LMNA is required for the positional stability of DNA repair foci.

<p>(<b>A</b>) Western blots showing the expression of lamin A/C, GFP-lamin A, dsRed-53BP1TD and H2AX in a set of matched murine embryonic fibroblasts. The antibody used to probe cell lysates is given in brackets. The dividing dashed lines divide images taken from the same gel and same exposure. (<b>B</b>) Example confocal image of a dsRed-53BP1TD expressing cell, scale bar 8 µm. (<b>C</b>) Example timepoints of live cell imaging of a single dsRed-53BP1TD LMNA+/+ cell; coloured crosses indicate the positions of six DNA repair foci from which positional information was taken, scale bar 7 µm. Brightness and contrast settings were altered for display purposes. (<b>D</b>) The graph shows the average cumulative distance moved by DNA repair foci in a population of cells (n = 12 cells +/− s.e.m), calculated as described in <b>methods</b>.</p

Altered lamin A/C-H2AX interaction after DNA damage.

<p>(<b>A</b>) Example immunofluorescent images of lamin-Histone H2AX interactions measured by proximity ligation assay in murine embryonic fibroblasts. Red dots represent the PLA signal, produced from antibodies in close proximity (<40 nm), and blue shows DNA stained by Hoechst 33342 dye. (<b>i</b>) Negative controls containing an antibody against only one interaction partner (either lamin A/C, H2AX or γH2AX). (<b>ii</b>) Sample containing both lamin A/C and H2AX antibodies (<b>iii</b>) Sample containing both lamin A/C and γH2AX antibodies. Scale bars 20 µm. (<b>B</b>) Automated microscopy was used to automatically quantitate PLA signal −/+ DNA damage in LMNA−/− GFP-lamin A cells stained with Hoechst 33342. The images show one example field of cells each for − and + DNA damage, in two different fluorescent channels containing Hoechst 33342 staining (top) and PLA signal (middle). Images are overlayed with automated identification of: cells as objects (blue), cell nuclei (green) and PLA signal (red dots). The bottom panel shows a colour overlay of PLA (green dots) and Hoescht staining (blue). (<b>C</b>) The histograms show the number of PLA dots +/− s.e.m in >2000 cells from two independent experiments. (<b>i</b>–<b>iii</b>) show results from murine embryonic fibroblasts and (<b>iv</b>) is from human cells. (<b>D</b>) The image shows the location of the PLA signal obtained from interaction between lamin A/C and γH2AX relative to chromocenters demarked by Hoechst 33342 staining in one single nucleus. (<b>E</b>) GFP-lamin A - H2AX complexes were found by co-immuno-pulldown. LMNA−/− GFP-lamin A cells were lysed in NP40 buffer and GFP-lamin A was isolated using protein A/G beads and anti-GFP antibody. The eluted complexes were probed by western blot with antibodies against H2AX, IgG and lamin A/C. (<b>F</b>) GFP-lamin A - γH2AX complexes were found by co-immuno-pulldown. 293T cells, transfected with either GFP-lamin A or GFP alone, were processed as described in (<b>E</b>) and the eluted samples were probed by western blot with antibodies specific to GFP and γH2AX.</p

DNA damage induces a pan-nuclear decrease in GFP-lamin A mobility.

<p>(<b>A</b>) FRAP data from the nucleoplasm of U2OS cells stably expressing GFP-lamin A. GFP-lamin A was bleached in a circle ∼1 µm in diameter using 100% laser power and fluorescence recovery was monitored using 3% laser power. The image shows four GFP-lamin A cells bleached three minutes previously with the shapes of the letters “LMNA”, demonstrating relatively little movement of the protein on this timescale. Scale bar 20 µm. (<b>B</b>) Shows the results of FRAP experiments on GFP-lamin A. In (<b>i</b>) three different bleach spot sizes were used, denoted ROI1, ROI2, ROI3. The rate of recovery into each bleach spot was monitored and plotted as the halftime of recovery, shown on the graph and calculated as described in <b>methods</b>. In (<b>ii</b>) half of the cell was bleached, and recovery was monitored at varying distances from the non-bleached area, denoted ROI1, ROI2, ROI3 and ROI4. (<b>C</b>) The graph plots the average fluorescence recovery curves (+/− s.e.m.) taken from 14 cells as described in (<b>A</b>), either before (black) or after (red) DNA damage induction using 10Gy IR. (<b>D</b>) GFP-lamin A FRAP recovery curves were taken at varying times after 10Gy IR. The bar graph shows the mean % recovery (+/− s.e.m) of GFP-lamin A FRAP recovery curves. (<b>E</b>) Fluorescence correlation spectroscopy measurements were taken in GFP-lamin A cells either untreated or after incubation with the DNA damaging drug etoposide. Each dot shows the average concentration of mobile GFP-lamin A in the cytoplasm of a single cell during a 45 second measurement, calculated from a fit of the G0 of the autocorrelation function as described in <b>methods</b>. (<b>F</b>) GFP-lamin A FRAP measurements were taken inside and outside regions of DNA damage marked by dsRed-53BP1TD, either in asynchronously growing cells or in cells damaged using a 365 nm laser as described in <b>Fig. 1C</b>. The images show example cells in which measurements were taken and the graphs show mean recovery curves +/− s.e.m taken from 15 cells in two independent experiments.</p

GFP-lamin A does not accumulate at sites of DNA damage.

