( 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 .

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 O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-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 complex ) ( F ) , and 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 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 analysis 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 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 - 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 .

O O O B-EXP_ASSAY I-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 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 B-GENEPROD 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 imaging time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), treated with control and KIF4A siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.


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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY 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 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 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 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 .

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


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 )

O 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 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)


Quantification of MT - flux in monopolar spindles subjected to indicated treatments . Graphs represent MT flux of individual cells with mean ± SD . N ( number of cells , number of independent experiments ) monopolar spindles : siControl ( 35 , 3 ) , siKIF15 ( 44 , 3 ) , siKID ( 38 , 3 ) , siKIF4A ( 44 , 3 ) , siKIF2A ( 38 , 3 ) , siCLASPs ( 12 , 2 ) , GSK923295 ( 33 , 3 ) .

O 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 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 - 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 .

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

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 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 .

O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O 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 . Quantification of MT - flux upon shRNA - mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi - resistant mCherry - KIF4A variants . The bars in graph represent mean ± SD . N ( number of cells , number of independent experiments ) : uninduced shKIF4A ( 48 , 4 ) , shKIF4A ( 60 , 4 ) , uninduced KIF4A WT ( 36 , 5 ) , shKIF4A + KIF4A WT ( 43 , 5 ) , uninduced KIF4A K94A ( 42 , 7 ) , shKIF4A + KIF4A K94A ( 38 , 6 ) , uninduced KIF4A ΔZip1 ( 32 , 4 ) , shKIF4A + ΔZip1 ( 30 , 4 ) .

O 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 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 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 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 ) .

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 O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O 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 B-SUBCELLULAR O B-SMALL_MOLECULE I-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 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 O B-SMALL_MOLECULE O 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 - intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions , immunostained with antibodies against KIF4A and α - tubulin . DNA was counterstained with DAPI . KIF4A in cyan and α - tubulin in red in merged image . Scale bar , 10 µm .

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

O O O B-EXP_ASSAY I-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 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 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 . 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 ) .

O O O O O O O O B-SUBCELLULAR O 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 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 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 . 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 ) .

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

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

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-SUBCELLULAR O O O B-CELL_LINE O O O O O O 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 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 . 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 ) .

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

O 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 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 B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-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 O O O O O O O O O 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 - 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 .

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

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 O B-EXP_ASSAY O 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 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 O O B-GENEPROD O 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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE 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 B-SUBCELLULAR 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 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 O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O 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 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 - cell time series of HeLa cells stably expressing CENP - E - GFP . White arrowheads highlight CENP - E - GFP at KTs , white arrows highlight CENP - E - GFP on interpolar MTs . Scale bar , 10 µm . Time , hour : min .

O 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 B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR O 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O 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 O O O O 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 B-SUBCELLULAR O O 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 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 . 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 ) .

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B, C. Quantification of MT-flux in prometaphase (B) and metaphase (C) bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. siNDC80, siNDC80 + GSK923295, early prometaphase, early prometaphase + GSK923295, siControl, and siKIF4A N (number of cells, number of independent experiments): siNDC80 + KIF15 + STLC (33, 5), siNDC80 + KIF15 + STLC + GSK923295 (25, 3), early prometaphase + KIF15 + STLC + GSK923295 (11, 2), siKIF15 + STLC (29, 3), siKIF4A + siKIF15 + STLC (10, 3).


D . Representative spinning disk confocal time series of U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , with indicated treatments ( left ) and corresponding kymographs ( right ) as described in ( A ) . Scale bars , 2 µm ( horizontal ) and 30 sec ( vertical ) .

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

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 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 .

O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O 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 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 B-SUBCELLULAR O O O B-SUBCELLULAR O 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 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 .

O O O B-EXP_ASSAY 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 B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE 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 . 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 ) .

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

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

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


( C - E ) MCF - 7 transfected with YFP - p27 or YFP - p27T198A mutant ( C , D ) or cotransfected with YFP - p27 and an empty RFP vector in a 5 : 1 ( YFP - p27 : RFP ) ratio ( E ) were treated with ( + ) or without ( − ) MG132 ( 10 μM ) for 12 h before western blot analysis ( C ) and fluorescence microscopy ( D , E ) .

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 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 O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-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 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 O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O 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 O B-SMALL_MOLECULE O 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 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 ) .

