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 .

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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 .

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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 1 . HEK293T cells were transfected with 200 nM siRNAs per well . After 24 h , the cells were infected with HIV - luc / 2019 - nCoV pseudoviruses . After washing and incubation with fresh medium for 48 h , the intracellular luciferase activity was measured . Ordinary one - way ANOVA test was used for this analysis . The results are shown as the mean and SEM . * P < 0 . 05 .

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Figure 1.HEK293T cells were transfected with 200 nM siRNAs per well. After 24 h, the cells were infected with HIV-luc/2019-nCoV pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.


Figure 2 . assay . B , Teicoplanin inhibit the entry of 2019 - nCoV and SARS - nCoV . Chemical structure of teicoplanin ( Left ) . A549 cells were seeded in a 96 - well plate , and 24 h later , the cells were infected with HIV - luc / 2019 - nCoV , HIV - luc / SARS - nCoV or HIV - luc / VSVG pseudoviruses . After washing and incubation with fresh medium for 48 h , the intracellular luciferase activity was measured . Ordinary one - way ANOVA test was used for this analysis . The results are shown as the mean and SEM . * P < 0 . 05 . C , A549 cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

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 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

Figure 2.assay.B, Teicoplanin inhibit the entry of 2019-nCoV and SARS-nCoV. Chemical structure of teicoplanin (Left). A549 cells were seeded in a 96-well plate, and 24 h later, the cells were infected with HIV-luc/2019-nCoV, HIV-luc/SARS-nCoV or HIV-luc/VSVG pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.C, A549 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.


Figure 3 . types . A , HEK293T cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software . Bsoftware . B , Huh7 cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software . CA549 cells were incubated with dalbavancin ( D ) at various concentrations and infected with HIV - luc / 2019 - nCoV pseudoviruses . The intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA test was used for this analysis . dalbavancinD , A549 cells were incubated with vancomycin at various concentrations and infected with HIV - luc / 2019 - nCoV pseudoviruses . The intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA test was used for this analysis . Left : Chemical structure of

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 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 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

Figure 3.types.A, HEK293T cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.Bsoftware.B, Huh7 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.CA549 cells were incubated with dalbavancin (D) at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. dalbavancinD, A549 cells were incubated with vancomycin at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. Left: Chemical structure of vancomycin


Figure 1Isosurface of the subtomogram average ( in pink ) with an applied C5 symmetry and 0 . 69 nm tomographic slices ( A - E ) at the indicated heights . Scale bars 10 nm . The average is shown in side view , whereas the slices represent perpendicular slices . The 5 pairs of pillars formed a narrow central channel and separated into 10 arches towards the base ( base not visible at the used threshold ) . The division of the structure into subparts was adapted from ( Durand et al . , 2015 ) . Subvolumes were extracted from cryotomograms of ghost cells and FIB - milled intact E . coli BL21 cells heterologously expressing TssJLM Slice ( 0 . 69 nm ) through the non - symmetrized average . Note that the C5 symmetry was

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Figure 1Isosurface of the subtomogram average (in pink) with an applied C5 symmetry and 0.69 nm tomographic slices (A-E) at the indicated heights. Scale bars 10 nm. The average is shown in side view, whereas the slices represent perpendicular slices. The 5 pairs of pillars formed a narrow central channel and separated into 10 arches towards the base (base not visible at the used threshold). The division of the structure into subparts was adapted from (Durand et al., 2015). Subvolumes were extracted from cryotomograms of ghost cells and FIB-milled intact E. coli BL21 cells heterologously expressing TssJLM Slice (0.69 nm) through the non-symmetrized average. Note that the C5 symmetry was visible


Figure 2Slice ( 9 . 7 nm ) through a cryotomogram of a FIB - milled E . coli BL21 cell expressing TssJLM . The average shown in Figure 1 was placed back at the positions and orientations of the individual subvolumes that were used to generate the final average . The zoomed - in area highlights the position of the TssJLM particles within the inner and the outer membrane . Some particles were found in cytoplasmic membrane invaginations , as indicated by white arrows . Scale bar 100 nmCryotomographic slices ( 9 . 7 nm ) showing side views of TssJLM . In these examples , the basal parts of TssJLM ( red arrows ) could be seen extending into the cytoplasm . Scale bar 10

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Figure 2Slice (9.7 nm) through a cryotomogram of a FIB-milled E. coli BL21 cell expressing TssJLM. The average shown in Figure 1 was placed back at the positions and orientations of the individual subvolumes that were used to generate the final average. The zoomed-in area highlights the position of the TssJLM particles within the inner and the outer membrane. Some particles were found in cytoplasmic membrane invaginations, as indicated by white arrows. Scale bar 100 nmCryotomographic slices (9.7 nm) showing side views of TssJLM. In these examples, the basal parts of TssJLM (red arrows) could be seen extending into the cytoplasm. Scale bar 10 nm


Figure 4Fluorescence of prey cells ( green ) indicating their survival against attacker cells after 4 h . The image of a representative bacterial competition spot is shown on the upper part . The relative fluorescent level ( in AU ) and the number of recovered E . coli recipient cells ( in log10 of colony forming units ( cfu ) ) are indicated in the lower graphs . The assays were performed from at least three independent cultures , with technical triplicates and a representative technical triplicate is shown . Error bars in the left graph represent ± standard deviation ( SD ) from technical triplicate . The circles in the right graph indicate values from technical triplicate , and the average is indicated by the bar . Time - lapse fluorescence microscopy recordings showing localization and dynamics of the sfGFPTssM fusion protein in the parental ( WT ) and TssJ mutated strains ( TssJ D97K ) . Individual images were taken every 60 seconds . The corresponding time of each micrograph is indicated in the lower right part of the image . Stable foci in the WT strain and less stable foci in the TssJ D97K strain are respectively indicated by white and blue arrowheads . Scale bars , 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 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

Figure 4Fluorescence of prey cells (green) indicating their survival against attacker cells after 4 h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar.Time-lapse fluorescence microscopy recordings showing localization and dynamics of the sfGFPTssM fusion protein in the parental (WT) and TssJ mutated strains (TssJ D97K). Individual images were taken every 60 seconds. The corresponding time of each micrograph is indicated in the lower right part of the image. Stable foci in the WT strain and less stable foci in the TssJ D97K strain are respectively indicated by white and blue arrowheads. Scale bars, 1 µm.


