( E ) CCFs in OIS are strongly positive for heterochromatic histone mark H3K27me3 but not for euchromatic mark H3K9ac . ( F ) from E were scored for indicated histone modifications in CCFs . A single representative experiment is shown at least 100 were scored per slide . Note majority of micronuclei in control are positive for both marks .

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(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 + CCF − from G were scored for colocalizing intranuclear γ - H2AX 53BP1 foci . Mean ± , n = 3 ; P < 0 . 019 . Bars ( in IF panels ) , 10 µm .

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(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.


- to - cytoplasm blebbing in senescent associated with depletion of lamin B1 . ( A ) Extrusion of GFP - H2B - positive fragment from nonmitotic RS nucleus . Confocal time - lapse microscopy of transiently transfected GFP - H2B - expressing , 60 × original magnification , 150 nm optical section , time in mins . Bars : 10 µm ; ( insert ) 2 µm .

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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 ) fragments blebbing out of nucleus are γ - H2AX positive . Bars : ( top ) 10 µm ; ( bottom ) 5 µm .

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(B) Chromatin fragments blebbing out of nucleus are γ-H2AX positive. Bars: (top) 10 µm; (bottom) 5 µm.


( C ) Down - regulation of lamin B1 lamin B receptor ( LBR ) in RS , assayed by Western blot . See Materials methods for time - course details .

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(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 , assayed by immunofluorescence . ( E ) A - scan of lamin B1 along arrows in D . Data shown are from a single representative experiment out of three repeats .

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(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 were assessed .

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. (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 ) . dotted shows basement between dermis epidermis . Bottom panels ( Epi Nevus ) are expanded from boxed areas on top . Confocal microscopy for DAPI ( blue ) , MelanA ( red ) , lamin B1 ( green ) stained tissue . Bars , 20 µm . ( I ) Quantitation of results from H for three different nevi . Epifluorescence of lamin B1 - MelanA - stained nevi were obtained epidermal or nevusMelanA - positive were scored for presence ( positive ) of a characteristic lamin B1 " ring . " At least 50 MelanA - expressing epidermalmelanocytes at least 100 nevusmelanocytes were scored . Average of three different nevi ± ; P < 0 . 0005 .

O O O O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O B-TISSUE O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD 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-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O

(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.


Blebbing of from nucleus to cytoplasm in senescent is associated with loss of envelope integrity . ( A ) Herniation of H3K27me3 - positive into cytoplasm of OIS . Yellow boxed 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 ± , n = 2 ; P < 0 . 01 .

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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 are permeable to both 70 - 500 - kD dextrans , indicating impairment of barrier function of envelope . nuclei exclude dextran . Bars , 10 µm . ( D ) Quantitation of results from C . Mean ± SD , n = 4 ; P < 0 . 000001 for both 70 - 500 - kD dextrans .

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(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 + SAHF − OIS . P < 1 . 9 × 10 − 5 . ( G ) In nuclei , extrusion dextran intrusion occurred in close physical proximity . Right - panels are expanded from yellow boxed areas on left . Bars : ( left ) 10 µm ; ( right ) 2 µm .

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(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.


histone is processed by a lysosomal / autophagy pathway . ( A ) Close juxtaposition of senescence - associated CCF ( RS ) with p62 bodies . Bars : 10 µm ; ( inset ) 1 µm . ( B ) Quantitation of control OIS with CCF overlapping p62 ( p62 + ) or not ( p62 − ) . Mean ± , n = 3 .

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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 RS with CCF overlapping p62 ( p62 + ) or not ( p62 − ) ubiquitination ( FK2 + ) or not ( FK2 − ) . Mean ± , n = 3 ; P < 0 . 0008 for FK2 + / p62 + CCF , comparing proliferating RS . ( D ) Ubiquitinated appear to surface in CCF . Yellow boxed areas are magnified in right - panels . Bars : ( left panels ) 10 µm ; ( right panels ) 1 µm .

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(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 . In contrast to RS , majority of cathepsin L in proliferating control exists in its inactive pro - cathepsin L form . Loading was normalized by number lamin A / C was used as a loading control . See Materials methods for details of time course .

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(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 . See Materials methods for details of time course . Western blotting was performed using same lysates as in Fig . S4 B , same lamin A / C panel is shown in both figures .

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(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 corresponding high mobility H3 in OIS .