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

O O O B-GENEPROD O B-GENEPROD B-CELL_LINE O 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 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 cells were cultured for 24 h following siRNA with or without FBS before western blot analysis ( a ) and cell - 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 O B-EXP_ASSAY I-EXP_ASSAY O O O 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 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 . ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 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 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 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 ) .

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 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 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 .

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

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

(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 the indicated cell lines .

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

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


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

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 B-TISSUE O O O O O

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


( c ) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild - type LKB1 cultured with or without glucose for 24 h .

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

(c) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild-type LKB1 cultured with or without glucose for 24 h.


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

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

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


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

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

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


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

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

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


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

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

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

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

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


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

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

(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 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 .

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

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 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 .

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 B-SUBCELLULAR 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-GENEPROD 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 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

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.


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

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

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


A Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . A minimum of 2 , 000 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 .

O O B-GENEPROD O O B-CELL_LINE O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR 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 B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD 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 O O O

A Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.


B Immortalized Brca1F / C61G MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . The frequency of end ‐ to ‐ end chromosome ‐ type fusions was analysed as in ( A ) .

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

B Immortalized Brca1F/C61G MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. The frequency of end‐to‐end chromosome‐type fusions was analysed as in (A).


C - F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and / or CtIP shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . GAPDH was used as a loading control . * non ‐ specific band .

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

C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band.


C - F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and / or CtIP shRNAs , followed by selection with puromycin for 72 h . Cells treated as in ( C ) and ( E ) were arrested in mitosis with colcemid , and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe ( D , F ) . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 .

O O O B-CELL_LINE O O O O O O O B-ORGANISM O B-GENEPROD O O O B-GENEPROD 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 B-SUBCELLULAR 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 B-SUBCELLULAR 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

C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cells treated as in (C) and (E) were arrested in mitosis with colcemid, and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe (D, F). The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05.


A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 was used as a loading control . * non ‐ specific band . B Quantification of the frequency of end ‐ to ‐ end chromosome ‐ type fusions of cells treated as in ( A ) represented as a percentage of fusions observed after TRF2 depletion . A minimum of 2 , 000 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . The P ‐ value was calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 .

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

A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.B Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (A) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.


Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . A minimum of 2 , 000 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 .

O B-GENEPROD O O B-CELL_LINE O O O B-ORGANISM O O O O O O O B-GENEPROD O O O B-SUBCELLULAR 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 B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD 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 O O O

Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.


D Immortalized Brca1F / − MEFs were infected with retroviruses expressing TRF2 shRNAs and / or Cre recombinase , together with a lentivirus expressing PARP1 shRNA , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 was used as a loading control . * non ‐ specific band . E Quantification of the frequency of end ‐ to ‐ end chromosome ‐ type fusions of cells treated as in ( D ) represented as a percentage of fusions observed after TRF2 depletion . A minimum of 1 , 500 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . The P ‐ value was calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 .

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

D Immortalized Brca1F/−MEFs were infected with retroviruses expressing TRF2 shRNAs and/or Cre recombinase, together with a lentivirus expressing PARP1 shRNA, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.E Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (D) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 1,500 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.


A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 and GAPDH were used as loading controls . * non ‐ specific band .

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

A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and GAPDH were used as loading controls. *non‐specific band.


B MboI ‐ and AluI ‐ digested DNA from cells treated as in ( A ) was resolved by pulsed ‐ field gel electrophoresis and probed with end ‐ labelled ( AACCCT ) 4 probe . Representative pulsed ‐ field gel samples run under native and denatured conditions are shown .

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

B MboI‐ and AluI‐digested DNA from cells treated as in (A) was resolved by pulsed‐field gel electrophoresis and probed with end‐labelled (AACCCT)4 probe. Representative pulsed‐field gel samples run under native and denatured conditions are shown.


C Quantification of the 3 ′ overhang in cells treated as in ( B ) . For each sample , the ss / total DNA ratios were expressed relative to the GFP shRNA ‐ treated control . Error bars represent SD of two independent experiments .

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

C Quantification of the 3′ overhang in cells treated as in (B). For each sample, the ss/total DNA ratios were expressed relative to the GFP shRNA‐treated control. Error bars represent SD of two independent experiments.


A - C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment . A minimum of 1 , 200 chromosomes 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 . Each graph represents a separate set of experiments .

O O O B-CELL_LINE O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR 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 B-SUBCELLULAR 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 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

A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes 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. Each graph represents a separate set of experiments.