Figure 5Fluorescence of prey cells ( green ) indicating their survival against attacker cells after 4h . The image of a representative bacterial competition spot is shown on the upper part . The relative fluorescent level ( in AU ) and the number of recovered E . coli recipient cells ( in log10 of colony forming units ( cfu ) ) are indicated in the lower graphs . The assays were performed from at least three independent cultures , with technical triplicates and a representative technical triplicate is shown . Error bars in the left graph represent ± standard deviation ( SD ) from technical triplicate . The circles in the right graph indicate values from technical triplicate , and the average is indicated by the bar . Fluorescence microscopy recordings showing sfGFPTssM foci in the parental ( WT ) and TssM mutated strains ( TssM Q779C / N780C , TssM ∆ 777 - 783 ) . TssM foci containing cells are indicated by arrowheads . Microscopy analyses were performed independently three times , each in technical triplicate , and a representative experiment is shown . Scale bars , 1 µm . Fluorescence microscopy recordings showing TssBsfGFP sheath in the parental ( WT ) and TssM mutated strains ( TssM Q779C / N780C , TssM ∆ 777 - 783 ) . Fluorescent sheath containing cells are indicated by arrowheads . Microscopy analyses were performed independently three times , each in technical triplicate , and a representative experiment is shown . Scale bars , 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 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 O O O O O O O O O O O O O O

Figure 5Fluorescence of prey cells (green) indicating their survival against attacker cells after 4h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar.Fluorescence microscopy recordings showing sfGFPTssM foci in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). TssM foci containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm.Fluorescence microscopy recordings showing TssBsfGFP sheath in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). Fluorescent sheath containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm.


Figure 1 ( B ) TLC analysis of ganglioside species in human serum . ( C ) Quantification by densitometry of major ganglioside species GM3 and GD1a in human serum . Data expressed as mean ± SD , n = 6 . ( E ) Quantification by LC - MS / MS analysis of serum GM3 species with differing acyl chain structures . Total abundance of 10 species was defined as 1 . Data expressed as mean ± SD , n = 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (B) TLC analysis of ganglioside species in human serum. (C) Quantification by densitometry of major ganglioside species GM3 and GD1a in human serum. Data expressed as mean ± SD, n = 6.  (E) Quantification by LC-MS/MS analysis of serum GM3 species with differing acyl chain structures. Total abundance of 10 species was defined as 1. Data expressed as mean ± SD, n = 6.


Figure 2 ( A ) Heat map analysis of serum GM3 species in various pathological phases : control ( n = 24 ) , VFA ( n = 38 ) , lipidemia ( n = 28 ) , glycemia ( n = 15 ) , and lipidemia + glycemia ( n = 17 ) . Colors indicate fold change average of each species relative to control ( defined as 1 ) , as shown in key at right . Order of pathological phases corresponds to increments of HOMA - IR and serum CRP . ( B - D ) Properties of various GM3 species as a function of pathological phases : control ( n = 24 ) , VFA ( n = 38 ) , lipidemia ( n = 28 ) , glycemia ( n = 15 ) , and lipidemia + glycemia ( n = 17 ) . Data shown are relative abundances of total LCFA species ( 16 : 0 , 18 : 0 , 20 : 0 ) ( B ) , total VLCFA species ( 22 : 0 , 23 : 0 , 24 : 0 , h24 : 0 ) ( C ) , and total unsaturated VLCFA species ( 22 : 1 , 24 : 1 , h24 : 1 ) ( D ) relative to total of ten major GM3 species ( defined as 1 ) in each subject . ( E - H ) Properties of various GM3 species as a function of BMI : LCFA - GM3 ( E ) , VLCFA - GM3 ( F ) , unsaturated VLCFA - GM3 ( G ) , and α - hydroxyVLCFA - GM3 ( h24 : 0 ) ( H ) . Colors indicate disease severity : light blue , no abnormal scores ( n = 25 ) ; orange , early - phase obesity ( n = 74 ) ; purple , severe obesity ( n = 23 ) . ( I , J ) Spearman ' s correlations for GM3 h24 : 0 vs . ALT ( I ) and vs . HOMA - IR ( J ) . ( K ) Plots of α - hydroxylation rate ( h24 : 0 / 24 : 0 ) vs . serum CRP . Colors indicate range of CRP value ( mg / dL ) : light blue , 0 . 01 - 0 . 02 ( n = 21 ) ; orange , 0 . 03 - 0 . 09 ( n = 56 ) ; gray , 0 . 10 - 0 . 29 ( n = 29 ) ; red , 0 . 3 - 1 . 0 ( diagnostically abnormal ; n = 15 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heat map analysis of serum GM3 species in various pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Colors indicate fold change average of each species relative to control (defined as 1), as shown in key at right. Order of pathological phases corresponds to increments of HOMA-IR and serum CRP.  (B-D) Properties of various GM3 species as a function of pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Data shown are relative abundances of total LCFA species (16:0, 18:0, 20:0) (B), total VLCFA species (22:0, 23:0, 24:0, h24:0) (C), and total unsaturated VLCFA species (22:1, 24:1, h24:1) (D) relative to total of ten major GM3 species (defined as 1) in each subject.  (E-H) Properties of various GM3 species as a function of BMI: LCFA-GM3 (E), VLCFA-GM3 (F), unsaturated VLCFA-GM3 (G), and α-hydroxyVLCFA-GM3 (h24:0) (H). Colors indicate disease severity: light blue, no abnormal scores (n=25); orange, early-phase obesity (n=74); purple, severe obesity (n=23).  (I, J) Spearman's correlations for GM3 h24:0 vs. ALT (I) and vs. HOMA-IR (J).  (K) Plots of α-hydroxylation rate (h24:0/24:0) vs. serum CRP. Colors indicate range of CRP value (mg/dL): light blue, 0.01-0.02 (n=21); orange, 0.03-0.09 (n=56); gray, 0.10-0.29 (n=29); red, 0.3-1.0 (diagnostically abnormal; n=15).


Figure 4 ( B , C ) GM3 - mediated enhancement and inhibition of proinflammatory cytokine production from LPS - stimulated monocytes ( LPS : 0 . 06 , 0 . 13 , 0 . 25 ng / mL ) . TNF - α ( B ) and IL - 6 ( C ) production in culture supernatant was measured by ELISA . ( D , E ) Co - stimulation of monocytes by LPS plus GM3 species or complex ganglioside species ( 1 . 5 , 3 . 0 , 4 . 5 µM ) . TNF - α ( D ) and IL - 6 ( E ) production were shown in heat maps . ( F ) Co - stimulation of monocytes by LPS plus GM3 species or precursor GSL species . TNF - α , IL - 6 , and IL - 12 / 23 production were shown in heat maps . ( G ) Inhibitory effect of LCFA and unsaturated VLCFA - GM3 on LVCFA - GM3 species . IL - 6 production in culture supernatant was measured by ELISA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C) GM3-mediated enhancement and inhibition of proinflammatory cytokine production from LPS-stimulated monocytes (LPS: 0.06, 0.13, 0.25 ng/mL). TNF-α (B) and IL-6 (C) production in culture supernatant was measured by ELISA.  (D, E) Co-stimulation of monocytes by LPS plus GM3 species or complex ganglioside species (1.5, 3.0, 4.5 µM). TNF-α (D) and IL-6 (E) production were shown in heat maps.  (F) Co-stimulation of monocytes by LPS plus GM3 species or precursor GSL species. TNF-α, IL-6, and IL-12/23 production were shown in heat maps.  (G) Inhibitory effect of LCFA and unsaturated VLCFA-GM3 on LVCFA-GM3 species. IL-6 production in culture supernatant was measured by ELISA.