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(G) Accumulation of H3cs.1 and corresponding high mobility H3 in OIS.


( H ) Predominant localization of H3cs . 1 in senescent . α - Tubulin lamin A / C were used as fractionation controls for fractions , respectively .

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(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.


Senescent in vitro in vivo contain reduced histone content . ( A ) Immunofluorescent confocal of histone H3 in control or OIS ( 11 d after activation of ER - RASG12V ) . Yellow arrows indicate with most pronounced SAHF . ( B ) Quantitative immunofluorescence of histone H3 in RS OIS . Representative of two independent experiments .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O

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 histones in senescent IMR90 in replicative senescence ( RS ) . Lysates were normalized by number an equal number of was loaded per each lane . Lamin A / C was used as a confirmatory loading control . See Materials methods for details of time course .

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(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 ) adjacent epidermis ( e ) . Right - panels are expanded from boxed areas on left . Note decreased nevus for H3 ( right bottom ) as compared with epidermis ( right top ) . Bars : ( left ) 100 µm ; ( right ) 50 µm .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O

(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 ) S100 ( melanocytes [ green ] ) in human benign nevus adjacent epidermis . Bars , 20 µm .

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(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 for H3 in nevus ( bottom panels ) , compared with epidermalmelanocytes ( top panels ) . Bars , 20 µm . ( G ) Quantitative immunofluorescence of H3 in epidermal nevusmelanocytes . were obtained in blue ( DAPI ) , green ( S100 ) , red ( H3 ) channels then H3 was measured in either S100 + epidermal , strictly adjacent to basal , or in S100 + nevusmelanocytes . H3 histograms represent distribution , combined from three nevi . At least 150 epidermal ( 50 per nevus ) 300 nevus ( 100 per nevus ) melanocytes were assessed .

O O O O O O O O O O O O O B-GENEPROD O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-TISSUE O O O O O O O O B-GENEPROD O B-TISSUE O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O O B-TISSUE O O O B-TISSUE O B-CELL_TYPE O O O

(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.


Senescence - associated loss of histones is V - ATPase dependent . ( A ) Bafilomycin A1 ( BafA1 ) blocks loss of nuclearhistone H3content in undergoing OIS . BafA1 was added to at 50 nM on day 5 after RASG12V induction . were harvested 24 h later , stained for histone H3 . Bars , 10 µm . ( B ) Quantitative histone H3immunofluorescence in from A . Single representative experiment out of three repeats .

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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 were treated with BafA1 as described in A then assessed for histones by Western blot . LC3 I / II has been used as a control for BafA1 activity . Lysates from 10 , 000 were loaded per well .

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(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 accumulation of H3cs . 1 in undergoing OIS .

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(D) BafA1 blocks the accumulation of H3cs.1 in cells undergoing OIS.


( E ) BafA1 concanamycin A ( Con A ) block accumulation of H3cs . 1 in RS .

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(E) BafA1 and concanamycin A (Con A) block the accumulation of H3cs.1 in RS cells.


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

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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.


. B , Teicoplanin inhibit entry of 2019 - nCoV SARS - nCoV . Chemical structure of teicoplanin ( Left ) . A549 were seeded in a 96 - well plate , 24 h later , were infected with HIV - luc / 2019 - nCoV , HIV - luc / SARS - nCoV or HIV - luc / VSVG pseudoviruses . After washing incubation with fresh medium for 48 h , intracellular luciferase activity was measured . Ordinary one - way ANOVA was used for this . results are shown as mean . * P < 0 . 05 .

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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 were infected with HIV - luc / 2019 - nCoV pseudoviruses then incubated with teicoplanin at various concentrations . intracellular luciferase activity was measured at 48 h post - infection . IC50 was calculated using GraphPad Prism software .

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, 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.


types . A , HEK293T were infected with HIV - luc / 2019 - nCoV pseudoviruses then incubated with teicoplanin at various concentrations . intracellular luciferase activity was measured at 48 h post - infection . IC50 was calculated using GraphPad Prism software .

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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.B


software . B , Huh7 were infected with HIV - luc / 2019 - nCoV pseudoviruses then incubated with teicoplanin at various concentrations . intracellular luciferase activity was measured at 48 h post - infection . IC50 was calculated using GraphPad Prism software .

software.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.C


A549 were incubated with dalbavancin ( D ) at various concentrations infected with HIV - luc / 2019 - nCoV pseudoviruses . intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA was used for this .

