Figure 1 . Cytoplasmic chromatin fragments in senescent cells . ( A ) Cytoplasmic chromatin fragments ( CCFs ) in senescent cells are strongly positive for histone H3 . Yellow arrow marks CCF . ( B ) Increased proportion of cells with CCFs in RS or OIS . Mean ± SEM , n = 3 ; P < 0 . 01 ( OIS ) , P < 0 . 002 ( RS ) . Assays were performed 10 d after induction with tamoxifen ( OIS ) or 4 wk after the last passage and > 90 % SA β - gal + ( RS ) . ( C ) CCFs in OIS cells are lamin A / C negative . ( D ) Cells from C were scored for lamin A / C + and lamin A / C − CCFs . Note that the majority of micronuclei in control cells are lamin A / C positive . Mean ± SEM , n = 3 ; P < 0 . 0006 for lamin A / C negative CCF con vs . OIS ; P < 0 . 6 for lamin A / C positive CCF con vs . OIS . ( E ) CCFs in OIS are strongly positive for heterochromatic histone mark H3K27me3 but not for euchromatic mark H3K9ac . ( F ) Cells from E were scored for indicated histone modifications in CCFs . A single representative experiment is shown and at least 100 cells were scored per slide . Note the majority of micronuclei in control cells are positive for both marks . ( G ) CCFs in RS contain γ - H2AX but not 53BP1 ( top ) . Intranuclear γ - H2AX foci colocalize with 53BP1 ( bottom ) . ( H ) CCF + and CCF − cells from G were scored for colocalizing intranuclear γ - H2AX and 53BP1 foci . Mean ± SEM , n = 3 ; P < 0 . 019 . Bars ( in IF panels ) , 10 µm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.Cytoplasmic chromatin fragments in senescent cells. (A) Cytoplasmic chromatin fragments (CCFs) in senescent cells are strongly positive for histone H3. Yellow arrow marks CCF. (B) Increased proportion of cells with CCFs in RS or OIS. Mean ± SEM, n = 3; P < 0.01 (OIS), P < 0.002 (RS). Assays were performed 10 d after induction with tamoxifen (OIS) or 4 wk after the last passage and >90% SA β-gal+ (RS).(C) CCFs in OIS cells are lamin A/C negative. (D) Cells from C were scored for lamin A/C+ and lamin A/C− CCFs. Note that the majority of micronuclei in control cells are lamin A/C positive. Mean ± SEM, n = 3; P < 0.0006 for lamin A/C negative CCF con vs. OIS; P < 0.6 for lamin A/C positive CCF con vs. OIS.(E) CCFs in OIS are strongly positive for heterochromatic histone mark H3K27me3 but not for euchromatic mark H3K9ac. (F) Cells from E were scored for indicated histone modifications in CCFs. A single representative experiment is shown and at least 100 cells were scored per slide. Note the majority of micronuclei in control cells are positive for both marks.(G) CCFs in RS contain γ-H2AX but not 53BP1 (top). Intranuclear γ-H2AX foci colocalize with 53BP1 (bottom). (H) CCF+ and CCF− cells from G were scored for colocalizing intranuclear γ-H2AX and 53BP1 foci. Mean ± SEM, n = 3; P < 0.019. Bars (in IF panels), 10 µm.


Figure 2 . Nuclear - to - cytoplasm chromatin blebbing in senescent cells associated with depletion of lamin B1 . ( A ) Extrusion of GFP - H2B - positive chromatin fragment from nonmitotic RS nucleus . Confocal time - lapse microscopy of transiently transfected GFP - H2B - expressing cell , 60 × original magnification , 150 nm optical section , time in mins . Bars : 10 µm ; ( insert ) 2 µm . ( B ) Chromatin fragments blebbing out of nucleus are γ - H2AX positive . Bars : ( top ) 10 µm ; ( bottom ) 5 µm . ( C ) Down - regulation of lamin B1 and lamin B receptor ( LBR ) in RS cells , assayed by Western blot . See Materials and methods for time - course details . ( D ) Depletion of lamin B1 from OIS cells , assayed by immunofluorescence . ( E ) A line - scan of lamin B1 fluorescence intensity along the arrows in D . Data shown are from a single representative experiment out of three repeats . . ( F ) Depletion of lamin B1 in BRAFV600E - induced OIS melanocytes in vitro . Bars , 10 µm . ( G ) Quantitative immunofluorescence of lamin B1 in melanocytes from F . A representative experiment out of two repeats is shown . At least 150 randomly selected cells were assessed . ( H ) Senescent nevusmelanocytes ( n ) stain less intensely for lamin B1 as compared with nonsenescent melanocytes in epidermis ( e ) . White dotted line shows the basement membrane between the dermis and epidermis . Bottom panels ( Epi and Nevus ) are expanded from white boxed areas on top . Confocal microscopy for DAPI ( blue ) , MelanA ( red ) , and lamin B1 ( green ) stained tissue . Bars , 20 µm . ( I ) Quantitation of results from H for three different nevi . Epifluorescence images of lamin B1 - and MelanA - stained nevi were obtained and epidermal or nevusMelanA - positive cells were scored for the presence ( positive ) of a characteristic lamin B1 " ring . " At least 50 MelanA - expressing epidermalmelanocytes and at least 100 nevusmelanocytes were scored . Average of three different nevi ± SEM ; P < 0 . 0005 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.Nuclear-to-cytoplasm chromatin blebbing in senescent cells associated with depletion of lamin B1. (A) Extrusion of GFP-H2B-positive chromatin fragment from nonmitotic RS nucleus. Confocal time-lapse microscopy of transiently transfected GFP-H2B-expressing cell, 60× original magnification, 150 nm optical section, time in mins. Bars: 10 µm; (insert) 2 µm.(B) Chromatin fragments blebbing out of nucleus are γ-H2AX positive. Bars: (top) 10 µm; (bottom) 5 µm.(C) Down-regulation of lamin B1 and lamin B receptor (LBR) in RS cells, assayed by Western blot. See Materials and methods for time-course details.(D) Depletion of lamin B1 from OIS cells, assayed by immunofluorescence. (E) A line-scan of lamin B1 fluorescence intensity along the arrows in D. Data shown are from a single representative experiment out of three repeats.. (F) Depletion of lamin B1 in BRAFV600E-induced OIS melanocytes in vitro. Bars, 10 µm. (G) Quantitative immunofluorescence of lamin B1 in melanocytes from F. A representative experiment out of two repeats is shown. At least 150 randomly selected cells were assessed.(H) Senescent nevusmelanocytes (n) stain less intensely for lamin B1 as compared with nonsenescent melanocytes in epidermis (e). White dotted line shows the basement membrane between the dermis and epidermis. Bottom panels (Epi and Nevus) are expanded from white boxed areas on top. Confocal microscopy for DAPI (blue), MelanA (red), and lamin B1 (green) stained tissue. Bars, 20 µm. (I) Quantitation of results from H for three different nevi. Epifluorescence images of lamin B1- and MelanA-stained nevi were obtained and epidermal or nevusMelanA-positive cells were scored for the presence (positive) of a characteristic lamin B1 "ring." At least 50 MelanA-expressing epidermalmelanocytes and at least 100 nevusmelanocytes were scored. Average of three different nevi ± SEM; P < 0.0005.


Figure 3 . Blebbing of chromatin from nucleus to cytoplasm in senescent cells is associated with loss of nuclear envelope integrity . ( A ) Herniation of H3K27me3 - positive chromatin into the cytoplasm of OIS cells . Yellow boxed area is magnified in bottom panels . Bars : ( top panels ) 10 µm ; ( bottom panels ) 5 µm . ( B ) Quantitation of lamin B1 gaps or herniations in OIS . Mean ± SEM , n = 2 ; P < 0 . 01 . ( C ) Isolated nuclei of senescent cells are permeable to both 70 - and 500 - kD dextrans , indicating impairment of the barrier function of nuclear envelope . Dark nuclei exclude fluorescent dextran . Bars , 10 µm . ( D ) Quantitation of results from C . Mean ± SD , n = 4 ; P < 0 . 000001 for both 70 - and 500 - kD dextrans . ( E ) Representative OIS SAHF + nucleus showing dextran permeability . Bars , 10 µm . ( F ) Quantitation of dextran 70 uptake in SAHF + and SAHF − OIS cells . P < 1 . 9 × 10 − 5 . ( G ) In individual nuclei , chromatin extrusion and dextran intrusion occurred in close physical proximity . Right - hand panels are expanded from yellow boxed areas on the left . Bars : ( left ) 10 µm ; ( right ) 2 µm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.Blebbing of chromatin from nucleus to cytoplasm in senescent cells is associated with loss of nuclear envelope integrity. (A) Herniation of H3K27me3-positive chromatin into the cytoplasm of OIS cells. Yellow boxed area is magnified in bottom panels. Bars: (top panels) 10 µm; (bottom panels) 5 µm. (B) Quantitation of lamin B1 gaps or herniations in OIS. Mean ± SEM, n = 2; P < 0.01.(C) Isolated nuclei of senescent cells are permeable to both 70- and 500-kD dextrans, indicating impairment of the barrier function of nuclear envelope. Dark nuclei exclude fluorescent dextran. Bars, 10 µm. (D) Quantitation of results from C. Mean ± SD, n = 4; P < 0.000001 for both 70- and 500-kD dextrans.(E) Representative OIS SAHF+ nucleus showing dextran permeability. Bars, 10 µm. (F) Quantitation of dextran 70 uptake in SAHF+ and SAHF− OIS cells. P < 1.9 × 10−5. (G) In individual nuclei, chromatin extrusion and dextran intrusion occurred in close physical proximity. Right-hand panels are expanded from yellow boxed areas on the left. Bars: (left) 10 µm; (right) 2 µm.


