG in representative tumors are shown. Scale bar = 20 μm. Cells with cytoplasm/nucleus staining (arrows) and cells with only plasma membrane staining (arrowheads) are indicated. ( b ) Cells with cytoplasm/nucleus staining were counted and the ratio in the overall cell count was calculated (N = 300). The data are represented as mean ± SD. *** P < 0.001 ( P = 0.00014).

Article Snippet: Gefitinib, MM-2206 and ICG-001 were purchased from Selleck chemicals (Houston, TX, USA).

Techniques: Injection, Derivative Assay, Staining, Immunohistochemical staining, Expressing, Control, Clinical Proteomics, Membrane, Cell Counting