Journal: Journal of Gastrointestinal Oncology

Article Title: Nestin overexpression reduces the sensitivity of gastric cancer cells to trastuzumab

doi: 10.21037/jgo-22-1048

Figure Lengend Snippet: Influence of Nestin on the activation of Nrf2/HO-1 and HER2/AKT/ERK1/2 of HER2-positive GC cells. The two cell lines received transfection by lentiviral-Nestin or lentiviral-empty, and then added to 10 μg/mL for 48 h. (A,B) Nestin mRNA level in MKN45 and NCI-N87 cells was detected by qRT-PCR. (C,D) WB was adopted to determine the protein level of Nestin, Nrf2, HO-1, NQO1, GST, AKT, ERK1/2, and HER2, in the MKN45 and NCI-N87 cells. (E,F) The cells were exposed to ARE luciferase reporter. 36 h post transfection, the luciferase activity was determined. Data are presented as the means ± SD of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. NTC, non-treated control; TRA, trastuzumab; TRA + NC, mice inoculated with cell infected with lentiviral-negative control were treated with TRA; TRA + Nestin, mice inoculated with cell infected with lentiviral-Nestin were treated with TRA; HO-1, hemeoxygenase-1; NQO1, NAD(P)H: quinone oxidoreductase-1; GST, glutathione S-transferase; AKT, protein kinase B; ERK1/2, extracellular regulated kinase 1/2; HER2, human epidermal growth factor receptor 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ARE, antioxidant response element; GC, gastric cancer; qRT-PCR, quantitative real-time polymerase chain reaction; WB, Western blotting; SD, standard deviation.

Article Snippet: After a day of culture, the cells were treated with 200 ng of ARE luciferase reporter plasmids using OriGene transfection reagent (Company, City, Country).

Techniques: Activation Assay, Transfection, Quantitative RT-PCR, Luciferase, Activity Assay, Control, Infection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Cancers

Article Title: Tetraspanin CD9 is Regulated by miR-518f-5p and Functions in Breast Cell Migration and In Vivo Tumor Growth

doi: 10.3390/cancers12040795

Figure Lengend Snippet: Transfection of miR-518f-5p mimic increases breast cell migration and decreases CD9 protein levels. ( A ). Cell migration in 184A1 cells following transfection of a miR-518f-5p mimic assessed using transwell migration assay. ( B ). Migration of MDA-MB-231 cells following transfection of miR-518f-5p mimic. Migration assay results are shown as fluorescence intensity (arbitrary units), * p = 0.02, ** p = 0.002. ( C ). Proliferation of 184A1 cells using the resazurin assay, after transfection of miR-518f-5p mimic. ( D ). MDA-MB-231 proliferation following transfection of a miR-518f-5p mimic, as measured using resazurin assay. Cell proliferation results are shown as fold change fluorescence relative to 24 h fluorescence (arbitrary units). ( E ). Changes in cell adhesion to basement membrane extract (BME) and fibronectin (FN) were assessed using an adhesion assay, following transfection with miR-518f-5p mimic in 184A1 cells. ( F ). Cell adhesion was assessed in MDA-MB-231 cells following transfection of miR-518f-5p mimic. Adhesion results are expressed as a ratio of adhesion (fluorescence intensity of adherent cells compared to total seeded cells, arbitrary units), FN: * p = 0.04, BME: * p = 0.01, n = 3. ( G ). Total CD9 protein expression was measured using SDS-PAGE and western blotting, following transfection of a miR-518f-5p mimic in 184A1 (left) and MDA-MB-231 cells (right) for 72 h. ( H ). Representative western blots of CD9 total protein expression following transfection with miR-518f-5p mimic for 72 h. Results shown as CD9 total protein normalized to beta actin protein expression, n = 3, * p = 0.04, ** p = 0.001. See for full western blotting images.

Article Snippet: Cells were seeded in 96 well plates and transfected with the CD9 3’UTR or empty 3’UTR renilla reporter vector (SwitchGear Genomics, Menlo Park, CA, USA) and pmiR-Report firefly luciferase transfection control vector (Promega) using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA).

Techniques: Transfection, Migration, Transwell Migration Assay, Fluorescence, Resazurin Assay, Cell Adhesion Assay, Expressing, SDS Page, Western Blot