( L - N ) B16N parental , O1C44 , O1DN were subcutaneously injected in right flank of syngeneic C57Bl6 mice revealing presence of O1C44 ( but not B16N - WT ) in abdominal organs within peritoneal wall ( L ) . ( M ) H & E stains of peritoneal wall revealing presence of melanoma in peritoneal wall . ( N ) Quantitative measurement of degree of invasion within through peritoneal wall . Data was analyzed using Kruskal - Wallis Rank Sum ( P < 0 . 001 ) . Within panels , differences established through multiple comparisons are labeled as * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L-N) B16N parental, O1C44, and O1DN cells were subcutaneously injected in the right flank of syngeneic C57Bl6 mice revealing the presence of O1C44 (but not B16N-WT) cells in abdominal organs and within the peritoneal wall (L). (M) H&E stains of the peritoneal wall revealing the presence of melanoma cells in the peritoneal wall. (N) Quantitative measurement of degree of invasion within and through the peritoneal wall. Data was analyzed using the Kruskal-Wallis Rank Sum test (P < 0.001). Within all panels, differences established through multiple comparisons are labeled as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( D - F ) Confluent layers of WM983 treated as indicated were ' scratched ' followed by a 20 hour incubation at 37 ° C with 5 % CO2 . ( D ) Representative of after being scratched after being incubated for 0 , 10 or 20 hours in absence ( - ) or presence ( + ) of 2uM IA65 . ( E - F ) - free was quantified by ImageJ ( N ≥ 8 biological replicates ) ; data displayed are mean ± . Data was analyzed by 2 - way ANOVA . In absence of IA65 , SOCE suppression effect was p < 0 . 05 ; effect of time was p < 0 . 0001 ; interaction was p < 0 . 05 . In presence of IA65 , SOCE suppression effect was p > 0 . 05 ; effect of time was p < 0 . 0001 ; interaction was p > 0 . 05 . Within panels , differences established through multiple comparisons are labeled as * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O 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 O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Confluent layers of WM983 cells treated as indicated were 'scratched' followed by a 20 hour incubation at 37°C with 5% CO2. (D) Representative images of cells after being scratched after being incubated for 0, 10 or 20 hours in the absence (-) or presence (+) of 2uM IA65. (E-F) The cell-free area was quantified by ImageJ (N≥8 biological replicates); data displayed are mean±SEM. Data was analyzed by 2-way ANOVA. In the absence of IA65, SOCE suppression effect was p<0.05; effect of time was p < 0.0001; interaction was p<0.05. In the presence of IA65, SOCE suppression effect was p>0.05; effect of time was p < 0.0001; interaction was p>0.05. Within all panels, differences established through multiple comparisons are labeled as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


WM983 UACC257 human melanoma were transfected with Perfringolysin O ( PFO ) - mCherry D4H cholesterol sensor plated on glass coverslips . were exposed to UV ( 0 or 175 J / m2 ) incubated for 0 , 2 or 5 as indicated . ( H ) Representative . ( I ) from analyzed in ( H ) was measured quantified using LASX software ( N ≥ 8 biological replicates ) ; data displayed are mean ± . Data was analyzed using 1 - way ANOVA with multiple comparisons ( WM983 , p < 0 . 001 ; UACC257 p > 0 . 05 ) . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

WM983 and UACC257 human melanoma cells were transfected with Perfringolysin O (PFO)-mCherry D4H cholesterol sensor and plated on glass coverslips. Cells were exposed to UV (0 or 175 J/m2) and incubated for 0, 2 or 5 weeks as indicated. (H) Representative cell images. (I) Fluorescence intensity from cells analyzed in (H) was measured and quantified using LASX analysis software (N≥8 biological replicates); data displayed are mean±SEM. Data was analyzed using 1-way ANOVA with multiple comparisons (WM983, p < 0.001; UACC257 p > 0.05). **p<0.01, ***p<0.001, ****p<0.0001.


