Figure 5A . 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 & quoC . 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 μmD . 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 presentedF . 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 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

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

Figure 5A. 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&quoC. 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 μmD. 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 presentedF. 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 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


Figure 6B . Representative fluorescence images of siRNA - control or siRNA - 6B injected SCNT embryos . Scale bar , 50 μmC . 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 tD . 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 μmE . 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 tF . Representative image of cloned mice derived by siRNA - 6B injected SCNT embryosG . 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 experimentsH . Immunostaining images of si6B - mdx - ntES expressed pluripotency markers . Scale bar , 50 μmI . 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 -

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

Figure 6B. Representative fluorescence images of siRNA-control or siRNA-6B injected SCNT embryos. Scale bar, 50 μmC. 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 tD. 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 μmE. 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
 tF. Representative image of cloned mice derived by siRNA-6B injected SCNT embryosG. 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 experimentsH. Immunostaining images of si6B-mdx-ntES expressed pluripotency markers. Scale bar, 50 μmI. 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-ntES


Figure 7B . 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 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 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 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 cells injected with siRNA - control , siRNA - Xist , or siRNA - Xist - 6B .

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

Figure 7B. 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 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 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 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 cells injected with siRNA-control, siRNA-Xist, or siRNA-Xist-6B.


Figure 1 ( 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 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 and Arg517 ( grey ) .

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

Figure 1(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 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 and Arg517 (grey).


Figure 2 ( 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 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 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 .

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

Figure 2(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 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 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.


Figure 3 ( 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 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 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 .

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

Figure 3(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 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 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.


Figure 4 ( 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 .

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

Figure 4(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.


Figure 5 ( 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 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 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 .

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

Figure 5(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 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 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.


Figure 6 ( 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 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 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 .

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

Figure 6(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 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 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.


Figure 7 ( 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 .

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

Figure 7(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.


Figure 8 ( 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 .

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

Figure 8(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.


Figure 1 ( 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 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 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 .

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

Figure 1(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 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 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 .


Figure 2 ( B ) Characterization of the diameter and zeta potential ( Z - potential ) with the addition of each layer . Graph below presenting NP size distribution .

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

Figure 2(B) Characterization of the diameter and zeta potential (Z-potential) with the addition of each layer. Graph below presenting NP size distribution.


Figure 3 ( 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 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 .

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

Figure 3(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 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.


Figure 4 ( 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 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 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 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 .

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

Figure 4(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 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 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 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.


Figure 5 ( 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 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 .

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

Figure 5(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 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.


Figure 6 ( 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 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 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 .

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

Figure 6(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 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 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.


Figure 2A ) 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 ( dark blue ) , Luminal B ( light blue ) , HER2 - enriched ( pink ) , basal - like ( red ) , Normal - like ( green ) and Unclassified ( gray ) . 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 .

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Figure 2A) 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 (dark blue), Luminal B (light blue), HER2-enriched (pink), basal-like (red), Normal-like (green) and Unclassified (gray). 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.


Figure 3Waterfall 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 .

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Figure 3Waterfall 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.


Figure 5 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 .

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Figure 5 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.


Figure 6 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 .

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Figure 6 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.


Figure 7 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 .

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Figure 7 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.


Figure 1E , 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 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 .

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Figure 1E, 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 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.


Figure 2B , 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 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 and WCE were analyzed by immunoblot using the indicated antibodies . Data information : For D , exemplary blot of two independent experiments . 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 .

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Figure 2B, 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 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 and WCE were analyzed by immunoblot using the indicated antibodies. Data information: For D, exemplary blot of two independent experiments.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.


Figure 3A , 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 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 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 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 .

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Figure 3A, 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 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 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 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.


Figure 4D , Immunoblot analysis of WCE obtained from a representative experiment depicted in ( C ) .

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Figure 4D, Immunoblot analysis of WCE obtained from a representative experiment depicted in (C).


Figure 5C , 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 ) ) .

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Figure 5C, 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)).


Figure 6A , 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 .

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Figure 6A, 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.


Figure 1Addition 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 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 ± 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 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 ( 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 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 ; 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 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 ; amp ; E section ( below ) . Scale bar , 25 μm .

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Figure 1Addition 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 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 ± 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 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 (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 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 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.


Figure 2Neutrophils 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 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 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 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 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 .

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Figure 2Neutrophils 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 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 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 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 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.


Figure 3Anti - 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 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 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 ± 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 .

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Figure 3Anti-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 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 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 ± 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.


Figure 4Treatment 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 .

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Figure 4Treatment 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.


Figure 5rhTNF 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 .

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Figure 5rhTNF 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.


Figure 1 ( 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 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 ) .

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Figure 1(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 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).


Figure 2 ( A ) L . pneumophila subpopulation proteomics . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) . After cell lysis , released intracellular bacteria were sorted by flow cytometry in mCherry - positive / GFP - negative and mCherry - positive / GFP - positive L . pneumophila subpopulations , and their proteome was determined and compared . Protein abundance in each subset is depicted as a volcano plot . Proteins enriched in the GFP - producing subpopulation have a positive log2 ratio ( q - value ) . ( D , E ) Expression of select L . pneumophila genes in amoebae . A . castellanii was infected ( MOI 5 ; 6 h and 48 h ) with L . pneumophila JR32 harboring reporter constructs for ( D ) PfleQ - mCherry - PflaA - gfp or ( E ) PlegC8 - mCherry - PflaA - gfp , respectively . The infected amoebae were fixed and imaged by confocal microscopy ( scale bars : 5 µm ) .

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Figure 2(A) L. pneumophila subpopulation proteomics. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp). After cell lysis, released intracellular bacteria were sorted by flow cytometry in mCherry-positive/GFP-negative and mCherry-positive/GFP-positive L. pneumophila subpopulations, and their proteome was determined and compared. Protein abundance in each subset is depicted as a volcano plot. Proteins enriched in the GFP-producing subpopulation have a positive log2 ratio (q-value).(D, E) Expression of select L. pneumophila genes in amoebae. A. castellanii was infected (MOI 5; 6 h and 48 h) with L. pneumophila JR32 harboring reporter constructs for (D) PfleQ-mCherry-PflaA-gfp or (E) PlegC8-mCherry-PflaA-gfp, respectively. The infected amoebae were fixed and imaged by confocal microscopy (scale bars: 5 µm).