Figure 3Western blot analysis ( probing with an anti - FLAG mAb ) to detect cleavage of HybO with ( + ) / without ( - ) chromosomal ( native ) or pBAD33 - encoded rhomboids ( plasmid ) . homboid substrates that are uncleaved , are marked by black , rrows , ild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of the catalytic serine and histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 . Western blot analysis to detect cleavage of HybA with ( + ) / without ( - ) chromosomal ( native ) or pBAD33 - encoded rhomboids ( plasmid ) . rhomboid substrates that are uncleaved , cleaved by GlpG , or by Rhom7 , are marked by black , red , and blue arrows , respectively . Wild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of the catalytic serine and histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 . Western blot analysis to detect cleavage of HybAG296F by endogenous or pBAD33 - encoded rhomboid . Wild - type S . sonnei , Ss . rhomboid substrates that are uncleaved , cleaved by GlpG , or by Rhom7 , re marked by black , red , and blue arrows , espectively . ild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of the catalytic serine and histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 . Western blot analysis to detect cleavage of HybAP300A by endogenous or pBAD33 - encoded rhomboid . rhomboid substrates that are uncleaved , cleaved by GlpG , or by Rhom7 , are marked by black , red , and blue arrows , respectively . Wild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of the catalytic serine and histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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

Figure 3Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of HybO with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). homboid substrates that are uncleaved, are marked by black, rrows, ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.Western blot analysis to detect cleavage of HybA with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.Western blot analysis to detect cleavage of HybAG296F by endogenous or pBAD33-encoded rhomboid. Wild-type S. sonnei, Ss. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, re marked by black, red, and blue arrows, espectively. ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.Western blot analysis to detect cleavage of HybAP300A by endogenous or pBAD33-encoded rhomboid. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.


Figure 4A Bacterial two - hybrid analysis with HybA and / or HybB fused chromosomally with the T25 and T18 domains of B . pertussis CyaA , respectively in the absence of endogenous CyaA . Bacteria were grown on LB agar containing 20 µg / ml X - gal at 37 ° C for 14 hours in the presence / absence of O2 . Scale bar , 1 cm . B Western blot analysis to detect HybA cleavage in S . sonneiΔrhom7 by wild - type ( + ) or inactive ( S201A ) GlpG expressed chromosomally ( native ) or from pUC19 ( plasmid ) with ( + ) / without ( - ) HybB . HybA was expressed from its native locus or a plasmid ( pHybA ) . HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows , respectively . C Quantification of the ratio of cleaved / uncleaved HybA in strains with ( + ) or without ( - ) HybB . mean ± S . D . of three experiments . * , p < 0 . 05 ; * * * * , p < 0 . 0001 ( One - way ANOVA ) . D Western blot analysis to detect GlpG - mediated cleavage of native tagged HybA at indicated times after blocking protein translation by the addition of chloramphenicol at T0 in the absence of HybB ( - ) . , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows , respectively . E Western blot analysis to detect GlpG - mediated cleavage of C - terminally sfCherry - 3xFLAG tagged HybA at times after blocking protein translation by the addition of chloramphenicol at T0 in the presence of HybB ( + ) . , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows , respectively . F Growth of bacteria lacking hydrogenases ( ΔhyaA - F ΔhycE + / - ΔhybO - G ) or GlpG ( ΔglpG ) , or expressing uncleavable HybB ( hybAG296F ) in 5 % H2G H2 consumption by S . sonnei . Bacteria were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0 . 5 % fumarate , 12 . 5 μg / ml nicotinic acid and 0 . 2 % casamino acids in a sealed glass chromatography vial . The headspace was purged with 10 % H2 / 90 % argon and cultures were incubated at 37 ° C with shaking at 180 rpm for 9 hours . H2 in the headspace was sampled and measured by gas chromatography . H S . sonnei growth in 10 % H2 with plasmid expressed wild - type or non - functional ( pglpGSAHA ) GlpG .

