Quantitative impairment in F53BP1 MPL responses in cells depleted of RNF8.

<p><b>A</b>) Endogenous 53BP1 and γH2AX focus formation in wild type MEFs transfected with control siRNA or siRNA directed against RNF8 1 hour after 3 Gy IR treatment. Quantification of the proportion of cells containing endogenous 53BP1 foci in control siRNA and siRNF8 treated cells. <b>B</b>) Quantification by RT-qPCR of the relative RNF8 mRNA level in cells transfected with control siRNA or siRNF8. <b>C</b>) Plot of peak fluorescence intensity for each responding cell. Bar represents the mean peak fluorescence intensity for each data set and error bars indicate SEM (p<0.004). Black circles: siLuc; Red squares: siRNF8. <b>D</b>) Plot of averaged mCherry-F53BP1 fluorescence accumulation over time normalized to a peak fluorescence intensity of 1.0 for each responding cell. <b>E</b>) Plot of slope of fluorescence accumulation at the inflection point for each responding cell. Bar represents the mean slope in fluorescence accumulation at the inflection point for each data set and error bars indicate SEM (p<0.013). <b>F</b>) Plot of lag-time in fluorescence accumulation for each responding cell. Bar represents the mean lag-time in fluorescence accumulation for each data set and error bars indicate SEM (p<0.0004). <b>G</b>) Plot of slope <i>vs</i>. peak fluorescence intensity for each responding cell. <b>H</b>) Plot of lag-time <i>vs</i>.peak fluorescence intensity for each responding cell. <b>I</b>) Plot of slope <i>vs</i>. lag-time for each responding cell.</p

Recruitment of mCherry-F53BP1 to sites of damage induced by multi-photon laser.

<p><b>A</b>) Endogenous 53BP1 and mCherry-F53BP1 focus formation in wild type MEFs 30 minutes after 3 Gy IR treatment. <b>B</b>) Representative images of mCherry-F53BP1 accumulation over time at site of MPL-induced DNA damage. <b>C</b>) Plot of accumulation kinetics of mCherry-F53BP1 to MPL-induced damage demonstrates the lag in 53BP1 recruitment. Blue diamonds represent raw data, red line represents mathematical (Gompertz) model fit to raw data. The mathematical model allows the parameterization of different kinetic behaviors. The “lag-time” is the time it takes for protein recruitment to reach the inflection point of the curve, where the rate of change of the slope is equal to 0. The “slope” of the curve is measured at the inflection point. <b>D</b>) Plot of the accumulation of GFP-MDC1 to MPL-induced damage over time is well fitted by first order kinetics (red line).</p

Interaction of maytansinoids with MCF7 cells.

<p>A. Binding of the maytansinoid ³[H]-<i>S</i>-methyl DM1 to the cells, a representative experiment. Cells were incubated in culture medium with various concentrations of ³[H]-<i>S</i>-methyl DM1 at 37° C for 4 h (equilibrium was reached within 4 h) with (squares) or without (circles) an excess (5 μM) of non-labeled <i>S</i>-methyl DM1, washed two times with medium, and cell-associated radioactivity was measured on a scintillation counter. The specific binding (triangles) was calculated as the difference between the two values. Cell-associated ³[H]-<i>S</i>-methyl DM1 was fully retained by cells after multiple washes, and thus the equilibrium was not distorted during the washes. B. Scatchard plot of A. C. The relationship between the cytotoxicity (IC₅₀) of a maytansinoid and its apparent affinity (ED₅₀) to cells. The IC₅₀ values were determined in clonogenic assays after a 4-h exposure of cells to a maytansinoid. Each point (the mean ± standard error) is the result of three independent experiments. The ED₅₀ is a concentration of a maytansinoid that decreased the binding of a trace amount of ³[H]-<i>S</i>-methyl DM1 by 50% in a competition binding assay. D. Structures of the maytansinoids. E. Efflux of ³[H]-<i>S</i>-methyl DM1 from MCF7 cells at 37° C in medium (filled symbols), or in the presence of an excess of non-labeled <i>S</i>-methyl DM1 (open symbols). Triangles and squares represent two independent experiments.</p

FCS Autocorrelation analysis of ECFP-tubulin in PTK2 cells treated with microtubule depolymerizing agents.

<p>Each compound was added into culture medium at the following final concentration: nocodazole (0.3 μM), <i>S</i>-methyl DM1 (0.2 μM), demecolcine (1.6 μM) and vinblastine (1 μM). Average molecular number (ECFP-tubulin) and average residence time (inversely related to diffusion constant) were determined by fitting time autocorrelation curves (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117523#pone.0117523.g004" target="_blank">Fig. 4A</a>) to a 3D diffusion model (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117523#sec002" target="_blank">Methods</a>).</p><p>FCS Autocorrelation analysis of ECFP-tubulin in PTK2 cells treated with microtubule depolymerizing agents.</p

Perturbed 53BP1 focus formation in cells depleted of H4K20me1.

