DNase I as a probe for unpolymerized actin: revisiting a classic tool for nuclear actin research

Figures

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  Fig. 1. Discovery of actin as a naturally occurring inhibitor of DNase I

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  Fig. 2. DNase I activity and staining patterns under different experimental conditions. A) Agarose gel electrophoresis of plasmid DNA after treatment with DNase I. Plasmid DNA was incubated without DNase I, with commercially available DNase I, and with labeled DNase I under different buffer conditions. Enzyme activity was observed in Mg2+-containing buffer (Mg2+) but not in PBS or PBS with EDTA. B) HeLa cells were washed with PBS and fixed in 4% PFA in PBS at the indicated temperatures. Cells were extracted using 0.2% Triton X in PBS and stained with Alexa Fluor 488–conjugated DNase I (1:500 in PBS, Invitrogen) and DAPI. A LSM800 (Zeiss) confocal laser scanning microscope equipped with a 40 × 1.3 oil immersion objective was used for microscopic analysis. The confocal pinhole was set to 1 Airy unit, and images were taken using a multi-track acquisition mode in the LSM Zen Blue software using the same settings for all images taken. Images were analyzed using the CellProfiler software. DAPI staining was used to identify the nuclear area. Cytoplasmic area was identified by DNase I staining omitting the nuclear area. The numbers in the bars indicate the number of cells analyzed. Data are…