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

Figure 4A Bacterial two-hybrid analysis with HybA and/or HybB fused chromosomally with the T25 and T18 domains of B. pertussis CyaA, respectively in the absence of endogenous CyaA. Bacteria were grown on LB agar containing 20 µg/ml X-gal at 37°C for 14 hours in the presence/absence of O2. Scale bar, 1 cm.B Western blot analysis to detect HybA cleavage in S. sonneiΔrhom7 by wild-type (+) or inactive (S201A) GlpG expressed chromosomally (native) or from pUC19 (plasmid) with (+)/without (-) HybB. HybA was expressed from its native locus or a plasmid (pHybA). HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.C Quantification of the ratio of cleaved/uncleaved HybA in strains with (+) or without (-) HybB. mean ± S.D. of three experiments. *, p<0.05; ****, p<0.0001 (One-way ANOVA).D Western blot analysis to detect GlpG-mediated cleavage of native tagged HybA at indicated times after blocking protein translation by the addition of chloramphenicol at T0 in the absence of HybB (-). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.E Western blot analysis to detect GlpG-mediated cleavage of C-terminally sfCherry-3xFLAG tagged HybA at times after blocking protein translation by the addition of chloramphenicol at T0 in the presence of HybB (+). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.F Growth of bacteria lacking hydrogenases (ΔhyaA-F ΔhycE +/- ΔhybO-G) or GlpG (ΔglpG), or expressing uncleavable HybB (hybAG296F) in 5% H2G H2 consumption by S. sonnei. Bacteria were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0.5% fumarate, 12.5 μg/ml nicotinic acid and 0.2% casamino acids in a sealed glass chromatography vial. The headspace was purged with 10% H2/90% argon and cultures were incubated at 37 °C with shaking at 180 rpm for 9 hours. H2 in the headspace was sampled and measured by gas chromatography.H S. sonnei growth in 10% H2 with plasmid expressed wild-type or non-functional (pglpGSAHA) GlpG.


Figure 5B Western blot analysis ( probing with an anti - FLAG mAb ) to detect cleavage of FdoH cleavage in S . sonneiΔrhom7 with ( + ) / without ( - ) FdoI . rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red arrows , respectively . C Western blot analysis to detect cleavage of FdnH in S . sonneiΔglpG with ( + ) or without ( - ) chromosomal Rhom7 ( native ) , or wild - type ( + ) / inactive ( SAHA ) Rhom7 expressed from pBAD33 in S . sonneiΔglpGΔrhom7 with ( + ) or without ( - ) FdnI . rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , and blue arrows , respectively . E Western blot analysis to detect cleavage of wild - type ( WT ) or uncleavable ( P300A ) HybA by active ( + ) or inactive ( S201A ) chromosomal GlpG in the presence ( + ) or absence ( - ) of HybB . rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red arrows , respectively . F Western blot analysis to detect cleavage of wild - type ( WT ) or modified ( P259A ) FdoH by active ( + ) or inactive ( S201A ) chromosomal GlpG in the presence ( + ) or absence ( - ) of FdoI . rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red arrows , respectively . G Western blot analysis to detect cleavage of wild - type ( WT ) or modified ( P259A ) FdnH with active ( + ) or inactive ( SAHA ) Rhom7 expressed from pBAD33 with ( + ) or without ( - ) FdnI . rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , blue arrows , respectively .

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

Figure 5B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of FdoH cleavage in S. sonneiΔrhom7 with (+)/without (-) FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.C Western blot analysis to detect cleavage of FdnH in S. sonneiΔglpG with (+) or without (-) chromosomal Rhom7 (native), or wild-type (+)/inactive (SAHA) Rhom7 expressed from pBAD33 in S. sonneiΔglpGΔrhom7 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively.E Western blot analysis to detect cleavage of wild-type (WT) or uncleavable (P300A) HybA by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of HybB. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.F Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdoH by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.G Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdnH with active (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.