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A549 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. dalbavancin


D , A549 were incubated with vancomycin at various concentrations infected with HIV - luc / 2019 - nCoV pseudoviruses . intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA was used for this . Left : Chemical structure of

O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

D, 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


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

O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

Isosurface 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


Slice ( 9 . 7 nm ) through a cryotomogram of a FIB - milled E . coli BL21 expressing TssJLM . average shown in Figure 1 was placed back at positions orientations of subvolumes that were used to generate final average . zoomed - in highlights position of TssJLM particles within inner outer . Some particles were found in invaginations , as indicated by arrows . Scale bar 100

O O O O O O O O O B-EXP_ASSAY O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-SUBCELLULAR O O O O 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-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

Slice (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 nm


Cryotomographic slices ( 9 . 7 nm ) showing side views of TssJLM . In these examples , basal parts of TssJLM ( red arrows ) could be seen extending into cytoplasm . Scale bar 10

B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O

Cryotomographic 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


of prey ( green ) indicating their survival against attacker after 4 h . image of a representative competition spot is shown on upper part . relative level ( in AU ) number of recovered E . coli recipient ( in log10 of colony forming units ( cfu ) ) are indicated in lower graphs . assays were performed from at least three independent cultures , with technical triplicates a representative technical triplicate is shown . Error bars in left graph represent ± standard deviation ( SD ) from technical triplicate . circles in right graph indicate values from technical triplicate , average is indicated by bar .

O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence 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 microscopy recordings showing localization dynamics of sfGFPTssM fusion in parental ( WT ) TssJ mutated strains ( TssJ D97K ) . were taken every 60 seconds . corresponding time of each micrograph is indicated in lower right part of image . Stable foci in WT less stable foci in TssJ D97K are respectively indicated by blue arrowheads . Scale bars , 1 µm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

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.


of prey ( green ) indicating their survival against attacker after 4h . image of a representative competition spot is shown on upper part . relative level ( in AU ) number of recovered E . coli recipient ( in log10 of colony forming units ( cfu ) ) are indicated in lower graphs . assays were performed from at least three independent cultures , with technical triplicates a representative technical triplicate is shown . Error bars in left graph represent ± standard deviation ( SD ) from technical triplicate . circles in right graph indicate values from technical triplicate , average is indicated by bar .

O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence 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.


microscopy recordings showing sfGFPTssM foci in parental ( WT ) TssM mutated strains ( TssM Q779C / N780C , TssM ∆ 777 - 783 ) . TssM foci containing are indicated by arrowheads . Microscopy analyses were performed independently three times , each in technical triplicate , a representative experiment is shown . Scale bars , 1 µm .

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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.


microscopy recordings showing TssBsfGFP sheath in parental ( WT ) TssM mutated strains ( TssM Q779C / N780C , TssM ∆ 777 - 783 ) . sheath containing are indicated by arrowheads . Microscopy analyses were performed independently three times , each in technical triplicate , a representative experiment is shown . Scale bars , 1 µm .

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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.


( B ) TLC of ganglioside species in human serum . ( C ) by densitometry of major ganglioside species GM3 GD1a in human serum . Data expressed as mean ± SD , n = 6 .

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(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.


( B ) TLC of ganglioside species in human serum .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O

(B) TLC analysis of ganglioside species in human serum.


( E ) by LC - MS / MS 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( A ) Heat map of serum GM3 species in various pathological phases : control ( n = 24 ) , VFA ( n = 38 ) , lipidemia ( n = 28 ) , glycemia ( n = 15 ) , 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 serum CRP .

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(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 ) , 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 ) , 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 .

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(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 ) , α - hydroxyVLCFA - GM3 ( h24 : 0 ) ( H ) . Colors indicate disease severity : blue , no abnormal scores ( n = 25 ) ; orange , early - phase obesity ( n = 74 ) ; purple , severe obesity ( n = 23 ) .

O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O 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-DISEASE O O O O O O O O O B-DISEASE O O O O O O

(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 ) vs . HOMA - IR ( J ) .

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(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 ) : 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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).


( B , C ) GM3 - mediated enhancement inhibition of proinflammatory cytokine production from LPS - stimulated monocytes ( LPS : 0 . 06 , 0 . 13 , 0 . 25 ng / mL ) . TNF - α ( B ) IL - 6 ( C ) production in culture supernatant was measured by ELISA .