Figure 4 . Cytoplasmic histone is processed by a lysosomal / autophagy pathway . ( A ) Close juxtaposition of senescence - associated CCF ( RS ) with p62 nuclear bodies . Bars : 10 µm ; ( inset ) 1 µm . ( B ) Quantitation of control and OIS cells with CCF overlapping p62 ( p62 + ) or not ( p62 − ) . Mean ± SEM , n = 3 . ( C ) Quantitation of proliferating and RS cells with CCF overlapping p62 ( p62 + ) or not ( p62 − ) and protein ubiquitination ( FK2 + ) or not ( FK2 − ) . Mean ± SEM , n = 3 ; P < 0 . 0008 for FK2 + / p62 + CCF , comparing proliferating and RS cells . ( D ) Ubiquitinated proteins appear to line chromatin surface in CCF . Yellow boxed areas are magnified in right - hand panels . Bars : ( left panels ) 10 µm ; ( right panels ) 1 µm . ( E ) Apparent activation of cathepsin L in RS IMR90 cells . In contrast to RS cells , the majority of cathepsin L in proliferating control cells exists in its inactive pro - cathepsin L form . Loading was normalized by cell number and lamin A / C was used as a loading control . See Materials and methods for details of time course . ( F ) Accumulation of H3cs . 1 , a specific N - terminal cleavage product of H3 , in OIS cells . See Materials and methods for details of time course . Western blotting was performed using the same cellular lysates as in Fig . S4 B , and the same lamin A / C panel is shown in both figures . ( G ) Accumulation of H3cs . 1 and corresponding high mobility H3 in OIS . ( H ) Predominant cytoplasmic localization of H3cs . 1 in senescent cells . α - Tubulin and lamin A / C were used as fractionation controls for cytoplasmic and nuclear fractions , respectively .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.Cytoplasmic histone is processed by a lysosomal/autophagy pathway. (A) Close juxtaposition of senescence-associated CCF (RS) with p62 nuclear bodies. Bars: 10 µm; (inset) 1 µm. (B) Quantitation of control and OIS cells with CCF overlapping p62 (p62+) or not (p62−). Mean ± SEM, n = 3.(C) Quantitation of proliferating and RS cells with CCF overlapping p62 (p62+) or not (p62−) and protein ubiquitination (FK2+) or not (FK2−). Mean ± SEM, n = 3; P < 0.0008 for FK2+/p62+ CCF, comparing proliferating and RS cells. (D) Ubiquitinated proteins appear to line chromatin surface in CCF. Yellow boxed areas are magnified in right-hand panels. Bars: (left panels) 10 µm; (right panels) 1 µm.(E) Apparent activation of cathepsin L in RS IMR90 cells. In contrast to RS cells, the majority of cathepsin L in proliferating control cells exists in its inactive pro-cathepsin L form. Loading was normalized by cell number and lamin A/C was used as a loading control. See Materials and methods for details of time course.(F) Accumulation of H3cs.1, a specific N-terminal cleavage product of H3, in OIS cells. See Materials and methods for details of time course. Western blotting was performed using the same cellular lysates as in Fig. S4 B, and the same lamin A/C panel is shown in both figures.(G) Accumulation of H3cs.1 and corresponding high mobility H3 in OIS.(H) Predominant cytoplasmic localization of H3cs.1 in senescent cells. α-Tubulin and lamin A/C were used as fractionation controls for cytoplasmic and nuclear fractions, respectively.


Figure 5 . Senescent cells in vitro and in vivo contain reduced histone content . ( A ) Immunofluorescent confocal images of histone H3 in control or OIS cells ( 11 d after activation of ER - RASG12V ) . Yellow arrows indicate cells with most pronounced SAHF . ( B ) Quantitative immunofluorescence analysis of histone H3 in RS and OIS cells . Representative of two independent experiments . ( C ) Progressive loss of core histones in senescent IMR90 cells in replicative senescence ( RS ) . Lysates were normalized by cell number and an equal number of cells was loaded per each lane . Lamin A / C was used as a confirmatory loading control . See Materials and methods for details of time course . ( D ) Immunohistochemistry for histone H3in human dermal nevus ( n ) and adjacent epidermis ( e ) . Right - hand panels are expanded from boxed areas on the left . Note decreased nevus staining for H3 ( right bottom ) as compared with epidermis ( right top ) . Bars : ( left ) 100 µm ; ( right ) 50 µm . ( E ) Immunofluorescence of histone H3 ( red ) and S100 ( melanocytes [ green ] ) in human benign nevus and adjacent epidermis . Bars , 20 µm . ( F ) Higher magnification of boxed regions in E shows reduced staining for H3 in nevus ( bottom panels ) , compared with epidermalmelanocytes ( top panels ) . Bars , 20 µm . ( G ) Quantitative immunofluorescence of H3 in epidermal and nevusmelanocytes . Images were obtained in blue ( DAPI ) , green ( S100 ) , and red ( H3 ) channels and then H3 intensity was measured in either S100 + epidermal cells , strictly adjacent to the basal membrane , or in S100 + nevusmelanocytes . H3 intensity histograms represent fluorescence intensity distribution , combined from three individual nevi . At least 150 epidermal ( 50 per nevus ) and 300 nevus ( 100 per nevus ) melanocytes were assessed .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5.Senescent cells in vitro and in vivo contain reduced histone content. (A) Immunofluorescent confocal images of histone H3 in control or OIS cells (11 d after activation of ER-RASG12V). Yellow arrows indicate cells with most pronounced SAHF. (B) Quantitative immunofluorescence analysis of histone H3 in RS and OIS cells. Representative of two independent experiments.(C) Progressive loss of core histones in senescent IMR90 cells in replicative senescence (RS). Lysates were normalized by cell number and an equal number of cells was loaded per each lane. Lamin A/C was used as a confirmatory loading control. See Materials and methods for details of time course.(D) Immunohistochemistry for histone H3in human dermal nevus (n) and adjacent epidermis (e). Right-hand panels are expanded from boxed areas on the left. Note decreased nevus staining for H3 (right bottom) as compared with epidermis (right top). Bars: (left) 100 µm; (right) 50 µm.(E) Immunofluorescence of histone H3(red) and S100 (melanocytes [green]) in human benign nevus and adjacent epidermis. Bars, 20 µm.(F) Higher magnification of boxed regions in E shows reduced staining for H3 in nevus (bottom panels), compared with epidermalmelanocytes (top panels). Bars, 20 µm. (G) Quantitative immunofluorescence of H3 in epidermal and nevusmelanocytes. Images were obtained in blue (DAPI), green (S100), and red (H3) channels and then H3 intensity was measured in either S100+epidermal cells, strictly adjacent to the basal membrane, or in S100+nevusmelanocytes. H3 intensity histograms represent fluorescence intensity distribution, combined from three individual nevi. At least 150 epidermal (50 per nevus) and 300 nevus (100 per nevus) melanocytes were assessed.


Figure 6 . Senescence - associated loss of histones is V - ATPase dependent . ( A ) Bafilomycin A1 ( BafA1 ) blocks the loss of nuclearhistone H3content in cells undergoing OIS . BafA1 was added to cells at 50 nM on day 5 after RASG12V induction . Cells were harvested 24 h later , and stained for histone H3 . Bars , 10 µm . ( B ) Quantitative histone H3immunofluorescence in cells from A . Single representative experiment out of three repeats . ( C ) RASG12V - induced OIS cells were treated with BafA1 as described in A and then assessed for histones by Western blot . LC3 I / II has been used as a control for BafA1 activity . Lysates from 10 , 000 cells were loaded per well . ( D ) BafA1 blocks the accumulation of H3cs . 1 in cells undergoing OIS . ( E ) BafA1 and concanamycin A ( Con A ) block the accumulation of H3cs . 1 in RS cells .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 6.Senescence-associated loss of histones is V-ATPase dependent. (A) Bafilomycin A1 (BafA1) blocks the loss of nuclearhistone H3content in cells undergoing OIS. BafA1 was added to cells at 50 nM on day 5 after RASG12V induction. Cells were harvested 24 h later, and stained for histone H3. Bars, 10 µm. (B) Quantitative histone H3immunofluorescence in cells from A. Single representative experiment out of three repeats.(C) RASG12V-induced OIS cells were treated with BafA1 as described in A and then assessed for histones by Western blot. LC3 I/II has been used as a control for BafA1 activity. Lysates from 10,000 cells were loaded per well.(D) BafA1 blocks the accumulation of H3cs.1 in cells undergoing OIS.(E) BafA1 and concanamycin A (Con A) block the accumulation of H3cs.1 in RS cells.


Figure 1Quantification by western - blot analysis of EZH2 and H3K27me3 levels at each stage of de novo transformation . UT : untransformed , PN : pre - neoplastic , TR : transformed . Histone H3 is used as a loading control . The graph on the right displays the H3 normalized densitometric values of western - blot band from untreated and DMSO treated cells . Values represent mean ± SEM from three biological replicates . One asterisk indicates p - value < 0 . 05 ( one - tailed unpaired Student ' s t - test ) . ns : non - significant . Volcano plot showing the relative enrichment of EZH2 binding at all sites detected in either untransformed or transformed cells . Numbers within the volcano plot indicate differential binding sites with a p - value ≤ 1e - 20 and a fold - change in normalised tag count ≥ 1 . 5 . Enrichment significance calculated based on a Poisson distribution using the ' getDifferentialPeaks ' function in homer ( see : methods ) . P - values of > 1e - 100 were set to 1e - 100 for display reasons . Tracks on the right show representative high - and low - magnitude EZH2 differential peaks . ChIP - seq signal normalised to sequencing depth is shown . Tracks are scaled to be of the same height to make samples comparable . Blue bars represent regions called as an EZH2 binding site . UT : untransformed , TR : transformed .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Quantification by western-blot analysis of EZH2 and H3K27me3 levels at each stage of de novo transformation. UT: untransformed, PN: pre-neoplastic, TR: transformed. Histone H3 is used as a loading control. The graph on the right displays the H3 normalized densitometric values of western-blot band from untreated and DMSO treated cells. Values represent mean ± SEM from three biological replicates. One asterisk indicates p-value < 0.05 (one-tailed unpaired Student's t-test). ns: non-significant.Volcano plot showing the relative enrichment of EZH2 binding at all sites detected in either untransformed or transformed cells. Numbers within the volcano plot indicate differential binding sites with a p-value ≤1e-20 and a fold-change in normalised tag count ≥1.5. Enrichment significance calculated based on a Poisson distribution using the 'getDifferentialPeaks' function in homer (see: methods). P-values of > 1e-100 were set to 1e-100 for display reasons. Tracks on the right show representative high- and low-magnitude EZH2 differential peaks. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. Blue bars represent regions called as an EZH2 binding site. UT: untransformed, TR: transformed.