Binding of recombinant ( r ) CEACAM1 ( CC1 ) - HIS ( 10 μg / mL ) to ( A ) a panel of Gram - positive bacteria , namely Enterococcus faecalis , Enterococcus faecium , Group A Streptococcus ( GAS ) , Group B Streptococcus ( GBS ) , Group C Streptococcus ( GCS ) , Group G Streptococcus ( GGS ) , Streptococcus pneumoniae , Staphylococcus aureus , ( B ) an expanded collection of GBS isolates , with serotype and carriage of bac gene shown . rCC1 binding to live bacteria was quantified by a secondary anti - His - FITC monoclonal antibody ( mAb ) . Mean and standard deviation ( SD ) values are reported for n = 3 independent experiments . In ( B ) , GBS strains from left to right are A909 , BS39 , 515 , BS22 , H36B , BS29 , 18RS21 , BM110 , COH1 , SBL3066 , NCTC10 / 84 , SB35 , SB10 , SB20 , BS26 .

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

Binding of recombinant (r)CEACAM1 (CC1)-HIS (10 μg/mL) to (A) a panel of Gram-positive bacteria, namely Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus (GAS), Group B Streptococcus (GBS), Group C Streptococcus (GCS), Group G Streptococcus (GGS), Streptococcus pneumoniae, Staphylococcus aureus, (B) an expanded collection of GBS isolates, with serotype and carriage of bac gene shown. rCC1 binding to live bacteria was quantified by a secondary anti-His-FITC monoclonal antibody (mAb). Mean and standard deviation (SD) values are reported for n = 3 independent experiments. In (B), GBS strains from left to right are A909, BS39, 515, BS22, H36B, BS29, 18RS21, BM110, COH1, SBL3066, NCTC10/84, SB35, SB10, SB20, BS26.


Western blot analysis of rCC1 - HIS ( 30 μg / mL ) binding to purified β protein , but not to control GBS surface proteins α and Rib . Panel on the left show staining of proteins after separation by SDS - PAGE .

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

Western blot analysis of rCC1-HIS (30 μg/mL) binding to purified β protein, but not to control GBS surface proteins α and Rib. Panel on the left show staining of proteins after separation by SDS-PAGE.


Inhibition of β - IgSF binding to DB . CC1 by anti - CC1 - N mAb ( clone CC1 / 3 / 5 - Sab ) , but not by anti - CC1 - A1B1 mAb ( clone B3 - 17 ) . β - IgSF - biotin was coupled to SPE . Mean and SD values are reported for n = 6 replicates .

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

Inhibition of β-IgSF binding to DB.CC1 by anti-CC1-N mAb (clone CC1/3/5-Sab), but not by anti-CC1-A1B1 mAb (clone B3-17). β-IgSF-biotin was coupled to SPE. Mean and SD values are reported for n = 6 replicates.


A ) Representative isothermal titration calorimetry ( ITC ) binding curves of CEACAM - N domains and β - IgSF duplicates . Experiments were performed using β - IgSF and N domains of ( r ) CEACAM1 ( CC1 ) , CEACAM3 ( CC3 ) , CEACAM5 ( CC5 ) , CEACAM6 ( CC6 ) and CEACAM8 ( CC8 ) . B ) Representative flow cytometry plots analyzing the interaction of rCC1 , CC3 , CC5 , CC6 or CC8 ( 10 μg / ml ) with GBS A909 . Fluorescence of bacteria was measured by flow cytometry . Representative data from n = 3 independent experiments .

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

A) Representative isothermal titration calorimetry (ITC) binding curves of CEACAM-N domains and β-IgSF duplicates. Experiments were performed using β-IgSF and N domains of (r)CEACAM1 (CC1), CEACAM3 (CC3), CEACAM5 (CC5), CEACAM6 (CC6) and CEACAM8 (CC8). B) Representative flow cytometry plots analyzing the interaction of rCC1, CC3, CC5, CC6 or CC8 (10 μg/ml) with GBS A909. Fluorescence of bacteria was measured by flow cytometry. Representative data from n = 3 independent experiments.


Adhesion of WT , ∆ bac and ∆ bac + pLZ . bac complemented A909 strains to human CC1 - or CC5 - expressing CHO transfectants , or empty vector control cells . Mean and SD from n = 5 independent replicates is displayed .