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(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 ganglioside species ( 1 . 5 , 3 . 0 , 4 . 5 µM ) . TNF - α ( D ) IL - 6 ( E ) production were shown in heat maps .

O O O O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

(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 , IL - 12 / 23 production were shown in heat maps .

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(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 unsaturated VLCFA - GM3 on LVCFA - GM3 species . IL - 6 production in culture supernatant was measured by ELISA .

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(G) Inhibitory effect of LCFA and unsaturated VLCFA-GM3 on LVCFA-GM3 species. IL-6 production in culture supernatant was measured by ELISA.


( 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 ) .

O O O O O O O B-ORGANISM B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O

(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 ) .

O O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O 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) 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 ) TNF - α ( D ) in culture supernatant following co - stimulation of monocytes by GM3 species plus human HMGB1 .

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(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 , co - stimulation by GM3 22 : 0 with LPS ( 5 ng / mL ) . TLR4 activation was monitored by NF - κB luciferase reporter ( termed " Relative Luc Activity " on Y - axis ) .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O

(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 )

O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O

(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 ) further addition of soluble human CD14 ( 1 µg / mL )

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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 by GM3 species .

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(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 B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O

(I) Co-stimulation of hTLR4/hMD-2 by LPS (5 ng/mL) plus various mixtures of GM3 species.


( 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 ) , CpG type - B ( 20 nM ) . TNF - α production in culture supernatant was quantified by ELISA .

O O O O O O O B-CELL_LINE B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-TISSUE B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O

(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 - 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 .

O O O O O O O B-CELL_LINE B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O

(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 ) , TNF - α production in culture supernatant .

O O O O O O O B-CELL_TYPE O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

(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 by GM3 species plus LPS ( 2 . 5 ng / mL ) , further addition of soluble mouse CD14 - Fc fusion ( 1 µg / mL ) .

O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O

(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 ganglioside species ( E ; 2 . 5 , 5 . 0 , 10 µM ) , ( shown in heat maps ) .

O O O O O O B-CELL_TYPE O O O O B-ORGANISM O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

(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 precursor GSL species ( F ; 2 . 5 , 5 . 0 , 10 µM ) ( shown in heat maps ) .

O O O O O O B-CELL_TYPE O O O O B-ORGANISM O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

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).


( A ) GM3 molecular species of 6 - wk - old control C57 / BL6 mice ob / ob mice were analyzed respectively by LC - MS / MS ( n = 3 ) .

O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(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 ) high - fat - diet ( HFD ) C57 / BL6 mice were analyzed by LC - MS / MS ( n = 4 ) .

O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(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 of acidic GSL fraction ( equivalent to 0 . 1 mg ) from epididymal fat pads of C3H / HeN ( A ) C3H / HeJ mice ( B ) on ND or HFD for 8 wk .

O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

(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 ) C3H / HeJ mice ( ND , HFD ) were analyzed by LC - MS / MS ( n = 4 ) .

O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(D) GM3 molecular species of C3H/HeN mice (ND, HFD) and C3H/HeJ mice (ND, HFD) were analyzed by LC-MS/MS (n=4).


( C , D ) Alanine scanning for basic residues involved in signal transduction via VLCFA - GM3 ( n = 5 ) ( C ) , combinations of effective mutations ( n = 6 ) ( D ) . Signal transduction was monitored by NF - κB reporter .

O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O 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-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O

(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 ) ( F ) , hTLR4 / hMD - 2 ( G ) ( n = 3 ) .

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(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 of recombinant mTLR4 ( extracellular domain ) / mMD - 2 complexed with GM3 18 : 0 , GM3 species enhancing mTLR4 activation .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O

(H) Cross-linked SDS-PAGE analysis of recombinant mTLR4 (extracellular domain)/mMD-2 complexed with GM3 18:0, GM3 species enhancing mTLR4 activation.


