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4 protocols using glass bottom dishes

1

Cellular FRET Dynamics with Imatinib Treatment

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Cells expressing either fluorescent protein-tagged or epitopetagged proteins were plated on 35-mm-diameter glass-bottom dishes (AGC Techno Glass, Shizuoka, Japan) coated with poly-Llysine-(for hematopoietic cells) or collagen-coated (for nonhema-topoietic cells). During the observation, cells were maintained in phenol red-free RPMI 1640 (for hematopoietic cells, Thermo Fisher Scientific) or phenol red-free DMEM/F12 (Thermo Fisher Scientific).
For imaging of FRET, cells were subjected to time-lapse, dualemission microscopy with an interval of every 30 sec. Beginning at 10 min, cells were treated with 2 μM imatinib. To represent FRET efficacy, the FRET/CFP emission ratio was calculated as the quotient of background-subtracted FRET and CFP images and normalized to that at time 0. In Fig. 3, FRET/CFP ratio images are shown in intensity-modulated display mode, in which eight colors from red to blue represent the normalized emission ratios, with the intensity of each color indicating the mean intensity of FRET.
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2

Visualizing Mitochondrial Morphology in H69 Cells

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The H69 cells, treated with 20 µM SFN or untreated control cells, were seeded into plates, at a density of 5×105 cells/ml, and incubated at 37°C for 96 h. To determine the mitochondrial morphology, the H69 cells were cultured in glass-bottom dishes (AGC Techno Glass Co., Ltd.) and transfected with a modified baculovirus vector (BacMam 2.0; Thermo Fisher Scientific, Inc.) encoding mitochondria-targeted green fluorescent protein according to the manufacturer's instructions. The vector (2 µl/10,000 cells) was transfected into the cells for 16 h at 37°C. The cells were incubated at 37°C in a humidified incubator with 5% CO2. The cell nuclei were stained with Hoechst33342 (5 µg/ml; Thermo Fisher Scientific, Inc.), at room temperature for 5 min in the dark box. Images of the cells were captured with an Olympus FV1000 confocal laser scanning microscope, using an α Plan-Apochromatic X ×100/1.46 NA objective with 3X digital zoom (Carl Zeiss AG).
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3

Mammalian Cell Expression Vectors and Reagents

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The expression vectors in mammalian cells, pAcGFP1-Actin, pAcGFP1-Tubulin, and pDsRed-Monomer-C1, were obtained from Clontech. Oligonucleotides used as PCR primers in the construction of expression plasmids for fusion proteins were custom-synthesized by Gene Design (Osaka, Japan). The WST-1 cell proliferation assay system was purchased from Takara Bio (Kyoto, Japan). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA). Glass-bottom dishes used for cell culture and imaging experiments were purchased from AGC Techno Glass (Shizuoka, Japan).
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4

Wound Healing Assay with Dynamin Inhibitors

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H1299 cells were cultured to confluence on glass-bottom dishes (35 mm diameter; AGC Techno Glass Co. Ltd., Tokyo, Japan) in DMEM supplemented with 0.2% FBS for 8 h. Thereafter, the cell layer was wounded with a plastic pipette tip as previously described (25 (link)). The cells were washed with DMEM supplemented with 0.2% FBS and incubated for 8 h in the presence of Dynasore, Dynole 34-2 or MitMAB at the indicated concentrations. For the negative control, cells were incubated with 1% dimethyl sulfoxide (DMSO). The cells were visualized by Giemsa staining, followed by the acquisition of phase contrast images from ≥20 randomly selected areas per dish. Areas filled with migrating cells were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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