E . Representative immunofluorescent of H3K27me3 in SCNT at 1 - stage . H3K27me3 enrichment in SCNT were observed with either 1 pseudo - pronucleus ( 1PPN ) , bipseudo - pronucleus ( 2PPN ) , or tripseudo - pronucleus ( 3PPN ) . BA , before activation ; AA , after activation . Representative from ≥ 55 analyzed in three independent micromanipulations are shown . Scale bar , 20

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E. Representative immunofluorescent staining of H3K27me3 in SCNT embryos at the 1-cell stage. H3K27me3 enrichment in the SCNT embryos were observed with either 1 pseudo-pronucleus (1PPN), bipseudo-pronucleus (2PPN), or tripseudo-pronucleus (3PPN). BA, before activation; AA, after activation. Representative images from ≥ 55 embryos analyzed in three independent micromanipulations are shown. Scale bar, 20 μm


F . Dynamic appearance of H3K27me3 during early preimplantation development . Shown are representative of stained with H3K27me3 . Negative of H3K27me3 could be observed in ICSI SCNT - tdTomato + at morula stage . Representative from ≥ 83 analyzed in four independent micromanipulations are shown . Scale bar , 20

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F. Dynamic appearance of H3K27me3 during early preimplantation development. Shown are representative images of embryos stained with DNA and H3K27me3. Negative staining of H3K27me3 could be observed in ICSI and SCNT-tdTomato+ embryo at morula stage. Representative images from ≥ 83 embryos analyzed in four independent micromanipulations are shown. Scale bar, 20 μm


G . Representative of H3K27me3 immunostainings on bovine intraspecies SCNT . IVF - derived bovine were used as a control for comparison . These are representative examples of shown in Fig 3H . Representative from ≥ 100 analyzed in four independent micromanipulations are shown ; Scale bar , 20 μm . H . bar chart shows percentages of strong moderate H3K27me3 modifications of bovine . Numbers of total analyzed from four independent micromanipulations are shown in bars .

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G. Representative images of H3K27me3 immunostainings on bovine intraspecies SCNT embryos. IVF-derived bovine embryos were used as a control for comparison. These images are representative examples of the quantification shown in Fig 3H. Representative images from ≥ 100 embryos analyzed in four independent micromanipulations are shown; Scale bar, 20 μm. H. The bar chart shows the percentages of strong and moderate H3K27me3 modifications of bovine embryos. Numbers of the total embryos analyzed from four independent micromanipulations are shown in the bars.


A , B RT - qPCR of KDM6A ( A ) KDM6B ( B ) mRNA in SCNT 2 - . Data shown are mean expression values relative to Gapdh . value in ICSI control was set as 1 . Error bars , s . e . m . , n ≥ 3 . * * P < 0 . 01 by Student " s

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A, B RT-qPCR analysis of KDM6A (A) and KDM6B (B) mRNA levels in SCNT 2-cell embryo. Data shown are mean expression values relative to Gapdh. The value in ICSI control was set as 1. Error bars, s.e.m., n ≥ 3. **P < 0.01 by Student"s t


C . sketch of KDM6A KDM6B in vitro transcription vector ( right ) , integrity of in vitro transcripted mRNA was confirmed by electrophoresis with formaldehyde gels ( left ) . M , marker ; T7 , in vitro transcription promoter ; HA , hemagglutinin epitope

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C. The sketch of KDM6A and KDM6B in vitro transcription vector (right), and the integrity of in vitro transcripted mRNA was confirmed by electrophoresis with formaldehyde gels (left). M, marker; T7, in vitro transcription promoter; HA, hemagglutinin epitope tag


D . Immunostaining of SCNT for H3K27me3 HA epitope tag after injection of different mRNA as indicated . Representative from ≥ 187 analyzed in four independent micromanipulations for each condition are shown . Scale bar , 20

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D. Immunostaining of SCNT embryo for H3K27me3 and HA epitope tag after injection of different mRNA as indicated. Representative images from ≥ 187 embryos analyzed in four independent micromanipulations for each condition are shown. Scale bar, 20 μm


F . Representative of SCNT at 115 h after injection of different mRNA as indicated . Scale bar , 50

F. Representative images of SCNT embryos at 115 h after injection of different mRNA as indicated. Scale bar, 50 μm


G . bar chart showing efficiency of blastocyst formation . Injection of KDM6A mRNA improved preimplantation development rate of SCNT . Error bars , s . e . m . , n ≥ 3 . * * P < 0 . 01 , * * * P < 0 . 001 by two - Student " s t - . n . s . , not

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G. The bar chart showing the efficiency of blastocyst formation. Injection of KDM6A mRNA improved the preimplantation development rate of SCNT embryos. Error bars, s.e.m., n ≥ 3. **P < 0.01, ***P < 0.001 by two-tailed Student"s
 t-test. n.s., not signif


I . retrieved rate of assessed at E8 . 5 . Five independent experiment replicates were performed . numbers at bottom of bars indicated total number of transferred

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I. The retrieved rate of embryo assessed at E8.5. Five independent experiment replicates were performed. The numbers at
 the bottom of the bars indicated the total number of transferred embryos


K . Preimplantation development rates in KDM6A - HA , KDM6B - HA , or KDM6A - cM / - nM / - ncM - HA mRNA - injected non - injected control SCNT groups . efficiency was calculated on number of cleavage . Error bars , s . d . , total numbers of cleavage in each condition ( KDM6A - HA , KDM6B - HA , KDM6A - cM / - nM / - ncM - HA , Control ) from three independent experiments were 275 , 199 , 290 , 221 , 244 , 286 , respectively .

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K. Preimplantation development rates in the KDM6A-HA, KDM6B-HA, or KDM6A-cM/-nM/-ncM-HA mRNA-injected and non-injected control SCNT groups. The efficiency was calculated based on the number of cleavage embryo. Error bars, s.d., the total numbers of cleavage embryos in each condition (KDM6A-HA, KDM6B-HA, KDM6A-cM/-nM/-ncM-HA, and Control) from three independent experiments were 275, 199, 290, 221, 244, and 286, respectively.


A . RT - qPCR of KDM6A / B in SCNT injected with siRNA - 6A or siRNA - 6B . SCNT were subject to injection of 10 μM or 20 μM siRNA as indicated . Data are mean expression relative to Gapdh with siRNA - control normalized to 1 . Error bars , s . e . m . , n = 3 . * * P < 0 . 01 , * * * P < 0 . 001 according to two - Student " s t - . n . s . , not significant . B . bar chart shows KDM6A / B transcript in SCNT with 20 μM siRNA - 6A / B . data are mean expression relative to Gapdh with control siRNA injected SCNT normalized to 1 . Error bars , s . e . m . , n = 3 , * * P < 0 . 01 by Student &

O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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. RT-qPCR analysis of KDM6A/B in SCNT embryos injected with siRNA-6A or siRNA-6B. The SCNT embryos were subject to injection of 10 μM or 20 μM
 siRNA as indicated. Data are mean expression relative to Gapdh with siRNA-control normalized to 1. Error bars, s.e.m., n = 3. **P < 0.01, ***P < 0.001 according to two-tailed Student"s t-test. n.s., not significant. B. The bar chart shows the KDM6A/B transcript levels in SCNT embryo with 20 μM siRNA-6A/B. All data are mean expression relative to Gapdh with control siRNA injected SCNT embryos normalized to 1.
 Error bars, s.e.m., n = 3, **P < 0.01 by Student&quo


C . Immunofluorescence results showing H3K27me3 of 2 - stage SCNT . was injected with siRNA as indicated . H3K27me3 between control double injected with siRNA - 6A - 6B cannot observe any difference , but a marked decrease was observed when injected with either siRNA - 6A or siRNA - 6B . Scale bar , 20

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C. Immunofluorescence staining results showing H3K27me3 of 2-stage SCNT embryos. Embryo was injected with siRNA as indicated. The H3K27me3 levels between control and double injected with siRNA-6A-6B cannot observe any difference, but a marked decrease was observed when injected with either siRNA-6A or siRNA-6B. Scale bar, 20 μm


D . Western blot showing expression of KDM6A KDM6B in SCNT after injection of different siRNA as indicated . lysates from 1 , 500 were loaded in each lane . results of one representative of two independent experiments are

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D. Western blot showing the expression of KDM6A and KDM6B in SCNT embryos after injection of different siRNA as indicated. Protein lysates from 1,500 embryos were loaded in each lane. The results of one representative of two independent experiments are presented


F . Representative of SCNT at 115 h after injection of different siRNA as indicated . These are representative examples of shown in Fig 5G . n ≥ 3 . Scale bar , 50 μm . G . Injection of siRNA - 6B improved preimplantation development rate of SCNT . Both cumulus , Sertoli , C57 MEF were used as donor . bovine intraspecies SCNT derived from bovine ear fibroblast . Shown is of that reached indicated stages . ♂ ♀ indicated , respectively . Error bars , s . d . , n ≥ 3 . H

