( D ) Dually labeled D . discoideum Ax3 producing GFP fusions of MCS components either mCherry - Sac1 ( pSV044 ) or mCherry - Sac1 _ ΔTMD ( pSV045 ) were infected ( MOI 5 , 2 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) fixed with 4 % PFA . Merged for analyzed time points are shown . Scale bars : 3 μm . ( E ) IFC of dually labeled D . discoideum Ax3 producing GFP fusions of MCS components either mCherry - Sac1 ( pSV044 ) or mCherry - Sac1 _ ΔTMD ( pSV045 ) , infected ( MOI 5 , 2 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . of ( B ) P4C - mCherry ( C ) Sac1 - GFP or ( E ) GFP fusions of MCS components localizing to LCVs at 2 h p . i . Due to lower resolution of IFC , " LCVs " designates LCV limiting tightly attached ER . Number of events per sample , n = 5000 . Data information E ) : Data represent mean of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - ) .

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

(D) Dually labeled D. discoideum Ax3 producing GFP fusions of MCS components and either mCherry-Sac1 (pSV044) or mCherry-Sac1_ΔTMD (pSV045) were infected (MOI 5, 2 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (E) IFC analysis of dually labeled D. discoideum Ax3 producing GFP fusions of MCS components and either mCherry-Sac1 (pSV044) or mCherry-Sac1_ΔTMD (pSV045), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32 (pAW014). Quantification of (B) P4C-mCherry (C) Sac1-GFP or (E) GFP fusions of MCS components localizing to LCVs at 2 h p.i. Due to the lower resolution of IFC, "LCVs" designates the LCV limiting membrane and tightly attached ER. Number of events per sample, n = 5000. Data information E): Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 producing GFP - D4H * ( pSV046 ) or P4C - GFP ( pWS034 ) P4C - mCherry ( pWS032 ) , CnxA - mCherry ( pAW012 ) , or AmtA - mCherry were infected ( MOI 5 , 15 - 120 min ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) fixed with 4 % PFA . Merged for analyzed time points are shown . Scale bars : 3 μm . ( B - C ) Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) is shown for ( B ) AmtA - mCherry with respect to P4C - GFP or GFP - D4H * ( n = 100 - 150 per time point ) , ( C ) GFP - D4H * with respect to P4C - mCherry or CnxA - mCherry ( n = 100 - 150 each per time point ) . Data represent mean of three independent biological replicates ( * P < 0 . 05 , Student ` s t - ) .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Dually labeled D. discoideum Ax3 producing GFP-D4H* (pSV046) or P4C-GFP (pWS034) and P4C-mCherry (pWS032), CnxA-mCherry (pAW012), or AmtA-mCherry were infected (MOI 5, 15 - 120 min) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B-C) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for (B) AmtA-mCherry with respect to P4C-GFP or GFP-D4H* (n=100-150 per time point), and (C) GFP-D4H* with respect to P4C-mCherry or CnxA-mCherry (n=100-150 each per time point). Data represent mean and SEM of three independent biological replicates (*P<0.05, Student`s t-test).


( D ) Single labeled D . discoideum Ax3 producing either CnxA - GFP ( pAW016 ) or P4C - GFP ( pWS034 ) were infected ( MOI 5 , 15 - 120 min ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , fixed with 4 % PFA stained with filipin . Merged for analyzed time points are shown . Scale bars : 3 μm . ( E ) Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) is shown for filipin with respect to CnxA - GFP or P4C - GFP ( n = 80 - 90 each per time point ) . Data represent mean of means of three independent biological replicates ( * * * P < 0 . 001 , Student ` s t - ) .