O1C78 WM983 expressing PFO - mCherry D4H cholesterol sensor were examined as in panels H I ( N ≥ 8 biological replicates ) ; data displayed are mean ± .

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

O1C78 WM983 cells expressing the PFO-mCherry D4H cholesterol sensor were examined as in panels H and I (N≥8 biological replicates); data displayed are mean±SEM.


surgical explants were received from biosample repository of FCCC . were isolated from tumors using 50 μM Medicons Medimachine system grown for 3 days before experimentation to eliminate non - melanoma . ( A - B ) Plated control or UV irradiated ( 175J / m2 ) were then incubated overnight on glass coverslips prior to Fura2 loading administration of Tg in Ca2 + - free media for 15 minutes . ( A ) Representative examples of SOCE responses in samples following addition of Ca2 + ( 1 mM ) . Each trace represents 5 to 10 ; solid represents mean dotted represents . ( B ) Each dot represents SOCE in obtained from a different sample ( n = 20 ; data displayed as mean ± ) . effect of UV on SOCE was determined by one way ANOVA ( p > 0 . 05 ) . ( C ) Comparison of SOCE in collected from both tumors metastatic lymph nodes from same ( from analyzed in panels A , B ) .

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

Patient surgical explants were received from the biosample repository of FCCC. Cells were isolated from tumors using 50 μM Medicons and the Medimachine system and grown for 3 days before experimentation to eliminate non-melanoma cells. (A-B) Plated control or UV irradiated (175J/m2) cells were then incubated overnight on glass coverslips prior to Fura2 loading and administration of Tg in Ca2+-free media for 15 minutes. (A) Representative examples of SOCE responses in patient samples following the addition of Ca2+ (1 mM). Each trace represents 5 to 10 individual cells; solid line represents the mean and the dotted line represents SEM. (B) Each dot represents SOCE in cells obtained from a different patient sample (n=20; data displayed as mean±SEM). The effect of UV on SOCE was determined by one way ANOVA (p > 0.05). (C) Comparison of SOCE in cells collected from both primary tumors and metastatic lymph nodes from the same patient (from patients analyzed in panels A, B).


( D ) SOCE in samples exhibiting NRAS ( 8 of 27 samples ) , BRAF ( 6 of 27 samples ) , TERT promoter ( 6 of 27 samples ) , NF1 ( 5 of 27 samples ) , CDKN2A ( 5 of 27 samples ) alterations were compared to SOCE in samples that expressed WT or unaltered gene . Data displayed as mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SOCE in primary patient samples exhibiting NRAS (8 of 27 samples), BRAF (6 of 27 samples), TERT promoter (6 of 27 samples), NF1 (5 of 27 samples), and CDKN2A (5 of 27 samples) alterations were compared to SOCE in samples that expressed WT or the unaltered gene. Data displayed as mean±SEM.


( E ) Single from samples were treated with or without indicated drug in presence of Tg in nominally Ca2 + - free media for 15 minutes prior to addition of Ca2 + ( 1 mM ) . number on each bar represents number of specimens tested . Data were compared against their matched control analyzed by unpaired T ( Tak475 , p = 0 . 015 ; compactin , p = 0 . 0049 ; IA65 , p = 0 . 0390 ) .

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 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 B-CONTROLLED_VAR O O O 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

(E) Single cells from patient samples were treated with or without the indicated drug in the presence of Tg in nominally Ca2+-free media for 15 minutes prior to the addition of Ca2+ (1 mM). The number on each bar represents the number of patient specimens tested. Data were compared against their matched control analyzed by the unpaired T test (Tak475, p = 0.015; compactin, p = 0.0049; IA65, p = 0.0390).