Figure 5 ( A ) Co - stimulation of human monocytes by GM3 species plus various TLR ligands : LPS ( 0 . 13 ng / mL ) , TLR4 / MD - 2 , Pam3CSK4 ( 0 . 5 µg / mL ) , TLR1 / 2 , Flagellin ( 50 ng / mL ) , TLR5 , R848 ( 0 . 5 µg / mL ) , TLR7 / 8 , MALP - 2 ( 1 . 0 ng / mL ) , TLR2 / 6 . IL - 6 production in culture supernatant was quantified by ELISA ( shown in a heat map ) . ( B ) Co - stimulation of monocytes by GM3 species ( 16 : 0 , 24 : 0 , 24 : 1 ) plus synthetic TLR4 ligands LA506 ( 15 ng / mL ) , LA505 ( 150 ng / mL ) , or LA504 ( 150 ng / mL ) . ( C , D ) Production of IL - 6 ( C ) and TNF - α ( D ) in culture supernatant following co - stimulation of monocytes by GM3 species plus human HMGB1 . ( E ) Overexpression of hTLR4 , hTLR4 / hMD - 2 , hTLR4 ( P714H ) / MD - 2 in HEK293T cells , and co - stimulation by GM3 22 : 0 with LPS ( 5 ng / mL ) . TLR4 activation was monitored by NF - κB luciferase reporter assay ( termed " Relative Luc Activity " on Y - axis ) . ( F , Co - stimulation of hTLR4 / hMD - 2 by GM3 species plus LPS ( 5 ng / mL ) G ) Co - stimulation of hTLR4 / hMD - 2 by GM3 species plus LPS ( 5 ng / mL ) and further addition of soluble human CD14 ( 1 µg / mL ) ( H ) Stimulation of Mal - overexpressing HEK293T cells by GM3 species . ( I ) Co - stimulation of hTLR4 / hMD - 2 by LPS ( 5 ng / mL ) plus various mixtures of GM3 species .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-stimulation of human monocytes by GM3 species plus various TLR ligands: LPS (0.13 ng/mL), TLR4/MD-2, Pam3CSK4 (0.5 µg/mL), TLR1/2, Flagellin (50 ng/mL), TLR5, R848 (0.5 µg/mL), TLR7/8, MALP-2 (1.0 ng/mL), TLR2/6. IL-6 production in culture supernatant was quantified by ELISA (shown in a heat map).  (B) Co-stimulation of monocytes by GM3 species (16:0, 24:0, 24:1) plus synthetic TLR4 ligands LA506 (15 ng/mL), LA505 (150 ng/mL), or LA504 (150 ng/mL).  (C, D) Production of IL-6 (C) and TNF-α (D) in culture supernatant following co-stimulation of monocytes by GM3 species plus human HMGB1.  (E) Overexpression of hTLR4, hTLR4/hMD-2, hTLR4 (P714H) /MD-2 in HEK293T cells, and co-stimulation by GM3 22:0 with LPS (5 ng/mL). TLR4 activation was monitored by NF-κB luciferase reporter assay (termed "Relative Luc Activity" on Y-axis).  (F, Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL)  G) Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL) and further addition of soluble human CD14 (1 µg/mL)  (H) Stimulation of Mal-overexpressing HEK293T cells by GM3 species.  (I) Co-stimulation of hTLR4/hMD-2 by LPS (5 ng/mL) plus various mixtures of GM3 species.


Figure 6 ( A ) Co - stimulation of RAW macrophages by GM3 species plus various TLR ligands : LPS ( 0 . 5 , 1 . 0 ng / mL ) , bovine thymus HMGB1 ( 0 . 25 µg / mL ) , Pam3CSK4 ( 50 ng / mL ) , Poly I : C ( 10 µg / mL ) , R848 ( 4 ng / mL ) , and CpG type - B ( 20 nM ) . TNF - α production in culture supernatant was quantified by ELISA . ( B ) Co - stimulation of RAW macrophages by low - and high - dose LPS ( 0 , 5 , 50 ng / mL ) plus GM3 22 : 0 ( 5 µM ) . Time course of TNF - α production in culture supernatant was quantified by ELISA . ( C ) Co - stimulation of BMDMs from C3H / HeN or C3H / HeJ mice by GM3 species plus LPS ( 0 . 5 ng / mL ) , and TNF - α production in culture supernatant . ( d ) Co - stimulation of mTLR4 / mMD - 2 - expressing HEK293T cells by GM3 species plus LPS ( 2 . 5 ng / mL ) , and further addition of soluble mouse CD14 - Fc fusion protein ( 1 µg / mL ) . ( E Co - stimulation of BMDMs from C3H / HeN mice by LPS plus GM3 species and complex ganglioside species ( E ; 2 . 5 , 5 . 0 , 10 µM ) , ( shown in heat maps ) . F ) Co - stimulation of BMDMs from C3H / HeN mice by LPS plus GM3 species and precursor GSL species ( F ; 2 . 5 , 5 . 0 , 10 µM ) ( shown in heat maps ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-stimulation of RAW macrophages by GM3 species plus various TLR ligands: LPS (0.5, 1.0 ng/mL), bovine thymus HMGB1 (0.25 µg/mL), Pam3CSK4 (50 ng/mL), Poly I:C (10 µg/mL), R848 (4 ng/mL), and CpG type-B (20 nM). TNF-α production in culture supernatant was quantified by ELISA.  (B) Co-stimulation of RAW macrophages by low- and high-dose LPS (0, 5, 50 ng/mL) plus GM3 22:0 (5 µM). Time course of TNF-α production in culture supernatant was quantified by ELISA.  (C) Co-stimulation of BMDMs from C3H/HeN or C3H/HeJ mice by GM3 species plus LPS (0.5 ng/mL), and TNF-α production in culture supernatant.  (d) Co-stimulation of mTLR4/mMD-2-expressing HEK293T cells by GM3 species plus LPS (2.5 ng/mL), and further addition of soluble mouse CD14-Fc fusion protein (1 µg/mL).  (E Co-stimulation of BMDMs from C3H/HeN mice by LPS plus GM3 species and complex ganglioside species (E; 2.5, 5.0, 10 µM), (shown in heat maps).  F) Co-stimulation of BMDMs from C3H/HeN mice by LPS plus GM3 species and precursor GSL species (F; 2.5, 5.0, 10 µM) (shown in heat maps).


