J Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals from the indicated time point ( in hours ) after the L4 stage . Animals were exposed to the indicated knockdown RNAi bacterial strains or a strain that only contained the empty RNAi vector since hatching . * P < 0 . 001 using ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point , with N = 20 animals per genotype and time point . Error bars indicate SEM .

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J Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) after the L4 stage. Animals were exposed to the indicated knockdown RNAi bacterial strains or a strain that only contained the empty RNAi vector since hatching. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM.


L Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals from the indicated time point ( in hours ) and the indicated genotypes after the L4 stage , as per ( J ) . * P < 0 . 001 using ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point , with N = 20 animals per genotype and time point . Error bars indicate SEM .

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L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) and the indicated genotypes after the L4 stage, as per (J). *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM.


K , L Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4 + 48 hours . * P < 0 . 001 using ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point , with N = 20 animals per genotype and time point . Data has been normalized to wild type . Error bars indicate SEM .

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K,L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Data has been normalized to wild type. Error bars indicate SEM.


B A separate SDS - PAGE and Western blot of the same lysates from ( A ) , probed with antibodies recognizing ubiquitin or actin as a loading control . The position of molecular weight markers is shown on the left of each blot . The position of UbG76V - GFP protein , as well as UbG76V - GFP with the indicated number of additional ubiquitin moieties , based on molecular weight , is indicated to the right of each blot . Endogenous poly - ubiquitinated proteins are also detected in the 80 - 200 kDa range , as indicated by the bracket . Twenty animals were loaded per lane for each indicated genotype .

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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR 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

B A separate SDS-PAGE and Western blot of the same lysates from (A), probed with antibodies recognizing ubiquitin or actin as a loading control. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated to the right of each blot. Endogenous poly-ubiquitinated proteins are also detected in the 80-200 kDa range, as indicated by the bracket. Twenty animals were loaded per lane for each indicated genotype.


B Heat map of log fold change ( logFC , color scale at graph bottom ) in the expression of C . elegans CYP genes between the indicated mutant and wild type . Genes whose expression is lower in a mutant relative to wild type are colored blue , whereas genes whose expression is higher in a mutant relative to wild type are colored yellow . Rows are labeled for the different CYP families and subfamilies . Specific genes ( rows ) are clustered according to sequence alignment ( CLUSTAL W ) to maintain the relationship of their molecular evolution

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B Heat map of log fold change (logFC, color scale at graph bottom) in the expression of C. elegans CYP genes between the indicated mutant and wild type. Genes whose expression is lower in a mutant relative to wild type are colored blue, whereas genes whose expression is higher in a mutant relative to wild type are colored yellow. Rows are labeled for the different CYP families and subfamilies. Specific genes (rows) are clustered according to sequence alignment (CLUSTAL W) to maintain the relationship of their molecular evolution


D Abundance of the indicated free ω6 PUFA - derived eicosanoid , normalized to total protein , for each indicated genotype . Red , gray , and blue stacked bars indicate hydroxides , epoxide , and epoxide diol forms of eicosanoids , respectively . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA with Dunnett ' s posthoc comparison to wild type . N = 3 trials . Error bars indicate SEM . E Abundance of the indicated free ω3 PUFA - derived eicosanoid , as in ( D ) , normalized to total protein , for each indicated genotype . Red , gray , and blue stacked bars indicate hydroxides , epoxide , and epoxide diol forms of eicosanoids , respectively . * * * * P < 0 . 0001 , ANOVA with Dunnett ' s posthoc comparison to wild type . N = 3 trials . Error bars indicate SEM

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D Abundance of the indicated free ω6 PUFA-derived eicosanoid, normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. E Abundance of the indicated free ω3 PUFA-derived eicosanoid, as in (D), normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM


B Relative abundance of mRNA for the cyp - 34A4 gene . Values are based on qRT - PCR analysis , with the relative levels for each gene normalized to the value observed in the wild - type control . * * * P < 0 . 001 , ANOVA with Dunnett ' s posthoc comparison to wild type . N = 3 trials . Error bars indicate SEM .

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B Relative abundance of mRNA for the cyp-34A4 gene. Values are based on qRT-PCR analysis, with the relative levels for each gene normalized to the value observed in the wild-type control. ***P<0.001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM.


C Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4 + 48 hours . * * * * P < 0 . 001 , ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point . N = 20 animals per genotype and time point . Error bars indicate SEM .

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

C Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. ****P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.


F Quantified fluorescence as in ( B ) for animals exposed to the indicated RNAi vector or empty vector ( EV ) control , and in either a wild type ( WT ) or fat - 1 ( wa9 ) genetic background . * P < 0 . 001 , ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point . N = 20 animals per genotype and time point . Error bars indicate SEM .