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

(D) Single labeled D. discoideum Ax3 producing either CnxA-GFP (pAW016) or P4C-GFP (pWS034) were infected (MOI 5, 15-120 min) with mCherry-producing L. pneumophila JR32 (pNP102), fixed with 4 % PFA and stained with filipin. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (E) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for filipin with respect to CnxA-GFP or P4C-GFP (n=80-90 each per time point). Data represent mean and SEM of the means of three independent biological replicates (***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 producing P4C - mCherry ( pWS032 ) either CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 5 , 1 - 8 h ) with mCerulean - producing L . pneumophila JR32 , ΔlepB or ΔsidC ( pNP99 ) fixed with 4 % PFA . Merged for analyzed time points are shown . Scale bars : 3 μm .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(A) Dually labeled D. discoideum Ax3 producing P4C-mCherry (pWS032) and either CnxA-GFP (pAW016), GFP-Sac1 (pLS037), or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 1-8 h) with mCerulean-producing L. pneumophila JR32, ΔlepB or ΔsidC (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm.


( C ) Imaging flow cytometry ( IFC ) of dually labeled D . discoideum Ax3 producing P4C - mCherry ( pWS032 ) either CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , or GFP - Sac1 _ ΔTMD ( pSV034 ) , infected ( MOI 5 , 1 - 8 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Number of events per sample , n = 5000 . Data represent mean of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - ) .

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

(C) Imaging flow cytometry (IFC) analysis of dually labeled D. discoideum Ax3 producing P4C-mCherry (pWS032) and either CnxA-GFP (pAW016), GFP-Sac1 (pLS037), or GFP-Sac1_ΔTMD (pSV034), infected (MOI 5, 1-8 h) with mPlum-producing L. pneumophila JR32 (pAW014). Number of events per sample, n = 5000. Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( E ) Representative western blot of scr or miR - 30c transfected TREx analysed for ORF65 expression with GAPDH as a loading control . Densitometry performed on n = 3 . Data information : data are presented as mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - ) . repeats are biological .

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

(E) Representative western blot of scr or miR-30c transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( F ) Representative western blot of scr or miR - 29b transfected TREx analysed for ORF65 expression with GAPDH as a loading control . Densitometry performed on n = 3 . Data information : data are presented as mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - ) . repeats are biological .

(F) Representative western blot of scr or miR-29b transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( C ) Representative western blot of ORF65 in scr circHIPK3 KD lines with GAPDH as a loading control . Densitometry of n = 3 western blots . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - ) . repeats are biological .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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 western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control. Densitometry analysis of n=3 western blots. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( B ) Representative western blot of DLL4 expression in TREx 0 24 hours post induction . GAPDH was used a loading control ( n = 3 ) . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - ) . repeats are biological .

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

(B) Representative western blot of DLL4 expression in TREx cells 0 and 24 hours post induction. GAPDH was used a loading control (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( D ) Representative western blot of DLL4 in scr circHIPK3 stable TREx at 0 24 hours post induction with GAPDH as a loading control . Densitometry of n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - ) . repeats are biological .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 western blot of DLL4 levels in scr and circHIPK3 stable TREx cells at 0 and 24 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( F ) Representative western blot of DLL4 in scr or miR - 30c transfected TREx at 0 , 24 48 hours post induction with GAPDH as a loading control . Densitometry of n = 3 Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - ) . repeats are biological .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 western blot of DLL4 levels in scr or miR-30c transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3 Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( J ) Representative western blot of DLL4 in scr or miR - 30c antagomiR transfected TREx at 0 , 24 48 hours post induction with GAPDH as a loading control . Densitometry of n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - ) . repeats are biological .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blot of DLL4 levels in scr or miR-30c antagomiR transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( B ) Representative western blot of DLL4 in scr DLL4 KD TREx with GAPDH as a loading control , densitometry is n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - ) . repeats are biological .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 western blot of DLL4 levels in scr and DLL4 KD TREx cells with GAPDH as a loading control, densitometry analysis is n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( C ) Representative western blot of ORF65 at 0 , 24 48 hours post induction in scr DLL4 KD TREx , GAPDH was used as a loading control densitometry performed on n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - ) . repeats are biological .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 western blot of ORF65 levels at 0, 24 and 48 hours post induction in scr and DLL4 KD TREx cells, GAPDH was used as a loading control and densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


B ChIP gel showing relative enrichment by H3K4me3 in controls ( n = 9 ) PD ( n = 18 ) . PCR amplified a 188 - bp region of SNCA from intron 1 where H3K4me3 peak was at its optimum . Mouse IgG ( mIgG ) was used as control bands were normalized by unbiased amplification from respective inputs . Data information : Data represent mean ± standard error of mean .