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F. Representative images of SCNT embryos at 115 h after injection of different siRNA as indicated. These images are representative examples of the quantification shown in Fig 5G. n ≥ 3. Scale bar, 50 μm. G. Injection of siRNA-6B improved the preimplantation development rate of SCNT embryos. Both cumulus cells, Sertoli cells, and C57 MEF cells were used as donor cells. The bovine intraspecies SCNT embryos derived from bovine ear fibroblast cells. Shown is the percentage of embryos that reached the indicated stages. ♂ and ♀ indicated the male and female, respectively. Error bars, s.d., n ≥ 3. H


RT - qPCR for select ZGA genes in ICSI SCNT . SCNT was injected with siRNA as indicated . Results were normalized on geometric mean of expression of two reference genes ( Ywhaz Gapdh ) . Error bars , s . e . m . , n = 3 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 according to two - Student " s t - . n . s . , not

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RT-qPCR analysis for select ZGA genes in ICSI and SCNT embryos. The SCNT embryo was injected with siRNA as indicated. Results were normalized based on the geometric mean of the expression levels of two reference genes (Ywhaz and Gapdh). Error bars, s.e.m., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 according to two-tailed Student"s t-test. n.s., not signif


B . Representative of siRNA - control or siRNA - 6B injected SCNT . Scale bar , 50

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B. Representative fluorescence images of siRNA-control or siRNA-6B injected SCNT embryos. Scale bar, 50 μm


C . of that expression tdTomato after injection with siRNA - control or siRNA - 6B . Numbers of observed are indicated . n ≥ 3 , Error bars , s . e . m . , * * * P < 0 . 001 according to two - Student " s

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C. Quantification of embryos that expression tdTomato after injection with siRNA-control or siRNA-6B. Numbers of observed embryos are indicated. n ≥ 3, Error bars, s.e.m., ***P < 0.001 according to two-tailed Student"s t


D . Representative of green in Oct4 : : EGFP reconstructed blastocysts derived from injected siRNA - control or siRNA - 6B SCNT . Representative from ≥ 25 analyzed in three independent micromanipulations are shown . Green indicates that Oct4 : : EGFP transgene has been expressed . Scale bar , 50

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D. Representative images of green fluorescence in Oct4::EGFP reconstructed blastocysts derived from injected siRNA-control or siRNA-6B SCNT embryos. Representative images from ≥ 25 embryos analyzed in three independent micromanipulations are shown. Green fluorescence indicates that the Oct4::EGFP transgene has been expressed. Scale bar, 50 μm


E . Oct4 : : EGFP was up - regulated significantly in ICSI compared with SCNT at blastocyst stages , as determined by RT - qPCR . Data shown are mean expression values relative to Gapdh . value in ICSI was set as 1 . Error bars , s . e . m . , n ≥ 3 . * P < 0 . 05 , * * * P < 0 . 001 by two - Student " s

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E. Oct4::EGFP was up-regulated significantly in ICSI embryos compared with SCNT embryos at blastocyst stages, as determined by RT-qPCR. Data shown are mean expression values relative to Gapdh. The value in ICSI embryos was set as 1. Error bars, s.e.m., n ≥ 3. *P < 0.05, ***P < 0.001 by two-tailed Student"s
 t


F . Representative image of cloned mice derived by siRNA - 6B injected SCNT

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F. Representative image of cloned mice derived by siRNA-6B injected SCNT embryos


G . Bar graph showing efficiency of attachment to feeder ( left ) si6B - mdx - ntES derivation ( right ) . mdx sick mice tail - tip fibroblasts as donors . N , total number of analyzed for each condition . Results are from four replicate

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G. Bar graph showing the efficiency of attachment to the feeder cells (left) and si6B-mdx-ntES derivation (right). The mdx sick mice tail-tip fibroblasts as nuclear donors. N, total number of embryos analyzed for each condition. Results are from four replicate experiments


H . Immunostaining of si6B - mdx - ntES expressed pluripotency markers . Scale bar , 50

H. Immunostaining images of si6B-mdx-ntES expressed pluripotency markers. Scale bar, 50 μm


I . si6B - mdx - ntES possessed multiple - differentiation potential , as shown in embryoid . Scale bar , 100 μm .

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I. The si6B-mdx-ntES possessed multiple-differentiation potential, as shown in embryoid body.
 Scale bar, 100 μm.


J . An image of a chimeric mouse derived from si6B - mdx -

J. An image of a chimeric mouse derived from si6B-mdx-ntES


B . comparing ZGA genes expression between WT - 2 NT - 2 si6B - NT ( FC > 5 , FPKM > 5 in each replicate ; left ) . A total of 1 , 813 DEGs are classified into two groups by unsupervised hierarchical clustering . KEGG GO of two groups by unsupervised hierarchical clustering ( right ) .

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B. Heatmap comparing ZGA genes expression between WT-2 and NT-2 and si6B-NT embryos (FC > 5, FPKM > 5 in each replicate; left). A total of 1,813 DEGs are classified into two groups by unsupervised hierarchical clustering. KEGG and GO analysis of the two groups by unsupervised hierarchical clustering (right).


E . Venn diagram showing overlap between genes that failed to be activated in SCNT 2 - derepressed in knock - down KDM6B ( left ) . Heat - map , KEGG GO enrichment showing expression pattern of 319 overlap genes ( FC > 5 , FPKM > 5 ; right ) .

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E. Venn diagram showing the overlap between the genes that failed to be activated in SCNT 2-cell embryos and derepressed in knock-down KDM6B (left). Heat-map, KEGG and GO enrichment showing the expression pattern of 319 overlap genes (FC > 5, FPKM >5; right).


G . Representative localization of Xist expression in nuclei of SCNT injected with siRNA - 6A , siRNA - 6B or siRNA - Xist ( left ) . Arrows indicate blastomeres enlarged in bottom panels . ratios of blastomeres classified according to positive or negative expression of Xist analyzed by - FISH ( right ) . Each bar represents a single .

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G. Representative localization of Xist expression in the nuclei of SCNT embryos injected with siRNA-6A, siRNA-6B or siRNA-Xist (left). Arrows indicate the blastomeres enlarged in the bottom panels. The ratios of blastomeres classified according to the positive or negative expression of Xist analyzed by RNA-FISH (right). Each bar represents a single embryo.


H . Large - scale qPCR of Xist eight X - linked genes in 4 - stage . Each rectangle bar represents expression value detected by single RT - qPCR . number of in each group is indicated . Coloured bars indicate expression .

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H. Large-scale qPCR analysis of Xist and eight X-linked genes in 4-cell stage male embryo. Each rectangle bar represents the expression value detected by single embryo RT-qPCR. The number of embryos in each group is indicated. Coloured bars indicate expression levels.


I . Image of full - term cloned pups derived from NT Sertoli injected with siRNA - control , siRNA - Xist , or siRNA - Xist - 6B .

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I. Image of full-term cloned pups derived from NT Sertoli cells injected with siRNA-control, siRNA-Xist, or siRNA-Xist-6B.


( B ) 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) thumb ( purple ) . polymerase is in with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . Key residues involved in stabilizing templating base ( Y505 R517 ) are shown in black .

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(B) The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). Key residues involved in stabilizing the templating base (Y505 and R517) are shown in black.


( C ) ( Left panel ) 8 - oxo - dG ( green ) preferentially adopts pro - mutagenic syn - conformation . A simulated annealing Fo - Fc omit density map is shown for 8 - oxo - dG ( contoured at 3σ ) . ( Right panel ) 8 - oxo - dG ( anti ) ( black , modeled using 2PFO ) was overlayed with Fo - Fc omit density map . In anti - conformation , C8 - carbonyl would directly clash with 5 ′ - phosphate ( red - dotted ) .

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(C) (Left panel) 8-oxo-dG (green) preferentially adopts the pro-mutagenic syn-conformation. A simulated annealing Fo-Fc omit density map is shown for 8-oxo-dG (contoured at 3σ). (Right panel) 8-oxo-dG(anti) (black, modeled using 2PFO)was overlayed with the Fo-Fc omit density map. In the anti-conformation, the C8-carbonyl would directly clash with the 5′-phosphate (red-dotted line).


( D ) 8 - oxo - dG ( syn ) ( green ) is stabilized by multiple interactions , including Tyr505 Arg517 ( grey ) .

O O O 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) 8-oxo-dG(syn) (green) is stabilized by multiple interactions, including Tyr505 and Arg517 (grey).


