Metavue software

To obtain recombinant GST-GFP-YB-1-C2 protein, pGST-GFP-YB-1-C2 was transformed into E. coli strain BL21(DE3). GST-GFP-YB-1-C2 overexpressed in bacterial cells was purified through glutathione–Sepharose (GE Healthcare) as described previously^{73 (link)}, and dialyzed against 20 mM HEPES (pH 7.3), 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 0.5 mM EGTA, 1 mM dithiothreitol, and 0.5 mM phenylmethyl sulfonyl fluoride. Ran-GDP and HeLa cytosol fractions were prepared as described previously^{5 (link),74 (link)}. In vitro transport assays were performed essentially as previously described^{5 (link)}. After protein mixtures containing GST-GFP-YB1-C2 proteins, as indicated in figure legends, were incubated with the permeabilized HeLa cells for 20 min at 30 °C, the cells were washed and fixed with 3.7% formaldehyde in transport buffer^{5 (link)}. Nuclear fluorescent proteins were detected by fluorescence microscopy (Olympus BX51), and images were captured using an ORCA-ER camera (Hamamatsu) controlled by the MetaVue software (Universal Imaging).

Preparations were evaluated using a Zeiss Axioskop fluorescence microscope fitted with appropriate filters. For DAPI-stained and immunostained cells, three-dimensional image stacks of different colour channels were sequentially recorded at 63× magnification with a monochrome SPOT camera (Diagnostic Instruments) using MetaVue software (Universal Imaging), deconvolved using AutoQuant (Media Cybernetics), and projected with ImageJ (http://imagej.nih.gov/ij/). False colours were assigned and merged to composite images using Photoshop (Adobe Systems). Centromere and telomere positions were determined on Cna1-stained and telFISH preparations at 100× magnification. Nuclear lengths were measured on electronic images of Schaudinn-fixed Giemsa-stained preparations using the measuring tool in ImageJ by manual tracing.

Proximal tibias were dissected and fixed for 18 hours in 10% neutral buffered formalin. Tibias were then decalcified with ethylenediamine tetraacetic acid (EDTA), dehydrated in a graded ethanol series, and embedded in paraffin. Five‐micrometer (5‐μm) sections were cut longitudinally onto glass slides and stained with hematoxylin and eosin (H&E) per standard protocols.
Sections were imaged using a Nikon Eclipse E‐800 (Nikon, Tokyo, Japan) and Universal Imaging Corporation's MetaVue software (version 7.4.6; Bedford Hills, NY, USA). Images were taken with a 4× objective (909 pixels/1 mm). A central section proximal tibia was selected for analyses. Adipocytes were manually segmented, counted, and measured using the iPad app YouDoodle and custom MATLAB (MathWorks, Natick, MA, USA) code. Measurements included mean adipocyte area (mm²), marrow cavity area (mm²), and adipocyte number density (number of adipocytes per marrow cavity area).
To confirm that the technique utilized to flush marrow from the humerus, additional humeri were assessed. These bones were dissected, harvested, and sections were prepared, imaged, and analyzed consistent with the description for tibias. Microscopy confirmed that marrow was almost entirely removed from the flushing step (Fig. S1).

Cell viability during impedance cell detection events was confirmed using brightfield and fluorescence video microscopy. Live and dead cell viability control treatments were conducted, wherein Calcein AM was used to assess cell viability. In the microdevice, fluorescent video microscopy acquisition was acquired at a frame rate of 3 frames per second (fps) and an exposure time of 250 ms. Fluorescence microscopy was performed using an inverted Eclipse TS100 Nikon fluorescence microscope(Nikon, Tokyo, Japan), and image stacks were recorded on a CoolSnapFX camera (Photometrics, Tucson, AZ, USA). Image stacks were processed using MetaVue software (Universal Imaging Corporation, West Chester, PA, USA) on a Windows computer.