Nanosecond electric pulses penetrate the nucleus and enhance speckle formation

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Abstract

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10 ns, 150 kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by ∼2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10 ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA–protein complexes.

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Materials and methods

Cell culture. Jurkat cells, a human T cell leukemia cell line [American Type Culture Collection (ATCC), Manassas, VA], were grown in RPMI-1640 medium (phenol red) containing 2 mM l-glutamine (ATCC, VA) and supplemented with 10% fetal bovine serum (ATCC, VA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma, St. Louis, MO), and were maintained at 37 °C with 5% CO2. The cells used in our experiments ranged between 30 and 50 passages. We verified that cell viability exceeded 90% daily and prior

Results

Using immune labeling, temporally, and spatially resolved images of nuclear speckles generated by 10 ns pulse-induced stimulation were measured by recording intra-nuclear fluorescence of Jurkat cells caused by the binding of Y12 antibody with FITC to the nuclear speckles. We examined the nuclear speckles before the pulse and at 10, 60, and 180 min post-pulse. Nuclear speckles were detected after pulsing by examining the cellular fluorescence corresponding to the predicted molecular weight of

Discussion

Compared to traditional electroporation, nsPEFs preferentially charge the membranes of subcellular organelles, thereby inducing distinct effects on cellular structure and function that are predominantly intracellular in nature [1], [2], [5], [8]. The resulting delayed plasma membrane permeabilization was likely secondary, arising due to subcellular effects [2] rather than direct electroporation [18], [19]. In previous confocal microscopic real time studies, we compared the nuclear and plasma

Acknowledgments

This work was supported in part by an Air Force Office of Scientific Research (AFOSR) MURI grant on Subcellular Response to Narrow Band and Wide Band Radio Frequency Radiation, administered by Old Dominion University. One author (A.L.G.) was supported by a National Defense Science and Engineering Graduate Fellowship sponsored by the Department of Defense.

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    Present address: GE Global Research Center, Niskayuna, NY 12309, USA.

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