( B ) Overview of 8 - oxo - dG : dATP insertion . 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) thumb ( purple ) . polymerase is in with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . primer terminal ddC incoming ddCTP are shown in cyan magenta , respectively . Key residues that form nascent base pair binding pocket ( N513 R517 ) are involved in catalysis ( D427 , D429 D490 ) are shown in black . catalytic metal A nucleotide binding metal B are shown in red neon , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming ddCTP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.


( C ) 8 - oxo - dG ( green ) establishes a Watson - Crick pair with ddCTP ( magenta ) . Binding of 8 - oxo - dG in anti - conformation is only possible because C8 - carbonyl is far enough away not to clash with 5 ′ - phosphate . Asn513 Arg517 ( purple ) interact with minor groove of nascent base pair . base pair geometry ( C1 ′ λ angles ) is indicated at bottom of figure . A simulated annealing Fo - Fc omit density map for pair is also shown ( contoured at 3σ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8-oxo-dG (green) establishes a Watson-Crick pair with ddCTP (magenta). Binding of the 8-oxo-dG in the anti-conformation is only possible because the C8-carbonyl is far enough away not to clash with the 5′-phosphate. Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of primer terminus active site with an undamaged reference structure ( 2PFO , black ) . catalytic aspartic are shown in yellow , primer terminus in cyan incoming dATP in magenta . A water molecule ( red ) Mg2 + ( neon ) are occupying metal A metal B sites , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and metal B sites, respectively.


( B ) Overview of 8 - oxo - dG : dATP insertion . 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) thumb ( purple ) . polymerase is in with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . primer terminal ddC incoming dATP are shown in cyan magenta , respectively . Key residues that form nascent base pair binding pocket ( N513 R517 ) are involved in catalysis ( D427 , D429 D490 ) are shown in black . catalytic metal A nucleotide binding metal B are shown in red neon , respectively .

(B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming dATP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.


( C ) 8 - oxo - dG ( green ) establishes a Hoogsteen base pair with dATP ( magenta ) . Asn513 Arg517 ( purple ) interact with minor groove of nascent base pair . base pair geometry ( C1 ′ λ angles ) is indicated at bottom of figure . A simulated annealing Fo - Fc omit density map for pair is also shown ( contoured at 3σ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8-oxo-dG (green) establishes a Hoogsteen base pair with dATP (magenta). Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of primer terminus active site with an undamaged reference structure ( 2PFO , black ) . catalytic aspartic are shown in yellow , primer terminus in cyan incoming dATP in magenta . A water molecule ( red ) Mg2 + ( neon ) are occupying metal A B sites , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and B sites, respectively.


( A B ) Overlay of nascent base pair in insertion structures with an undamaged reference structure ( 2PFO , black ) . Binding of 8 - oxo - dG ( green ) in both anti - ( A ) or syn - ( B ) conformation involves a repositioning of 5 ′ - phosphate ( red arrow ) . Furthermore , Arg514 side chain ( purple ) that normally stacks with templating base interacts with 5 ′ - phosphate ( blue - dotted ) , also repositions ( black arrow ) . In both structures , Arg514 interacts with C6 - carbonyl of templating 8 - oxo - dG ( anti ) ( A ) or C6 - group of incoming dATP ( B ) . incoming nucleotide is shown in magenta .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Overlay of the nascent base pair in the insertion structures with an undamaged reference structure (2PFO, black). Binding of 8-oxo-dG (green) in both the anti- (A) or syn-(B) conformation involves a repositioning of the 5′-phosphate (red arrow). Furthermore, the Arg514 side chain (purple) that normally stacks with the templating base and interacts with the 5′-phosphate (blue-dotted line), also repositions (black arrow). In both structures, Arg514 interacts with the C6-carbonyl of the templating 8-oxo-dG(anti) (A) or the C6-amino group of the incoming dATP (B). The incoming nucleotide is shown in magenta.


( B ) Overview of 8 - oxo - dG : dC extension . 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) thumb ( purple ) . polymerase is in with a 1 - nt gapped 16 - mer oligo ( grey ) an incoming dUMPNPP ( magenta ) . primer terminal dC 8 - oxo - dG are shown in cyan magenta , respectively . Key residues that form primer terminal base pair binding pocket ( N513 , R517 Glu529 ) are involved in catalysis ( D427 , D429 D490 ) are shown in black . nucleotide binding metal ( B ) is shown in neon .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dC and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513, R517 and Glu529) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.


( C ) 8 - oxo - dG ( orange ) establishes a Watson - Crick base pair with dC ( cyan ) at primer terminus . Tyr505 , Arg517 , Glu529 ( purple ) interact with minor groove of base pair . base pair geometry ( C1 ′ λ angles ) is indicated at bottom of figure . A simulated annealing Fo - Fc omit density map for pair is also shown ( contoured at 3σ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8-oxo-dG (orange) establishes a Watson-Crick base pair with dC (cyan) at the primer terminus. Tyr505, Arg517, and Glu529 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of primer terminus active site with an undamaged reference structure ( 2PFO , black ) . catalytic aspartic are shown in yellow , incoming dUMPNPP in magenta , primer terminal dC in cyan . Expectedly , Mg2 + ( neon ) is occupying metal B site . However , metal A is absent in structure , resulting in a non - catalytic position of 3 ′ - OH . This is consistent with other structures containing an incoming non - hydrolyzable analog .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dC in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.


( B ) Overview of 8 - oxo - dG : dC extension . 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) thumb ( purple ) . polymerase is in with a 1 - nt gapped 16 - mer oligo ( grey ) an incoming dUMPNPP ( magenta ) . primer terminal dA 8 - oxo - dG are shown in cyan magenta , respectively . Key residues that form primer terminal base pair binding pocket ( N513 R517 ) are involved in catalysis ( D427 , D429 D490 ) are shown in black . nucleotide binding metal ( B ) is shown in neon .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dA and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.


( C ) 8 - oxo - dG ( orange ) establishes a Hoogsteen base pair with dA ( cyan ) at primer terminus . Tyr505 Arg517 ( purple ) interact with minor groove of base pair . base pair geometry ( C1 ′ λ angles ) is indicated at bottom of figure . A simulated annealing Fo - Fc omit density map for pair is also shown ( contoured at 3σ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8-oxo-dG (orange) establishes a Hoogsteen base pair with dA (cyan) at the primer terminus. Tyr505 and Arg517 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of primer terminus active site with an undamaged reference structure ( 2PFO , black ) . catalytic aspartic are shown in yellow , incoming dUMPNPP in magenta , primer terminal dA in cyan . Expectedly , Mg2 + ( neon ) is occupying metal B site . However , metal A is absent in structure , resulting in a non - catalytic position of 3 ′ - OH . This is consistent with other structures containing an incoming non - hydrolyzable analog .

(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dA in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.


( A ) Glu529 ( purple ) forms a hydrogen bond with C2 - group of primer terminal templating 8 - oxo - dG ( orange ) in anti - conformation . ( B ) This interaction is abolished when 8 - oxo - dG ( orange ) adopts syn - conformation . An overlay with 8 - oxo - dG ( anti ) - containing structure reveals that Glu529 ( black ) would likely clash ( red - dotted ) with C8 - carbonyl of 8 - oxo - dG ( syn ) . Repositioning of Glu529 ( purple ) places side chain 4 . 5 Å away from C8 - carbonyl . ( C ) Comparison of catalytic efficiencies for extension past an 8 - oxo - dG ( anti ) : dC , 8 - oxo - dG ( syn ) : dA dG : dC base pairs at primer terminus using WT E529A Pol λ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O

(A) Glu529 (purple) forms a hydrogen bond with the C2-amino group of the primer terminal templating 8-oxo-dG (orange) in the anti-conformation. (B) This interaction is abolished when 8-oxo-dG (orange) adopts the syn-conformation. An overlay with the 8-oxo-dG(anti)-containing structure reveals that Glu529 (black) would likely clash (red-dotted line) with the C8-carbonyl of 8-oxo-dG(syn). Repositioning of Glu529 (purple) places the side chain 4.5 Å away from the C8-carbonyl. (C) Comparison of catalytic efficiencies for extension past an 8-oxo-dG(anti):dC, 8-oxo-dG(syn):dA and dG:dC base pairs at the primer terminus using WT and E529A Pol λ.


( A ) Undamaged structure containing a templating dG at primer terminus ( 2PFO ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at primer terminus . ( B ) Undamaged structure containing a templating dA at primer terminus ( 1XSN ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at primer terminus .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Undamaged structure containing a templating dG at the primer terminus (2PFO) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus. (B) Undamaged structure containing a templating dA at the primer terminus (1XSN) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus.


