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Additional Supporting Information may be found in the online version of this article.

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CYTO_22265_sm_SuppFig1.tif8543KSupporting Information Figure 1: Single-staining analysis of the targeted parameters. Bivariate DNA distribution and mean intensity per pixel per cell of the indicated parameters in the blank (DNA alone plus secondary antibodies), or single stained samples validating the distributions reported in the 7 parameter combined analysis. The observed cross talk in the Pacific Orange channel due to the EdU-Pacific Blue staining did not significantly contribute to the detection of the 53BP1 protein targeted in the channel (even after subtraction of the spill-over the percentage of midN 53BP1 positive remained essentially unaltered).
CYTO_22265_sm_SuppFig2.tif12525KSupporting Information Figure 2: Determination of a reference intensity for arbitrary discrimination of low and high expressing cell populations. Histograms report the smoothed distribution of the targeted parameters (light blue line) and the Gaussian fit calculated from values up to the first peak (red line). The Gaussian curve was used to determine the position of the level discriminating between low and high intensity populations.
CYTO_22265_sm_SuppFig3.tif11818KSupporting Information Figure 3: Comparison of different estimation-parameters of cell-cycle related phosphorylation levels of H2AX histone. Dot-plots report the bivariate distribution of DNA content versus mean intensity per pixel per nucleus (A), total intensity per nucleus (B) and integrated intensity of foci per nucleus (C) of the gammaH2AX associated fluorescence. The S-phase associated increase was detected independently from the adopted estimating parameter.
CYTO_22265_sm_SuppFig4.tif12840KSupporting Information Figure 4: A persistent fraction of quiescent cells in an exponentially growing population expresses p63, a stem-cell putative marker. KI67 proliferation antigen staining was employed to target the G0 fraction of the cell population (Magenta). A) Combined molecular phenotyping reveals that together with the CDKI p21, quiescent cells expresses high levels of 53BP1 and of the putative stem cell marker p63 as shown in the reported dot plots. B) Representative images of the selected population.
CYTO_22265_sm_SuppFig5.tif15346KSupporting Information Figure 5: Analysis of highly-expressing cell populations unmasks cell-cycle modulated relations between checkpoints mediators and DDR. The expression levels of p21, p53, 53BP1, gammaH2AX and 53BP1 foci were compared according to their ploidy. Each column reports the distribution of the corresponding highly expressing populations in all mentioned channels (e.g. first column shows the distribution of the p21 high-expressing population (Magenta) in the bivariate plots DNA content vs i) p21 ii) p53 iii) 53BP1 as mean intensity per pixel per cell and iv) gammaH2AX and v) 53BP1 foci total intensity per cell)). The fraction of cells present in the corresponding highly expressing fraction of the analysed parameter is reported (e.g. in the first row second column plot 91% of the p53 Highly expressing cells is part of the p21 highly expressing populations). The colored line report the average value of the corresponding populations (e.g. in the first row second column plot the magenta line reports the p21 average content of the p21 high expressing fraction and the red one the p21 average content of the p53 highly expressing population). Each panel reports the values calculated for the 2N, midN and 4N DNA content populations.
CYTO_22265_sm_SuppFig6.tif3867KSupporting Information Figure 6: gammaH2AX and 53BP1 foci population analysis unmasks different hierarchical positions in the DDR network. The gammaH2AX and 53BP1 mean fluorescence of gammaH2AX (Red) and 53BP1 (Green) populations of foci were plotted. While 53BP1 foci are almost homogenously distributed, a distinct fraction of gammaH2AX foci distributed along the X axis shows a 53BP1 independent damage recognition.
CYTO_22265_sm_SuppFig7.tif6016KSupporting Information Figure 7: gammaH2AX cell-cycle distribution is conserved among different cell lines. The integrated intensity of gammaH2AX foci per cell was plotted according to the cell-cycle phase (identified by DNA versus EdU content). Increased levels of gammaH2AX were detected during DNA replication, independently of the normal (MRC5), immortalized (BJTert, HaCat) and malignant (HCT116, U2OS) biological background.
CYTO_22265_sm_SuppFig8.tif7104KSupporting Information Figure 8: 53BP1 foci cell-cycle distribution is conserved among different cell lines. The integrated intensity of 53BP1 foci per cell was plotted according to the cell-cycle phase (identified by DNA versus EdU content). Increased intensity of 53BP1 foci was detected in G1 and G2 but not during DNA replication, independently of the normal (MRC5), immortalized (BJTert, HaCat) and malignant (HCT116, U2OS) biological background.
CYTO_22265_sm_SuppFig9.tif7104KSupporting Information Figure 9: p21 cell-cycle distribution is conserved among different cell lines. The mean p21 fluorescence intensity per pixel per cell was plotted according to the cell-cycle phase (identified by DNA versus EdU content). Increased intensity was detected in G1 and G2 but not during DNA replication, independently of the normal (MRC5), immortalized (BJTert, HaCat) and malignant (HCT116, U2OS) biological background.
CYTO_22265_sm_SuppFig10.tif14304KSupporting Information Figure 10: p21 increased expression in G1 is not always associated with DDR activation in different cell lines. GammaH2AX foci versus 53BP1 foci integrated intensity per cell reporting the distribution of 2N p21 high-expressing cells (red dots). A variable, but consistent, fraction of cells showing low DDR activation has been detected in all the analyzed cell lines.
CYTO_22265_sm_SuppFig11.tif12777KSupporting Information Figure 11: Quiescence is not associated to p53 and DDR activation in p53-wild-type non-transformed biological background. Quiescent cells in normal fibroblasts were identified as KI67 negative cells (first row). DNA versus p53 content (second row) and GammaH2AX foci versus 53BP1 foci integrated intensity per cell distribution showed absence of p53 and DDR activation in this sub-population.
CYTO_22265_sm_SuppTab1.doc41KSupporting Information Table 1: Fluorescence Filter Configuration for seven fluorescence parameter combination. The table reports the features of the employed fluorescence filters. The excitation source was a 150 W Me-Xe Lamp.
CYTO_22265_sm_SuppTab2.doc44KSupporting Information Table 2:High-content 7-parameter analysis of normal, immortalized and transformed cell lines. Several cell lines were analyzed to verify the general biological representation of the phenotypes and cell-cycle distribution measured in MCF10A cells. p53 expression profiling was not included due to the heterogeneous genetic background of the TP53 locus. pH2AX and 53BP1 columns refers to the properties described in the text (e.g. increased pH2AX foci intensity in S phase, 53BP1 foci peaking, replication-specific downregulation and DDR independent p21-overexpression, p53 and DDR-independent KI67-negativity). ND: not detectable. NA: not analysed. Known genetic and/or functional alterations of the analyzed cell line were reported in the Notes column.

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