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F Quantified fluorescence as in (B) for animals exposed to the indicated RNAi vector or empty vector (EV) control, and in either a wild type (WT) or fat-1(wa9) genetic background. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.


G Quantified fluorescence as in ( B ) for animals of the indicated genotype at the indicated time point . * P < 0 . 001 , ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point . N = 20 animals per genotype and time point . Error bars indicate SEM .

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G Quantified fluorescence as in (B) for animals of the indicated genotype at the indicated time point. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.


B . Representative immunofluorescence and live - cell images of dynamics MERVL : : tdTomato and Gag expression during embryos preimplantation development ( left ) . Quantification of tdTomato and Gag intensity ( right ) . For the live - cell images , average intensity of tdTomato signal intensities relative to 2 - cell stage embryos . For the immunofluorescence images , bar graphs showing the relative intensities of Gag / DAPI signal ratio . N , total number of embryos analyzed for each condition . Error bars , s . d . , n ≥ 4 . * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed Student " s t - test . Scale bar ,

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 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 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

B. Representative immunofluorescence and live-cell images of dynamics MERVL::tdTomato and Gag expression during embryos preimplantation development (left). Quantification of tdTomato and Gag intensity (right). For the live-cell images, average intensity of tdTomato signal intensities relative to 2-cell stage embryos. For the immunofluorescence images, bar graphs showing the relative intensities of Gag/DAPI signal ratio. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 4. **P < 0.01, ***P < 0.001 by two-tailed Student"s
 t-test. Scale bar,


C . Western blot analysis MERVL : : tdTomato levels in MII oocyte and embryos at the indicated stages ( upper ) . GAPDH was used as a loading control . Numbers below the Western blots indicate band intensity ( normalized to total GAPDH ) measured by using ImageJ software . Quantification of Western blot results ( bottom ) . MII , MII oocyte ; M , morula ; B , blastocyst . Error bars , s . d . , n = 3 . * * P < 0 . 01 , * * * P < 0 . 001 by two - tailed Student " s

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C. Western blot analysis MERVL::tdTomato levels in MII oocyte and embryos at the indicated stages (upper). GAPDH was used as a loading control. Numbers below the Western blots indicate band intensity (normalized to total GAPDH) measured by using ImageJ software. Quantification of Western blot results (bottom). MII, MII oocyte; M, morula; B, blastocyst. Error bars, s.d., n = 3. **P < 0.01, ***P < 0.001 by two-tailed Student"s
 t


D . Representative fluorescence image of 2 - cell embryos derived from ICSI or SCNT . The SCNT embryos produced by transfer of MERVL : : tdTomato cumulus cell into WT enucleated oocytes . ICSI embryos produced by MERVL : : tdTomato sperm and MII oocytes from the littermates of transgenic mice . Scale bar , 50

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D. Representative fluorescence image of 2-cell embryos derived from ICSI or SCNT. The SCNT embryos produced by transfer of MERVL::tdTomato cumulus cell into WT enucleated oocytes. ICSI embryos produced by MERVL::tdTomato sperm and MII oocytes from the littermates of transgenic mice. Scale bar, 50 μm


E . The summary of tdTomato + 2 - cell embryos derived from ICSI or SCNT . N , total number of embryos analyzed for each condition . Error bars , s . d . , n ≥ 3 . * * * P < 0 . 001 by two - tailed Student " s

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E. The summary of tdTomato+ 2-cell embryos derived from ICSI or SCNT. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 3. ***P < 0.001 by two-tailed Student"s
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F . The endogenous MERVL was up - regulated significantly in ICSI embryos compared with SCNT embryos at 2 - cell stages , as determined by RT - qPCR . Error bars , s . e . m . , n ≥

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F. The endogenous MERVL was up-regulated significantly in ICSI embryos compared with SCNT embryos at 2-cell stages, as determined by RT-qPCR. Error bars, s.e.m., n ≥ 3


G . RT - qPCR data for select ZGA genes activated following MERVL : : tdTomato expression in mouse 2 - cell embryos derived from ICSI or SCNT . 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 . 001 by two - tailed Student " s

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

G. RT-qPCR data for select ZGA genes activated following MERVL::tdTomato expression in mouse 2-cell embryos derived from ICSI or SCNT. 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 < 0.001 by two-tailed Student"s
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H . Representative images of tdTomato + and tdTomato - embryos derived from ICSI or SCNT , after 4 . 5 days of culturing in vitro . Scale bar , 100

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H. Representative images of tdTomato+ and tdTomato- embryos derived from ICSI or SCNT, after 4.5 days of culturing in vitro. Scale bar, 100 μm


I . Preimplantation development rates in the tdTomato + and tdTomato - embryos derived from ICSI or SCNT . The efficiency was calculated based on the number of 2 - cell embryo that have been divided into tdTomato + and tdTomato - groups . Error bars , s . d . , n ≥ 3 .