( B ) YTS KIR2DL1 were transfected with either N . S siRNA ( blue curve ) or with SHP - 1 Cbls siRNA ( red curve ) . After 48 hours , were loaded with calcium - sensitive Fluo - 3 - AM analyzed for basal intracellular calcium for 1 min . NK were then mixed with 721 . 221 Cw4 target at 37 ° C monitoring of calcium was continued . data shown are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 cells were transfected with either N.S siRNA (blue curve) or with SHP-1 and Cbls siRNA (red curve). After 48 hours, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C and monitoring of calcium levels was continued. The data shown are representative of three independent experiments.


( C ) YTS KIR2DL1 were incubated with either 721 . 221 Cw4 or Cw7 target for 2 hours analyzed by flow cytometry to determine expression of CD107a . Expression of CD107a was measured by mean ( MFI ) . Relative expression of CD107a MFI was normalized to mock - transfected sample following Cw4 incubation . Data are means ± of five independent experiments . ( n = 5 ) . P values were calculated by One - sample t - One way ANOVA following Tukey ' s post hoc are indicated by asterisks * P < 0 . 05 , * * * * P < 0 . 0001 .

O O O 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-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the mock-transfected sample following Cw4 incubation. Data are means ± SEM of five independent experiments.(n=5). P values were calculated by One-sample t-tests and One way ANOVA following Tukey's post hoc analysis are indicated by asterisks *P < 0.05, ****P<0.0001 .


( E ) Killing . YTS KIR2DL1 were mock - transfected or transfected with N . S siRNA or SHP - 1 Cbls siRNA . After 48 hours , were incubated with [ 35S ] Met - loaded 721 . 221 Cw4 or Cw7 target . After 5 hours of co - culture , supernatant was collected , radioactive signal was measured . Data are means ± of three independent experiments . P values were calculated by One - way ANOVA following Tukey ' s post hoc are indicated by asterisks * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Killing assay. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 hours, cells were incubated with [35S] Met-loaded 721.221 Cw4 or Cw7 target cells. After 5 hours of co-culture, the supernatant was collected, and the radioactive signal was measured. Data are means ± SEM of three independent experiments. P values were calculated by One-way ANOVA following Tukey's post hoc analysis and are indicated by asterisks *P < 0.05, **P < 0.01 .


( B ) Characterization of diameter zeta potential ( Z - potential ) with addition of each layer . Graph below presenting NP distribution .

O 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) Characterization of the diameter and zeta potential (Z-potential) with the addition of each layer. Graph below presenting NP size distribution.


( A ) NK92 - NKp46low , NK92 - NKp46high , YTS KIR2DL1 , K562 721 . 221 HLA - Cw4 were stained with anti - NKp46 monoclonal antibody followed by with Alexa568 - Fluor goat anti - mouse IgG secondary antibody . were then analyzed using flow cytometry . ( B ) NK92 - NKp46low , NK92 - NKp46 + , YTS KIR2DL1 , K562 , 721 . 221 HLA - Cw4 were incubated with 50 µg rhodamine - labeled NPs analyzed using flow cytometry to confirm internalization of coated NPs in a NKp46 - specific manner .

O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O

(A) NK92-NKp46low, NK92-NKp46high, YTS KIR2DL1, K562 and 721.221 HLA-Cw4 cells were stained with anti-NKp46 monoclonal antibody followed by staining with Alexa568-Fluor goat anti-mouse IgG secondary antibody. Cells were then analyzed using flow cytometry. (B) NK92-NKp46low, NK92-NKp46+, YTS KIR2DL1, K562, and 721.221 HLA-Cw4 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using flow cytometry to confirm the internalization of the coated NPs in a NKp46-specific manner.


( C - F ) ( C ) NK92 - NKp46low , ( D ) NK92 - NKp46high , ( E ) YTS KIR2DL1 ( F ) K562 were incubated with 50 µg rhodamine - labeled NPs analyzed using confocal microscopy . Upper panel shows wide field . Lower panel shows single - . data shown are representative of three 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 O O O O O O O O O O O O O O O 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) (C) NK92-NKp46low, (D) NK92-NKp46high, (E) YTS KIR2DL1 and (F) K562 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using confocal microscopy. Upper panel shows wide field images. Lower panel shows single-cell images. The data shown are representative of three independent experiments.


( A ) YTS KIR2DL1 NK were incubated with NPs loaded with Cbl - b , c - Cbl SHP - 1 siRNA , or NPs loaded with 1500 pmol N . S siRNA . Following 48 hours of incubation , were lysed , was blotted with anti - Cbl - b , anti - c - Cbl or anti - SHP - 1 antibodies . GAPDH served as loading control . Densitometric of bands was performed using ImageJ normalized to GAPDH densitometry values . by ImageJ densitometry is summarized in graph below . Data are means ± of four independent experiments . ( n = 4 ) . P values were calculated by One - sample t - are indicated by asterisks . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 and primary NK cells were incubated with NPs loaded with Cbl-b, c-Cbl and SHP-1 siRNA, or NPs loaded with 1500 pmol N.S siRNA. Following 48 hours of incubation, cells were lysed, and the membrane was blotted with anti-Cbl-b, anti-c-Cbl or anti-SHP-1 antibodies. GAPDH served as loading control. Densitometric analysis of the bands was performed using ImageJ and normalized to the GAPDH densitometry values. Analysis by ImageJ densitometry is summarized in the graph below. Data are means ± SEM of four independent experiments. (n=4). P values were calculated by One-sample t-tests and are indicated by asterisks. *P < 0.05, **P < 0.01.


( B ) YTS KIR2DL1 were incubated with SHP - 1 Cbls siRNAs - loaded NPs ( red curve ) , with NS siRNA loaded NPs ( green curve ) or with empty NPs ( blue curve ) . After 48 hours , were loaded with calcium - sensitive Fluo - 3 - AM analyzed for basal intracellular calcium for 1 min . NK were then mixed with 721 . 221 Cw4 target at 37 ° C , monitoring of calcium was continued . data shown are representative of three independent experiments .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 cells were incubated with SHP-1 and Cbls siRNAs-loaded NPs (red curve), with NS siRNA loaded NPs (green curve) or with empty NPs (blue curve). After 48 hours, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C, and monitoring of calcium levels was continued. The data shown are representative of three independent experiments.


( D ) YTS KIR2DL1 were treated with SHP - 1 Cbls siRNAs - loaded NPs or N . S siRNA - loaded NPs . After 48 hours , were incubated with either 721 . 221 Cw4 or Cw7 target , supernatant was then collected , granzyme B were evaluated using ELISA sandwich . of granzyme B were quantified on a standard curve . Data are means ± of five independent experiments . ( n = 5 ) . P values were calculated by One way ANOVA following Tukey ' s post hoc , are indicated by asterisks . * * P < 0 . 01 .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 cells were treated with SHP-1 and Cbls siRNAs-loaded NPs or N.S siRNA-loaded NPs. After 48 hours, cells were incubated with either 721.221 Cw4 or Cw7 target cells, the supernatant was then collected, and granzyme B levels were evaluated using ELISA sandwich assay. The levels of granzyme B were quantified based on a standard curve. Data are means ± SEM of five independent experiments. (n=5). P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. **P < 0.01.


( F ) pNK were pretreated with either SHP - 1 Cbls siRNA - loaded NPs or N . S siRNA - loaded NPs . After 48 hours , were incubated with either 721 . 221 Cw4 or Cw7 target for 2 hours analyzed by flow cytometry to determine expression of CD107a . Expression of CD107a was quantitated by mean ( MFI ) . data represent five independent experiments ( n = 5 ) Data are shown as mean ± . ) ( n = 5 ) . P values were calculated by One - sample t - One way ANOVA following Tukey ' s post hoc , are indicated by asterisks . * P ≤ 0 . 05 .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) pNK cells were pretreated with either SHP-1 and Cbls siRNA-loaded NPs or N.S siRNA-loaded NPs. After 48 hours, cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was quantitated by mean fluorescence intensity (MFI). The data represent five independent experiments (n=5) Data are shown as mean ± SEM.) (n=5). P values were calculated by One-sample t-tests and One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. *P≤0.05.