( F ) were placed in Matrigel transwell inserts in OptiMEM reduced serum media with or without TAK475 ( 10uM ) , compactin ( 2μM ) , IA65 ( 2μM ) or OSMI1 ( 40μM ) ; DMEM with 10 % FBS was used as a chemoattractant . Crystal violet - stained invaded were measured 20 hrs after initiation of . Each dot represents 1 sample . Data analyzed by 2 - way ANOVA ( cholesterol inhibition was p < 0 . 0001 ; samples were p < 0 . 001 ; interaction was p < 0 . 01 ) . For IA65 , ( inhibition was p < 0 . 0001 ; samples were p < 0 . 01 ; interaction was p < 0 . 0001 ) . For OSMI1 , ( inhibition was p < 0 . 05 ; samples were p > 0 . 05 ; interaction was p < 0 . 05 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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 B-CONTROLLED_VAR 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 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 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

(F) Patient cells were placed in Matrigel transwell inserts in OptiMEM reduced serum media with or without TAK475 (10uM), compactin (2μM), IA65 (2μM) or OSMI1 (40μM); DMEM with 10% FBS was used as a chemoattractant. Crystal violet-stained invaded cells were measured 20 hrs after initiation of assay. Each dot represents 1 patient sample. Data analyzed by 2-way ANOVA (cholesterol inhibition was p < 0.0001; patient samples were p < 0.001; interaction was p < 0.01). For IA65, (inhibition was p < 0.0001; patient samples were p < 0.01; interaction was p < 0.0001). For OSMI1, (inhibition was p < 0.05; patient samples were p > 0.05; interaction was p < 0.05). *p<0.05, **p<0.01, ****p<0.0001.


ZO - 1 GAPDH assessed in Epi by Western blot of whole extracts .

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

ZO-1 and GAPDH protein levels assessed in Epi cells by Western blot of whole protein extracts.


ZO - 1 of IF performed using Fiji software bar - plotted as normalized integrated densities per ( grey dots ) with bars corresponding to Mean ± Standard Error of Mean ( ) for at least 11 high - field units of at least 100 ; * P - value < 0 . 05 ( Student ' s t - , N = 11 ) .

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

ZO-1 quantification of IF images performed using the Fiji software and bar-plotted as normalized integrated densities per cell (grey dots) with bars corresponding to Mean ± Standard Error of the Mean (SEM) for at least 11 high-field units of at least 100 cells; * P-value < 0.05 (Student's t-test, N=11).


LMS ( red ) HMS ( black ) gene expression in Epi by random - primed RT - qPCR relative to RPL11 represented as log2 fold change to CTR . Each dot corresponds to an independent replicate , bars represent Mean ± Standard Deviation values .

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

LMS (red) and HMS (black) gene expression quantification in Epi cells by random-primed RT-qPCR relative to RPL11 and represented as log2 fold change to CTR. Each dot corresponds to an independent replicate, bars represent Mean ± Standard Deviation values.


Metagene profiling within 10 kb window around TSS of H3K27ac , LSD1 H3K4me2 over IgG in Epi expressing none ( CTR ) or different HOTAIR variants ( HOT , HOTΔP HOTΔL ) . bp stands for base pairs .

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

Metagene profiling within the 10 kb window around TSS of H3K27ac, LSD1 and H3K4me2 over IgG in Epi cells expressing none (CTR) or different HOTAIR variants (HOT, HOTΔP and HOTΔL). bp stands for base pairs.


of unsupervised hierarchical clustering on H3K27ac mean values at gene promoters showing separation of two phenotypic groups .

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

Heatmap of unsupervised hierarchical clustering based on H3K27ac mean values at gene promoters showing separation of two phenotypic groups.


Metagene profiling of H3K27ac , LSD1 H3K4me2 over IgG within 5 kb window of enhancers in Epi expressing none ( CTR ) or different HOTAIR variants ( HOT , HOTΔP HOTΔL ) . bp stands for base pairs .

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

Metagene profiling of H3K27ac, LSD1 and H3K4me2 over IgG within the 5 kb window of enhancers in Epi cells expressing none (CTR) or different HOTAIR variants (HOT, HOTΔP and HOTΔL). bp stands for base pairs.