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

B ChIP gel images showing the relative enrichment by H3K4me3 in controls (n=9) and PD patients (n= 18). PCR amplified a 188-bp region of SNCA from intron 1 where H3K4me3 peak was at its optimum. Mouse IgG (mIgG) was used as control and the bands were normalized by unbiased amplification from respective inputs. Data information: Data represent mean ± standard error of the mean.


C normalized of α - synuclein in entire cohort were divided into high ( n = 12 ) low . cut off value for determining threshold was set at median from subjects . high of α - synuclein were plotted with corresponding H3K4me3 values of those subjects . corresponding ID of each sample is shown next to each point in graph . There was a significant correlation between high of α - synuclein H3K4me3 . Data information : Data represent mean ± standard error of mean . Spearman rank correlation was performed in ( C ) . Two p value was calculated with Fisher ' s exact p .

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

C The normalized levels of α-synuclein in the entire cohort were divided into high (n = 12) and low levels. The cut off value for determining the threshold was set at the median from all the subjects. The high levels of α-synuclein were plotted with corresponding H3K4me3 values of those subjects. The corresponding ID of each sample is shown next to each point in the graph. There was a significant correlation between high levels of α-synuclein and H3K4me3. Data information: Data represent mean ± standard error of the mean. Spearman rank correlation test was performed in (C). Two tailed p value was calculated with Fisher's exact p.


D graph represents statistically significant difference of relative H3K4me3 enrichment between PD ( n = 7 ) controls ( n = 6 ) . Neuronal nuclei from PD brain samples show significantly higher enrichment of H3K4me3 at SNCA intron 1 ( p = 0 . 01 ) compared to controls . * P < 0 . 05 . Data were analyzed using non - parametric t - followed by Mann - Whitney post - hoc corrections . Two - p - values were calculated for . Data information : Data represent mean ± standard error of mean .

O O O O O O O O B-GENEPROD O O B-DISEASE O O O O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O B-DISEASE B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D The graph represents the statistically significant difference of relative H3K4me3 enrichment between PD (n=7) and controls (n=6). Neuronal nuclei from PD brain samples show significantly higher enrichment of H3K4me3 at SNCA intron 1 (p=0.01) compared to controls. *P < 0.05. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated for all. Data information: Data represent mean ± standard error of the mean.


corresponding α - synuclein C ) were evaluated in SH - SY5Y expressing dCas9 - 5xGCN4 with ( sgA - dCas9 ) or without scFV - sfGFP - JARID1A ( sgA - dCas9 - JA ) . C α - SYN in SH - SY5Y were also evaluated using western blot . of α - SYN was normalized to β - actin . Significant reduction of α - SYN was observed in expressing JARID1A . Three independent repeats were performed . of reduction is shown in graph . * * P < 0 . 01 . Data were analyzed using non - parametric t - followed by Mann - Whitney post - hoc corrections . Two - p - values were calculated . Data information : data are presented as mean ± .

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

The corresponding α-synuclein levels C) were evaluated in SH-SY5Y cells expressing dCas9-5xGCN4 with (sgA-dCas9) or without scFV-sfGFP-JARID1A (sgA-dCas9-JA). C α-SYN levels in SH-SY5Y cells were also evaluated using western blot. The levels of α-SYN was normalized to β-actin. Significant reduction of α-SYN was observed in cells expressing JARID1A. Three independent repeats were performed. Percentage of reduction is shown in the graph. **P <0.01. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated. Data information: All data are presented as mean ± SEM.