B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O B-ORGANISM B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O

Adhesion of WT, ∆bac and ∆bac + pLZ.bac complemented A909 strains to human CC1- or CC5-expressing CHO transfectants, or empty vector control cells. Mean and SD from n = 5 independent replicates is displayed.


Binding of rCC1 - N to dynabeads ( DB ) coated with β - IgI3 wild type ( WT ) or mutants . Mutation of β - IgI3 residues 42 , 46 and 53 lead to significant changes in CC1 - N binding capacity .

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Binding of rCC1-N to dynabeads (DB) coated with β-IgI3 wild type (WT) or mutants. Mutation of β-IgI3 residues 42, 46 and 53 lead to significant changes in CC1-N binding capacity.


Binding of β - IgI3 coupled to Streptavidin - PE to DB coated with rCC1 - N wildtype or variants . Mutation of CC1 - N residues 29 , 44 , 89 , 91 and 95 lead to significant changes in β - IgI3 binding capacity .

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Binding of β-IgI3 coupled to Streptavidin-PE to DB coated with rCC1-N wildtype or variants. Mutation of CC1-N residues 29, 44, 89, 91 and 95 lead to significant changes in β-IgI3 binding capacity.


Binding of recombinant ( r ) CC1 - HIS to dynabeads ( DB ) coated with β - IgI3 , R28 - IgI3 or control . Mean and SD is shown for n = 6 independent replicates .

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Binding of recombinant (r)CC1-HIS to dynabeads (DB) coated with β-IgI3, R28-IgI3 or control. Mean and SD is shown for n = 6 independent replicates.


Binding of β - IgI3 , R28 - IgI3 or human serum albumin ( HSA ) coupled to PE - conjugated streptavidin ( SPE ) , to DB coated with CC1 ( DB . CC1 ) . Mean and SD is shown for n = 6 independent replicates .

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Binding of β-IgI3, R28-IgI3 or human serum albumin (HSA) coupled to PE-conjugated streptavidin (SPE), to DB coated with CC1 (DB.CC1). Mean and SD is shown for n = 6 independent replicates.


( A ) Quantitative analysis of Rnf114 mRNA by realtime - PCR in various mouse tissues and organs shows that Rnf114 was predominately expressed in oocytes .

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(A) Quantitative analysis of Rnf114 mRNA by realtime-PCR in various mouse tissues and organs shows that Rnf114 was predominately expressed in oocytes.


( B ) Immunohistochemistry analysis of RNF114 in mouse ovary shows that RNF114 was mainly expressed in the oocytes ( Scale bar = 100um ) .

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(B) Immunohistochemistry analysis of RNF114 in mouse ovary shows that RNF114 was mainly expressed in the oocytes (Scale bar = 100um).


( C ) Knockdown efficiency of Rnf114 in the 2 - cell stage embryos derived from Rnf114 siRNA # 1 or siRNA # 2 injected zygotes was verified by realtime - PCR and western blotting . NC indicates negative control injected with non - silencing siRNA . Three independent experiment replicates were performed for realtime - PCR , error bars represent s . e . m . ; * * : P < 0 . 01 , * * * : P < 0 . 01 in unpaired two - tailed t - test .

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(C) Knockdown efficiency of Rnf114 in the 2-cell stage embryos derived from Rnf114 siRNA #1 or siRNA #2 injected zygotes was verified by realtime-PCR and western blotting. NC indicates negative control injected with non-silencing siRNA. Three independent experiment replicates were performed for realtime-PCR, error bars represent s.e.m.; **:P<0.01, ***:P<0.01 in unpaired two-tailed t-test.


( D ) RNF114 depletion impaired the early embryonic development . Rnf114 siRNA # 1 or siRNA # 2 injected zygotes were cultured and quantified ( siRNA1 # , n = 120 ; siRNA2 # , n = 128 ; three independent experiments ) ; NC indicates negative control ( n = 193 ; six independent experiments ) . The error bars represent s . d . , * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 in unpaired two - tailed t - test .

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(D) RNF114 depletion impaired the early embryonic development. Rnf114 siRNA #1 or siRNA #2 injected zygotes were cultured and quantified (siRNA1#, n = 120; siRNA2#, n = 128; three independent experiments); NC indicates negative control (n = 193; six independent experiments). The error bars represent s.d., *:P<0.05, **:P<0.01, ***:P<0.001 in unpaired two-tailed t-test.