Figure 7 ( A ) GM3 molecular species of 6 - wk - old control C57 / BL6 mice and ob / ob mice were analyzed respectively by LC - MS / MS ( n = 3 ) . ( B ) GM3 molecular species of normal - diet ( ND ) and high - fat - diet ( HFD ) C57 / BL6 mice were analyzed by LC - MS / MS ( n = 4 ) . ( C ) TLC analysis of acidic GSL fraction ( equivalent to 0 . 1 mg protein ) from epididymal fat pads of C3H / HeN ( A ) and C3H / HeJ mice ( B ) on ND or HFD for 8 wk . ( D ) GM3 molecular species of C3H / HeN mice ( ND , HFD ) and C3H / HeJ mice ( ND , HFD ) were analyzed by LC - MS / MS ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GM3 molecular species of 6-wk-old control C57/BL6 mice and ob/ob mice were analyzed respectively by LC-MS/MS (n=3).  (B) GM3 molecular species of normal-diet (ND) and high-fat-diet (HFD) C57/BL6 mice were analyzed by LC-MS/MS (n=4).  (C) TLC analysis of acidic GSL fraction (equivalent to 0.1 mg protein) from epididymal fat pads of C3H/HeN (A) and C3H/HeJ mice (B) on ND or HFD for 8 wk.  (D) GM3 molecular species of C3H/HeN mice (ND, HFD) and C3H/HeJ mice (ND, HFD) were analyzed by LC-MS/MS (n=4).


Figure 8 ( C , D ) Alanine scanning for basic residues involved in signal transduction via VLCFA - GM3 ( n = 5 ) ( C ) , and combinations of effective mutations ( n = 6 ) ( D ) . Signal transduction was monitored by NF - κB reporter assay . ( E - G ) Comparative inhibitory effects of GM3 16 : 0 on mTLR4 / mMD - 2 ( E ) , mTLR4 / hMD - 2 ( domain - swapped complex ) ( F ) , and hTLR4 / hMD - 2 ( G ) ( n = 3 ) . ( H ) Cross - linked SDS - PAGE analysis of recombinant mTLR4 ( extracellular domain ) / mMD - 2 complexed with GM3 18 : 0 , GM3 species enhancing mTLR4 activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (C, D) Alanine scanning for basic residues involved in signal transduction via VLCFA-GM3 (n=5) (C), and combinations of effective mutations (n=6) (D). Signal transduction was monitored by NF-κB reporter assay.  (E-G) Comparative inhibitory effects of GM3 16:0 on mTLR4/mMD-2 (E), mTLR4/hMD-2 (domain-swapped complex) (F), and hTLR4/hMD-2 (G) (n=3).  (H) Cross-linked SDS-PAGE analysis of recombinant mTLR4 (extracellular domain)/mMD-2 complexed with GM3 18:0, GM3 species enhancing mTLR4 activation.


Figure 1A . Representative spinning disk confocal live - cell imaging time series of U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , treated with control and KIF4A siRNAs . White arrowheads highlight poleward motion of the photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec . B . Representative spinning disk confocal live - cell imaging time series images of S - trityl - L - cysteine ( STLC ) - treated U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) . Note that MT - flux is abrogated in presence of taxol ( lower panel ) . Scale bars , 10 µm . Time , min : sec . C . Illustration of 405nm laser - photoactivated regions in monopolar spindles ( blue circles , left ) . The effect of photoactivation and region selected for kymograph generation ( dashed white rectangle , right ) . Scale bar , 10 µm . D . Corresponding kymograph profiles of the photoactivated regions in bipolar and monopolar spindles used for quantification of the flux rates ( red dotted lines highlight MT - flux slopes ) . Scale bars , 30 sec . Quantification of MT - flux in bipolar spindles subjected to indicated treatments . Graphs represent MT flux of individual cells with mean ± SD . N ( number of cells , number of independent experiments ) : - bipolar spindles : siControl ( 49 , 5 ) , STLC ( 31 , 3 ) , siKIF15 ( 39 , 3 ) , siKID ( 32 , 3 ) , siKIF4A ( 28 , 3 ) , siKIF2A ( 36 , 3 ) , siCLASPs ( 21 , 2 ) , GSK923295 ( 44 , 3 ) Quantification of MT - flux in monopolar spindles subjected to indicated treatments . Graphs represent MT flux of individual cells with mean ± SD . N ( number of cells , number of independent experiments ) monopolar spindles : siControl ( 35 , 3 ) , siKIF15 ( 44 , 3 ) , siKID ( 38 , 3 ) , siKIF4A ( 44 , 3 ) , siKIF2A ( 38 , 3 ) , siCLASPs ( 12 , 2 ) , GSK923295 ( 33 , 3 ) .

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 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 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 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

Figure 1A. Representative spinning disk confocal live-cell imaging time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), treated with control and KIF4A siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.B. Representative spinning disk confocal live-cell imaging time series images of S-trityl-L-cysteine (STLC)-treated U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red). Note that MT-flux is abrogated in presence of taxol (lower panel). Scale bars, 10 µm. Time, min:sec.C. Illustration of 405nm laser-photoactivated regions in monopolar spindles (blue circles, left). The effect of photoactivation and region selected for kymograph generation (dashed white rectangle, right). Scale bar, 10 µm. D. Corresponding kymograph profiles of the photoactivated regions in bipolar and monopolar spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 30 sec. Quantification of MT-flux in bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. N (number of cells, number of independent experiments): - bipolar spindles: siControl (49, 5), STLC (31, 3), siKIF15 (39, 3), siKID (32, 3), siKIF4A (28, 3), siKIF2A (36, 3), siCLASPs (21, 2), GSK923295 (44, 3)Quantification of MT-flux in monopolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. N (number of cells, number of independent experiments) monopolar spindles: siControl (35, 3), siKIF15 (44, 3), siKID (38, 3), siKIF4A (44, 3), siKIF2A (38, 3), siCLASPs (12, 2), GSK923295 (33, 3).


Figure 2A . Representative spinning disk confocal live - cell imaging time series images of U2OS PA - GFP - α - tubulin cells conditionally co - expressing KIF4A shRNA and RNAi - resistant mCherry - Kif4A variants induced using doxycycline . Chromosomes were stained using SiR - DNA . White arrowheads highlight poleward flux of the photoactivated regions . Scale bar , 10 µm . Time , min : sec . B . Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi - resistant mCherry - KIF4A variants . The anti - KIF4A antibody was used , together with anti - vinculin antibody as a loading control . C . Quantification of MT - flux upon shRNA - mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi - resistant mCherry - KIF4A variants . The bars in graph represent mean ± SD . N ( number of cells , number of independent experiments ) : uninduced shKIF4A ( 48 , 4 ) , shKIF4A ( 60 , 4 ) , uninduced KIF4A WT ( 36 , 5 ) , shKIF4A + KIF4A WT ( 43 , 5 ) , uninduced KIF4A K94A ( 42 , 7 ) , shKIF4A + KIF4A K94A ( 38 , 6 ) , uninduced KIF4A ΔZip1 ( 32 , 4 ) , shKIF4A + ΔZip1 ( 30 , 4 ) .