Figure 2Western - blot analysis of H3K27me3 levels after treatment of cells with an EZH2 inhibitor ( EZH2i ) or DMSO as control for 12 days . Histone H3 is used as a loading control . Values represent H3 - normalized densitometric values of the H3K27me3 bands , and are expressed relative to the relevant DMSO control . UT : untransformed , PN : pre - neoplastic , TR : transformed . Quantification of differentially expressed genes ( false discovery rate ( FDR ) ≤ 0 . 01 , Log2FC ≥ 1 / ≤ - 1 and maximal transcripts per million ( maxTPM ) ≥ 1 ) detected by RNA - seq in each cellular state upon EZH2i treatment . UT : untransformed , PN : pre - neoplastic , TR : transformed . Heatmap showing the relative expression of genes identified by the multi - step filtering strategy in the indicated cellular states . UT : untransformed , PN : pre - neoplastic , TR : transformed . Colours represent log2 - transformed , row - normalised TPM values .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Western-blot analysis of H3K27me3 levels after treatment of cells with an EZH2 inhibitor (EZH2i) or DMSO as control for 12 days. Histone H3 is used as a loading control. Values represent H3-normalized densitometric values of the H3K27me3 bands, and are expressed relative to the relevant DMSO control. UT: untransformed, PN: pre-neoplastic, TR: transformed.Quantification of differentially expressed genes (false discovery rate (FDR) ≤ 0.01, Log2FC ≥ 1/≤-1 and maximal transcripts per million (maxTPM) ≥ 1) detected by RNA-seq in each cellular state upon EZH2i treatment. UT: untransformed, PN: pre-neoplastic, TR: transformed.Heatmap showing the relative expression of genes identified by the multi-step filtering strategy in the indicated cellular states. UT: untransformed, PN: pre-neoplastic, TR: transformed. Colours represent log2-transformed, row-normalised TPM values.


Figure 3ChIP - seq signal for EZH2 and H3K27me3 ( left ) at EMX2 and HOXB9 loci . ChIP - seq signal normalised to sequencing depth is shown . Tracks are scaled to be of the same height to make samples comparable . mRNA expression of EMX2 and HOXB9 ( right ) as detected by RNA - seq . Expression values represent mean ± SEM from three biological replicates . UT : untransformed , PN : pre - neoplastic , TR : transformed . TPM : transcripts per million . qRT - PCR showing the expression levels of EMX2 and HOXB9 in the indicated cells treated with EZH2i or a DMSO control for 12 days . Values represent mean ± SEM from three biological replicates . Two asterisks indicate p - value < 0 . 01 ( one - tailed unpaired Student ' s t - test ) . UT : untransformed , TR : transformed . Expression levels of EMX2 and HOXB9 in primary human neural cells , as detected by RNA - seq . Data sourced from Brainseq2 . Expression values represent mean ± SEM from four , twelve , one and five biological replicates of fetal astrocytes , mature astrocytes , neurons and oligodendrocytes , respectively . FPKM : fragments per kilobase of transcript per million . Expression levels of EMX2 and HOXB9 in 62 glioma cell lines . Data sourced from CCLE . Every dot represents a cell line . RPKM : reads per kilobase of transcript per million . In the boxplot , the top , middle and bottom box delimiters represent the 75th , 50th and 25th percentiles of the data , respectively . Top and bottom whiskers show the 75th percentile + 1 . 5 * interquartile range and 25th percentile - 1 . 5 * interquartile range , respectively . Quantification of EZH2 binding at EMX2 , HOXB9 and GAPDH ( negative control ) promoters by ChIP - qPCR in M059K GBM cells . Values from two biological replicates are shown . Three asterisks indicate p - value < 0 . 001 of EZH2 samples relative to the negative control GAPDH ( two - way ANOVA followed by pairwise comparisons using Holm - Sidak method ) . Ns : non - significant . Quantification of EMX2 levels by qRT - PCR in five different GBM cell lines upon treatment with 1μM , 3μM or 10μM of EZH2i for eight days , or a DMSO control . Values represent mean ± SEM from three technical replicates . The SEM is indicated to show reliability of the RT - PCR values , due to the low endogenous levels of EMX2 . Two asterisks indicate p - value < 0 . 01 comparing EZH2i - and DMSO - treated cells ( Two - way ANOVA ) . Relationship between the extent of EMX2 upregulation induced by EZH2i and the DNA methylation level at the EMX2 promoter in GBM cell lines . Values for EMX2 upregulation are the fold - change in mRNA expression induced upon treatment with 10μM EZH2i ( see : panel G ) , and are expressed relative to a DMSO control . Values represent mean ± SEM from three technical replicates . The SEM is indicated to show reliability of the RT - PCR values , due to the low endogenous levels of EMX2 . Methylation values are averages of DNA methylation at CpGs across the promoter of EMX2 in GBM cell lines . All data sourced from the CCLE . The significance of the anti - correlation between mRNA levels and DNA methylation levels across cell lines is indicated ( Spearman rank correlation ) . Heatmap visualizing the fold change in EMX2 expression as detected by qRT - PCR after treatment of the indicated PDX - derived ( blue ) and established cancer cell lines ( black ) with EZH2i for eight days . Values are from three technical replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3ChIP-seq signal for EZH2 and H3K27me3 (left) at EMX2 and HOXB9 loci. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. mRNA expression of EMX2 and HOXB9 (right) as detected by RNA-seq. Expression values represent mean ± SEM from three biological replicates. UT: untransformed, PN: pre-neoplastic, TR: transformed. TPM: transcripts per million.qRT-PCR showing the expression levels of EMX2 and HOXB9 in the indicated cells treated with EZH2i or a DMSO control for 12 days. Values represent mean ± SEM from three biological replicates. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). UT: untransformed, TR: transformed.Expression levels of EMX2 and HOXB9 in primary human neural cells, as detected by RNA-seq. Data sourced from Brainseq2. Expression values represent mean ± SEM from four, twelve, one and five biological replicates of fetal astrocytes, mature astrocytes, neurons and oligodendrocytes, respectively. FPKM: fragments per kilobase of transcript per million.Expression levels of EMX2 and HOXB9 in 62 glioma cell lines. Data sourced from CCLE. Every dot represents a cell line. RPKM: reads per kilobase of transcript per million. In the boxplot, the top, middle and bottom box delimiters represent the 75th, 50th and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile - 1.5*interquartile range, respectively.Quantification of EZH2 binding at EMX2, HOXB9 and GAPDH (negative control) promoters by ChIP-qPCR in M059K GBM cells. Values from two biological replicates are shown. Three asterisks indicate p-value < 0.001 of EZH2 samples relative to the negative control GAPDH (two-way ANOVA followed by pairwise comparisons using Holm-Sidak method). Ns: non-significant.Quantification of EMX2 levels by qRT-PCR in five different GBM cell lines upon treatment with 1μM, 3μM or 10μM of EZH2i for eight days, or a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Two asterisks indicate p-value <0.01 comparing EZH2i- and DMSO-treated cells (Two-way ANOVA).Relationship between the extent of EMX2 upregulation induced by EZH2i and the DNA methylation level at the EMX2 promoter in GBM cell lines. Values for EMX2 upregulation are the fold-change in mRNA expression induced upon treatment with 10μM EZH2i (see: panel G), and are expressed relative to a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Methylation values are averages of DNA methylation at CpGs across the promoter of EMX2 in GBM cell lines. All data sourced from the CCLE. The significance of the anti-correlation between mRNA levels and DNA methylation levels across cell lines is indicated (Spearman rank correlation).Heatmap visualizing the fold change in EMX2 expression as detected by qRT-PCR after treatment of the indicated PDX-derived (blue) and established cancer cell lines (black) with EZH2i for eight days. Values are from three technical replicates.