( E ) Representative images to show early embryonic development defects . The embryos arrested at two - cell stage are indicated by arrows ( Scale bar = 100um ) .

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(E) Representative images to show early embryonic development defects. The embryos arrested at two-cell stage are indicated by arrows (Scale bar = 100um).


( A ) In vitro ubiquitination assay . Purified His - RNF114 was subjected to ubiquitination assays with the indicated components and the samples were analyzed by immunoblotting with anti - FLAG ( Ub ) antibody . Lanes 1 - 4 indicate negative controls . The results show that recombinant RNF114 efficiently catalyzed the formation of polyubiquitin chains .

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(A) In vitro ubiquitination assay. Purified His-RNF114 was subjected to ubiquitination assays with the indicated components and the samples were analyzed by immunoblotting with anti-FLAG (Ub) antibody. Lanes 1-4 indicate negative controls. The results show that recombinant RNF114 efficiently catalyzed the formation of polyubiquitin chains.


( B , C ) Purified wild - type and RING mutated His - RNF114 were stained by Coomassie Blue ( B ) . Purified wild - type and RING mutated His - RNF114 were subjected to ubiquitination assays together with E1 , Ubc4 , FLAG - Ub , and ATP . The results show that mutant RNF114 lacked ubiquitination activity ( C ) .

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(B, C) Purified wild-type and RING mutated His-RNF114 were stained by Coomassie Blue (B). Purified wild-type and RING mutated His-RNF114 were subjected to ubiquitination assays together with E1, Ubc4, FLAG-Ub, and ATP. The results show that mutant RNF114 lacked ubiquitination activity (C).


( D ) Screening for proteins ubiquitinated by RNF114 on a proteome array . Putative substrates of RNF114 were identified on Ubiquitin Ligase Substrate Identification on ProtoArray ® Human Protein Microarrays v5 . 0 . Ubiquitination reactions were performed on quintuplicate arrays using the indicated components , the five sub - array are respectively negative controls , E1 + E2 ( no E3 ) , WT RNF114 ( two arrays , duplicate ) , and mutated RNF114 .

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(D) Screening for proteins ubiquitinated by RNF114 on a proteome array. Putative substrates of RNF114 were identified on Ubiquitin Ligase Substrate Identification on ProtoArray® Human Protein Microarrays v5.0. Ubiquitination reactions were performed on quintuplicate arrays using the indicated components, the five sub-array are respectively negative controls, E1+E2 (no E3), WT RNF114 (two arrays, duplicate), and mutated RNF114.


( E ) A heat map comparing hits between E1 + E2 , E1 + E2 + WT RNF114 , and E1 + E2 + mutated RNF114 reactions . Candidate substrates of RNF114 selected for further characterization are indicated in red font ; HIP1 , indicated by blue font , was considered to be a false positive because it was also detected in the mutated RNF114 reaction . Black font indicates proteins with ubiquitin - binding domains such as UIM , UBA or CUE or proteins without clearly reported functions , which were excluded from further investigation .

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(E) A heat map comparing hits between E1+E2, E1+E2+WT RNF114, and E1+E2+mutated RNF114 reactions. Candidate substrates of RNF114 selected for further characterization are indicated in red font; HIP1, indicated by blue font, was considered to be a false positive because it was also detected in the mutated RNF114 reaction. Black font indicates proteins with ubiquitin-binding domains such as UIM, UBA or CUE or proteins without clearly reported functions, which were excluded from further investigation.


( A ) RNF114 substrate validation . HEK293 cells were co - transfected with constructs expressing FLAG - tagged substrates , wild - type or mutated His - RNF114 and EGFP , and western blot was performed with anti - FLAG and anti - His antibodies . The results show that the protein levels of CD74 , TAB1 , OPTN , TNIP1 , APPL1 , PSAT1 , and RALGPS1 were reduced in the presence of wild - type RNF114 compared to co - transfecting with mutant RNF114 .

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(A) RNF114 substrate validation. HEK293 cells were co-transfected with constructs expressing FLAG-tagged substrates, wild-type or mutated His-RNF114 and EGFP, and western blot was performed with anti-FLAG and anti-His antibodies. The results show that the protein levels of CD74, TAB1, OPTN, TNIP1, APPL1, PSAT1, and RALGPS1 were reduced in the presence of wild-type RNF114 compared to co-transfecting with mutant RNF114.