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 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

Figure 2A. Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α-tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec.B. Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi-resistant mCherry-KIF4A variants. The anti-KIF4A antibody was used, together with anti-vinculin antibody as a loading control.C. Quantification of MT-flux upon shRNA-mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi-resistant mCherry-KIF4A variants. The bars in graph represent mean ± SD. N (number of cells, number of independent experiments): uninduced shKIF4A (48, 4), shKIF4A (60, 4), uninduced KIF4A WT (36, 5), shKIF4A + KIF4A WT (43, 5), uninduced KIF4A K94A (42, 7), shKIF4A + KIF4A K94A (38, 6), uninduced KIF4A ΔZip1 (32, 4), shKIF4A + ΔZip1 (30, 4).


Figure 3A . Representative spinning disk confocal time series of MT - flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes ( MUGs ) . U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , labeled for chromosomes with SiR - DNA ( grey ) are shown , with 5 mM caffeine used as a control . Scale bar , 10 µm . Time , min : sec . B . Quantification of the MT - flux rates from indicated conditions . MT - flux values with mean ± SD are plotted . N ( number of cells , number of independent experiments ) : Caffeine - only control ( 30 , 3 ) and MUGs ( 34 , 3 ) . C . Representative point - scanning confocal maximum - intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions , immunostained with antibodies against KIF4A and α - tubulin . DNA was counterstained with DAPI . KIF4A in cyan and α - tubulin in red in merged image . Scale bar , 10 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 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

Figure 3A. Representative spinning disk confocal time series of MT-flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes (MUGs). U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), labeled for chromosomes with SiR-DNA (grey) are shown, with 5 mM caffeine used as a control. Scale bar, 10 µm. Time, min:sec. B. Quantification of the MT-flux rates from indicated conditions. MT-flux values with mean ± SD are plotted. N (number of cells, number of independent experiments): Caffeine-only control (30, 3) and MUGs (34, 3).C. Representative point-scanning confocal maximum-intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions, immunostained with antibodies against KIF4A and α-tubulin. DNA was counterstained with DAPI. KIF4A in cyan and α-tubulin in red in merged image. Scale bar, 10 µm.


Figure 4A . Representative spinning disk confocal live - cell image series of MT - flux in U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) treated with indicated siRNAs . White arrowheads highlight poleward motion of the photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec . B . Quantification of the impact of the MT - crosslinking proteins on MT - flux in U2OS PA - GFP / mCherry - α - tubulin cells transfected with respective siRNAs . Graph represent MT - flux values with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 49 , 5 ) , siPRC1 ( 45 , 3 ) , siNuMA ( 40 , 3 ) , siHSET ( 37 , 3 ) , siHSET + siNuMA ( 39 , 3 ) . C . Quantification of the impact of the motor activity of HSET on MT - flux in U2OS cells stably expressing mEOS - α - tubulin treated with control or HSET 3 ′ UTR siRNAs in presence or absence of the respective RNAi - resistant GFP - HSET constructs . Graph represent MT - flux values with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 23 , 3 ) , siHSET 3 ′ UTR ( 33 , 3 ) , siHSET 3 ′ UTR + HSET WT ( 29 , 5 ) , siHSET 3 ′ UTR + HSET N593K ( 30 , 4 ) . D . Representative kymographs of the photoactivated spindles displaying asynchronous flux motion ( split of the two red dashed lines towards individual poles ) upon RNAi - mediated depletion of MT - crosslinkers . E . Percentage of cells with asynchronous flux

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 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 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 4A. Representative spinning disk confocal live-cell image series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.B. Quantification of the impact of the MT-crosslinking proteins on MT-flux in U2OS PA-GFP/mCherry-α-tubulin cells transfected with respective siRNAs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siPRC1 (45, 3), siNuMA (40, 3), siHSET (37, 3), siHSET + siNuMA (39, 3).C. Quantification of the impact of the motor activity of HSET on MT-flux in U2OS cells stably expressing mEOS-α-tubulin treated with control or HSET 3′UTR siRNAs in presence or absence of the respective RNAi-resistant GFP-HSET constructs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (23, 3), siHSET 3′UTR (33, 3), siHSET 3′UTR + HSET WT (29, 5), siHSET 3′UTR + HSET N593K (30, 4).D. Representative kymographs of the photoactivated spindles displaying asynchronous flux motion (split of the two red dashed lines towards individual poles) upon RNAi-mediated depletion of MT-crosslinkers. E. Percentage of cells with asynchronous flux movements


Figure 5A . Representative spinning disk confocal live - cell time series of MT - flux in U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) treated with indicated conditions . White arrowheads follow the poleward motion of the photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec . B . Quantification of the impact of stable end - on KT - MT attachments on MT - flux in NDC80 - depleted cells under indicated conditions and in early prometaphase cells with and without the CENP - E inhibitor GSK923295 . MT - flux values are plotted with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 49 , 5 ) , siNDC80 ( 59 , 6 ) , siNDC80 + siKIF4A ( 41 , 3 ) , siNDC80 + STLC ( bipol . ) ( 28 , 3 ) , siNDC80 + siKIF15 ( 36 , 4 ) , siNDC80 + siCLASPs ( 33 , 3 ) , siNDC80 + siKIF2A ( 38 , 4 ) , siNDC80 + GSK923295 ( 38 , 3 ) , early prometaphase ( 23 , 3 ) , early prometaphase + GSK923295 ( 28 , 3 ) . C . Kymograph profiles ( left ) highlighting that the photoactivated regions in prometaphase ( upper panel ) split and move towards both poles , whereas metaphase spindles ( lower panel ) flux uniformly towards individual poles ( indicated by red dotted lines ) . Illustration of the photoactivated regions ( right ) in prometaphase and metaphase spindles , highlighting that interpolar MTs overlap over a broader region in early mitosis , becoming focused at the spindle equator in metaphase . Scale bar , 30 sec . D . Percentage of cells with parallel and antiparallel flux movements during prometaphase and metaphase . N ( number of cells , number of independent experiments ) : prometaphase ( 23 , 3 ) , metaphase ( 22 , 3 ) .