( A ) YTS KIR2DL1 were incubated with SHP - 1 Cbls siRNA - loaded NPs ( red curve ) or empty NPs ( purple curve ) . After 48 hours , were loaded with Fluo - 3 - AM analyzed for intracellular calcium for 20 min at 37 ° C . YTS KIR2DL1 that were treated with PBS served as a negative control ( green curve ) . NK that were mixed with 721 . 221 Cw7 target served as a positive control ( blue curve ) . Calcium were analyzed by spectrofluorometry . ( n = 3 )

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 cells were incubated with SHP-1 and Cbls siRNA-loaded NPs (red curve) or empty NPs (purple curve). After 48 hours, cells were loaded with Fluo-3-AM and analyzed for intracellular calcium levels for 20 min at 37°C. YTS KIR2DL1 cells that were treated with PBS served as a negative control (green curve). NK cells that were mixed with 721.221 Cw7 target cells served as a positive control (blue curve). Calcium levels were analyzed by spectrofluorometry. (n=3)


( B ) Freshly isolated PBMCs were treated with SHP - 1 Cbls siRNA - loaded NPs , empty NPs , PBS as negative control or PHA as a positive control . Following incubation , supernatant from each sample was collected of human cytokines TNF - α , IL - 6 , IL - 10 IFN - γ were determined using ELISA . data represent four independent experiments ( n = 4 ) . P values were calculated by One way ANOVA following Tukey ' s post hoc , are indicated by asterisks . * P ≤ 0 . 008 . Data are shown as mean ± .

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(B) Freshly isolated PBMCs were treated with SHP-1 and Cbls siRNA-loaded NPs, empty NPs, PBS as negative control or PHA as a positive control. Following incubation, supernatant from each sample was collected and the levels of human cytokines TNF-α, IL-6, IL-10 and IFN-γ were determined using ELISA. The data represent four independent experiments (n=4). P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. *P≤0.008. Data are shown as mean ± SEM.


( C ) Average growth rates of tumors from first treatment to end point from same experimental groups of mice as indicated in B . P value was calculated by two - Student ' s t - is indicated within graph P = 0 . 001 . Data are shown as mean ± .

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(C) Average growth rates of tumors from the first treatment to the end point from same experimental groups of mice as indicated in B. P value was calculated by two-tailed Student's t-test and is indicated within the graph P =0.001. Data are shown as mean ± SEM.


( E ) Following 24 hours after last treatment , extracted from tumors of either SHP - 1 Cbls or N . S siRNA loaded NKp46 - NPs treated mice were analyzed by flow cytometry . Expression of CD107a was measured by mean ( MFI ) . Relative expression of CD107a MFI was normalized to sample treated with N . S siRNA loaded NPs . data represent three independent experiments . Data are shown as mean ± SEMn = 3 ; P value was calculated by one sample t - is indicated within graph P = 0 . 009 .

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(E) Following 24 hours after the last treatment, extracted cells from tumors of either SHP-1 and Cbls or N.S siRNA loaded NKp46-NPs treated mice were analyzed by flow cytometry. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the sample treated with N.S siRNA loaded NPs. The data represent three independent experiments. Data are shown as mean ± SEMn=3; P value was calculated by one sample t-test and is indicated within the graph P =0.009.


( F ) Survival in mice treated with NK ' gene silenced for SHP - 1 Cbls using siRNA - loaded NPs ( n = 18 ) or N . S siRNA - loaded NPs ( n = 18 ) as demonstrated by Kaplan - Meier survival curve . P values were calculated using log - rank is indicated within graph P = 0 . 00009 .

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(F) Survival analysis in mice treated with NK cells' gene silenced for SHP-1 and Cbls using siRNA-loaded NPs (n=18) or N.S siRNA-loaded NPs (n=18) as demonstrated by Kaplan-Meier survival curve. P values were calculated using the log-rank test and is indicated within the graph P =0.00009.


A ) Contribution of base change types to overall SNV composition in ABiM cohort for captured regions mRNA in captured regions , as well as SCAN - B whole mRNA .

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A) Contribution of base change types to the overall SNV composition in the ABiM cohort for captured DNA regions and mRNA in the captured DNA regions, as well as SCAN-B whole mRNA.


B ) Number of non - synonymous mutations per sample . Bars are colored by PAM50 subtypes Luminal A ( blue ) , Luminal B ( blue ) , HER2 - enriched ( pink ) , basal - like ( red ) , Normal - like ( green ) Unclassified ( gray ) .

B) Number of non-synonymous mutations per sample. Bars are colored by PAM50 subtypes Luminal A (dark blue), Luminal B (light blue), HER2-enriched (pink), basal-like (red), Normal-like (green) and Unclassified (gray).


C - F ) Lollipop plots showing location , abundance , impact of SNVs in C ) TP53 , D ) PIK3CA , E ) PTEN , F ) ERBB2 on respective encoded . change labels are shown for most mutated acid positions , with residues ordered left to right by mutation frequency within each label .

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C-F) Lollipop plots showing the location, abundance, and impact of SNVs in C) TP53, D) PIK3CA, E) PTEN, and F) ERBB2 on the respective encoded protein. Protein change labels are shown for the most mutated amino acid positions, with residues ordered left to right by mutation frequency within each label.


Waterfall plot of 20 most frequently mutated genes ( rows ) across 3 , 217 SCAN - B samples ( columns ) . Genes are ranked from top to bottom by mutation frequency . Samples are sorted by histological subtype alteration occurrence . Mutations are colored by predicted functional impact .

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Waterfall plot of the 20 most frequently mutated genes (rows) across 3,217 SCAN-B samples (columns). Genes are ranked from top to bottom by mutation frequency. Samples are sorted by histological subtype and alteration occurrence. Mutations are colored by predicted functional impact.


A - E ) Overall survival ( OS ) of with tumors containing mutations in genes A ) TP53 , B ) PIK3CA , C ) ERBB2 , D ) PTEN . E ) OS by PTEN - MutExp genotype ( " low " defined as PTEN mutation or PTEN expression in lower quartile across cohort , " normal " otherwise ) stratified by groups receiving no systemic treatment ( n = 336 ) , endocrine therapy only ( Endo only ; n = 1 , 579 ) , endocrine chemotherapy ( Endo + Chemo ± any ; n = 914 ) , as well as HER2 treatment with any other treatment or none ( HER2 ± any ; n = 348 ) . Specific treatments in these groups are detailed in Table EV5 . In each Kaplan - Meier plot , wildtype ( wt ) normal cases are plotted in blue , mutated ( mut ) low cases are plotted in red , log - rank P value is given , hazard ( HR ) for mutation / low is given with a 95 % CI after uni - variable multi - variable ( MV ) Cox regression adjustment . Covariables included in MV were at diagnosis , lymph node status , , variables denoted by following symbols : ¶ , ER , PgR , HER2 , NHG ; ¤ , ER , PgR , NHG ; $ , HER2 NHG . Abbreviations : ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade .

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A-E) Overall survival (OS) of patients with tumors containing mutations in the genes A) TP53, B) PIK3CA, C) ERBB2, and D) PTEN. E) OS by PTEN-MutExp genotype ("low" defined as PTEN mutation or PTEN expression in the lower quartile across the cohort, "normal" otherwise) stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemotherapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV5. In each Kaplan-Meier plot, wildtype (wt) and normal cases are plotted in blue, mutated (mut) and low cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation/low is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.


A - E ) Overall survival of with tumors containing mutations in pathways A ) WNT signaling , B ) Hedgehog signaling , C ) NOTCH2 signaling , D ) p53 independent damage repair , E ) TGFβ signaling , stratified by groups receiving no systemic treatment ( n = 336 ) , endocrine therapy only ( Endo only ; n = 1 , 579 ) , endocrine chemo therapy ( Endo + Chemo ± any ; n = 914 ) , as well as HER2 treatment with any other treatment or none ( HER2 ± any ; n = 348 ) . Specific treatments in these groups are detailed in Table EV4 . In each Kaplan - Meier plot , wildtype ( wt ) cases are plotted in blue , mutated ( mut ) cases are plotted in red , log - rank P value is given , hazard ( HR ) for mutation is given with a 95 % CI after uni - variable multi - variable ( MV ) Cox regression adjustment . Covariables included in MV were at diagnosis , lymph node status , , variables denoted by following symbols : ¶ , ER , PgR , HER2 , NHG ; ¤ , ER , PgR , NHG ; $ , HER2 NHG . Abbreviations : ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade .

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A-E) Overall survival of patients with tumors containing mutations in pathways A) WNT signaling, B) Hedgehog signaling, C) NOTCH2 signaling, D) p53 independent DNA damage repair, E) TGFβ signaling, stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemo therapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV4. In each Kaplan-Meier plot, wildtype (wt) cases are plotted in blue, mutated (mut) cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.


Overall survival stratified by mutational burden ( TMB ) across treatment groups in 3 , 217 . Samples were classified as TMB - high if amount of non - synonymous mutations per expressed MB ( rnaMB ) was ≥ median number of non - synonymous mutations per rnaMB across whole SCAN - B cohort ( 0 . 082 mutations per rnaMB ) TMB - low otherwise . In each Kaplan - Meier plot , TMB - low cases are plotted in blue , TMB - high cases are plotted in red , log - rank P value is given , hazard ( HR ) for TMB - high is given with a 95 % CI after uni - variable multi - variable ( MV ) Cox regression adjustment . Covariables included in MV were at diagnosis , lymph node status , , variables denoted by following symbols : ¶ , ER , PgR , HER2 , NHG ; ¤ , ER , PgR , NHG ; $ , HER2 NHG ; # , NHG . Abbreviations : TMB , mutational burden ; ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade ; HoR , hormone receptor ; TNBC , triple - negative breast cancer .