( B ) RNF114 - dependent in vitro ubiquitination of substrates . Substrate ubiquitination assays were performed with purified RNF114 , the reaction products were immunoblotted using anti - His antibody to detect the ubiquitin ( upper panel ) or anti - FLAG antibody detecting the substrate protein ( middle panel ) , and the lower panel showed the merged signals . The results show that clear polyubiquitin chains were formed in the systems containing recombinant CD74 , TAB1 , TNIP1 , PSAT1 or RALGPS1 .

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(B) RNF114-dependent in vitro ubiquitination of substrates. Substrate ubiquitination assays were performed with purified RNF114, the reaction products were immunoblotted using anti-His antibody to detect the ubiquitin (upper panel) or anti-FLAG antibody detecting the substrate protein (middle panel), and the lower panel showed the merged signals. The results show that clear polyubiquitin chains were formed in the systems containing recombinant CD74, TAB1, TNIP1, PSAT1 or RALGPS1.


( A ) Mouse zygotes were microinjected with sterile ddH2O ( control group ) or Ralgps1 , Cd74 , Tnip1 , Psat1 , or Tab1 mRNA . During 96 - 100 h culturing , the percentages of embryos at various stages were counted under microscopy . The results show that overexpression of Tab1 impaired the early embryonic development . Three independent experiment replicates were performed , error bars represent s . d . ; * : P < 0 . 05 , * * * : P < 0 . 001 in unpaired two - tailed t - test .

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(A) Mouse zygotes were microinjected with sterile ddH2O (control group) or Ralgps1, Cd74, Tnip1, Psat1, or Tab1 mRNA. During 96-100 h culturing, the percentages of embryos at various stages were counted under microscopy. The results show that overexpression of Tab1 impaired the early embryonic development. Three independent experiment replicates were performed, error bars represent s.d.; *:P<0.05, ***:P<0.001 in unpaired two-tailed t-test.


( B ) TAB1 reduced with mouse early embryonic development , while abnormally accumulated at 2 - cell stage once treated with Rnf114 siRNAs . TAB1 was detected by western blotting with anti - TAB1 antibody . The relative level of TAB1 protein after Rnf114 siRNAs treatment were presented as mean ± s . e . m . ( * : P < 0 . 05 in unpaired two - tailed t - test , four independent experiment replicates ) .

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(B) TAB1 reduced with mouse early embryonic development, while abnormally accumulated at 2-cell stage once treated with Rnf114 siRNAs. TAB1 was detected by western blotting with anti-TAB1 antibody. The relative level of TAB1 protein after Rnf114 siRNAs treatment were presented as mean±s.e.m. (*:P<0.05 in unpaired two-tailed t-test, four independent experiment replicates).


( C ) Detection of endogenous TAB1 ubiquitination at 1 - cell or 2 - cell stages . Endogenous TAB1 proteins were immunoprecipated by anti - TAB1 antibody at either 1 - cell or 2 - cell stages , the immunoprecipates were detected by either anti - TAB1 antibody or anti - ubiquitin antibody . The results show that more Ub chains on TAB1 were detected in the two cell stage embryos than in the zygotes , when the TAB1 proteins were at comparable level .

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(C) Detection of endogenous TAB1 ubiquitination at 1-cell or 2-cell stages. Endogenous TAB1 proteins were immunoprecipated by anti-TAB1 antibody at either 1-cell or 2-cell stages, the immunoprecipates were detected by either anti-TAB1 antibody or anti-ubiquitin antibody. The results show that more Ub chains on TAB1 were detected in the two cell stage embryos than in the zygotes, when the TAB1 proteins were at comparable level.


( D ) RNF114 - dependent TAB1 degradation during early embryo development . ( a - c ) Representive time lapse embryo images were shown for each treatment . Scale bar = 20um . ( a ' - c ' ) Quantification of the GFP fluorescent signals for respective treatments in ( a - c ) . n = 14 , 9 or 12 for the NC , Rnf114 siRNA1 # or Rnf114 siRNA2 # treatment group respectively . Gray traces : Tab1 - GFP fluorescent signals were normalized to the first time point ; Black trace : average signal of all embryos for each treatment .