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 O O O O 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

Figure 5A. Representative spinning disk confocal live-cell time series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated conditions. White arrowheads follow the poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.B. Quantification of the impact of stable end-on KT-MT attachments on MT-flux in NDC80-depleted cells under indicated conditions and in early prometaphase cells with and without the CENP-E inhibitor GSK923295. MT-flux values are plotted with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siNDC80 (59, 6), siNDC80 + siKIF4A (41, 3), siNDC80 + STLC (bipol.) (28, 3), siNDC80 + siKIF15 (36, 4), siNDC80 + siCLASPs (33, 3), siNDC80 + siKIF2A (38, 4), siNDC80 + GSK923295 (38, 3), early prometaphase (23, 3), early prometaphase + GSK923295 (28, 3).C. Kymograph profiles (left) highlighting that the photoactivated regions in prometaphase (upper panel) split and move towards both poles, whereas metaphase spindles (lower panel) flux uniformly towards individual poles (indicated by red dotted lines). Illustration of the photoactivated regions (right) in prometaphase and metaphase spindles, highlighting that interpolar MTs overlap over a broader region in early mitosis, becoming focused at the spindle equator in metaphase. Scale bar, 30 sec. D. Percentage of cells with parallel and antiparallel flux movements during prometaphase and metaphase. N (number of cells, number of independent experiments): prometaphase (23, 3), metaphase (22, 3).


Figure 6A . Representative maximum - intensity projected coherent - hybrid STED ( CH - STED ) images of mitotic spindles in U2OS cells treated with control and NDC80 siRNAs and stained for DNA ( DAPI ) , KTs ( CENP - C , magenta ) , α - tubulin ( red ) and CENP - E ( green ) . Scale bar , 10 µm . B . Model illustrating potential mechanisms for CENP - E mediated MT - flux forces either through its KT - or antiparallel MT - based localization and sliding . CENP - E localized at KTs laterally interacting with MTs ( white arrowheads in 1 , 2 ) , and CENP - E localized at interpolar MTs ( white arrows in 3 , 4 ) , in control and NDC80 - depleted mitotic cells . Scale bars , 2 µm . C . CH - STED analyses of HeLa cells stably expressing CENP - E - GFP illustrating bipolar spindles in prometaphase , metaphase and just after sister - chromatid separation at anaphase onset . Approximate x - y resolution is 70 nm . The expected / described localization of CENP - E - GFP ( green ) at KTs is shown . In addition , CENP - E - GFP was found associated with interpolar MTs ( ipMTs ) , including regions of overlapping anti - parallel MTs , and k - fibers . Chromosomes ( confocal mode only ) were revealed in the larger panels with DAPI ( cyan ) and MTs ( magenta ) were detected with an anti - α - tubulin antibody . Arrows indicate examples of clear MT bundles , including regions of overlapping anti - parallel MTs . Scale bar in all panels is 5 μm . D . Representative spinning disk confocal live - cell time series of HeLa cells stably expressing CENP - E - GFP . White arrowheads highlight CENP - E - GFP at KTs , white arrows highlight CENP - E - GFP on interpolar MTs . Scale bar , 10 µm . Time , hour : min .

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 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 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

Figure 6A. Representative maximum-intensity projected coherent-hybrid STED (CH-STED) images of mitotic spindles in U2OS cells treated with control and NDC80 siRNAs and stained for DNA (DAPI), KTs (CENP-C, magenta), α-tubulin (red) and CENP-E (green). Scale bar, 10 µm.B. Model illustrating potential mechanisms for CENP-E mediated MT-flux forces either through its KT- or antiparallel MT- based localization and sliding. CENP-E localized at KTs laterally interacting with MTs (white arrowheads in 1, 2), and CENP-E localized at interpolar MTs (white arrows in 3, 4), in control and NDC80-depleted mitotic cells. Scale bars, 2 µm.C. CH-STED analyses of HeLa cells stably expressing CENP-E-GFP illustrating bipolar spindles in prometaphase, metaphase and just after sister-chromatid separation at anaphase onset. Approximate x-y resolution is 70 nm. The expected/described localization of CENP-E-GFP (green) at KTs is shown. In addition, CENP-E-GFP was found associated with interpolar MTs (ipMTs), including regions of overlapping anti-parallel MTs, and k-fibers. Chromosomes (confocal mode only) were revealed in the larger panels with DAPI (cyan) and MTs (magenta) were detected with an anti-α-tubulin antibody. Arrows indicate examples of clear MT bundles, including regions of overlapping anti-parallel MTs. Scale bar in all panels is 5 μm.D. Representative spinning disk confocal live-cell time series of HeLa cells stably expressing CENP-E-GFP. White arrowheads highlight CENP-E-GFP at KTs, white arrows highlight CENP-E-GFP on interpolar MTs. Scale bar, 10 µm. Time, hour:min.


Figure 7A . Representative spinning disk confocal time series of U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , with indicated treatments ( left ) . White arrowheads highlight poleward flux of the photoactivated regions . Scale bar , 10 µm . Time , min : sec . Right panels display corresponding kymographs of the photoactivated spindles used for quantification of the flux rates ( red dotted lines highlight MT - flux slopes ) . Scale bars , 2 µm ( horizontal ) and 30 sec ( vertical ) . B , C . Quantification of MT - flux in prometaphase ( B ) and metaphase ( C ) bipolar spindles subjected to indicated treatments . Graphs represent MT flux of individual cells with mean ± SD . siNDC80 , siNDC80 + GSK923295 , early prometaphase , early prometaphase + GSK923295 , siControl , and siKIF4A N ( number of cells , number of independent experiments ) : siNDC80 + KIF15 + STLC ( 33 , 5 ) , siNDC80 + KIF15 + STLC + GSK923295 ( 25 , 3 ) , early prometaphase + KIF15 + STLC + GSK923295 ( 11 , 2 ) , siKIF15 + STLC ( 29 , 3 ) , siKIF4A + siKIF15 + STLC ( 10 , 3 ) . D . Representative spinning disk confocal time series of U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , with indicated treatments ( left ) and corresponding kymographs ( right ) as described in ( A ) . Scale bars , 2 µm ( horizontal ) and 30 sec ( vertical ) .

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 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 O O O O O 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 7A. Representative spinning disk confocal time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), with indicated treatments (left). White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec. Right panels display corresponding kymographs of the photoactivated spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 2 µm (horizontal) and 30 sec (vertical).B, C. Quantification of MT-flux in prometaphase (B) and metaphase (C) bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. siNDC80, siNDC80 + GSK923295, early prometaphase, early prometaphase + GSK923295, siControl, and siKIF4A N (number of cells, number of independent experiments): siNDC80 + KIF15 + STLC (33, 5), siNDC80 + KIF15 + STLC + GSK923295 (25, 3), early prometaphase + KIF15 + STLC + GSK923295 (11, 2), siKIF15 + STLC (29, 3), siKIF4A + siKIF15 + STLC (10, 3).D. Representative spinning disk confocal time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), with indicated treatments (left) and corresponding kymographs (right) as described in (A). Scale bars, 2 µm (horizontal) and 30 sec (vertical).