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Overall survival stratified by tumor mutational burden (TMB) across treatment groups in 3,217 patients. Samples were classified as TMB-high if the amount of non-synonymous mutations per expressed MB (rnaMB) was ≥ the median number of non-synonymous mutations per rnaMB across the whole SCAN-B cohort (0.082 mutations per rnaMB) and TMB-low otherwise. In each Kaplan-Meier plot, TMB-low cases are plotted in blue, TMB-high cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for TMB-high is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG; #, NHG. Abbreviations: TMB, tumor mutational burden; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade; HoR, hormone receptor; TNBC, triple-negative breast cancer.


E , Immunoblot of described in ( D ) , harvested at indicated time points after G1 / S release . Exemplary blot from three independent experiments .

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E, Immunoblot analysis of cells described in (D), harvested at the indicated time points after G1/S release. Exemplary blot from three independent experiments.


F , DUB activity for OTUD6B in asynchronous , G1 / S - or mitotically synchronized released A549 using HA - ubiquitin vinyl - sulfone to isolate active forms of DUBs . was performed by immunoblotting using indicated antibodies .

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F, DUB activity assay for OTUD6B in asynchronous, G1/S- or mitotically synchronized and released A549 cells using HA-ubiquitin vinyl-sulfone to isolate active forms of DUBs. Analysis was performed by immunoblotting using the indicated antibodies.


B , In - vivo ubiquitylation analyses of LIN28B in HEK293T in which OTUD6B was silenced by siRNA . were transfected with indicated siRNAs overexpression constructs , then treated with MG132 for 3 hrs . Lysis IP was done under denaturing conditions followed by WB . Data information : For B , exemplary blot of two independent experiments .

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B, In-vivo ubiquitylation analyses of LIN28B in HEK293T cells in which OTUD6B was silenced by siRNA. Cells were transfected with the indicated siRNAs and overexpression constructs, then treated with MG132 for 3 hrs. Lysis and IP was done under denaturing conditions followed by WB analysis. Data information: For B, exemplary blot of two independent experiments.


C , In - vivo ubiquitylation of LIN28B in OTUD6B a catalytically inactive variant of OTUD6B ( OTUD6B - C158A ) overexpressing . HEK293T were transfected with indicated combinations of FLAG - LIN28B , HA - Ubiquitin , OTUD6B , OTUD6B - C158A EV control treated with MG132 for 3 hrs 24 hrs later . Denatured WCE were subjected to FLAG - IP . WCE IP were analyzed by immunoblotting including a K48 - specific ubiquitin antibody . Exemplary blots from three independent experiments are shown .

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C, In-vivo ubiquitylation assay of LIN28B in OTUD6B and a catalytically inactive variant of OTUD6B (OTUD6B-C158A) overexpressing cells. HEK293T cells were transfected with indicated combinations of FLAG-LIN28B, HA-Ubiquitin, OTUD6B, OTUD6B-C158A and EV control and treated with MG132 for 3 hrs 24 hrs later. Denatured WCE were subjected to FLAG-IP. WCE and IP were analyzed by immunoblotting including a K48-specific ubiquitin antibody. Exemplary blots from three independent experiments are shown.


D , Pull - down of endogenous LIN28B from HEK293T WCE using bacterially purified GST - OTUD6B . Pulldowns WCE were analyzed by immunoblot using indicated antibodies . Data information : For D , exemplary blot of two independent experiments .

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D, Pull-down of endogenous LIN28B from HEK293T WCE using bacterially purified GST-OTUD6B. Pulldowns and WCE were analyzed by immunoblot using the indicated antibodies. Data information: For D, exemplary blot of two independent experiments.


E , Immunoblot of an in - vitro deubiquitylation using GST - purified OTUD6B from FLAG - purified ubiquitylated LIN28B from HEK293T . FLAG - IP was performed under denaturing conditions from HEK293T WCEs expressing FLAG - LIN28B HA - Ubiquitin . Purified FLAG - LIN28B was eluted from beads incubated with GST or GST - OTUD6B followed by immunoblot . Data information : For E : exemplary blot of two independent experiments .

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E, Immunoblot analysis of an in-vitro deubiquitylation assay using GST-purified OTUD6B from bacteria and FLAG-purified ubiquitylated LIN28B from HEK293T. FLAG-IP was performed under denaturing conditions from HEK293T WCEs expressing FLAG-LIN28B and HA-Ubiquitin. Purified FLAG-LIN28B was eluted from the beads and incubated with GST or GST-OTUD6B proteins followed by immunoblot analysis. Data information: For E: exemplary blot of two independent experiments.


A , Co - IP of FLAG - OTUD6B with endogenous LIN28B from MM1 . S synchronized in G1 / S 12 hrs post release . MM1 . S stably expressing FLAG - OTUD6B or FLAG - EV were synchronized at G1 / S harvested at indicated time points after release . IPs were preformed analyzed together with WCE by immunoblotting . Data information : blots are representations of three independent experiments .

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A, Co-IP of FLAG-OTUD6B with endogenous LIN28B from MM1.S cells synchronized in G1/S and 12 hrs post release. MM1.S cells stably expressing FLAG-OTUD6B or FLAG-EV were synchronized at G1/S and harvested at the indicated time points after release. IPs were preformed and analyzed together with WCE by immunoblotting. Data information: All blots are representations of three independent experiments.


B , Immunoblot of LIN28B OTUD6B throughout cycle . RPMI ( B ) were synchronized at G1 / S transition by a double thymidine block or in mitosis by a sequential thymidine / nocodazole block . Synchronized were released , harvested at indicated time points analyzed by immunoblot using indicated antibodies . Data information : blots are representations of three independent experiments .

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B, Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle. RPMI (B) cells were synchronized at the G1/S transition by a double thymidine block or in mitosis by a sequential thymidine/nocodazole block. Synchronized cells were released, harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies. Data information: All blots are representations of three independent experiments.


C , Immunoblot of LIN28B OTUD6B throughout cycle . A549 ( C ) were synchronized at G1 / S transition by a double thymidine block or in mitosis by a sequential thymidine / nocodazole block . Synchronized were released , harvested at indicated time points analyzed by immunoblot using indicated antibodies . Data information : blots are representations of three independent experiments .

C, Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle. A549 (C) cells were synchronized at the G1/S transition by a double thymidine block or in mitosis by a sequential thymidine/nocodazole block. Synchronized cells were released, harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies. Data information: All blots are representations of three independent experiments.


D , Immunoblot of LIN28B half - life in G1 / S - synchronized ( upper panel ) asynchronous ( lower panel ) A549 upon OTUD6B depletion . transfected with respective siRNAs were synchronized in G1 / S - phase or not treated with cycloheximide ( CHX ) as indicated . Data information : blots are representations of three independent experiments .

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D, Immunoblot analysis of LIN28B protein half-life in G1/S-synchronized (upper panel) and asynchronous (lower panel) A549 cells upon OTUD6B depletion. Cells transfected with the respective siRNAs were synchronized in G1/S-phase or not and treated with cycloheximide (CHX) as indicated. Data information: All blots are representations of three independent experiments.


D , Immunoblot of WCE obtained from a representative experiment depicted in ( C ) .

D, Immunoblot analysis of WCE obtained from a representative experiment depicted in (C).


C , Immunoblot of RPMI8226 , KMS12BM MM1 . S expressing indicated shRNAs . Depicted are examples of three independent experiments per . Specification of MYC rearrangements : RPMI8226 ( der ( 17 ) t ( ? 8 ; 17 ) ( q21 . 2 ; q25 ) ) , KMS12BM ( der ( 1 ; 8 ) ( q10 ; q10 ) x2 ) , MM1 . S ( der3t ( 3 ; 8 ) ) .

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C, Immunoblot analysis of RPMI8226, KMS12BM and MM1.S cells expressing the indicated shRNAs. Depicted are examples of three independent experiments per cell line. Specification of MYC rearrangements: RPMI8226 (der(17)t(?8;17)(q21.2;q25)), KMS12BM (der(1;8)(q10;q10)x2), MM1.S (der3t(3;8)).


A , Correlation between MYC OTUD6B expression in MM samples at diagnosis . mRNA expression in CD138 + MM was quantified by real - time qPCR ( n = 89 ) . Data are fit by linear regression ( black ) ; Pearson r , Pearson correlation coefficient ; P < 0 . 0001 ; by linear regression Pearson correlation .