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(D) RNF114-dependent TAB1 degradation during early embryo development. (a-c) Representive time lapse embryo images were shown for each treatment. Scale bar = 20um. (a'-c') Quantification of the GFP fluorescent signals for respective treatments in (a-c). n=14, 9 or 12 for the NC, Rnf114 siRNA1# or Rnf114 siRNA2# treatment group respectively. Gray traces: Tab1-GFP fluorescent signals were normalized to the first time point; Black trace: average signal of all embryos for each treatment.


( E ) Tab1 knockdown partly rescued the early embryo development defect observed with Rnf114 knockdown . Both Rnf114 and Tab1 siRNAs were injected into the GV oocytes , then the oocytes matured , and were fertilized by IVF , and the fertilized eggs were transferred into KSOM media , cultured for 48 - 50 h . The percentages of embryos arrested at 2 - cell stage were counted . Three independent experiment replicates were performed , error bars represent s . d . ; * * : P < 0 . 01 in unpaired two - tailed t - test .

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(E) Tab1 knockdown partly rescued the early embryo development defect observed with Rnf114 knockdown. Both Rnf114 and Tab1 siRNAs were injected into the GV oocytes, then the oocytes matured, and were fertilized by IVF, and the fertilized eggs were transferred into KSOM media, cultured for 48-50 h. The percentages of embryos arrested at 2-cell stage were counted. Three independent experiment replicates were performed, error bars represent s.d.; **:P<0.01 in unpaired two-tailed t-test.


( A ) NF - κB activation during MZT . Immunofluorescence staining of p65 shows nuclear translocation of p65 at the two - cell stage and thereafter . Scale bar = 25um .

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(A) NF-κB activation during MZT. Immunofluorescence staining of p65 shows nuclear translocation of p65 at the two-cell stage and thereafter. Scale bar = 25um.


( B ) The protein levels of IκBα and its phosphorylated form ( p - IκBα ) during mouse early embryonic development were detected by western blotting , representative western blotting of IκBα and p - IκBα was shown on the left and the quantitation analysis ( right panel ) indicated that the ratio of p - IκBα / total IκBα was gradually increased from MII oocytes to 4 - cell stage embryos ( three independent experiment replicates were performed , error bars represent s . e . m . ) .

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(B) The protein levels of IκBα and its phosphorylated form (p-IκBα) during mouse early embryonic development were detected by western blotting, representative western blotting of IκBα and p-IκBα was shown on the left and the quantitation analysis (right panel) indicated that the ratio of p-IκBα/total IκBα was gradually increased from MII oocytes to 4-cell stage embryos (three independent experiment replicates were performed, error bars represent s.e.m.).


( C ) NF - κB pathway inhibition using the specific inhibitor Bay11 - 7082 resulted in two cell stage arrest . Three independent experiment replicates were performed , error bars represent s . d . ; * * * : P < 0 . 001 in unpaired two - tailed t - test .

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(C) NF-κB pathway inhibition using the specific inhibitor Bay11-7082 resulted in two cell stage arrest. Three independent experiment replicates were performed, error bars represent s.d.; ***:P<0.001 in unpaired two-tailed t-test.


( D , E ) Representive immunofluorescence staining of p65 after knockdown of Rnf114 by Rnf114 siRNA # 1 or siRNA # 2 or overexpression of Tab1 by Tab1 mRNA injection ( shown in D . Scale bar = 25um . ) , the percentages of two - cell stage embryos with p65 nuclear translocation ( shown in E . ) were significantly decreased after Rnf114 knockdown or Tab1 overexpression ( chi - square test was performed for statistical analysis ) .

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(D, E) Representive immunofluorescence staining of p65 after knockdown of Rnf114 by Rnf114 siRNA #1 or siRNA #2 or overexpression of Tab1 by Tab1 mRNA injection (shown in D. Scale bar = 25um.), the percentages of two-cell stage embryos with p65 nuclear translocation (shown in E.) were significantly decreased after Rnf114 knockdown or Tab1 overexpression (chi-square test was performed for statistical analysis).


( F ) Western blot analysis at 2 - cell stage showed the levels of p - IκBα were downregulated in the Rnf114 siRNA # 1 , siRNA # 2 injected groups .

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(F) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the Rnf114 siRNA #1, siRNA #2 injected groups.


( G ) Western blot analysis at 2 - cell stage showed the levels of p - IκBα were downregulated in the exogenous Tab1 mRNA injected groups , ddH2O was injected as control . Dotted line indicates a cropped lane .

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(G) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the exogenous Tab1 mRNA injected groups, ddH2O was injected as control. Dotted line indicates a cropped lane.