Figure 4Expression of EMX2 in normal brain and GBM patient samples . Data sourced from the Repository of Molecular Brain Neoplasia Data ( REMBRANDT ) . Four asterisks indicate p - value < 0 . 0001 ( two - tailed Mann Whitney U test ) . Bars represent median ± interquartile range . Values are expressed relative to the mean of normal brain samples . N : 21 for normal brain , 214 for GBM . Note that the extent of EMX2 repression in GBM patients is likely underestimated due to the possible presence of normal adjacent tissue in the analysed samples , and due to the intrinsic background noise of microarrays , which limits detection of truly silenced genes . Relative expression of HOX genes in normal brain and GBM patient samples as detected by microarray analysis . Data sourced from REMBRANDT . Values are expressed relative to the median of normal brain samples . In the boxplots , the top , middle and bottom box delimiters represent the 75th , 50th and 25th percentiles of the data , respectively . Top and bottom whiskers show the 75th percentile + 1 . 5 * interquartile range and 25th percentile - 1 . 5 * interquartile range , respectively . One asterisk indicates p - value < 0 . 05 ( two - tailed Mann Whitney U test corrected for multiple comparison using Holm ' s method ) . N : 21 for normal brain , 214 for GBM . Data was not available for HOXD8 . Covariance between EMX2 and EZH2 expression levels in GBM patient samples , as detected by microarray analysis . Data sourced from REMBRANDT . P - value and correlation coefficient ( r ) of the covariance are shown ( Spearman rank correlation ) . Every dot is a patient . N : 214 . Expression levels of EMX2 ( top ) and EZH2 ( bottom ) in tumour or adjacent normal regions laser microdissected from human GBM tumours as detected by RNA - seq . Data sourced from the Ivy Glioblastoma Atlas . FPKM : fragments per kilobase of transcript per million . The significance of the differential expression in normal and tumour regions across patients is indicated ( two - way ANOVA ) . Bars represent mean ± SEM . N : 3 regions sampled for each GBM tumour , 2 , 3 , 2 , 3 , 2 , 3 , 3 and 1 regions for normal tissue of patients 1 - 8 , respectively . Hematoxylin and eosin ( H & E ) staining of a human GBM tumour . The black square represents the approximate location of the field shown in G ( left image ) . Serial sections were used for H & E and RNA - FISH . Scale bar : 300 µm . Visualisation of EZH2 ( green ) and EMX2 mRNA ( red ) in a human GBM tumour by RNA FISH . Nuclei were counterstained with DAPI . Scale bar : 20 µm . Expression of EMX2 ( left ) and EZH2 ( right ) in glioma patient samples , grouped according to tumour grade , as detected by RNA - seq . Data sourced from the Chinese Glioma Genome Atlas . Four asterisks indicate p - value < 0 . 0001 ( Kruskal - Wallis test ) . Bars represent median ± interquartile range . N : 109 for grade II - , 72 for grade III , 144 for grade IV . RPKM : Reads per kilobase of transcript per million .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Expression of EMX2 in normal brain and GBM patient samples. Data sourced from the Repository of Molecular Brain Neoplasia Data (REMBRANDT). Four asterisks indicate p-value < 0.0001 (two-tailed Mann Whitney U test). Bars represent median ± interquartile range. Values are expressed relative to the mean of normal brain samples. N: 21 for normal brain, 214 for GBM. Note that the extent of EMX2 repression in GBM patients is likely underestimated due to the possible presence of normal adjacent tissue in the analysed samples, and due to the intrinsic background noise of microarrays, which limits detection of truly silenced genes.Relative expression of HOX genes in normal brain and GBM patient samples as detected by microarray analysis. Data sourced from REMBRANDT. Values are expressed relative to the median of normal brain samples. In the boxplots, the top, middle and bottom box delimiters represent the 75th, 50th and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile - 1.5*interquartile range, respectively. One asterisk indicates p-value <0.05 (two-tailed Mann Whitney U test corrected for multiple comparison using Holm's method). N: 21 for normal brain, 214 for GBM. Data was not available for HOXD8.Covariance between EMX2 and EZH2 expression levels in GBM patient samples, as detected by microarray analysis. Data sourced from REMBRANDT. P-value and correlation coefficient (r) of the covariance are shown (Spearman rank correlation). Every dot is a patient. N: 214.Expression levels of EMX2 (top) and EZH2 (bottom) in tumour or adjacent normal regions laser microdissected from human GBM tumours as detected by RNA-seq. Data sourced from the Ivy Glioblastoma Atlas. FPKM: fragments per kilobase of transcript per million. The significance of the differential expression in normal and tumour regions across patients is indicated (two-way ANOVA). Bars represent mean ± SEM. N: 3 regions sampled for each GBM tumour, 2, 3, 2, 3, 2, 3, 3 and 1 regions for normal tissue of patients 1-8, respectively.Hematoxylin and eosin (H&E) staining of a human GBM tumour. The black square represents the approximate location of the field shown in G (left image). Serial sections were used for H&E and RNA-FISH. Scale bar: 300 µm.Visualisation of EZH2 (green) and EMX2 mRNA (red) in a human GBM tumour by RNA FISH. Nuclei were counterstained with DAPI. Scale bar: 20 µm.Expression of EMX2 (left) and EZH2 (right) in glioma patient samples, grouped according to tumour grade, as detected by RNA-seq. Data sourced from the Chinese Glioma Genome Atlas. Four asterisks indicate p-value < 0.0001 (Kruskal-Wallis test). Bars represent median ± interquartile range. N: 109 for grade II -, 72 for grade III, 144 for grade IV. RPKM: Reads per kilobase of transcript per million.


Figure 5Proliferation assay examining the effect of EMX2 ectopic expression in U - 87 MG and DBTRG - 05MG GBM cells , RFP is used as a control . Values represent mean ± SEM from three biological replicates . The fold change in cell number after eight days of proliferation whilst expressing the exogenous protein is shown relative to an uninduced control . Two asterisks indicate p - value < 0 . 01 ( one - tailed unpaired Student ' s t - test ) . Similar results were reported in [ 103 ] using other GBM cell lines . Transplantation assay comparing the growth kinetics of subcutaneous DBTRG - 05MG - induced tumours expressing EMX2 at levels comparable to those expressed in normal cells , or expressing RFP as a control . Values represent mean ± SEM from six tumours . Three asterisks indicate p - value < 0 . 001 ( two - tailed single sample Student ' s t - test ) . Similar results were obtained in an independent experiment using a distinct batch of transduced cells . MRI scans showing representative brain tumours six weeks post - injection of DBTRG - 05MG cells expressing EMX2 or RFP . Axial anatomical scan and coronal signal intensity map indicate location of tumour in RFP control ( yellow arrow ) . Contrast enhancement in post - contrast images indicates tumour blood brain barrier breakdown . Signal was not present in pre - contrast images . Images show the same MRI slice position between mice . Scale bar : 2mm . Quantification of brain tumour size in mice injected with EMX2 - or RFP - expressing DBTRG - 05MG cells . Values represent mean ± SEM from four control and three EMX2 tumours . Five mice per conditions were injected but three ( one for RFP , and two for EMX2 ) had to be excluded from the study due to complications from the procedure .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Proliferation assay examining the effect of EMX2 ectopic expression in U-87 MG and DBTRG-05MG GBM cells, RFP is used as a control. Values represent mean ± SEM from three biological replicates. The fold change in cell number after eight days of proliferation whilst expressing the exogenous protein is shown relative to an uninduced control. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). Similar results were reported in [103] using other GBM cell lines.Transplantation assay comparing the growth kinetics of subcutaneous DBTRG-05MG-induced tumours expressing EMX2 at levels comparable to those expressed in normal cells, or expressing RFP as a control. Values represent mean ± SEM from six tumours. Three asterisks indicate p-value < 0.001 (two-tailed single sample Student's t-test). Similar results were obtained in an independent experiment using a distinct batch of transduced cells.MRI scans showing representative brain tumours six weeks post-injection of DBTRG-05MG cells expressing EMX2 or RFP. Axial anatomical scan and coronal signal intensity map indicate location of tumour in RFP control (yellow arrow). Contrast enhancement in post-contrast images indicates tumour blood brain barrier breakdown. Signal was not present in pre-contrast images. Images show the same MRI slice position between mice. Scale bar: 2mm. Quantification of brain tumour size in mice injected with EMX2- or RFP- expressing DBTRG-05MG cells. Values represent mean ± SEM from four control and three EMX2 tumours. Five mice per conditions were injected but three (one for RFP, and two for EMX2) had to be excluded from the study due to complications from the procedure.


Figure 1B . Representative image of primary microglia simultaneously treated with Aβ oligomer - bound SN ( oAβ - SN ; in pHrodo red ( magenta ) ) and control SN ( Ctrl - SN ; in pHrodo deep red ( cyan ) ) . Scale bar , 50 μm . F . Time - lapse images of primary Homer1 - eGFP neurons ( green ) treated with 50 nM oAβ versus vehicle control . Yellow arrows indicate increasing PSVue550 ( magenta ) signal on dendritic spines with Aβ oligomer treatment over 45 min . Scale bar , 2 μm . I . Quantification of colocalized Homer1 - eGFP with PSVue among total Homer1 . 3 ROIs per neuron , 4 - 5 neurons , n = 3 independent experiments . J . Quantification of colocalized PSVue with Homer1 - eGFP among total PSVue . 3 ROIs per neuron , 4 - 5 neurons , n = 3 independent experiments . Data information : Data shown as mean ± SD Each shaded point represents 1 ROI and each open point represents the mean unpaired ( I and J ) t - test . P - values shown as * P < 0 . 05 ; * * < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Representative image of primary microglia simultaneously treated with Aβ oligomer-bound SN (oAβ-SN; in pHrodo red (magenta)) and control SN (Ctrl-SN; in pHrodo deep red (cyan)). Scale bar, 50 μm.F. Time-lapse images of primary Homer1-eGFP neurons (green) treated with 50 nM oAβ versus vehicle control. Yellow arrows indicate increasing PSVue550 (magenta) signal on dendritic spines with Aβ oligomer treatment over 45 min. Scale bar, 2 μm.I. Quantification of colocalized Homer1-eGFP with PSVue among total Homer1. 3 ROIs per neuron, 4-5 neurons, n=3 independent experiments. J. Quantification of colocalized PSVue with Homer1-eGFP among total PSVue. 3 ROIs per neuron, 4-5 neurons, n=3 independent experiments. Data information: Data shown as mean ± SD Each shaded point represents 1 ROI and each open point represents the mean unpaired (I and J) t-test. P-values shown as *P<0.05; **<0.01; ***P<0.001; ****P<0.0001.