I. Preimplantation development rates in the tdTomato+ and tdTomato- embryos derived from ICSI or SCNT. The efficiency was calculated based on the number of 2-cell embryo that have been divided into tdTomato+ and tdTomato- groups. Error bars, s.d., n ≥ 3.


B . Representative live - cell images of dynamics MERVL : : tdTomato expression during SCNT embryos preimplantation development . The selected images from a series acquired every 15 min . The time after starting observation is shown on the upper right corner of each image . Scale bar , 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative live-cell images of dynamics MERVL::tdTomato expression during SCNT embryos preimplantation development. The selected images from a series acquired every 15 min. The time after starting observation is shown on the upper right corner of each image. Scale bar, 50 μm


C . Representative DAPI staining of blastocysts of tdTomato + and tdTomato - SCNT embryos after 115 hr . of culture in vitro . Scale bar , 50 μm . D . The tdTomato + and tdTomato - SCNT blastocyst cell numbers were determined by counting the DAPI - stained cells . Three independent experiment replicates were performed . N , total number of embryos analyzed for each condition . Red bars indicated the mean value . E

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C. Representative DAPI staining of blastocysts of tdTomato+ and tdTomato- SCNT embryos after 115 hr. of culture in vitro. Scale bar, 50 μm. D. The tdTomato+ and tdTomato- SCNT blastocyst cell numbers were determined by counting the DAPI-stained cells. Three independent experiment replicates were performed. N, total number of embryos analyzed for each condition. Red bars indicated the mean value. E


E . Immunofluorescence images of tdTomato + and tdTomato - blastocysts derived from SCNT . Nanog ( ICM ) and Cdx2 ( TE ) were used as lineage markers . Representative images from ≥ 55 embryos analyzed in four independent micromanipulations are shown . Scale bar , 50

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E. Immunofluorescence images of tdTomato+ and tdTomato- blastocysts derived from SCNT. Nanog (ICM) and Cdx2 (TE) were used as lineage markers. Representative images from ≥ 55 embryos analyzed in four independent micromanipulations are shown. Scale bar, 50 μm


F . Representative image of tdTomato + and tdTomato - SCNT embryos retrieved at E6 . 5 . The tdTomato + SCNT embryo displays normal egg - cylinder morphology . By contrast , the tdTomato - SCNT embryo shows abnormal morphology . Epi , embryonic epiblast ; ExEm , extraembryonic ectoderm ; EPC , ectoplacental cone . Scale bar , 50

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F. Representative image of tdTomato+ and tdTomato- SCNT embryos retrieved at E6.5. The tdTomato+ SCNT embryo displays normal egg-cylinder morphology. By contrast, the tdTomato- SCNT embryo shows abnormal morphology. Epi, embryonic epiblast; ExEm, extraembryonic ectoderm; EPC, ectoplacental cone. Scale bar, 50 μm


G . The bar chart showing the efficiency of attachment to the feeder cells of SCNT blastocysts . The efficiency was calculated based on the total number of blastocysts used for ntES derivation . N , total number of embryos analyzed for each condition . Error bars , s . d . , n ≥ 3 . * * P < 0 . 01 by two - tailed Student " s

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G. The bar chart showing the efficiency of attachment to the feeder cells of SCNT blastocysts. The efficiency was calculated based on the total number of blastocysts used for ntES derivation. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 3. **P < 0.01 by two-tailed Student"s
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H . The bar chart shows the efficiency of ntES derivation . The efficiency was calculated based on the total number of attached blastocysts for ntES derivation . N , total number of embryos analyzed for each condition . Error bars , s . d . , n ≥ 3 . * * P < 0 . 01 by two - tailed Student " s

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H. The bar chart shows the efficiency of ntES derivation. The efficiency was calculated based on the total number of attached blastocysts for ntES derivation. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 3. **P < 0.01 by two-tailed Student"s
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I . RT - qPCR analysis for somatic cell genes in SCNT 1 - cell embryos , 2 - cell tdTomato + , and 2 - cell tdTomato - embryos . 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 . 001 by two - tailed Student " s