( A ) Confocal observation of mitochondrial potential after TMRM staining of LMP1 - negative or - positive NPC cells . Red fluorescence , which indicates normal mitochondrial potential , was converted into green fluorescence after a reduction in mitochondrial potential . Images were analyzed using Image J software ( scale bar , 10 μm ) . Data are presented as means ± S . E . M . ( paired t - test , n = 6 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) .

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(A) Confocal observation of mitochondrial potential after TMRM staining of LMP1-negative or -positive NPC cells. Red fluorescence, which indicates normal mitochondrial potential, was converted into green fluorescence after a reduction in mitochondrial potential. Images were analyzed using Image J software (scale bar, 10 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).


( C ) Extent of Ca2 + - mediated mitochondrial swelling in NPC cells . Data are presented as means ± S . E . M . ( paired t - test , n = 6 , biological replicates per group , * * p < 0 . 01 ) .

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(C) Extent of Ca2+-mediated mitochondrial swelling in NPC cells. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).


( A ) LMP1 is localized to the inner mitochondrial membrane . Isolated mitochondria were incubated with proteinase K ( 40 μM ) for 30 min before LMP1 expression was detected ( left ) ; the mitochondria were further subjected to submitochondrial fractionation . VDAC1 , TIM23 , and HSP60 were used to represent MOM , MIM , and mitochondrial matrix protein , respectively ( right ) .

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

(A) LMP1 is localized to the inner mitochondrial membrane. Isolated mitochondria were incubated with proteinase K (40 μM) for 30 min before LMP1 expression was detected (left); the mitochondria were further subjected to submitochondrial fractionation. VDAC1, TIM23, and HSP60 were used to represent MOM, MIM, and mitochondrial matrix protein, respectively (right).


( B ) The interaction of LMP1 and endogenous ANT1 was analyzed by an immunofluorescence confocal assay . HK1 - LMP1 cells were immunostained with anti - LMP1 ( green ) and anti - ANT1 ( red ) antibodies and then subjected to confocal microscopy ， and use imageJ to calculate the co - location level of both . The nuclei were stained with DAPI ( scale bar , 5 μm ) . The quantified graph on the top right shows the percentage of yellow fluorescence ( merge ) to red fluorescence ( ANT1 ) in HK1 - LMP1 cells . Data are presented as means ± S . E . M . ( n = 6 , biological replicates per group ) .

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

(B) The interaction of LMP1 and endogenous ANT1 was analyzed by an immunofluorescence confocal assay. HK1-LMP1 cells were immunostained with anti-LMP1 (green) and anti-ANT1 (red) antibodies and then subjected to confocal microscopy，and use imageJ to calculate the co-location level of both. The nuclei were stained with DAPI (scale bar, 5 μm). The quantified graph on the top right shows the percentage of yellow fluorescence (merge) to red fluorescence (ANT1) in HK1-LMP1 cells. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).


( G ) LMP1 binds directly to ANT1 dependent on its transmembrane domain in 293T cells . IP / IB was used to detect the interaction between Flag - tagged LMP1 mutants and Myc - tagged ANT1 . IP of cell lysates using a Flag or Myc antibody .

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

(G) LMP1 binds directly to ANT1 dependent on its transmembrane domain in 293T cells. IP/IB was used to detect the interaction between Flag-tagged LMP1 mutants and Myc-tagged ANT1. IP of cell lysates using a Flag or Myc antibody.


( A Effect of inhibitors BKA and CATR on ANT1 - VDAC1 binding in 293T cells .

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

(A Effect of inhibitors BKA and CATR on ANT1-VDAC1 binding in 293T cells.


B ) Effect of inhibitors BKA and CATR on ANT1 - VDAC1 binding in 293T cells . Data are presented as means ± S . E . M . ( paired t - test , n = 3 , biological replicates per group , * * p < 0 . 01 ) .

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O 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) Effect of inhibitors BKA and CATR on ANT1-VDAC1 binding in 293T cells. Data are presented as means ± S.E.M. (paired t-test, n = 3, biological replicates per group, ** p < 0.01).


( C The effect of different amounts of EBV - LMP1 on the binding of ANT1 - VDAC1 was detected by CO - IP .

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

(C The effect of different amounts of EBV-LMP1 on the binding of ANT1-VDAC1 was detected by CO-IP.