Figure 6A Western blot analysis ( probing with an anti - V5 mAb ) to detect degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P300A ) HybA in S . sonneiΔrhom7 chromosomally expressing wild - type ( + ) or inactive ( S201A ) GlpG with ( + ) or without ( - ) HybB . Rhomboid substrates that are uncleaved , cleaved by GlpG , , are marked by black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows . B Degradation of V5 - tagged HybA at times after blocking protein translation at T0 in the presence ( + ) or absence ( - ) of HybB . Rhomboid substrates that are uncleaved , cleaved by GlpG , black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows . C Western blot analysis of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdoH in S . sonneiΔrhom7 chromosomally expressing wild - type ( + ) or inactive ( S201A ) GlpG with ( + ) / without ( - ) FdoI . Rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red , arrows , respectively . D Western blot analysis of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdnH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( SAHA ) Rhom7 expressed from pBAD33 with ( + ) / without ( - ) FdnI . Rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , blue arrows , respectively . Degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdoH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( S201A , SAHA ) GlpG expressed chromosomally ( native ) or from pUC19 ( plasmid ) without FdoI , + / - exposure to 400 µM CuCl2 for 30 min . : Rhomboid substrates that are uncleaved , cleaved by GlpG , are marked by black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows . F Degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdnH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( S201A , SAHA ) GlpG expressed from pUC19 ( plasmid ) without FdnI ( - ) , , + / - exposure to 400 µM CuCl2 for 30 min . Rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , and blue arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

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

Figure 6A Western blot analysis (probing with an anti-V5 mAb) to detect degradation of N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+) or without (-) HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, , are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.B Degradation of V5-tagged HybA at times after blocking protein translation at T0 in the presence (+) or absence (-) of HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.C Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+)/without (-) FdoI. Rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively.D Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+)/without (-) FdnI. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI , +/- exposure to 400 µM CuCl2 for 30 min. : Rhomboid substrates that are uncleaved, cleaved by GlpG, are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.F Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed from pUC19 (plasmid) without FdnI (-), , +/- exposure to 400 µM CuCl2 for 30 min. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.


Figure 7A Western blot analysis probing the localisation and status of plasmid - encoded N - terminally V5 - tagged wild - type ( WT ) or modified ( P300A ) HybA in S . sonneiΔrhom7ΔhybB with wild - type ( + ) or inactive ( SAHA ) GlpG expressed from pUC19 . Whole cell lysate ( Whole cell . ) , Sol ( Sol . ) , Mem ( detergent - solubilised , Mem . ) , and the Aggregate ( Agg . ) fractions are shown . HybA that is uncleaved , cleaved only by GlpG , and further degraded post GlpG cleavage is marked by black , red , and green arrows , respectively .

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

Figure 7A Western blot analysis probing the localisation and status of plasmid-encoded N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7ΔhybB with wild-type (+) or inactive (SAHA) GlpG expressed from pUC19. Whole cell lysate (Whole cell.), Sol (Sol.), Mem (detergent-solubilised, Mem.), and the Aggregate (Agg.) fractions are shown. HybA that is uncleaved, cleaved only by GlpG, and further degraded post GlpG cleavage is marked by black, red, and green arrows, respectively.


Figure 1B Identification of sec homozygotes . LB represents the left border primer of the T - DNA insertion . LP and RP represent the left and right genomic primers , respectively . C qRT - PCR analysis of SEC mRNA levels in sec - 4 and sec - 5 mutants . The expression level was normalized to that of TUBULIN , a reference gene for qRT - PCR . Independent biological experiments were repeated three times , and one representative result is shown here . Data shown are means ± standard deviation ( s . d . ) , n = 3 . D The early flowering phenotype of sec - 5 mutant under long - day ( LD ) conditions . Scale bar : 1 cm . E qRT - PCR analysis of the expression levels of FLC , SOC1 , and MAF1 - 3 in 12 - d - old vegetative Col - 0 , sec - 4 and sec - 5 plants . Independent biological experiments were repeated three times , and one representative result is shown here . The expression level was normalized to that of TUBULIN . Data are means ± s . d . , n = 3 . ( * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 05 . F The sec - 5 mutant exhibits an early flowering phenotype under short - day ( SD ) conditions . Scale bar : 1 cm . G qRT - PCR analysis of FLC and SOC1 expression in Col - 0 and sec - 5 plants under SD conditions . The expression level was normalized to that of TUBULIN . Experiments were repeated three times , and one representative result is shown here . Data are mean ± s . d . , n = 3 . ( * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 05 .

