( B - D ) The Pearson ' s correlation coefficients were generated using Coloc 2 from Fiji ( ImageJ ) and are shown for MCS components fused to GFP with respect to ( B ) CnxA - mCherry ( n = 146 ) , ( C ) P4C - mCherry ( n = 138 ) , or ( D ) AmtA - mCherry ( n = 159 ) . Data represent mean and SEM of three independent biological replicates ( statistics refer to JR32 - infected D . discoideum producing GFP - Sac1 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(B-D) The Pearson's correlation coefficients were generated using Coloc 2 from Fiji (ImageJ) and are shown for MCS components fused to GFP with respect to (B) CnxA-mCherry (n=146), (C) P4C-mCherry (n=138), or (D) AmtA-mCherry (n=159). Data represent mean and SEM of three independent biological replicates (statistics refer to JR32-infected D. discoideum producing GFP-Sac1; *P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 producing mCherry - Vap ( pSV048 ) and OSBP8 - GFP ( pMIB89 ) or GFP - OSBP11 ( pMIB39 ) were infected ( MOI 5 , 1 - 8 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 ( insets : 1 μm ) .

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

(A) Dually labeled D. discoideum Ax3 producing mCherry-Vap (pSV048) and OSBP8-GFP (pMIB89) or GFP-OSBP11 (pMIB39) were infected (MOI 5, 1-8 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 (insets: 1 μm).


( B ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for OSBP8 or OSBP11 fused to GFP with respect to mCherry - Vap ( n = 100 - 150 each per time point ) . Data represent mean and SEM of three independent biological replicates ( * * P < 0 . 01 , Student ` s t - test ) .

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

(B) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for OSBP8 or OSBP11 fused to GFP with respect to mCherry-Vap (n=100-150 each per time point). Data represent mean and SEM of three independent biological replicates (**P<0.01, Student`s t-test).


( A ) D . discoideum Ax3 , or the ∆ vap , ∆ osbG , ∆ osbH , or ∆ osbK mutant strains producing GFP ( pDM317 ) or GFP fusions of the MCS components were infected ( MOI 1 ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to JR32 infected D . discoideum Ax3 producing GFP ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) . To improve clarity , the different mutant strains are shown in separate graphs , each depicting the same data for Ax3 / GFP .

O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) D. discoideum Ax3, or the ∆vap, ∆osbG, ∆osbH, or ∆osbK mutant strains producing GFP (pDM317) or GFP fusions of the MCS components were infected (MOI 1) with mCherry-producing L. pneumophila JR32 (pNP102), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to JR32 infected D. discoideum Ax3 producing GFP; *P<0.05; **P<0.01; ***P<0.001, Student`s t-test). To improve clarity, the different mutant strains are shown in separate graphs, each depicting the same data for Ax3/GFP.


( B ) D . discoideum Ax3 producing CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , GFP - Sac1 _ C383S ( pSV015 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 1 ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to JR32 infected D . discoideum Ax3 producing GFP - Sac1 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(B) D. discoideum Ax3 producing CnxA-GFP (pAW016), GFP-Sac1 (pLS037), GFP-Sac1_C383S (pSV015) or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 1) with mCherry-producing L. pneumophila JR32 (pNP102), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to JR32 infected D. discoideum Ax3 producing GFP-Sac1; ***P<0.001, Student`s t-test).


( C ) RAW 264 . 7 macrophages were treated with increasing concentrations of OSW - 1 ( 0 - 100 nM , 1 - 60 h ) , infected ( MOI 1 ) with GFP - producing L . pneumophila JR32 ( pNT28 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to infected cells treated with 0 nM OSW - 1 ; * P < 0 . 05 ; * * * P < 0 . 001 , Student ` s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RAW 264.7 macrophages were treated with increasing concentrations of OSW-1 (0-100 nM, 1-60 h), infected (MOI 1) with GFP-producing L. pneumophila JR32 (pNT28), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to infected cells treated with 0 nM OSW-1; *P<0.05; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and CnxA - GFP ( pAW016 ) , or Ax3 producing P4C - mCherry and either GFP - Sac1 ( pLS037 ) , GFP - Sac1 _ C383S ( pSV015 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 5 , 2 - 16 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 . ( B ) LCV area was measured using ImageJ ( n = 100 - 200 per condition from 3 independent biological replicates ) . Means and SEM of single cells are shown ( * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(A) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and CnxA-GFP (pAW016), or Ax3 producing P4C-mCherry and either GFP-Sac1 (pLS037), GFP-Sac1_C383S (pSV015) or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 2-16 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. (B) LCV area was measured using ImageJ (n=100-200 per condition from 3 independent biological replicates). Means and SEM of single cells are shown (**P<0.01; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and GFP - Sac1 ( pLS037 ) were infected ( MOI 5 , 2 - 16 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 . ( B , C ) Imaging flow cytometry ( IFC ) analysis of dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and either GFP - Sac1 ( pLS037 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) , infected ( MOI 5 , 2 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Data information ( B , C , : Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(A) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and GFP-Sac1 (pLS037) were infected (MOI 5, 2-16 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. (B, C) Imaging flow cytometry (IFC) analysis of dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and either GFP-Sac1 (pLS037) or GFP-Sac1_ΔTMD (pSV034), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32 (pAW014). Data information (B, C, : Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( 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 ) .

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

(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 ) 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 ) .

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

(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 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 ) .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 ) .

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

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

(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 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 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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

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

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

(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 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 .

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

(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 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 .

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

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 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 .

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

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 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 .

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

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.


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 .

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

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 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

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

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 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 .

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

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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O 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.


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 ) .

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

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


( D ) SOCE in 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 .

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

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


( E ) Single 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and GAPDH protein levels assessed in Epi cells by Western blot of whole protein extracts .

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

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


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 ) .

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

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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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


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

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

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


Metagene profiling of H3K27ac , LSD1 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 .

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

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


J Quantified 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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


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

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


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

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

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


D Abundance of 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

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

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


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

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

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


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

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

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

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

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


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

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


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

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

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


E . 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

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

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

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


G . RT - qPCR data for select ZGA genes activated following MERVL : : tdTomato expression in mouse 2 - cell embryos derived from ICSI or SCNT . Results were normalized based on the geometric mean of the expression levels of two reference genes ( Ywhaz and Gapdh ) . Error bars , s . e . m . , n = 3 . * * * P < 0 . 001 by two - tailed Student " s

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G. RT-qPCR data for select ZGA genes activated following MERVL::tdTomato expression in mouse 2-cell embryos derived from ICSI or SCNT. Results were normalized based on the geometric mean of the expression levels of two reference genes (Ywhaz and Gapdh). Error bars, s.e.m., n = 3. ***P < 0.001 by two-tailed Student"s
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H . Representative images of tdTomato + and tdTomato - embryos derived from ICSI or SCNT , after 4 . 5 days of culturing in vitro . Scale bar , 100

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


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

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


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

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

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


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

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


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

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


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

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


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

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

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H. The bar chart shows the efficiency of ntES derivation. The efficiency was calculated based on the total number of attached blastocysts for ntES derivation. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 3. **P < 0.01 by two-tailed Student"s
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I . RT - qPCR analysis for somatic cell genes in SCNT 1 - cell embryos , 2 - cell tdTomato + , and 2 - cell tdTomato - embryos . Results were normalized based on the geometric mean of the expression levels of two reference genes ( Ywhaz and Gapdh ) . Error bars , s . e . m . , n = 3 . * * * P < 0 . 001 by two - tailed Student " s

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

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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( C ) ( Left panel ) 8 - oxo - dG ( green ) preferentially adopts 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 ) .

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


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

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