Imaris

MDA-MB-231 cells grown on SecureSlip coverslips (Sigma-Aldrich) were fixed with 4% paraformaldehyde (PFA), then permeabilized with 0.1% Triton X-100 in HBSS for 15 min and washed with HBSS. Cells were incubated with 3% BSA in HBSS for 1 h and then with TOM20 primary antibody diluted for 1:100 overnight at 4 °C. Cells washed three times with HBSS were incubated with fluorochrome-conjugated secondary antibody diluted for 1 or 2 h at RT in the dark. Coverslips were mounted on a glass slide after DAPI in Fluoroshield Mounting Medium was added to the top of the cells. Fluorescence images were taken with Zeiss LSM510 META with IMARIS (Zeiss).

A z-series of super-resolution images was acquired with a super-resolution spinning-disk confocal microscope (SpinSR10, Olympus) and was subjected to deconvolution and calculation of Manders overlap with imaging software (cellSens, Olympus). The images were also visualized with three-dimensional image construction software (Imaris, Zeiss).

The Dnmt1-KO ESCs stably expressing hKO1-HP1γ was established by the transfection of the linearized CAG- hKO1-HP1γ expressing plasmid. Time-lapse fluorescence microscopy was performed using an upright OLYMPUS confocal microscope (SpinSR10) with an x100 oil immersion lens. Images were taken every 2 min and processed using cellSens (OLYMPUS) and IMARIS (Carl Zeiss) software.

For image analysis, Imaris (version 8, RRID:SCR_007370), ZEN (ZEISS Efficient Navigation, Carl Zeiss), ImageJ (version 1.53) distributed by Fiji (RRID:SCR_002285) (Schindelin et al., 2012 (link)), and TRI/3DBON (FCS64, Ratoc System Engineering, Japan) software were used. For visualization of myofibrils, the plasma membrane of muscle cells was traced with nine-pixel-width lines on serial cross sections of laboratory-based Zernike X-ray tomographic microscopy. Using the mask of plasma membrane, we performed segmentation of myofibrils. To generate a schematic model, FreeCAD (version 0.19, available from http://www.freecadweb.org) was used. Adobe Photoshop (RRID:SCR_014199) was used for pseudo-coloring of TEM images.

RD-SCARB2 cells and RD cells were seeded onto 16-well Lab-Tek Chamber Slide (Nunc). One day after seeding, purified EV71 were added at an MOI of 25 at 4°C to allow viral attachment to the cell surface without entry. In selected cases, cells were pretreated with or without 40 mM NH₄Cl or 5 mM 3-MA for 30 min. Cells were then shifted to 37°C (designated as time 0), washed with PBS (-) with or without NH₄Cl or 3-MA, fixed in PBS containing 4% paraformaldehyde (PFA) for 15 min and washed four times with cold PBS before further processing. In Fig. 3, to detect EGF localization, we added 2 μg/ml of biotinylated EGF bound to AlexaFluor555 streptavidin (Invitrogen) in the medium. Fixed cells were incubated with PBS containing 0.05% saponin and 5% bovine serum albumin fraction V (Sigma-Aldrich) to permeabilize the membrane and block nonspecific reactions. Samples were incubated overnight at 4°C with primary antibodies. After being washed with PBS (-), samples were then incubated with the secondary antibodies for 90 min at room temperature, and after another PBS (-) wash, mounted in Vectashield with DAPI Mounting Medium (Vector Laboratories). Samples were imaged with a laser-scanning microscope (TCS SP2, Leica Microsystems). Images were analyzed by Imaris (Zeiss).