Figure 2A . Time - lapse images of microglia ( cyan , labelled with IB4 - 647 , 3D rendered ) internalizing ( yellow arrowheads ) PSVue + Homer1 - eGFP dendritic spines . Note PSVue - Homer1 - eGFP dendritic spines are left behind ( empty yellow arrowheads ) . Scale bar , 5 μm . C . SRM images of Homer1 - eGFP and CD68 ( magenta ) lysosomes in microglia . Inset shows orthogonal view of colocalized Homer1 - eGFP and CD68 . Scale bar , 5 μm . G . ( Top panel ) Representative traces of spontaneous GCaMP7 signals at 48 h post - treatment with Aβ oligomer in dendritic spines from neuron - only culture ( cyan ) , neuron - microglia co - culture ( magenta ) , and neuron - microglia co - culture treated with AnnxV ( yellow ) . ( Bottom panel ) number of calcium transients per minute before ( 0 h ) , at 24 h and 48 h post - treatment of Aβ oligomer . ∼ 10 spines per neuron , ∼ 10 neurons per experiment , from 3 independent experiments . Data information : Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ( G ) P - values shown ns P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . J - K . Number of PSVue puncta per 100 μm2 ( J ) and mean intensities of PSVue signal ( K ) in neuron - only culture , and neuron - microglia co - culture with or without AnnxV . 16 ROIs per experiment , n = 3 independent experiments . Data information : Data shown as box plots ( J and K ) . The top and the bottom of the boxes represent the 75th and 25th percentiles , respectively , and the lines in the middle represent the median . The whiskers represent the highest and lowest values that are not outliers . one - way ANOVA followed by Bonferroni post - hoc test ( J and K ) . P - values shown ns P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Time-lapse images of microglia (cyan, labelled with IB4-647, 3D rendered) internalizing (yellow arrowheads) PSVue+ Homer1-eGFP dendritic spines. Note PSVue- Homer1-eGFP dendritic spines are left behind (empty yellow arrowheads). Scale bar, 5 μm.C. SRM images of Homer1-eGFP and CD68 (magenta) lysosomes in microglia. Inset shows orthogonal view of colocalized Homer1-eGFP and CD68. Scale bar, 5 μm.G. (Top panel) Representative traces of spontaneous GCaMP7 signals at 48 h post-treatment with Aβ oligomer in dendritic spines from neuron-only culture (cyan), neuron-microglia co-culture (magenta), and neuron-microglia co-culture treated with AnnxV (yellow). (Bottom panel) number of calcium transients per minute before (0 h), at 24 h and 48 h post-treatment of Aβ oligomer. ∼10 spines per neuron, ∼10 neurons per experiment, from 3 independent experiments. Data information: Kruskal-Wallis test followed by Dunn's multiple comparisons test (G) P-values shown ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. J-K. Number of PSVue puncta per 100 μm2 (J) and mean intensities of PSVue signal (K) in neuron-only culture, and neuron-microglia co-culture with or without AnnxV. 16 ROIs per experiment, n=3 independent experiments. Data information: Data shown as box plots (J and K). The top and the bottom of the boxes represent the 75th and 25th percentiles, respectively, and the lines in the middle represent the median. The whiskers represent the highest and lowest values that are not outliers. one-way ANOVA followed by Bonferroni post-hoc test (J and K). P-values shown ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.


Figure 3A . Trem2 CV and Trem2 R47H KI primary microglia treated simultaneously with oAβ - bound synaptosomes ( SN ) conjugated with pHrodo red ( magenta ) and control SN , conjugated with pHrodo deep red ( cyan ) . Scale bar , 50 μm . B . pHrodo fluorescence with time shown as AUC at 3 h . AUC of oAβ - SN is higher compared to Ctrl - SN in Trem2 CV but not in Trem2 R47H KI microglia . ∼ 40 microglia per ROI , 2 ROIs per well , 2 - 3 wells per experiment , n = 3 independent experiments . C . Quantification of the percent of C1qa area covered normalized to WT showing an increase in the NL - F but not NL - F KI ; Trem2 R47H KI . N = 3 - 4 animals per genotype . Data information : Data shown as mean ± SEM . Each shaded point represents 1 ROI , and each open point represents the mean of each independent experiment . Two - way ANOVA followed by Bonferroni ' s post - hoc test . P - values shown as ns P > 0 . 05 ; * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . Hippocampal CA1 SR of 6 mo WT , Trem2 R47H KI , NL - F KI and NL - F KI ; Trem2 R47H KI mice immunostained for P2Y12 ( red ) , CD68 ( magenta ) and Homer1 ( green ) . G . Representative 3D surface rendering reconstructions showing increased Homer1 in microglia in NL - F KI compared to WT , Trem2 R47H KI , and NL - F KI ; Trem2 R47H KI mice . Scale bar , 10 μm . I . SRM images from the hippocampal CA1 SR of 6 mo WT , Trem2 R47H KI , NL - F KI and NL - F KI ; Trem2 R47H KI mice immunostained for Synaptotagmin ( Syt1 / 2 , magenta ) and Homer1 ( green ) , pre - and post - synaptic puncta , respectively . Scale bar , 1 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Trem2 CV and Trem2 R47H KI primary microglia treated simultaneously with oAβ-bound synaptosomes (SN) conjugated with pHrodo red (magenta) and control SN, conjugated with pHrodo deep red (cyan). Scale bar, 50 μm. B. pHrodo fluorescence with time shown as AUC at 3 h. AUC of oAβ-SN is higher compared to Ctrl-SN in Trem2 CV but not in Trem2 R47H KI microglia. ∼40 microglia per ROI, 2 ROIs per well, 2-3 wells per experiment, n=3 independent experiments. C. Quantification of the percent of C1qa area covered normalized to WT showing an increase in the NL-F but not NL-F KI; Trem2 R47H KI. N=3-4 animals per genotype. Data information: Data shown as mean ± SEM. Each shaded point represents 1 ROI, and each open point represents the mean of each independent experiment. Two-way ANOVA followed by Bonferroni's post-hoc test. P-values shown as ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001.Hippocampal CA1 SR of 6 mo WT, Trem2 R47H KI, NL-F KI and NL-F KI; Trem2 R47H KI mice immunostained for P2Y12 (red), CD68 (magenta) and Homer1 (green). G. Representative 3D surface rendering reconstructions showing increased Homer1 in microglia in NL-F KI compared to WT, Trem2 R47H KI, and NL-F KI; Trem2 R47H KI mice. Scale bar, 10 μm.I. SRM images from the hippocampal CA1 SR of 6 mo WT, Trem2 R47H KI, NL-F KI and NL-F KI; Trem2 R47H KI mice immunostained for Synaptotagmin (Syt1/2, magenta) and Homer1 (green), pre- and post-synaptic puncta, respectively. Scale bar, 1 μm.


Figure 4B . Percentage of synaptic Homer1 - immunoreactive puncta found co - localized with PSVue in NL - F KI ; Trem2 R47H compared to NL - F KI . 3 ROIs per animal , n = 3 - 4 animals per genotype . Data information : Data shown as mean ± SEM . Unpaired t - test ( B ) P - values shown as ns P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 , * * * * P < 0 . 0001 . D . Number of spontaneous calcium transients per minute at 48 h post - treatment of Aβ oligomer in neuron - only culture ( cyan ) , neuron - Trem2 CV microglia co - culture ( magenta ) or neuron - Trem2 R47H KI microglia co - culture ( purple ) . ∼ 10 spines per neuron , ∼ 10 neurons per experiment , from 3 - 4 independent experiments . Data information : Data shown as mean ± SEM . Each shaded point represents 1 ROI , and each open point represents the mean ( or median for GCaMP studies ) of each independent experiment . Central bands of the violin plot ( D ) represent median and quartiles . Kruskal - Wallis test followed by Dunn ' s test ( D ) , P - values shown as ns P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 , * * * * P < 0 . 0001 . F . SRM 3D images from the hippocampal CA1 SR of 6 mo WT , Trem2 R47H KI , NL - F KI and NL - F KI ; Trem2 R47H KI mice immunostained for Bassoon ( cyan ) , a presynaptic marker enriched in functional active zones . Scale bar , 1 μm . G . Representative western blots comparing levels of cleaved caspase - 3 ( 17 / 19 kDa ) and procaspase - 3 ( 35 kDa ) with respect to GAPDH ( 38 kDa ) loading control in synaptosomes isolated from human NDC , AD , NDC TREM2 and AD TREM2 brains . 1 lane represents 1 patient . H . Western blot densitometry analysis showing cleaved caspase - 3 levels ( left graph ) and the ratio of cleaved caspase - 3 / procaspase - 3 ( right graph ) in synaptosomes ( SN ) isolated from human NDC , AD , TREM2 NDC and TREM2 variants . N = 9 NDC , 11 AD , 5 TREM2 NDC , 10 TREM2 AD cases . Data information : Data shown as mean ± SEM . The top and the bottom of the box plot ( H ) represent the 75th and 25th percentiles , respectively , and the line represents the median . The whiskers represent the highest and lowest values that are not outliers . One - way ( H ) ANOVA followed by Bonferroni ' s post - hoc test . P - values shown as ns P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 , * * * * P < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. Percentage of synaptic Homer1-immunoreactive puncta found co-localized with PSVue in NL-F KI; Trem2 R47H compared to NL-F KI. 3 ROIs per animal, n=3-4 animals per genotype. Data information: Data shown as mean ± SEM. Unpaired t-test (B) P-values shown as ns P>0.05; *P<0.05; **P<0.01, ****P<0.0001.D. Number of spontaneous calcium transients per minute at 48 h post-treatment of Aβ oligomer in neuron-only culture (cyan), neuron- Trem2 CV microglia co-culture (magenta) or neuron- Trem2 R47H KI microglia co-culture (purple). ∼10 spines per neuron, ∼10 neurons per experiment, from 3-4 independent experiments. Data information: Data shown as mean ± SEM. Each shaded point represents 1 ROI, and each open point represents the mean (or median for GCaMP studies) of each independent experiment. Central bands of the violin plot (D) represent median and quartiles. Kruskal-Wallis test followed by Dunn's test (D), P-values shown as ns P>0.05; *P<0.05; **P<0.01, ****P<0.0001.F. SRM 3D images from the hippocampal CA1 SR of 6 mo WT, Trem2 R47H KI, NL-F KI and NL-F KI; Trem2 R47H KI mice immunostained for Bassoon (cyan), a presynaptic marker enriched in functional active zones. Scale bar, 1 μm.G. Representative western blots comparing levels of cleaved caspase-3 (17/19 kDa) and procaspase-3 (35 kDa) with respect to GAPDH (38 kDa) loading control in synaptosomes isolated from human NDC, AD, NDC TREM2 and AD TREM2 brains. 1 lane represents 1 patient.H. Western blot densitometry analysis showing cleaved caspase-3 levels (left graph) and the ratio of cleaved caspase-3/procaspase-3 (right graph) in synaptosomes (SN) isolated from human NDC, AD, TREM2 NDC and TREM2 variants. N= 9 NDC, 11 AD, 5 TREM2 NDC, 10 TREM2 AD cases. Data information: Data shown as mean ± SEM. The top and the bottom of the box plot (H) represent the 75th and 25th percentiles, respectively, and the line represents the median. The whiskers represent the highest and lowest values that are not outliers. One-way (H) ANOVA followed by Bonferroni's post-hoc test. P-values shown as ns P>0.05; *P<0.05; **P<0.01, ****P<0.0001.