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

I. RT-qPCR analysis for somatic cell genes in SCNT 1-cell embryos, 2-cell tdTomato+, and 2-cell tdTomato- embryos. 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 < 0.001 by two-tailed Student"s
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A . Immunofluorescence images of ICSI , SCNT - tdTomato + and SCNT - tdTomato - embryos . Embryos approximately 26hr after activation were fixed and processed for immunostaining with antibodies to H3K27me3 and Gag protein . ICSI embryos produced by MERVL : : tdTomato sperm from the littermates of male mice . The images are representative examples of the quantification shown in Fig 3B . Representative images from ≥ 128 embryos analyzed in five independent micromanipulations are shown . Scale bar , 20 μm . B . Percentage of 2 - cell ICSI and SCNT embryos with strong H3K27me3 staining and moderate staining ( left ) . Numbers of the total embryos analyzed from five independent micromanipulations are shown in the bars . Boxplots ( right ) for relative intensities of H3K27me3 from five independent experiments for each expression as in Fig 3A . Error bars , s . e . m . , * * * P < 0 . 001 by two - tailed Student " s

O O O O O O O O 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 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 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 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

A. Immunofluorescence images of ICSI, SCNT-tdTomato+ and SCNT-tdTomato- embryos. Embryos approximately 26hr after activation were fixed and processed for immunostaining with antibodies to H3K27me3 and Gag protein. ICSI embryos produced by MERVL::tdTomato sperm from the littermates of male mice. The images are representative examples of the quantification shown in Fig 3B. Representative images from ≥ 128 embryos analyzed in five independent micromanipulations are shown. Scale bar, 20 μm. B. Percentage of 2-cell ICSI and SCNT embryos with strong H3K27me3 staining and moderate staining (left). Numbers of the total embryos analyzed from five independent micromanipulations are shown in the bars. Boxplots (right) for relative intensities of H3K27me3 from five independent experiments for each expression as in Fig 3A. Error bars, s.e.m., ***P < 0.001 by two-tailed Student"s
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C . Western blot results showing H3K27me3 levels in SCNT and ICSI embryos . α - Tubulin ( TUBB ) was blotted as a loading

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C. Western blot results showing H3K27me3 levels in SCNT and ICSI embryos. α-Tubulin (TUBB) was blotted as a loading control


D . Representative immunofluorescent staining of H3K27me3 in ICSI embryos at the 1 - cell stage . ICSI 1 - cell embryos are characterised by the visualisation of two distinct pronuclei ( 2PN ) 6 hours after sperm injection . H3K27me3 enrichment in the female - PN . The identity of the pronuclei was determined by their size and position relative to the polar body . Representative images from ≥ 95 embryos analyzed in four independent micromanipulations are shown SY , syngamy . Scale bar , 20

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D. Representative immunofluorescent staining of H3K27me3 in ICSI embryos at the 1-cell stage. ICSI 1-cell embryos are characterised by the visualisation of two distinct pronuclei (2PN) 6 hours after sperm injection. H3K27me3 enrichment in the female-PN. The identity of the pronuclei was determined by their size and position relative to the polar body. Representative images from ≥ 95 embryos analyzed in four independent micromanipulations are shown SY, syngamy. Scale bar, 20 μm


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

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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 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

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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 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 .

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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 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

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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 . 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

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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 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

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

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


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

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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 . 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

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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 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 .

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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 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 &

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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 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

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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 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

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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 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

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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 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

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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 fluorescence images of siRNA - control or siRNA - 6B injected SCNT embryos . 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 . 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

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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 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

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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 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

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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 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

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

G. 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 images 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 . The si6B - mdx - ntES possessed multiple - differentiation potential , as shown in embryoid body . 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 . 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 cells 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 ) 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 .

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

(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 ) .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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

(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 ) .

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


( 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 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(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 ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 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 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 O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR I-MEASURED_VAR 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 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 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(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 ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(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 O O O O O O O O O O O O O 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 B-MEASURED_VAR 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

(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 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 O O 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 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 O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(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 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 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR 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

(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 ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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 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 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 O O O O O O 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 B-MEASURED_VAR 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

(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 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 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-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

(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 ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(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 .

(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 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 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 I-MEASURED_VAR I-MEASURED_VAR 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 I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR 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 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 O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 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 .

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 I-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 B-MEASURED_VAR 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 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 .

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

O O O O 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 B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR 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 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 O O O O O O O O O O O O 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

(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 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 .

O O O 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 O B-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 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 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 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 .

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(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 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR O O O O 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 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 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 O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 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 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 )

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 B-MEASURED_VAR 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 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 O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(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 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 .

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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 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 .

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 O B-CONTROLLED_VAR 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 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

(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 O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

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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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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 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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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 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 .

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

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 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 .

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

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.