E ChIP on H3K4me3 at SNCA promoter between sPD1 - 1 lines after locus - specific epigenomic modulation . Differentiated were transiently transfected with either sgA - dCas9 5xGCN4 - scFV - JARID1A or sg A - dCas9 5xGCN4 - scFV - empty backbone vectors . A significant reduction in H3K4me3 at SNCA promoter was observed in transfected with JARID1A . Data information : Three independent repeats were performed for both ChIP experiments . * P < 0 . 05 ; * * * * P < 0 . 0001 . Data were analyzed using non - parametric t - followed by Mann - Whitney post - hoc corrections . One - p - values were calculated for ChIP

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

E ChIP on H3K4me3 at the SNCA promoter between sPD1-1 lines after locus-specific epigenomic modulation. Differentiated cells were transiently transfected with either sgA-dCas9 5xGCN4-scFV-JARID1A or sg A-dCas9 5xGCN4-scFV-empty backbone vectors. A significant reduction in H3K4me3 at the SNCA promoter was observed in cells transfected with JARID1A. Data information: Three independent repeats were performed for both ChIP experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. One-tailed p-values were calculated for ChIP analysis


F Western blot of α - synuclein ( α - SYN ) in under same conditions as analyzed in ( E ) . A significant decrease ( 56 - 66 % ) in α - SYN was observed in transfected with JARID1A . normalized relative expression of α - SYN in JARID1A transfected are shown as a of control ( transfected by empty backbone vector ) . Data information : Three independent repeats were performed for western blot experiments . * P < 0 . 05 ; * * * * P < 0 . 0001 . Data were analyzed using non - parametric t - followed by Mann - Whitney post - hoc corrections . two - p - value was calculated for western blot experiments . data are presented as mean ± .

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

F Western blot analysis of α-synuclein (α-SYN) levels in the cells under the same conditions as analyzed in (E). A significant decrease (56-66%) in α-SYN levels was observed in cells transfected with JARID1A. The normalized and relative expression of α-SYN in JARID1A transfected cells are shown as a percentage of control (transfected by empty backbone vector). Data information: Three independent repeats were performed for western blot experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. two-tailed p-value was calculated for western blot experiments. All data are presented as mean ± SEM.


( 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 B-CELL_LINE O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O B-ORGANISM O O O B-CELL_LINE O O O B-CELL_LINE O O O O B-TISSUE O O B-TISSUE I-TISSUE O O O O O O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O B-DISEASE O B-TISSUE I-TISSUE O O O O O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-CELL_LINE I-CELL_LINE B-ORGANISM B-DISEASE O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O 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 ± .

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

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 B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-DISEASE B-TISSUE I-TISSUE 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 B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

(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 B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O

(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 B-TISSUE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY 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-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD 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-GENEPROD 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 .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD 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-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

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 B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O 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 B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O B-GENEPROD B-CELL_TYPE O B-CELL_TYPE O O O B-ORGANISM 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

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

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

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

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 .

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

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

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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O B-GENEPROD O B-TISSUE O B-GENEPROD O B-TISSUE 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE 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 B-EXP_ASSAY O B-CELL_TYPE O O B-TISSUE O O O O O O B-EXP_ASSAY O B-TISSUE O O B-CELL_TYPE O O O O O O O O O O O O O O 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 B-CELL_TYPE O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-TISSUE O B-CELL_TYPE O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE B-GENEPROD O O B-TISSUE I-TISSUE B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY 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 B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE 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 B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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


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


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

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

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

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-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O B-ORGANISM O O O O B-ORGANISM B-TISSUE B-CELL_TYPE O O O O 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

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

O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O B-DISEASE O B-CELL_TYPE O O O O O B-DISEASE O B-ORGANISM O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O

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


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

O O B-EXP_ASSAY O O O B-DISEASE O B-CELL_TYPE B-EXP_ASSAY O O O O O O O

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 .

O O O O B-DISEASE O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O O O O

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 -

O O O B-EXP_ASSAY O O O B-ORGANISM O O O O B-DISEASE O

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 ) .

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

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

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 B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY O B-GENEPROD O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY O O O O O B-ORGANISM O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

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


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

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


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

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

(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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.