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A, Correlation between MYC and OTUD6B expression in MM patient samples at diagnosis. mRNA expression in primary CD138+ MM cells was quantified by real-time qPCR (n = 89 patients). Data are fit by linear regression (black line); Pearson r, Pearson correlation coefficient; P < 0.0001; by linear regression and Pearson correlation.


Addition of rhTNF at fracture site on days 0 1 led to augmented healing , indicated by increased % callus mineralization , at day 28 after operation in a dose - dependent manner . Data are presented as mean ± . Treatment with 1 ng TNF versus PBS control , * * * * P < 0 . 0001 by one - way ANOVA with Dunnett ' s multiple comparisons .

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Addition of rhTNF at the fracture site on days 0 and 1 led to augmented healing, indicated by increased % callus mineralization, at day 28 after operation in a dose-dependent manner. Data are presented as mean ± SEM. Treatment with 1 ng TNF versus PBS control, ****P < 0.0001 by one-way ANOVA with Dunnett's multiple comparisons test.


Addition of rhTNF at fracture site must be given within first 24 h to augment healing , indicated by % callus mineralization . Data are presented as mean ± . 1 ng TNF versus PBS control treatment on days 0 1 , * * * P = 0 . 0009 by one - way ANOVA with Dunnett ' s multiple comparisons .

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Addition of rhTNF at the fracture site must be given within the first 24 h to augment healing, indicated by % callus mineralization. Data are presented as mean ± SEM. 1 ng TNF versus PBS control treatment on days 0 and 1, ***P = 0.0009 by one-way ANOVA with Dunnett's multiple comparisons test.


Treatment with systemic anti - TNF or local rmIL - 10 led to impaired fracture repair at day 28 , indicated by % callus mineralization . Data are presented as mean ± . Treatment with PBS control versus anti - TNF , * P = 0 . 037 , PBS control versus rmIL - 10 , * P = 0 . 037 , by unpaired two - sided t - .

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Treatment with systemic anti-TNF or local rmIL-10 led to impaired fracture repair at day 28, indicated by % callus mineralization. Data are presented as mean ± SEM. Treatment with PBS control versus anti-TNF, *P = 0.037, PBS control versus rmIL-10, *P = 0.037, by unpaired two-sided t-tests.


Representative micro - CT at fracture site showing ( from left to right ) : lateral view of tibia , cross - section , cross longitudinal sections with color overlay 3D reconstruction . In color overlays , shade of blue corresponds to mineralization : blue denotes soft immature callus , whereas blue denotes hard mineralized callus . Scale bar , 2 mm .

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Representative micro-CT images at the fracture site showing (from left to right): lateral view of tibia, cross-section, and cross and longitudinal sections with color overlay and 3D reconstruction. In the color overlays, the shade of blue corresponds to percentage mineralization: light blue denotes soft immature callus, whereas dark blue denotes hard mineralized callus. Scale bar, 2 mm.


Representative ISH image ( field ) showing TNF expression at murine fracture site at 24 h after fracture . Scale bar , 250 μm . Red box indicates region of interest .

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Representative ISH image (light field) showing TNF expression at the murine fracture site at 24 h after fracture. Scale bar, 250 μm. Red box indicates region of interest.


High - power of region of interest from ( E ) : at 24 h after fracture , mTNF expression ( signal on field , above ) co - localized with polymorphonuclear found on adjacent H & amp ; amp ; E section ( below ) . Scale bar , 25 μm . Neutrophils were identified by their polymorphonuclear as well as positive brown with anti - neutrophil elastase .

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High-power micrographs of region of interest from (E): at 24 h after fracture, mTNF expression (white signal on dark field, above) co-localized with polymorphonuclear cells found on the adjacent H&amp;E section (below). Scale bar, 25 μm. Neutrophils were identified by their polymorphonuclear morphology as well as positive dark brown staining with anti-neutrophil elastase.


High - power : at 7 days , TNF expression ( signal on field , above ) co - localized with F4 / 80 - positive ( stained brown ) extravasating from a blood vessel on adjacent H & amp ; amp ; E section ( below ) . Scale bar , 25 μm .

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High-power micrographs: at 7 days, TNF expression (white signal on dark field, above) co-localized with F4/80-positive cells (stained dark brown) extravasating from a blood vessel on the adjacent H&amp;E section (below). Scale bar, 25 μm.


Neutrophils were mobilized into murine systemic circulation within 30 min of injury in our murine fracture model . Compared to control number of blood neutrophils at 3 h * * * P = 0 . 0006 at 6 h * P = 0 . 042 , by one - way ANOVA with Dunnett correction .

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Neutrophils were mobilized into the murine systemic circulation within 30 min of injury in our murine fracture model. Compared to control and number of blood neutrophils at 3 h ***P = 0.0006 and at 6 h *P = 0.042, by one-way ANOVA with Dunnett correction.


Depletion of neutrophils using anti - Ly6G . Intravenous anti - Ly6G treatment led to depletion of neutrophils in systemic circulation ( left ) as well local peri - fracture soft tissues ( right ) . Left : of neutrophils in blood harvested by cardiac puncture at 3 h post - injury . Data are presented as mean ± ( * * P = 0 . 0074 using unpaired one - t - ) . Right : of positively stained infiltrative neutrophils in adjacent muscle to fracture site comparing neutrophil depletion with anti - Ly6G antibody versus IgG control at day 1 day 7 post - fracture . At day 1 , number of neutrophils in control group as detected by Ly6G NE in control versus treatment : * * * * P = 0 . 0001 for both Ly6G NE , by unpaired two - t - . At day 7 , number of neutrophils in control group as detected by Ly6G NE in control versus treatment groups : * * * * P = 0 . 0001 for Ly6G * P = 0 . 020 for NE , by unpaired two - t - . Number of neutrophils at day 1 versus day 7 in control groups , * P = 0 . 037 for Ly6G * * * P = 0 . 0006 for NE , by unpaired two - t - .

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Depletion of neutrophils using anti-Ly6G. Intravenous anti-Ly6G treatment led to depletion of neutrophils in the systemic circulation (left) as well the local peri-fracture soft tissues (right). Left: counts of neutrophils in the blood harvested by cardiac puncture at 3 h post-injury. Data are presented as mean ± SEM (**P = 0.0074 using unpaired one-tailed t-test). Right: counts of positively stained infiltrative neutrophils in the adjacent muscle to the fracture site comparing neutrophil depletion with anti-Ly6G antibody versus IgG control at day 1 and day 7 post-fracture. At day 1, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment: ****P = 0.0001 for both Ly6G and NE, by unpaired two-tailed t-test. At day 7, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment groups: ****P = 0.0001 for Ly6G and *P = 0.020 for NE, by unpaired two-tailed t-test. Number of neutrophils at day 1 versus day 7 in control groups, *P = 0.037 for Ly6G and ***P = 0.0006 for NE, by unpaired two-tailed t-test.


Representative sections showing local infiltration of neutrophils in adjacent muscle stained using anti - Ly6G anti - neutrophil elastase antibodies at day 1 post - fracture following treatment with anti - Ly6G antibody or IgG control . Scale bar , 100 μm .

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Representative sections showing local infiltration of neutrophils in the adjacent muscle stained using anti-Ly6G and anti-neutrophil elastase primary antibodies at day 1 post-fracture following treatment with anti-Ly6G antibody or IgG control. Scale bar, 100 μm.


Depletion of neutrophils using anti - Ly6G led to impaired fracture healing . Representative section stained with Masson ' s trichrome at day 14 comparing fracture healing in mouse treated with isotype control ( top ) versus anti - Ly6G ( bottom ) . Black in right column are identical to color in left column ; they have been labeled to clearly demonstrate anatomical structures . Control section shows advanced mineralized callus , while treatment section shows a large immature unmineralized callus . Muscle : red ; collagen , bone , mineralized callus : green . Scale bar , 1 mm .

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Depletion of neutrophils using anti-Ly6G led to impaired fracture healing. Representative section stained with Masson's trichrome at day 14 comparing fracture healing in mouse treated with isotype control (top) versus anti-Ly6G (bottom). Black and white images in the right column are identical to the color images in the left column; they have been labeled to clearly demonstrate the anatomical structures. Control section shows advanced mineralized callus, while treatment section shows a large immature unmineralized callus. Muscle fibers: red; collagen, bone, and mineralized callus: green. Scale bar, 1 mm.


Anti - Ly6G treatment led to impaired fracture healing as shown by reduced % callus mineralization at day 28 after surgery representative micro - CT . Anti - Ly6G treatment led to delayed mineralization remodeling of fracture callus compared to control . Data are presented as mean ± . * * * P = 0 . 0006 by unpaired two - sided t - . Scale Bar , 2 mm .