Figure 8A - C . Graph depicting a direct correlation between MT - flux and mitotic spindle where mean spindle length is plotted over mean MT - flux rates for all indicated conditions ( A ) , mitotic conditions containing stable end - on attachments ( B ) , and NDC80 depleted conditions lacking stable end - on attachments ( C ) . Solid and dotted lines represent the linear regression and 95 % confidence interval , respectively . Pearson correlation coefficient ( r ) , coefficient of determination ( r2 ) and corresponding p values are indicated . The error bars over x and y axis represent the standard deviation of MT - flux and spindle length , respectively . D . Representative images from spinning disk confocal live - cell time series of U2OS PA - GFP - α - tubulin / mCherry - α - tubulin cells under indicated treatments , with SiR - DNA - labelled DNA . Microtubules are shown in red and DNA in cyan in the merged image . Scale bar : 10 µm . E . Quantification of spindle lengths at metaphase following indicated treatments . Graph represents the spindle length of individual cells with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 49 , 5 ) , siMCAK ( 34 , 3 ) , siKIF4A + siKIF15 + STLC ( 46 , 6 ) , siKIF4A + siKIF15 + STLC + siMCAK ( 43 , 3 ) , siKIF4A + siKIF15 + STLC + siKIF2A ( 22 , 3 ) , siCLASPs ( 64 , 6 ) , siCLASPs + siMCAK ( 33 , 3 ) , siCLASPs + siKIF2A ( 32 , 3 ) .

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Figure 8A-C. Graph depicting a direct correlation between MT-flux and mitotic spindle where mean spindle length is plotted over mean MT-flux rates for all indicated conditions (A), mitotic conditions containing stable end-on attachments (B), and NDC80 depleted conditions lacking stable end-on attachments (C). Solid and dotted lines represent the linear regression and 95% confidence interval, respectively. Pearson correlation coefficient (r), coefficient of determination (r2) and corresponding p values are indicated. The error bars over x and y axis represent the standard deviation of MT-flux and spindle length, respectively.D. Representative images from spinning disk confocal live-cell time series of U2OS PA-GFP-α-tubulin/mCherry-α-tubulin cells under indicated treatments, with SiR-DNA-labelled DNA. Microtubules are shown in red and DNA in cyan in the merged image. Scale bar: 10 µm.E. Quantification of spindle lengths at metaphase following indicated treatments. Graph represents the spindle length of individual cells with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siMCAK (34,3), siKIF4A + siKIF15 + STLC (46, 6), siKIF4A + siKIF15 + STLC + siMCAK (43, 3), siKIF4A + siKIF15 + STLC + siKIF2A (22, 3), siCLASPs (64, 6), siCLASPs + siMCAK (33, 3), siCLASPs + siKIF2A (32, 3).


figf1 ( A , B ) Two - dimensional tryptic mapping of YFP - p27 , YFP - p27T198A and a mixture of both ( A ) . Phosphoamino - acid analysis of spots a and b ( B ) . The migration of phosphoserine ( S ) , phosphothreonine ( T ) and phosphotyrosine ( Y ) is marked with circles in B . The asterisk indicates the origin of migration . ( C - E ) MCF - 7 transfected with YFP - p27 or YFP - p27T198A mutant ( C , D ) or cotransfected with YFP - p27 and an empty RFP vector in a 5 : 1 ( YFP - p27 : RFP ) ratio ( E ) were treated with ( + ) or without ( − ) MG132 ( 10 μM ) for 12 h before western blot analysis ( C ) and fluorescence microscopy ( D , E ) . ( C - E ) MCF - 7 transfected with YFP - p27 or YFP - p27T198A mutant ( C , D ) or cotransfected with YFP - p27 and an empty RFP vector in a 5 : 1 ( YFP - p27 : RFP ) ratio ( E ) were treated with ( + ) or without ( − ) MG132 ( 10 μM ) for 12 h before western blot analysis ( C ) and fluorescence microscopy ( D , E ) . ( F ) Western blots of protein extracts from MCF - 7 transfected with YFP - p27 , YFP - p27T198A or YFP - p27T198D mutants following incubation with cycloheximide ( CHX ) for the indicated times . Uncropped images of the blots are shown in the Supplementary Information , Fig . S5 .

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 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

figf1(A, B) Two-dimensional tryptic mapping of YFP-p27, YFP-p27T198A and a mixture of both (A). Phosphoamino-acid analysis of spots a and b (B). The migration of phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) is marked with circles in B. The asterisk indicates the origin of migration.(C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E).(C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E).(F) Western blots of protein extracts from MCF-7 transfected with YFP-p27, YFP-p27T198A or YFP-p27T198D mutants following incubation with cycloheximide (CHX) for the indicated times. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.


figf2 ( a ) Crystal violet staining of MCF - 7 cell transfected with YFP ( C1 ) , YFP - p27 , YFP - p27T198A or YFP - p27T198D and selected with G418 for three weeks . Data are presented as mean ± s . d . ( n = 3 ) . ( b ) Flow cytometry analysis of MCF - 7 cells transfected with YFP ( C1 ) or YFP - p27 , YFP - p27T198A and YFP - p27T198D for 48 h . Data are presented as mean ± s . d . ( n = 3 ) . ( c ) GFP - LC3 U2OS cells 48 h post - transfection with RFP , RFP - p27 , RFP - p27T198A or RFP - p27T198D . Data represent results of three independent experiments , from which a total of ∼ 200 cells were counted ( s . d . , 10 ∼ 15 % ) . The scale bars represent 50 μm in c .

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figf2(a) Crystal violet staining of MCF-7 cell transfected with YFP (C1), YFP-p27, YFP-p27T198A or YFP-p27T198D and selected with G418 for three weeks. Data are presented as mean ± s.d. (n = 3).(b) Flow cytometry analysis of MCF-7 cells transfected with YFP (C1) or YFP-p27, YFP-p27T198A and YFP-p27T198D for 48 h. Data are presented as mean ± s.d. (n = 3).(c) GFP-LC3 U2OS cells 48 h post-transfection with RFP, RFP-p27, RFP-p27T198A or RFP-p27T198D. Data represent results of three independent experiments, from which a total of ∼200 cells were counted (s.d., 10∼15%). The scale bars represent 50 μm in c.


figf3 ( a , b ) MCF - 7 cells were cultured for 24 h following siRNA with or without FBS before western blot analysis ( a ) and cell - viability assays ( b ) . ( c ) MCF - 7 cells were transfected with vector alone ( C1 ) , YFP - p27 ( WT ) , or the siRNA - resistant constructs WT - R and T198A - R 24 h before RNA interference ( RNAi ) . PARP cleavage was determined by western blotting and densitometric scanning . Data represent three independent experiments ( mean ± s . d . ) . ( d ) Western blots of lysates of MCF - 7 cells transfected with siRNA before serum - starvation or treatment with LY294002 ( LY , 10 μM ) or rapamycin ( Rap , 100 nM ) for 48 h with or without methylpyruvate ( MP , 10 mM ) . ( e ) MCF - 7 cells treated with or without LY294002 for 48 h following RNAi . p27 siRNA pool - 1 , siRNA1 , 2 and 3 ; arrows , membrane blebbing ; triangles , apoptotic bodies . The scale bar represents 50 μm . ( f ) Western blots of protein extracts from MCF - 7 cultured with ( + ) or without ( − ) glucose for 24 h following RNAi . Fr , fragment ; percentage PARP cleavage = 100 × PARPFr / ( PARP + PARPFr ) .