Figure 1 ( A ) NIH - 3T3 cells were transduced with retroviral vectors expressing M45 or GFP . Two days after transduction cells were stimulated with the TLR4 agonist LPS ( 10 µg / ml ) , the TLR2 agonist LTA - SA ( 10 µg / ml ) , or IL - 1β ( 20 ng / ml ) . IκBα levels were determined by immunoblotting . ( B ) NIH - 3T3 cells were transduced with an M45 - expressing or an empty retroviral vector . 30 min after stimulation with IL - 1β ( 10 ng / ml ) cells were fixed , and subcellular localization of the NF - κB p65 subunit was analyzed by immunofluorescence . ( C ) NIH - 3T3 cells expressing an NF - κB - dependent secreted alkaline phosphatase ( SEAP ) reporter were transduced with retroviral vectors expressing M45 or GFP and stimulated 78 h later with the TLR2 agonist Pam3CSK4 ( Pam . , 1 µg / ml ) , the TLR4 agonist LPS ( 0 . 1 µg / ml ) , or IL - 1β ( 5 ng / ml ) . SEAP activity in the supernatant was quantified 15 h after stimulation and is shown as fold induction of SEAP activity of stimulated cells compared to non - stimulated cells ( mean ± SD ) ( D ) NIH - 3T3 cells transduced with retroviral vectors expressing M45 or GFP were stimulated with IL - 1β ( 20 ng / ml , 15 min ) or TNFα ( 10 ng / ml , 5 min ) . Phosphorylated and total p38 in cell lysates was detected by immunoblotting .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) NIH-3T3 cells were transduced with retroviral vectors expressing M45 or GFP. Two days after transduction cells were stimulated with the TLR4 agonist LPS (10 µg/ml), the TLR2 agonist LTA-SA (10 µg/ml), or IL-1β (20 ng/ml). IκBα levels were determined by immunoblotting.(B) NIH-3T3 cells were transduced with an M45-expressing or an empty retroviral vector. 30 min after stimulation with IL-1β (10 ng/ml) cells were fixed, and subcellular localization of the NF-κB p65 subunit was analyzed by immunofluorescence.(C) NIH-3T3 cells expressing an NF-κB-dependent secreted alkaline phosphatase (SEAP) reporter were transduced with retroviral vectors expressing M45 or GFP and stimulated 78 h later with the TLR2 agonist Pam3CSK4 (Pam.,1 µg/ml), the TLR4 agonist LPS (0.1 µg/ml), or IL-1β (5 ng/ml). SEAP activity in the supernatant was quantified 15 h after stimulation and is shown as fold induction of SEAP activity of stimulated cells compared to non-stimulated cells (mean ± SD)(D) NIH-3T3 cells transduced with retroviral vectors expressing M45 or GFP were stimulated with IL-1β (20 ng/ml, 15 min) or TNFα (10 ng/ml, 5 min). Phosphorylated and total p38 in cell lysates was detected by immunoblotting.


Figure 2 ( A ) NIH - 3T3 cells were infected with wt MCMV - GFP , an M45 deletion mutant ( ΔM45 ) or a revertant virus ( RM45 ) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam3CSK4 ( Pam . , 0 . 1 µg / ml ) , the TLR4 agonist LPS ( 10 µg / ml ) , or IL - 1β ( 20 ng / ml ) for the indicated times . Levels of the indicated proteins were analyzed by immunoblotting . ( B ) BMDMs were infected with wt MCMV - GFP , ΔM45 , or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 ( 0 . 1 µM ) . TNFα and IL - 6 levels in the supernatant were determined by ELISA ( mean ± SD ) . ( C ) RAW264 . 7 macrophages were infected with wt MCMV - GFP or ΔM45 at an MOI of 0 . 1 , stimulated 24 h postinfection for 4 hours with TLR agonists Pam3CSK4 ( Pam . ) or Malp - 2 ( TLR2 ) , LPS ( TLR4 ) , R837 ( TLR7 ) , or CpG ( TLR9 ) , in the presence of brefeldin A . Cells were fixed , permeabilized , and stained with a TNFα - specific antibody . The percentages of TNFα - positive cells within infected ( GFP - positive ) cell populations were determined by FACS analysis ( mean ± SD ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam3CSK4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting.(B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD).(C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam3CSK4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).


Figure 3 ( A ) 293A cells were transfected with plasmids expressing HA - tagged M45 or an unrelated MCMV control protein ( m143 ) and Flag - tagged IKK subunits . Lysates were subjected to immunoprecipitation ( IP ) using an anti - HA antibody . Immunoprecipitates were analyzed by immunoblotting ( IB ) using anti - Flag antibody . Whole cell lysates ( WCL ) were immunoblotted using Flag - , HA - , and actin - specific antibodies , respectively . ( B ) Nemo − / − MEFs were transfected with HA - tagged IKK subunits and HA - tagged M45 or an unrelated MCMV control protein ( m142 ) , respectively . IP and IB were performed using anti - HA and anti - Flag antibodies , respectively . WCL were immunoblotted with anti - NEMO antibody and as described for panel A . ( C ) rip1 − / − MEFs were transfected with HA - tagged M45 or an unrelated MCMV control protein ( m142 ) and Flag - tagged NEMO . IP was done with an anti - HA antibody . Immunoprecipitates and WCL were analyzed by immunoblotting using the indicated antibodies . ( D ) 293A cells were transfected with plasmids expressing HA - tagged M45 or m143 ( as a control ) . Lysates were subjected to anti - HA immunoprecipitation . Immunoblotting of precipitates and the WCL was done with antibodies as indicated . ( E ) NIH - 3T3 cells were infected for 4 hours with RM45 ( MCMV expressing HA - tagged M45 ) , ΔM45 , or Rm142 ( MCMV expressing HA - tagged m142 ) at an MOI of 5 . Cell lysates were subjected to IP and IB with antibodies as indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) 293A cells were transfected with plasmids expressing HA-tagged M45 or an unrelated MCMV control protein (m143) and Flag-tagged IKK subunits. Lysates were subjected to immunoprecipitation (IP) using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting (IB) using anti-Flag antibody. Whole cell lysates (WCL) were immunoblotted using Flag-, HA-, and actin-specific antibodies, respectively.(B) Nemo−/− MEFs were transfected with HA-tagged IKK subunits and HA-tagged M45 or an unrelated MCMV control protein ( m142), respectively. IP and IB were performed using anti-HA and anti-Flag antibodies, respectively. WCL were immunoblotted with anti-NEMO antibody and as described for panel A.(C) rip1−/− MEFs were transfected with HA-tagged M45 or an unrelated MCMV control protein (m142) and Flag-tagged NEMO. IP was done with an anti-HA antibody. Immunoprecipitates and WCL were analyzed by immunoblotting using the indicated antibodies.(D) 293A cells were transfected with plasmids expressing HA-tagged M45 or m143 (as a control). Lysates were subjected to anti-HA immunoprecipitation. Immunoblotting of precipitates and the WCL was done with antibodies as indicated.(E) NIH-3T3 cells were infected for 4 hours with RM45 (MCMV expressing HA-tagged M45), ΔM45, or Rm142 (MCMV expressing HA-tagged m142) at an MOI of 5. Cell lysates were subjected to IP and IB with antibodies as indicated.