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

Figure 1B Identification of sec homozygotes. LB represents the left border primer of the T-DNA insertion. LP and RP represent the left and right genomic primers, respectively.C qRT-PCR analysis of SEC mRNA levels in sec-4 and sec-5 mutants. The expression level was normalized to that of TUBULIN, a reference gene for qRT-PCR. Independent biological experiments were repeated three times, and one representative result is shown here. Data shown are means ± standard deviation (s.d.), n = 3.D The early flowering phenotype of sec-5 mutant under long-day (LD) conditions. Scale bar: 1 cm.E qRT-PCR analysis of the expression levels of FLC, SOC1, and MAF1-3 in 12-d-old vegetative Col-0, sec-4 and sec-5 plants. Independent biological experiments were repeated three times, and one representative result is shown here. The expression level was normalized to that of TUBULIN. Data are means ± s.d., n = 3. (*), Statistical significance (two-tailed t-test) with P < 0.05.F The sec-5 mutant exhibits an early flowering phenotype under short-day (SD) conditions. Scale bar: 1 cm.G qRT-PCR analysis of FLC and SOC1 expression in Col-0 and sec-5 plants under SD conditions. The expression level was normalized to that of TUBULIN. Experiments were repeated three times, and one representative result is shown here. Data are mean ± s.d., n = 3. (*), Statistical significance (two-tailed t-test) with P < 0.05.


Figure 2B ChIP - qPCR assay of H3K4me2 levels of indicated regions at FLC chromatin . C ChIP - qPCR assay of H3K4me3 levels of indicated regions at FLC chromatin . D ChIP - qPCR assay of H3K36me3 levels of indicated regions at FLC chromatin . E ChIP - qPCR assay of H3K27me3 levels of indicated regions at FLC chromatin . Data information : For ChIP analysis , 12 - d - old plants were collected and three independent experiments were conducted . Each bar represents the mean ± s . d . of three independent experiments , n = 3 . The relative abundance was normalized to the input .

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

Figure 2B ChIP-qPCR assay of H3K4me2 levels of indicated regions at FLC chromatin. C ChIP-qPCR assay of H3K4me3 levels of indicated regions at FLC chromatin. D ChIP-qPCR assay of H3K36me3 levels of indicated regions at FLC chromatin. E ChIP-qPCR assay of H3K27me3 levels of indicated regions at FLC chromatin .Data information: For ChIP analysis, 12-d-old plants were collected and three independent experiments were conducted. Each bar represents the mean ± s.d. of three independent experiments, n=3. The relative abundance was normalized to the input.


Figure 3A Yeast two - hybrid assay of the interaction between ATX1 and SEC . The full - length coding sequence ( CDS ) of ATX1 was cloned into the bait vector pGBKT7 . A full - length SEC CDS and a truncated fragment were cloned into the prey vector pGADT7 to express complete or truncated SEC - N ( including only TPR domains at the N - terminus ) , respectively . AD vectors expressing SEC or SEC - N were each cotransformed with BD - ATX1 . SD / 2 , SD / - Leu / - Trp medium ; SD / 4 , SD / - Ade / - His / - Leu / - Trp medium . Cotransformed pGBKT7 - 53 / pGADT7 - T was used as a positive control . B Immunoblot analysis for O - GlcNAcylation of ATX1 by SEC using anti - CTD110 . 6 antibody . HA - tagged ATX1 was expressed alone or together with Flag - tagged SEC in tobacco leaves . Nuclear proteins were extracted from tobacco mesophyll cells , and expression of FLAG - SEC and ATX1 - HA was confirmed by western blotting . C Flowering phenotype of 35S : : ATX1 - HA overexpression transgenic lines in Col - 0 and sec - 5 backgrounds , respectively . Scale bar = 1 cm . D Immunoblot analysis using anti - HA antibody to confirm ATX1 - HA expression in Col - 0 and 35S : : ATX1 - HA - overexpressing lines ACTIN was used as a loading control . Flowering time phenotype analysis for Col - 0 , sec - 5 and 35S : : ATX1 - HA transgenic lines Experiments were repeated three times , for each line , a total of 62 plants was counted for statistical analysis of rosette leaf numbers Data are mean ± s . d . , ( * * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 01 . n . s . , not significant . Flowering time phenotype analysis for Col - 0 , sec - 5 and 35S : : ATX1 - HA transgenic lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination , respectively . Experiments were repeated three times , for each line , a total of 62 plants was counted for statistical analysis of visible buds percent and bolting rates . Data are mean ± s . d . , ( * * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 01 . n . s . , not significant .