<p>(<b>A</b>) Western blot analysis of U2OS cells stably co-expressing GFP-lamin A and dsRed-53BP1TD, using multiple different antibodies as indicated. The whole gel is shown to indicate the absence of erroneously truncated forms of the proteins which might complicate analyses, and horizontal dashed lines indicate where the membrane was cut to incubate with different antibodies. (<b>B</b>) DNA damage was induced focally using a 405 nm laser following Hoechst 43332 pre-sensitisation, in one spot per cell (denoted by the white circles). GFP-lamin A and dsRed-53BP1TD were followed simultaneously by time-lapse imaging at one minute intervals. The images show example timepoints and the line profile graphs quantitate the fluorescence intensity of both channels in the zoomed region entitled “detail”. (<b>C</b>) DNA damage was induced focally using a 365 nm micropoint laser (Andor) and GFP-lamin A dsRed-53BP1TD were followed with time-lapse imaging using one minute intervals. The images are maximum intensity projections from confocal z-stacks, showing timepoints 60–161 min after DNA damage induction. The white arrows and white dashed line denote the route taken by the 365 nm laser used to induce DNA damage prior to time-lapse imaging. (<b>D</b>) Representative confocal image and line intensity profile of a fixed GFP-lamin A/dsRed-53BP1TD cell stained for DNA with Hoechst 33342 after treatment with the radio-mimetic topoisomerase inhibitor etoposide (5 µM). The black arrows on the x-axis of the line intensity profile graph indicate the position of the lamina, and the white dashed opaque line on the image indicates the position of the line profile shown in the graph. Scale bar 10 µm. (<b>E</b>) Confocal images of a cell damaged focally with a micropoint 365 nm laser (denoted by white arrows) and stained for the marker of DNA damage γH2AX and endogenous lamin A/C.</p

Overexpression of LSD1 is observed only in basal-like breast cancer.

<p>This is a plot of LSD1 expression from the TCGA dataset (expression of mRNA as log₂ TMM-normalized raw counts in the y-axis) plotted across different subtypes of breast cancer (x-axis). Significant differences in mRNA between intrinsic subtypes were calculated using edgeR. The number of samples were as shown in the scatter plots. LSD1 is amplified in basal-like breast cancer when compared to other subtypes, in this dataset.</p

mRNA expression of LSD1 in subtypes of breast cancer.

<p>False discovery rate calculated by edgeR are also shown as q-value.</p><p>mRNA expression of LSD1 in subtypes of breast cancer.</p

Overexpression of LSD1 is linked to poor outcome in triple negative breast cancer.

<p>Representative images of immunohistochemistry of LSD1 in 20 clinical samples are shown (A and B, LSD1 high and low, respectively). The Kaplan Meir curve compares the recurrence free survival of cancer with high or low level LSD1 protein products (C). Definition of LSD1 high is samples that have IHC scores above 5.</p

Protein expression of BRCA1 and LSD1 in triple negative breast cancer.

<p>IHC score are shown.</p><p>IHC score = SI*PP (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118002#sec002" target="_blank">material and methods</a>)</p><p>Protein expression of BRCA1 and LSD1 in triple negative breast cancer.</p

mRNA expression of LSD1 in subtypes of breast cancer.

<p>False discovery rate calculated by edgeR are also shown as q-value.</p><p>mRNA expression of LSD1 in subtypes of breast cancer.</p

Overexpression of LSD1 reduces BRCA1 expression in triple negative or in basal-like breast cancer and increases sensitivity to PARP inhibitor in basal-like breast cancer.

<p>Histogram shows protein expression of BRCA1 in triple negative breast cancer with high or low level of LSD1 protein (A). Y-axis indicates IHC score of BRCA1. Basal-like breast cancer cell lines (MDA-MB-231, MDA-MB-157 and HCC70) was transfected as indicated. Cell lysates were subjected for Western Blots with indicated antibodies (B). Cell viability of basal-like breast cancer cell lines are assessed by colony formation assay. Percent survival is shown in different doses of PARP inhibitor. Error bars show standard deviation of three independent experiments (C-E).</p