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 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 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 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

figf3(a, b) MCF-7 cells were cultured for 24 h following siRNA with or without FBS before western blot analysis (a) and cell-viability assays (b).(c) MCF-7 cells were transfected with vector alone (C1), YFP-p27 (WT), or the siRNA-resistant constructs WT-R and T198A-R 24 h before RNA interference (RNAi). PARP cleavage was determined by western blotting and densitometric scanning. Data represent three independent experiments (mean ± s.d.).(d) Western blots of lysates of MCF-7 cells transfected with siRNA before serum-starvation or treatment with LY294002 (LY, 10 μM) or rapamycin (Rap, 100 nM) for 48 h with or without methylpyruvate (MP, 10 mM).(e) MCF-7 cells treated with or without LY294002 for 48 h following RNAi. p27 siRNA pool-1, siRNA1, 2 and 3; arrows, membrane blebbing; triangles, apoptotic bodies. The scale bar represents 50 μm.(f) Western blots of protein extracts from MCF-7 cultured with (+) or without (−) glucose for 24 h following RNAi. Fr, fragment; percentage PARP cleavage = 100 × PARPFr / (PARP + PARPFr).


figf4 ( a ) Western blots of lysates from the indicated cell lines . ( b ) Pearson correlation analysis of p27 and LKB1 protein levels quantified by reverse phase protein array assays in 74 randomly selected primary breast tumors ( MDACC ) . ( c ) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild - type LKB1 cultured with or without glucose for 24 h . ( d - f ) Western blots of protein extracts from MEFs treated with AICAR ( d and e ) , ( d - f ) Western blots of protein extracts from MEFs treated with AICAR ( d and e ) ( d - f ) Western blots of protein extractsfrom 3T3L1 cells cultured with or without glucose for 30 min ( f ) . ( g ) Western blot analysis of 3T3L1 cells 72 h post - transfection RNAi with or without glucose starvation for 1 h before analysis . Densitometric quantification of western blots is shown . Uncropped images of the blots are shown in the Supplementary Information , Fig . S5 .

O B-PANEL_START 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 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 B-PANEL_START 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 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

figf4(a) Western blots of lysates from the indicated cell lines.(b) Pearson correlation analysis of p27 and LKB1 protein levels quantified by reverse phase protein array assays in 74 randomly selected primary breast tumors (MDACC).(c) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild-type LKB1 cultured with or without glucose for 24 h.(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e),(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e)(d-f) Western blots of protein extractsfrom 3T3L1 cells cultured with or without glucose for 30 min (f).(g) Western blot analysis of 3T3L1 cells 72 h post-transfection RNAi with or without glucose starvation for 1 h before analysis. Densitometric quantification of western blots is shown. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.


figf5 ( A ) FACS analysis of TUNEL positive p27 + / + and p27 − / − cells treated with AICAR for 20 h . Data represent mean ± s . d . of three independent experiments . ( B , C ) Electron microscopy of p27 + / + ( a , c and e ) and p27 − / − ( b , d and f ) MEFs treated with ( c and d ) , or without ( a and b ) AICAR ( 1 mM ) , or without glucose ( e and f ) for 14 h . Magnified images of the boxed areas were shown on the left ( a ′ , b ′ , c ′ , d ′ , e ′ and f ′ ) with arrowheads indicating autophagic vacuoles including autolysosomes ( black arrowheads ) . The scale bars represent 10 μm in a - f and 0 . 5 μm in a ′ - f ′ . N , nucleus . Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s . d . ( C ) . ( B , C ) Electron microscopy of p27 + / + ( a , c and e ) and p27 − / − ( b , d and f ) MEFs treated with ( c and d ) , or without ( a and b ) AICAR ( 1 mM ) , or without glucose ( e and f ) for 14 h . Magnified images of the boxed areas were shown on the left ( a ′ , b ′ , c ′ , d ′ , e ′ and f ′ ) with arrowheads indicating autophagic vacuoles including autolysosomes ( black arrowheads ) . The scale bars represent 10 μm in a - f and 0 . 5 μm in a ′ - f ′ . N , nucleus . Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s . d . ( C ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

figf5(A) FACS analysis of TUNEL positive p27+/+ and p27−/− cells treated with AICAR for 20 h. Data represent mean ± s.d. of three independent experiments.(B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).(B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).


Figure 1A Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 and tubulin were used as loading controls . * non ‐ specific band . B Cells treated as in ( A ) were fixed 48 h after selection and stained with an anti ‐ 53BP1 antibody ( green ) . Telomeres were visualized with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe ( red ) . Yellow arrowheads point to 53BP1 foci that co ‐ localize with telomeres . C Quantification of TIFs in cells treated as in ( B ) . A minimum of 200 nuclei were scored for each sample . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; NS , P > 0 . 05 .

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Figure 1A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and tubulin were used as loading controls. *non‐specific band.B Cells treated as in (A) were fixed 48 h after selection and stained with an anti‐53BP1 antibody (green). Telomeres were visualized with a Cy3‐conjugated (CCCTAA)3‐PNA probe (red). Yellow arrowheads point to 53BP1 foci that co‐localize with telomeres.C Quantification of TIFs in cells treated as in (B). A minimum of 200 nuclei were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05.


Figure 2A Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO ‐ FISH analysis . Metaphase chromosome spreads were stained with Cy3 ‐ conjugated leading strand telomeric PNA probe ( red ) and FITC ‐ conjugated lagging strand telomeric PNA probe ( green ) . DNA was counter ‐ stained with DAPI ( blue ) . B The frequency of chromosome ‐ type telomeric fusions in cells treated as in ( A ) was quantified as a percentage of total number of chromosomes ( illustrated in Supplementary Fig S2A ) . A minimum of 2 , 000 chromosomes were scored for each treatment . Error bars represent SD of at least two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; NS , P > 0 . 05 .

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Figure 2A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO‐FISH analysis. Metaphase chromosome spreads were stained with Cy3‐conjugated leading strand telomeric PNA probe (red) and FITC‐conjugated lagging strand telomeric PNA probe (green). DNA was counter‐stained with DAPI (blue).B The frequency of chromosome‐type telomeric fusions in cells treated as in (A) was quantified as a percentage of total number of chromosomes (illustrated in Supplementary Fig S2A). A minimum of 2,000 chromosomes were scored for each treatment. Error bars represent SD of at least two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; **P ≤ 0.01; NS, P > 0.05.