<p><b>A</b>) Top: Immunoblot for HA-PR-Set7 proteins (WT = wild type, CD = catalytically dead) and β-actin loading control in wild type and <i>Suv4-20h1/2</i> null cells. Bottom: Immunoblot for H4K20me1 and H4 loading control in <i>Suv4-20h1/2</i> null cells infected with empty, PR-Set7 WT and PR-Set7 CD retrovirus. <b>B</b>) 53BP1 focus formation and HA immunofluorescence staining in <i>Suv4-20h1/2</i> null cells expressing empty or HA-PR-Set7 WT retrovirus, 30 minutes after 3 Gy IR treatment.</p

Fluorescent microscopy photographs of ECFP-PTK2 cells treated with various microtubule depolymerizing agents.

<p>Each compound was added into culture medium with the following final concentration and incubated for the following time period: nocodazole (0.3 μM for 60 min), <i>S</i>-methyl DM1 (0.2 μM for 90 min), demecolcine (1.6 μM for 90 min) and vinblastine (1 μM for 90 min). Scale bars are 10 μm for main images and 5 μm for sub-region images.</p

Impact of WHSC1 inactivation on 53BP1 DSB responses.

<p><b>A</b>)–<b>C</b>). Comparison of wild type (WT) vs. <i>WHSC1^mut/mut</i> (Whsc1 mut) MEFs. <b>D</b>)–<b>F</b>) Comparison of wild type MEFs transfected with siRNA to Luciferase (siLuc; control sample) or WHSC1 (siWHSC1). <b>A</b>) and <b>D</b>) Plot of averaged mCherry-F53BP1 fluorescence accumulation over time normalized to a peak fluorescence intensity of 1.0 for each responding cell. Error bars indicate s.d.. <b>B</b>) and <b>E</b>) Endogenous 53BP1 and γH2AX focus formation in cultures 30 minutes following treatment with 3 Gy IR. <b>C</b>) and <b>F</b>) Immunoblot with antibody specific for WHSC1 N-terminus and for β-actin (loading control) in test cultures. FL = full length WHSC1, Tr = likely truncated <i>WHSC1</i> gene product, * = unidentified band, a potential <i>MHSC1</i> gene product in view of its depletion by siWHSC1. Panel <b>C</b> also shows abundance of mono- and di-methylated H4K20 in WT vs. <i>WHSC1</i>^mut/mut MEFs, with histone H4 as a loading control. Note slightly increased abundance of H4K20me1 in <i>WHSC1</i>^mut/mut MEFs.</p

Impact of altered H4K20 methylation on recruitment kinetics of F53BP1 to MPL-induced DSBs.

<p><b>A</b>)–<b>D</b>) Comparison of wild type (WT) vs. <i>Suv4-20h1/2</i> null (h1/2 null) MEFs. <b>E</b>)–<b>H</b>) Comparison of <i>Suv4-20h1/2</i> null MEFs expressing empty retrovirus vs. PR-SET7 retrovirus. <b>A</b>) and <b>E</b>) Plot of peak fluorescence intensity for each responding cell. Note that the high proportion of non-responsive cells in PR-SET7 cultures are not represented in this analysis. Bar represents the mean peak fluorescence intensity for each data set and error bars indicate s.e.m. (<b>A</b>: p<0.45; <b>E</b>: p<0.09). <b>B</b>) and <b>F</b>) Plot of averaged mCherry-F53BP1 fluorescence accumulation over time, normalized to a peak fluorescence intensity of 1.0 for each responding cell. <b>C</b>) and <b>G</b>) Plot of slope in fluorescence accumulation at the inflection point for each cell imaged. Bar represents the mean slope and error bars indicate s.e.m. (<b>C</b>: p<0.18; <b>G</b>: p<0.0026). <b>D</b>) and <b>H</b>) Plot of lag-time in fluorescence accumulation for each cell imaged. Bar represents the mean lag-time and error bars indicate s.e.m. (<b>D</b>: p<0.4; <b>H</b>: p<0.0001).</p

Intracellular accumulation of <i>S-</i>methyl DM1 in cells <i>via</i> its low-affinity interaction tubulin.

<p>The large number of intracellular tubulin molecules leads to accumulation of <i>S-</i>methyl DM1 inside cells at low external concentrations. <i>S-</i>methyl DM1 loosely associated with tubulin is available for binding to the tips of mitotic spindle microtubules, which leads to suppression of their dynamic instability, interfering with faithful chromosome segregation during mitosis. This in turn induces cell cycle arrest and subsequent cell death.</p

<i>S</i>-methyl DM1 does not induce oligomerization of ECFP tubulin in ECFP-PTK2 cells.

<p>A. Intracellular fluorescence autocorrelation curves for ECFP tubulin in ECFP-PTK2 cells treated with various microtubule depolymerizing agents. Each curve was the average of individual cell measurements: a total of 30 cells and 144 measurements were collected for average for non-treated cells, a total of 12 cells and 60 measurements for nocodazole, a total of 9 cells and 54 measurements for both <i>S</i>-methyl DM1 and demecolcine, and a total of 11 cells and 52 measurements for vinblastine. B. Diffusion coefficients of cytoplasmic tubulin obtained from the FCS measurements in living cells treated with microtubule depolymerizing agents. Whiskers indicate minimum to maximum and box median value (line within the box).</p