A . Representative spinning disk confocal live - imaging time series of U2OS stably co - expressing PA - GFP - α - tubulin ( cyan ) mCherry - α - tubulin ( red ) , treated with control KIF4A siRNAs . arrowheads highlight poleward motion of photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

A. 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 - imaging time series of S - trityl - L - cysteine ( STLC ) - treated U2OS stably co - expressing PA - GFP - α - tubulin ( cyan ) mCherry - α - tubulin ( red ) . Note that MT - flux is abrogated in presence of taxol ( lower panel ) . Scale bars , 10 µm . Time , min : sec .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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 - photoactivated regions in monopolar spindles ( blue circles , left ) . effect of photoactivation region selected for kymograph generation ( dashed rectangle , right ) . Scale bar , 10 µm . D . Corresponding kymograph profiles of photoactivated regions in bipolar monopolar spindles used for of flux rates ( red dotted lines highlight MT - flux slopes ) . Scale bars , 30 sec .

O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O

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.


of MT - flux in bipolar spindles subjected to indicated treatments . Graphs represent MT flux of with mean ± SD . N ( number of , 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 )

O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O

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)


of MT - flux in monopolar spindles subjected to indicated treatments . Graphs represent MT flux of with mean ± SD . N ( number of , 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-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O

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).


A . Representative spinning disk confocal live - imaging time series of U2OS PA - GFP - α - tubulin conditionally co - expressing KIF4A shRNA RNAi - resistant mCherry - Kif4A variants induced using doxycycline . Chromosomes were stained using SiR - . arrowheads highlight poleward flux of photoactivated regions . Scale bar , 10 µm . Time , min : sec .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

A. 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 lysates obtained before after doxycycline induction to validate efficiency of KIF4A shRNA construct expression of RNAi - resistant mCherry - KIF4A variants . anti - KIF4A antibody was used , together with anti - vinculin antibody as a loading control .

O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O

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 . of MT - flux upon shRNA - mediated depletion of KIF4A alone or in combination with conditional expression of RNAi - resistant mCherry - KIF4A variants . bars in graph represent mean ± SD . N ( number of , 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 O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

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).


A . Representative spinning disk confocal time series of MT - flux in bipolar spindles in control undergoing mitosis with unreplicated genomes ( MUGs ) . U2OS stably co - expressing PA - GFP - α - tubulin ( cyan ) mCherry - α - tubulin ( red ) , labeled for chromosomes with SiR - ( grey ) are shown , with 5 mM caffeine used as a control . Scale bar , 10 µm . Time , min : sec . B . of MT - flux rates from indicated conditions . MT - flux values with mean ± SD are plotted . N ( number of , number of independent experiments ) : Caffeine - only control ( 30 , 3 ) MUGs ( 34 , 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

A. 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 - projected of mitotic spindles in U2OS subjected to indicated conditions , immunostained with antibodies against KIF4A α - tubulin . was counterstained with DAPI . KIF4A in cyan α - tubulin in red in merged image . Scale bar , 10 µm .

O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O

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.


A . Representative spinning disk confocal live - image series of MT - flux in U2OS stably co - expressing PA - GFP - α - tubulin ( cyan ) mCherry - α - tubulin ( red ) treated with indicated siRNAs . arrowheads highlight poleward motion of photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

A. 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 . of impact of MT - crosslinking on MT - flux in U2OS PA - GFP / mCherry - α - tubulin transfected with respective siRNAs . Graph represent MT - flux values with mean ± SD . N ( number of , number of independent experiments ) : siControl ( 49 , 5 ) , siPRC1 ( 45 , 3 ) , siNuMA ( 40 , 3 ) , siHSET ( 37 , 3 ) , siHSET + siNuMA ( 39 , 3 ) .

O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . of impact of motor activity of HSET on MT - flux in U2OS stably expressing mEOS - α - tubulin treated with control or HSET 3 ′ UTR siRNAs in presence or absence of respective RNAi - resistant GFP - HSET constructs . Graph represent MT - flux values with mean ± SD . N ( number of , 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 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O

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 photoactivated spindles displaying asynchronous flux motion ( split of two red dashed lines towards poles ) upon RNAi - mediated depletion of MT - crosslinkers . E . of with asynchronous flux

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O

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


A . Representative spinning disk confocal live - time series of MT - flux in U2OS stably co - expressing PA - GFP - α - tubulin ( cyan ) mCherry - α - tubulin ( red ) treated with indicated conditions . arrowheads follow poleward motion of photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

A. 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 . of impact of stable end - on KT - MT attachments on MT - flux in NDC80 - depleted under indicated conditions in early prometaphase with without CENP - E inhibitor GSK923295 . MT - flux values are plotted with mean ± SD . N ( number of , 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 ) .