Figure 4 ( B ) NIH - 3T3 cells were transduced with retroviruses expressing full length M45 , truncated M45 proteins , or GFP . After stimulation with IL - 1β ( 20 ng / ml , 15 min ) , IκBα levels were determined by immunoblotting . ( C ) 293A cells were transfected with plasmids encoding Flag - tagged NEMO and HA - tagged full - length M45 , truncated M45 , or an unrelated MCMV control protein ( m142 ) , respectively . Lysates were subjected to immunoprecipitation ( IP ) with an anti - HA antibody . Immunoprecipitates and the whole cell lysates ( WCL ) were analyzed by immunoblotting ( IB ) with the indicated antibodies . ( D ) Primary BMDMs were mock infected or infected with GFP - expressing wt MCMV ( wt ) , ΔM45 mutant ( ΔM45 ) , M45 revertant ( RM45 ) , or MCMVs expressing Ct or Nt3 ( RCt and RNt3 ) at an MOI of 1 . 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG ( 0 . 5 µM ) or TLR7 agonist R838 ( 0 . 1 µM ) in the presence of brefeldin A . Cells were fixed , permeabilized , and stained with a TNFα - specific antibody . The percentages of TNFα - positive cells within infected ( GFP - positive ) cell populations were determined by FACS analysis ( mean ± SEM ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) NIH-3T3 cells were transduced with retroviruses expressing full length M45, truncated M45 proteins, or GFP. After stimulation with IL-1β (20 ng/ml, 15 min), IκBα levels were determined by immunoblotting.(C) 293A cells were transfected with plasmids encoding Flag-tagged NEMO and HA-tagged full-length M45, truncated M45, or an unrelated MCMV control protein (m142), respectively. Lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody. Immunoprecipitates and the whole cell lysates (WCL) were analyzed by immunoblotting (IB) with the indicated antibodies.(D) Primary BMDMs were mock infected or infected with GFP-expressing wt MCMV (wt), ΔM45 mutant (ΔM45), M45 revertant (RM45), or MCMVs expressing Ct or Nt3 (RCt and RNt3) at an MOI of 1. 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG (0.5 µM) or TLR7 agonist R838 (0.1 µM) in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SEM).


Figure 5 ( A ) NIH - 3T3 cells were infected with MCMV ΔM45 or the revertant virus , RM45 , at an MOI of 3 . Cells were harvested at indicated time points postinfection , and the levels of IKK subunits , M45 , IE1 , and actin in lysates were analyzed by immunoblotting . ( B ) Murine fibroblasts ( NIH - 3T3 , 10 . 1 ) or human embryonic kidney 293A cells were transduced with retroviral vectors expressing M45 or GFP , harvested 72 hours later , and analyzed by immunoblotting . ( C ) ts20 cells harboring a immunoblotting - sensitive E1 enzyme were incubated for 14 h at a permissive ( 35 ° C ) or a restrictive ( 40 . 5 ° C ) temperature . Cells were then mock - infected or infected with RM45 , and analyzed 10 h postinfection by immunoblotting ( D ) NIH - 3T3 cells were mock infected or infected with RM45 ( MOI of 5 ) and treated 2 hpi with 10 mM ammonium chloride ( NH4Cl ) or a mix of lysosomal protease inhibitors ( PI - mix ) . 9 hpi , levels of IKK subunits , M45 , IE1 , and actin were determined by immunoblotting . ( ( E ) NIH - 3T3 cells were stably transduced with retroviral vectors expressing M45 or GFP , and treated for the indicated times with PI - mix , 10 mM NH4Cl , or 5 µM lactacystin , respectively . Levels of the indicated proteins were analyzed by immunoblotting .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) NIH-3T3 cells were infected with MCMV ΔM45 or the revertant virus, RM45, at an MOI of 3. Cells were harvested at indicated time points postinfection, and the levels of IKK subunits, M45, IE1, and actin in lysates were analyzed by immunoblotting.(B) Murine fibroblasts (NIH-3T3, 10.1) or human embryonic kidney 293A cells were transduced with retroviral vectors expressing M45 or GFP, harvested 72 hours later, and analyzed by immunoblotting.(C) ts20 cells harboring a immunoblotting-sensitive E1 enzyme were incubated for 14 h at a permissive (35°C) or a restrictive (40.5°C) temperature. Cells were then mock-infected or infected with RM45, and analyzed 10 h postinfection by immunoblotting(D) NIH-3T3 cells were mock infected or infected with RM45 (MOI of 5) and treated 2 hpi with 10 mM ammonium chloride (NH4Cl) or a mix of lysosomal protease inhibitors (PI-mix). 9 hpi, levels of IKK subunits, M45, IE1, and actin were determined by immunoblotting. ((E) NIH-3T3 cells were stably transduced with retroviral vectors expressing M45 or GFP, and treated for the indicated times with PI-mix, 10 mM NH4Cl, or 5 µM lactacystin, respectively. Levels of the indicated proteins were analyzed by immunoblotting.


Figure 6 ( A ) NIH - 3T3 cells stably expressing Flag - tagged NEMO were mock infected or infected with wt MCMV - GFP or MCMV - GFP - ΔM45 at an MOI of 6 . Cells were fixed 6 hpi , and NEMO distribution was analyzed by immunofluorescence . The arrow indicates the region which is shown in higher magnification in the right corner of the picture . ( B ) NIH - 3T3 cells were transfected with Flag - NEMO and M45 - HA expressing plasmids as indicated . 24 h later , cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags . Regions shown in higher magnification in the left corner of the pictures are indicated by arrows . ( C ) NIH - 3T3 cells stably expressing Flag - NEMO were mock infected or infected with MCMV - M45 - HA or MCMV - ΔM45 at an MOI of 7 . Seven hpi , cells were fixed and analyzed as described for panel B . ( D ) NIH - 3T3 cells were transfected with expression plasmids for Flag - NEMO and M45 - HA or empty vector as indicated . Plasmids encoding GFP - tagged LC3 , Rab5 , or Rab7 were cotransfected . 24 hours posttransfection cells were fixed and used for anti - Flag immunofluorescence staining .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) NIH-3T3 cells stably expressing Flag-tagged NEMO were mock infected or infected with wt MCMV-GFP or MCMV-GFP-ΔM45 at an MOI of 6. Cells were fixed 6 hpi, and NEMO distribution was analyzed by immunofluorescence. The arrow indicates the region which is shown in higher magnification in the right corner of the picture.(B) NIH-3T3 cells were transfected with Flag-NEMO and M45-HA expressing plasmids as indicated. 24 h later, cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags. Regions shown in higher magnification in the left corner of the pictures are indicated by arrows.(C) NIH-3T3 cells stably expressing Flag-NEMO were mock infected or infected with MCMV-M45-HA or MCMV-ΔM45 at an MOI of 7. Seven hpi, cells were fixed and analyzed as described for panel B.(D) NIH-3T3 cells were transfected with expression plasmids for Flag-NEMO and M45-HA or empty vector as indicated. Plasmids encoding GFP-tagged LC3, Rab5, or Rab7 were cotransfected. 24 hours posttransfection cells were fixed and used for anti-Flag immunofluorescence staining.


Figure 7 ( A ) atg5 − / − and atg5 + / + MEFs were mock infected or infected with MCMVΔM45 or the revertant virus , RM45 , at an MOI of 10 . Eight hpi cells were harvested and levels of the indicated proteins were analyzed by immunoblotting . ( B ) 10 . 1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5 . Cells were left untreated or treated 30 min after infection with 10 mM NH4Cl to block lysosomal degradation of LC3 - II . Cells were harvested at the indicated time points , and LC3 - II levels in cell lysates were analyzed by immunoblotting using an LC3 - II - specific antibody . LC3 - II levels were quantified by densitometric analysis and normalized using the corresponding actin levels . Fold increases are shown relative to mock - infected cells .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) atg5−/− and atg5+/+ MEFs were mock infected or infected with MCMVΔM45 or the revertant virus, RM45, at an MOI of 10. Eight hpi cells were harvested and levels of the indicated proteins were analyzed by immunoblotting.(B) 10.1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5. Cells were left untreated or treated 30 min after infection with 10 mM NH4Cl to block lysosomal degradation of LC3-II. Cells were harvested at the indicated time points, and LC3-II levels in cell lysates were analyzed by immunoblotting using an LC3-II-specific antibody. LC3-II levels were quantified by densitometric analysis and normalized using the corresponding actin levels. Fold increases are shown relative to mock-infected cells.


Figure 8 ( A ) NIH - 3T3 cells stably expressing GFP - LC3 were transduced with retroviral vectors expressing M45 or Ct3 . Three days later , cells were analyzed by confocal laser scanning microscopy . GFP - LC3 dots were counted in 50 cells per sample . Results are shown as box and whisker plots . The bottom and top of the box represent the first and third quartile , respectively . The horizontal line within the box represents the median . The minima and maxima within 1 . 5 - fold interquartile range are shown as whiskers . Dots represent outliers . Significance was determined using ANOVA ( * * * , p < 0 . 001 ) . M45 and Ct3 were detected by immunoblot ( B ) Fluorescence images of cells described above . Large GFP - LC3 aggregates are indicated by arrow heads . ( C ) Electron micrograph of a double - membrane autophagosome in an M45 - expressing cell . ( D ) Autophagosomes were counted in 10 randomly selected M45 - and Ct3 - expressing cells ( 2 - 3 TEM sections per cell ) . Results are shown in box and whisker plots as described for panel A . Significance was determined using the student ' s t - test ( * * * , p < 0 . 001 ) . ( E ) Epifluorescence image of an M45 - expressing cell with large GFP - LC3 structures ( F ) TEM image of the same cell reveals large aggregates marked with ' A ' . ( G , H ) Successive enlargement of the region marked in panel F shows an example of an aggregate - associated autophagosome .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) NIH-3T3 cells stably expressing GFP-LC3 were transduced with retroviral vectors expressing M45 or Ct3. Three days later, cells were analyzed by confocal laser scanning microscopy. GFP-LC3 dots were counted in 50 cells per sample. Results are shown as box and whisker plots. The bottom and top of the box represent the first and third quartile, respectively. The horizontal line within the box represents the median. The minima and maxima within 1.5-fold interquartile range are shown as whiskers. Dots represent outliers. Significance was determined using ANOVA (***, p<0.001). M45 and Ct3 were detected by immunoblot(B) Fluorescence images of cells described above. Large GFP-LC3 aggregates are indicated by arrow heads.(C) Electron micrograph of a double-membrane autophagosome in an M45-expressing cell.(D) Autophagosomes were counted in 10 randomly selected M45- and Ct3-expressing cells (2-3 TEM sections per cell). Results are shown in box and whisker plots as described for panel A. Significance was determined using the student's t-test (***, p<0.001).(E) Epifluorescence image of an M45-expressing cell with large GFP-LC3 structures(F) TEM image of the same cell reveals large aggregates marked with 'A'. (G, H) Successive enlargement of the region marked in panel F shows an example of an aggregate-associated autophagosome.