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

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 O O

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

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

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

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

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 Western blot of same lysates from ( A ) , probed with antibodies recognizing ubiquitin or actin as a loading control . position of molecular markers is shown on left of each blot . position of UbG76V - GFP , as well as UbG76V - GFP with indicated number of additional ubiquitin moieties , on molecular , is indicated to right of each blot . Endogenous poly - ubiquitinated are also detected in 80 - 200 kDa range , as indicated by bracket . Twenty 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 B-MEASURED_VAR 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 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

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 expression of C . elegans CYP genes between indicated mutant 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 different CYP families subfamilies . Specific genes ( rows ) are clustered according to alignment ( CLUSTAL W ) to maintain relationship of their molecular evolution

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

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

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

O O O 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

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

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

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

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

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

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

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

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 MERVL : : tdTomato in MII oocyte at indicated stages ( upper ) . GAPDH was used as a loading control . Numbers below Western blots indicate band ( normalized to total GAPDH ) measured by using ImageJ software . 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 - 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 O O O O O O O O O O O O O O O O O

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 image of 2 - derived from ICSI or SCNT . SCNT produced by transfer of MERVL : : tdTomato cumulus into WT enucleated oocytes . ICSI produced by MERVL : : tdTomato sperm MII oocytes from of transgenic mice . 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

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

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
 t


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

O O O O O O O O O O O O O O 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. 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 - derived from ICSI or SCNT . Results were normalized on geometric mean of expression of two reference genes ( Ywhaz Gapdh ) . Error bars , s . e . m . , n = 3 . * * * P < 0 . 001 by two - 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

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
 t


H . Representative of tdTomato + tdTomato - derived from ICSI or SCNT , after 4 . 5 days of culturing in vitro . Scale bar , 100

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

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 tdTomato + tdTomato - derived from ICSI or SCNT . efficiency was calculated on number of 2 - that have been divided into tdTomato + 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 - of dynamics MERVL : : tdTomato expression during SCNT preimplantation development . selected from a series acquired every 15 min . time after starting observation is shown on 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

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

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

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

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 + tdTomato - SCNT retrieved at E6 . 5 . tdTomato + SCNT displays normal egg - cylinder . By contrast , tdTomato - SCNT shows abnormal . Epi , embryonic epiblast ; ExEm , extraembryonic ectoderm ; EPC , ectoplacental cone . 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 O O

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

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
 t


H . bar chart shows efficiency of ntES derivation . efficiency was calculated on total number of attached blastocysts for ntES derivation . N , total number of analyzed for each condition . Error bars , s . d . , n ≥ 3 . * * P < 0 . 01 by two - 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

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
 t


I . RT - qPCR for somatic genes in SCNT 1 - , 2 - tdTomato + , 2 - tdTomato - . Results were normalized on geometric mean of expression of two reference genes ( Ywhaz Gapdh ) . Error bars , s . e . m . , n = 3 . * * * P < 0 . 001 by two - 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

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
 t


A . Immunofluorescence of ICSI , SCNT - tdTomato + SCNT - tdTomato - . approximately 26hr after activation were fixed processed for immunostaining with antibodies to H3K27me3 Gag . ICSI produced by MERVL : : tdTomato sperm from of mice . are representative examples of shown in Fig 3B . Representative from ≥ 128 analyzed in five independent micromanipulations are shown . Scale bar , 20 μm . B . of 2 - ICSI SCNT with strong H3K27me3 moderate ( left ) . Numbers of total analyzed from five independent micromanipulations are shown in 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 - 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 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 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 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

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
 t


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

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

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


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

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 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

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

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

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


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

O O O O O B-MEASURED_VAR 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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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 O O O O O O

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

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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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 B-CONTROLLED_VAR O

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


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

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

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


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

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 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

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


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

O O O 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

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


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

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

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


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

O O O 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 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

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


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

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

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


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

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

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


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

O O O 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

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


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

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

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


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

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

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


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

O O O O O 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 B-MEASURED_VAR 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

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


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

O O O 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

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


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

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

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


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

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

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


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

O O O 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

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


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

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


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

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

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


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

O O O O O O O O B-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 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 B-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 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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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