O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

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 photoactivated regions in prometaphase ( upper panel ) split move towards both poles , whereas metaphase spindles ( lower panel ) flux uniformly towards poles ( indicated by red dotted lines ) . Illustration of photoactivated regions ( right ) in prometaphase metaphase spindles , highlighting that interpolar MTs overlap over a broader region in early mitosis , becoming focused at spindle equator in metaphase . Scale bar , 30 sec . D . of with parallel antiparallel flux movements during prometaphase metaphase . N ( number of , number of independent experiments ) : prometaphase ( 23 , 3 ) , metaphase ( 22 , 3 ) .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O 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-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


A . Representative maximum - projected coherent - hybrid STED ( CH - STED ) of mitotic spindles in U2OS treated with control NDC80 siRNAs stained for ( DAPI ) , KTs ( CENP - C , magenta ) , α - tubulin ( red ) CENP - E ( green ) . Scale bar , 10 µm .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O B-GENEPROD O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

A. 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 - localization sliding . CENP - E localized at KTs laterally interacting with MTs ( arrowheads in 1 , 2 ) , CENP - E localized at interpolar MTs ( arrows in 3 , 4 ) , in control NDC80 - depleted mitotic . Scale bars , 2 µm .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O O O

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 stably expressing CENP - E - GFP illustrating bipolar spindles in prometaphase , metaphase just after sister - chromatid separation at anaphase onset . Approximate x - y resolution is 70 nm . 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 , k - . Chromosomes ( confocal mode only ) were revealed in larger panels with DAPI ( cyan ) 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 panels is 5 μm .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O

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 - time series of HeLa stably expressing CENP - E - GFP . arrowheads highlight CENP - E - GFP at KTs , arrows highlight CENP - E - GFP on interpolar MTs . Scale bar , 10 µm . Time , hour : min .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O

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.


A . Representative spinning disk confocal time series of U2OS stably co - expressing PA - GFP - α - tubulin ( cyan ) mCherry - α - tubulin ( red ) , with indicated treatments ( left ) . arrowheads highlight poleward flux of photoactivated regions . Scale bar , 10 µm . Time , min : sec . Right panels display corresponding kymographs of photoactivated spindles used for of flux rates ( red dotted lines highlight MT - flux slopes ) . Scale bars , 2 µm ( horizontal ) 30 sec ( vertical ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

A. 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 . of MT - flux in prometaphase ( B ) metaphase ( C ) bipolar spindles subjected to indicated treatments . Graphs represent MT flux of with mean ± SD . siNDC80 , siNDC80 + GSK923295 , early prometaphase , early prometaphase + GSK923295 , siControl , siKIF4A N ( number of , 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 ) .

O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

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 stably co - expressing PA - GFP - α - tubulin ( cyan ) mCherry - α - tubulin ( red ) , with indicated treatments ( left ) corresponding kymographs ( right ) as described in ( A ) . Scale bars , 2 µm ( horizontal ) 30 sec ( vertical ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

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).


A - C . Graph depicting a direct correlation between MT - flux mitotic spindle where mean spindle is plotted over mean MT - flux rates for indicated conditions ( A ) , mitotic conditions containing stable end - on attachments ( B ) , NDC80 depleted conditions lacking stable end - on attachments ( C ) . Solid dotted lines represent linear regression 95 % confidence interval , respectively . Pearson correlation coefficient ( r ) , coefficient of determination ( r2 ) corresponding p values are indicated . error bars over x y axis represent standard deviation of MT - flux spindle , respectively .

O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O B-SUBCELLULAR O O O

A-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 from spinning disk confocal live - time series of U2OS PA - GFP - α - tubulin / mCherry - α - tubulin under indicated treatments , with SiR - - labelled . Microtubules are shown in red in cyan in merged image . Scale bar : 10 µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O O O O O

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 . of spindle lengths at metaphase following indicated treatments . Graph represents spindle of with mean ± SD . N ( number of , 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 ) .