Figure 1Representative images of γH2AX immuno - FISH in troponin - C / α - actinin ( top left and bottom left panels , respectively ) positive mouse CMs ( white - troponin - C / α - actinin ; red - telo - FISH ; green - γH2AX ) . Images are Z - projections of 0 . 1μm stacks taken with a 100x oil objective . Scale bar : 20μm . Right panels represent a single Z - plane where co - localisation between a γH2AX foci and telomere was observed , Scale bars 1μm . ( Below ) 3D reconstruction of Immuno - FISH using STED microscopy for γH2A . X and telomeres in a CM from 30 month old mice . Scale bars as indicated and scale the same for both individual TAF examples using STED . Mean number of TAF α - actinin positive CMs from C57BL / 6 mice . Data are mean ± SEM of n = 4 - 7 mice per age group . α - actinin positive CMs from C57BL / 6 mice . Data are mean ± SEM of n = 4 - 7 mice per age group . 100 α - actinin positive CMs were quantified per mouse . Mean number of total γH2A . X foci in α - actinin positive CMs from C57BL / 6 mice . Data are mean ± SEM of n = 4 - 7 mice per age group . 100 α - actinin positive CMs were quantified per mouse . % of γH2A . X foci - positive nuclei ( E ) in α - actinin positive CMs from C57BL / 6 mice . Data are mean ± SEM of n = 4 - 7 mice per age group . 100 α - actinin positive CMs were quantified per mouse . Fold enrichment of γH2AX at telomere repeats by real - time PCR . Graph represents fold enrichment of γH2AX at telomeric repeats between IgG control , 3 and 30 month old whole mouse hearts . Data are mean ± SEM of n = 3 mice per age group . Quantitative PCR - ELISA TRAP assay comparing telomerase activity of 3 and 30 month old C57BL / 6 mice whole heart lysates . Data are mean ± SEM of n = 4 mice per age group . Histograms displaying distributions of telomere intensity in CMs analysed by 3D Q - FISH in young ( 4 months ) and old ( 30 months ) wild - type mice . Data are from n = 3 mice . > 100 CMs were analysed per mouse . % of telomere FISH signal loss in 30 months old wild - type mice and late generation Terc - / - mice ( 6 months old ) in comparison to 4 months old wild - type mice . Data are from n = 3 mice . > 100 CMs were analysed per mouse .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative images of γH2AX immuno-FISH in troponin-C/α-actinin (top left and bottom left panels, respectively) positive mouse CMs (white - troponin-C/α-actinin; red - telo-FISH; green - γH2AX). Images are Z-projections of 0.1μm stacks taken with a 100x oil objective. Scale bar: 20μm. Right panels represent a single Z-plane where co-localisation between a γH2AX foci and telomere was observed, Scale bars 1μm. (Below) 3D reconstruction of Immuno-FISH using STED microscopy for γH2A.X and telomeres in a CM from 30 month old mice. Scale bars as indicated and scale the same for both individual TAF examples using STED.Mean number of TAF α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group.α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.Mean number of total γH2A.X foci in α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.% of γH2A.X foci -positive nuclei (E) in α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.Fold enrichment of γH2AX at telomere repeats by real-time PCR. Graph represents fold enrichment of γH2AX at telomeric repeats between IgG control, 3 and 30 month old whole mouse hearts. Data are mean ± SEM of n=3 mice per age group.Quantitative PCR-ELISA TRAP assay comparing telomerase activity of 3 and 30 month old C57BL/6 mice whole heart lysates. Data are mean ± SEM of n=4 mice per age group.Histograms displaying distributions of telomere intensity in CMs analysed by 3D Q-FISH in young (4 months) and old (30 months) wild-type mice. Data are from n=3 mice. > 100 CMs were analysed per mouse.% of telomere FISH signal loss in 30 months old wild-type mice and late generation Terc-/- mice (6 months old) in comparison to 4 months old wild-type mice. Data are from n=3 mice. >100 CMs were analysed per mouse.


Figure 2Representative images of mouse embryonic cardiomyocytes at days 0 , 3 , 5 and 10 days following 10Gy X - irradiation . Left panels represent troponin C - positive embryonic cardiomyocytes ( troponin C - magenta ; DAPI - light blue ) . Middle panels display γH2AX foci ( green ) and telomeres ( red ) in Z projections of 0 . 1µm slices , with white arrows indicating co - localisation . Co - localising foci are amplified in the right - hand panels ( amplified images represent a single Z - plane where co - localisation was observed ) . Scale bars represent 10μm . Scale bars in single plane images 500nm . ( Left ) Mean number of both TAF and non - TAF in troponin I - positive mouse embryonic cardiomyocytes at days 0 , 3 , 5 and 10 following 10Gy X - irradiation . Data are mean ± S . E . M of n = 3 independent experiments ; 30 - 50 troponin positive cardiomyocytes were analysed per experiment . ( Right ) Mean percentage of γH2AX foci co - localising with telomeres ( % TAF ) in troponin C - positive mouse embryonic cardiomyocytes at days 0 , 3 , 5 and 10 following 10Gy X - irradiation . Statistical analysis performed using one - way ANOVA ( Holm - Sidak method ) ; * P < 0 . 05 . Significant differences were found for mean number of non - TAF , but not for mean number of TAF . Mean number of both TAF and non - TAF in neonatal rat cardiomyocytes at days 0 , 3 , 5 , 10 days following treatment for 24h with H2O2 . Data are mean ± S . E . M of n = 3 . > 50 cells were quantified per condition . ( Left ) Representative images of γH2AX immuno - FISH in H9C2 myoblasts 3 days 10Gy X - irradiation ( red - telo - FISH ; green - γH2AX ) . White arrows identify areas shown in higher magnification panels . ( Right ) Mean number of both TAF and non - TAF in H9C2 myoblasts at days 3 and 5 following 10Gy X - irradiation . Data are mean ± S . E . M of n = 3 . > 50 cells were quantified per condition . Statistical analysis performed using one - way ANOVA ( Holm - Sidak method ) ; * * * P < 0 . 001 . Scale bars represent 1μm . Scale bars in single plane images 500nm . Representative time - lapse images of H9C2 rat cardiomyoblasts expressing AcGFP - 53BP1 from 3 days after 10Gy irradiation at the indicated times ( mins ) . Images are maximum intensity projections with a 6 . 7µm focal depth . Kaplan - Meyer survival curves for AcGFP - 53BP1c DDR foci in H9C2 cells 3 days after 10Gy irradiation at 10 minute intervals for 24 hours . > 500 foci from 10 cells were tracked per condition . Schematic illustration showing 1 month old C57BL / 6 mice treated with 2Gy whole body X - irradiation , followed by a recovery period of 11 months before culling at 12 months of age . Mean number of TAF in α - actinin positive cardiomyocytes . Data are mean ± SEM of n = 3 mice per group . 90 α - actinin positive cardiomyocytes were quantified per condition .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative images of mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 days following 10Gy X-irradiation. Left panels represent troponin C-positive embryonic cardiomyocytes (troponin C - magenta; DAPI - light blue). Middle panels display γH2AX foci (green) and telomeres (red) in Z projections of 0.1µm slices, with white arrows indicating co-localisation. Co-localising foci are amplified in the right-hand panels (amplified images represent a single Z-plane where co-localisation was observed). Scale bars represent 10μm. Scale bars in single plane images 500nm.(Left) Mean number of both TAF and non-TAF in troponin I-positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10Gy X-irradiation. Data are mean ±S.E.M of n=3 independent experiments; 30-50 troponin positive cardiomyocytes were analysed per experiment. (Right) Mean percentage of γH2AX foci co-localising with telomeres (% TAF) in troponin C-positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10Gy X-irradiation. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); * P<0.05. Significant differences were found for mean number of non-TAF, but not for mean number of TAF.Mean number of both TAF and non-TAF in neonatal rat cardiomyocytes at days 0, 3, 5, 10 days following treatment for 24h with H2O2. Data are mean ±S.E.M of n=3. >50 cells were quantified per condition.(Left) Representative images of γH2AX immuno-FISH in H9C2 myoblasts 3 days 10Gy X-irradiation (red - telo-FISH; green - γH2AX). White arrows identify areas shown in higher magnification panels. (Right) Mean number of both TAF and non-TAF in H9C2 myoblasts at days 3 and 5 following 10Gy X-irradiation. Data are mean ±S.E.M of n=3. >50 cells were quantified per condition. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); ***P<0.001. Scale bars represent 1μm. Scale bars in single plane images 500nm.Representative time-lapse images of H9C2 rat cardiomyoblasts expressing AcGFP-53BP1 from 3 days after 10Gy irradiation at the indicated times (mins). Images are maximum intensity projections with a 6.7µm focal depth.Kaplan-Meyer survival curves for AcGFP-53BP1c DDR foci in H9C2 cells 3 days after 10Gy irradiation at 10 minute intervals for 24 hours. >500 foci from 10 cells were tracked per condition.Schematic illustration showing 1 month old C57BL/6 mice treated with 2Gy whole body X-irradiation, followed by a recovery period of 11 months before culling at 12 months of age. Mean number of TAF in α-actinin positive cardiomyocytes. Data are mean ± SEM of n=3 mice per group. 90 α-actinin positive cardiomyocytes were quantified per condition.