Laser micro-irradiation experiment for Y14-silenced U2OS cells

Published: 20 February 2019| Version 1 | DOI: 10.17632/yym8r56fgf.1
Contributors:
Pei-Yu WU,
Chi-Chieh Lee,
Woan-Yuh Tarn

Description

U2OS cells that stably express the GFP-tagged MDC1, Ku70 or Ku80 were transiently transfected with or without the Y14 shRNA-mCherry-expressing vectors. Cells were subjected to laser-microirradiation with 405 nm UV, followed by live-cell imaging using a confocal microscopy. The representative confocal images of each experimental condition were in the folder "sequential_images". Images of the green fluorescence show accumulation of GFP-fusion proteins at sites of laser-microirradiation at indicated time points. Images of the red fluorescence show the expression of mCherry as the indication of shY14-expressing cells. Fluorescence intensities of GFP-fusion proteins at irradiated region in each tested cells were quantified periodically, normalized, and presented as mean ± S.D. for at least 10 cells in each experiment. Quantitative results were summarized in ".xlsx" files.

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Steps to reproduce

Laser micro-irradiation experiment: The cDNAs of Ku70 and Ku80 were fused with GFP cDNA, cloned into a retrovirus vector pLNCX (Clontech), and subjected to transduce U2OS cells as previously described (Pan et al., 2015). U2OS cells stably expressing GFP-fused MDC1 were from a previous study (Chang et al., 2015). Cells were mounted onto the incubation stage in a Leica SP5 X inverted confocal microscope (Leica Microsystems) supplemented with humidified 5% CO2 at 37 °C. Laser-microirradiation was carried out using a 405 nm laser diode with full output settings. Ten scans were applied to generate DNA damages restricted to the laser path with minimal cellular toxicity. Live-cell images were taken every 15 s using the same confocal microscope with the minimum-detectable output of a 488 nm laser diode. Quantification and statistics: To measure the recruitment of GFP-tagged proteins at laser micro-irradiation sites, fluorescence intensities in the cell nucleus were respectively measured by using ImageJ (National Institutes of Health). The Average of the intensities of two regions at the irradiated site at the time point (t) was designated as I(t), and that of two regions at non-irradiated background was designated as B(t). Relative Fluorescence was thus calculated using a normalization equation: [I(t)/I(preIR)+(1-B(t)/B(preIR)], where preIR indicates the intensities before laser micro-irradiation. Significance of results was calculated using two-tailed student T-test.