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Anti-Ly6G treatment led to impaired fracture healing as shown by the reduced % callus mineralization at day 28 after surgery and representative micro-CT images. Anti-Ly6G treatment led to delayed mineralization and remodeling of the fracture callus compared to control. Data are presented as mean ± SEM. ***P = 0.0006 by unpaired two-sided t-test. Scale Bar, 2 mm.


Anti - Ly6G treatment depletes local monocytes / macrophages at fracture site . of positively stained infiltrative F4 / 80 + in adjacent muscle to fracture site following treatment with control or anti - Ly6G at day 1 day 7 . Data are presented as mean ± . * * P = 0 . 005 at day 1 * * P = 0 . 002 at day 7 , * P = 0 . 013 for control at day 1 versus control at day 7 , by unpaired two - sided t - .

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Anti-Ly6G treatment depletes local monocytes/macrophages at the fracture site. Counts of positively stained infiltrative F4/80+ cells in the adjacent muscle to the fracture site following treatment with control or anti-Ly6G at day 1 and day 7. Data are presented as mean ± SEM. **P = 0.005 at day 1 and **P = 0.002 at day 7, and *P = 0.013 for control at day 1 versus control at day 7, by unpaired two-sided t-test.


Addition of 1 ng rhTNF to fracture supernatant in air pouch model led to increased numbers of neutrophils ( Ly6G + , CD11b + ) monocytes / macrophages ( Ly6G − , CD11b + , CD115 + ) in infiltrate . These effects were abrogated by addition of anti - CCL2 . Neutrophils : FS versus FS + TNF * P = 0 . 015 , FS + TNF versus FS + TNF + anti - CCL2 * P = 0 . 028 , FS + TNF + anti - CCL2 versus FS + TNF + IgG * P = 0 . 042 by one - way ANOVA followed by Bonferroni ' s multiple comparison . Monocytes / macrophages : FS versus FS + TNF * * P = 0 . 0063 , FS + TNF versus FS + TNF + anti - CCL2 * * * * P < 0 . 0001 , FS + TNF + anti - CCL2 v FS + TNF + IgG * P = 0 . 015 by one - way ANOVA followed by Bonferroni ' s multiple comparisons .

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Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) in the cellular infiltrate. These effects were abrogated by the addition of anti-CCL2. Neutrophils: FS versus FS + TNF *P = 0.015, FS + TNF versus FS + TNF + anti-CCL2 *P = 0.028, and FS + TNF + anti-CCL2 versus FS + TNF + IgG *P = 0.042 by one-way ANOVA followed by Bonferroni's multiple comparison test. Monocytes/macrophages: FS versus FS + TNF **P = 0.0063, FS + TNF versus FS + TNF + anti-CCL2 ****P < 0.0001, and FS + TNF + anti-CCL2 v FS + TNF + IgG *P = 0.015 by one-way ANOVA followed by Bonferroni's multiple comparisons test.


Addition of 1 ng rhTNF to fracture supernatant increased CCL2 in air pouch . " Neat " indicates level of CCL2 in fracture supernatant before injection into air pouch . Data are presented as mean ± . FS versus FS + TNF * * P = 0 . 0020 by one - way ANOVA with Bonferroni ' s multiple comparisons .

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Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch. "Neat" indicates level of CCL2 in fracture supernatant before injection into air pouch. Data are presented as mean ± SEM. FS versus FS + TNF **P = 0.0020 by one-way ANOVA with Bonferroni's multiple comparisons test.


Addition of rhTNF to enriched bone marrow - derived murine neutrophils pre - exposed to fracture supernatant in vitro led to up - regulation of CCL2 production at 1 h . Data are presented as mean ± . No TNF versus TNF 0 . 01 ng / ml * P = 0 . 012 , TNF 0 . 1 ng / ml * * * P < 0 . 0001 , TNF 1 . 0 ng * * * P = 0 . 0001 , TNF 10 ng / ml * * * P < 0 . 0001 , TNF 100 ng / ml * P = 0 . 023 by one - way ANOVA with Dunnett ' s multiple comparisons .

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Addition of rhTNF to enriched bone marrow-derived murine neutrophils pre-exposed to fracture supernatant in vitro led to up-regulation of CCL2 production at 1 h. Data are presented as mean ± SEM. No TNF versus TNF 0.01 ng/ml *P = 0.012, TNF 0.1 ng/ml ***P < 0.0001, TNF 1.0 ng ***P = 0.0001, TNF 10 ng/ml ***P < 0.0001, and TNF 100 ng/ml *P = 0.023 by one-way ANOVA with Dunnett's multiple comparisons test.


Treatment with CCR2 antagonist , INCB3344 , led to impaired fracture healing compared to vehicle control as shown by reduced % callus mineralization at day 28 after operation . Data are presented as mean ± . * P = 0 . 011 unpaired two - sided t - . Representative micro - CT are shown . Scale bar , 2 mm .

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Treatment with CCR2 antagonist, INCB3344, led to impaired fracture healing compared to vehicle control as shown by the reduced % callus mineralization at day 28 after operation. Data are presented as mean ± SEM. *P = 0.011 unpaired two-sided t-test. Representative micro-CT images are shown. Scale bar, 2 mm.


rhTNF treatment augmented early phase fracture healing in osteoporotic mice . Treatment with 1 ng rhTNF at fracture site on days 0 1 led to increased % callus mineralization at day 14 but no difference at day 28 indicating accelerated early healing but same final result at day 28 . Data are presented as mean ± . At day 14 , PBS control versus TNF , * * P = 0 . 0099 ; at day 28 , PBS control versus TNF , nsP = 0 . 37 , by unpaired two - sided t - . Representative micro - CT at day 14 are shown . rhTNF treatment led to mature callus bridging across fracture which was absent in PBS - treated control group . Scale bar , 2 mm .

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rhTNF treatment augmented early phase fracture healing in osteoporotic mice. Treatment with 1 ng rhTNF at the fracture site on days 0 and 1 led to increased % callus mineralization at day 14 but no difference at day 28 indicating accelerated early healing but the same final result at day 28. Data are presented as mean ± SEM. At day 14, PBS control versus TNF, **P = 0.0099; at day 28, PBS control versus TNF, nsP = 0.37, by unpaired two-sided t-test. Representative micro-CT images at day 14 are shown. rhTNF treatment led to mature callus bridging across the fracture which was absent in the PBS-treated control group. Scale bar, 2 mm.


( A - D ) A . castellanii was infected ( MOI 5 ) with L . pneumophila JR32 producing ( A ) GFP under control of PflaA ( Ptac - mCherry - PflaA - gfp ) mCherry constitutively , ( B ) GFP under control of PralF ( PralF - gfp ) stained with DAPI , ( C ) GFP under control of PsidC ( PsidC - gfp ) stained with DAPI , or ( D ) Timer constitutively ( pNP107 ) . infected amoebae were fixed at different time points post - infection , analyzed by confocal microscopy 3D reconstructed ( scale bars : 5 µm ) . Motile virulent ( GFP - positive ) emerge at colony cluster periphery only after replication has ceased ( red Timer ) .

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(A-D) A. castellanii was infected (MOI 5) with L. pneumophila JR32 producing (A) GFP under the control of PflaA (Ptac-mCherry-PflaA-gfp) and mCherry constitutively, (B) GFP under the control of PralF (PralF-gfp) and stained with DAPI, (C) GFP under the control of PsidC (PsidC-gfp) and stained with DAPI, or (D) Timer constitutively (pNP107). The infected amoebae were fixed at different time points post-infection, analyzed by confocal microscopy and 3D reconstructed (scale bars: 5 µm). Motile and virulent bacteria (GFP-positive) emerge at the colony cluster periphery only after bacterial replication has ceased (red fluorescent Timer).


( G ) Transmission electron of A . castellanii infected with L . pneumophila . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila wild - type JR32 subjected to high - pressure freezing electron microscopy . Many LCVs were still intact at 42 h post infection ( left panel ; arrow : intact LCV , arrowhead plasma ) , occasionally , were flagellated ( middle panel , arrow ) . Some LCVs were already ruptured , were released into host cytosol ( right panel ; arrows : ruptured LCV ) . Scale bars : 2 µm , 0 . 5 µm ( insets ) .

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(G) Transmission electron micrographs of A. castellanii infected with L. pneumophila. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila wild-type strain JR32 and subjected to high-pressure freezing and electron microscopy. Many LCVs were still intact at 42 h post infection (left panel; arrow: intact LCV membrane, arrowhead plasma membrane), and occasionally, the bacteria were flagellated (middle panel, arrow). Some LCVs were already ruptured, and the bacteria were released into the host cell cytosol (right panel; arrows: ruptured LCV membrane). Scale bars: 2 µm, 0.5 µm (insets).