Figure 2.

γH2AX activation in response to camptothecin is restricted to S-phase cells

Asynchronously growing U-2 OS cells were left untreated or treated with 1 μM of camptothecin (CPT) for 1 h. Cells were subsequently treated with extraction buffer (CSK-Triton 0.5%) before fixation with paraformaldehyde and staining for DNA content, EdU and γH2AX signal for quantitative image-based cytometry analysis. (A) Representative images for DAPI, EdU and γH2AX (invert LUT) staining are presented. Right panels: nuclei segmented with the in house developed Fiji-script (02_StarDist-QIBC_v12.ijm) are outlined in yellow and nuclei cell-cycle phase is indicated. The acquired images full size of acquired images is 2034 × 2034 pixels, an 800 × 800 pixels sector is presented for each wavelength. Scale bar: 20 μM. (B) Multivariate scatter plots displaying QIBC analysis from (A) representing the relationship among nuclei total DAPI intensity (X axis), mean EdU intensity (Y axis) and γH2AX mean intensity (color gradient). Number of analyzed nuclei (n) is indicated for each sample. (C) γH2AX signal intensity as a function of the cell-cycle phase in nuclei of untreated cells or cells treated with CPT. The bounds of the box are 25–75th percentile, center shows the median, whiskers indicate the 5–95 percentiles and the data points outside of this range are drawn as individual dots. Statistical analysis was performed with Prism (GraphPad) software. Data were analyzed with the two-tailed non-parametric Kruskal-Wallis test followed by a two-stage linear set-up procedure of Bonferroni, Kreiger and Yekutieliwas.