O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


( A , B ) Two - dimensional tryptic mapping of YFP - p27 , YFP - p27T198A a mixture of both ( A ) . Phosphoamino - acid of spots a b ( B ) . migration of phosphoserine ( S ) , phosphothreonine ( T ) phosphotyrosine ( Y ) is marked with circles in B . asterisk indicates origin of migration .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

(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 an empty RFP vector in a 5 : 1 ( YFP - p27 : RFP ) ( E ) were treated with ( + ) or without ( − ) MG132 ( 10 μM ) for 12 h before western blot ( C ) microscopy ( D , E ) .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O

(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).


O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O


( F ) Western blots of extracts from MCF - 7 transfected with YFP - p27 , YFP - p27T198A or YFP - p27T198D mutants following incubation with cycloheximide ( CHX ) for indicated times . Uncropped of blots are shown in Supplementary Information , Fig . S5 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(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.


( a ) Crystal violet of MCF - 7 transfected with YFP ( C1 ) , YFP - p27 , YFP - p27T198A or YFP - p27T198D selected with G418 for three . Data are presented as mean ± s . d . ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(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 of MCF - 7 transfected with YFP ( C1 ) or YFP - p27 , YFP - p27T198A YFP - p27T198D for 48 h . Data are presented as mean ± s . d . ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(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 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 were counted ( s . d . , 10 ∼ 15 % ) . scale bars represent 50 μm in c .

O O O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( a , b ) MCF - 7 were cultured for 24 h following siRNA with or without FBS before western blot ( a ) - viability assays ( b ) .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O

(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 were transfected with vector alone ( C1 ) , YFP - p27 ( WT ) , or siRNA - resistant constructs WT - R T198A - R 24 h before interference ( RNAi ) . PARP cleavage was determined by western blotting densitometric scanning . Data represent three independent experiments ( mean ± s . d . ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O

(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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

(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 treated with or without LY294002 for 48 h following RNAi . p27 siRNA pool - 1 , siRNA1 , 2 3 ; arrows , blebbing ; triangles , apoptotic bodies . scale bar represents 50 μm .

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(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 extracts from MCF - 7 cultured with ( + ) or without ( − ) glucose for 24 h following RNAi . Fr , fragment ; PARP cleavage = 100 × PARPFr / ( PARP + PARPFr ) .

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(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).


( a ) Western blots of lysates from indicated lines .

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(a) Western blots of lysates from the indicated cell lines.


( b ) Pearson correlation of p27 LKB1 quantified by reverse phase array assays in 74 randomly selected breast tumors ( MDACC ) .

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(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 lysates from Hela transfected with either pCDNA3 or wild - type LKB1 cultured with or without glucose for 24 h .

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(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 extracts from MEFs treated with AICAR ( d e ) ,

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(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e),


( d - f ) Western blots of extracts from MEFs treated with AICAR ( d e )

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(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e)


( d - f ) Western blots of extractsfrom 3T3L1 cultured with or without glucose for 30 min ( f ) .

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(d-f) Western blots of protein extractsfrom 3T3L1 cells cultured with or without glucose for 30 min (f).


( g ) Western blot of 3T3L1 72 h post - transfection RNAi with or without glucose starvation for 1 h before . Densitometric of western blots is shown . Uncropped of blots are shown in Supplementary Information , Fig . S5 .

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(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.


( A ) FACS of TUNEL positive p27 + / + p27 − / − treated with AICAR for 20 h . Data represent mean ± s . d . of three independent experiments .

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(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 e ) p27 − / − ( b , d f ) MEFs treated with ( c d ) , or without ( a b ) AICAR ( 1 mM ) , or without glucose ( e f ) for 14 h . Magnified of boxed areas were shown on left ( a ′ , b ′ , c ′ , d ′ , e ′ f ′ ) with arrowheads indicating autophagic vacuoles including autolysosomes ( black arrowheads ) . scale bars represent 10 μm in a - f 0 . 5 μm in a ′ - f ′ . N , nucleus . Autophagic vacuoles were counted for three randomly selected from each electron microscopy section data represent mean ± s . d . ( C ) .

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(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).


A Immortalized Brca1F / − MEFs were infected with retroviruses expressing indicated shRNAs / or Cre recombinase , followed by selection with puromycin for 72 h . extracts were prepared 48 h later analysed by Western blotting as indicated . SMC1 tubulin were used as loading controls . * non ‐ specific band .

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A 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 treated as in ( A ) were fixed 48 h after selection 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 of TIFs in 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 ‐ t ‐ . * P ≤ 0 . 05 ; NS , P > 0 . 05 .

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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.