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

Figure 3A Yeast two-hybrid assay of the interaction between ATX1 and SEC. The full-length coding sequence (CDS) of ATX1 was cloned into the bait vector pGBKT7. A full-length SEC CDS and a truncated fragment were cloned into the prey vector pGADT7 to express complete or truncated SEC-N (including only TPR domains at the N-terminus), respectively. AD vectors expressing SEC or SEC-N were each cotransformed with BD-ATX1. SD/2, SD/-Leu/-Trp medium; SD/4, SD/-Ade/-His/-Leu/-Trp medium. Cotransformed pGBKT7-53/pGADT7-T was used as a positive control.B Immunoblot analysis for O-GlcNAcylation of ATX1 by SEC using anti-CTD110.6 antibody. HA-tagged ATX1 was expressed alone or together with Flag-tagged SEC in tobacco leaves. Nuclear proteins were extracted from tobacco mesophyll cells, and expression of FLAG-SEC and ATX1-HA was confirmed by western blotting.C Flowering phenotype of 35S::ATX1-HA overexpression transgenic lines in Col-0 and sec-5 backgrounds, respectively. Scale bar = 1 cm.D Immunoblot analysis using anti-HA antibody to confirm ATX1-HA expression in Col-0 and 35S::ATX1-HA-overexpressing lines ACTIN was used as a loading control.Flowering time phenotype analysis for Col-0, sec-5 and 35S::ATX1-HA transgenic lines Experiments were repeated three times, for each line, a total of 62 plants was counted for statistical analysis of rosette leaf numbers Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. n.s., not significant.Flowering time phenotype analysis for Col-0, sec-5 and 35S::ATX1-HA transgenic lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Experiments were repeated three times, for each line, a total of 62 plants was counted for statistical analysis of visible buds percent and bolting rates. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. n.s., not significant.


Figure 4A Detection and conformation of O - GlcNAc modification of His - ATX1ΔN catalyzed by His - SECΔN in vitro . His - ATX1ΔN ( expressing residues 592 - 952 of the C - terminus ) and His - SECΔN ( expressing residues 801 - 1062 of the C - terminus ) were recombinantly expressed and affinity purified separately . O - GlcNAcylation of His - ATX1ΔN was detected by anti - CTD110 . 6 antibody , and further conformed by β - elimination analysis . B His - SECΔN activates His - ATX1ΔN in vitro . Histone methyltransferase activity of ATX1ΔN was detected with or without recombinant SECΔN . H3K4A : mutated H3 in which the fourth amino acid , a lysine ( K ) , was replaced with alanine ( A ) . C Analysis of ATX1 O - GlcNAc modification in wild - type and sec - 5 plants . Total soluble protein extracts from 12 - d - old seedlings were subjected to SDS - PAGE followed by immunoblotting using the indicated antibodies . CBB : Coomassie brilliant blue staining , showing relative protein loading amount . D Loss of SEC function reduced ATX1 activity in Arabidopsis . Nuclear proteins were extracted and ATX1 was immunoprecipitated with anti - ATX1 antibody from wild - type and sec - 5 mutant plants , respectively , and then used for histone H3K4 methyltransferase activity analysis with recombinant H3 as catalyzing substrate . Band intensities were quantified with Image J . The H3 signal was first normalized by input signal and then was used for H3K4me3 signal normalization . E Comparison of ATX1 protein levels in Col - 0 and sec - 5 plants at stages before and after flowering . Seedlings were collected for protein extraction at 21 days ( before flowering ) and 35 days ( after flowering ) after seeds were planted on plates . Anti - ATX1 antibody was used for immunoblot assay .

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

Figure 4A Detection and conformation of O-GlcNAc modification of His-ATX1ΔN catalyzed by His-SECΔN in vitro. His-ATX1ΔN (expressing residues 592-952 of the C-terminus) and His-SECΔN (expressing residues 801-1062 of the C-terminus) were recombinantly expressed and affinity purified separately. O-GlcNAcylation of His-ATX1ΔN was detected by anti-CTD110.6 antibody, and further conformed by β-elimination analysis.B His-SECΔN activates His-ATX1ΔN in vitro. Histone methyltransferase activity of ATX1ΔN was detected with or without recombinant SECΔN. H3K4A: mutated H3 in which the fourth amino acid, a lysine (K), was replaced with alanine (A).C Analysis of ATX1 O-GlcNAc modification in wild-type and sec-5 plants. Total soluble protein extracts from 12-d-old seedlings were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. CBB: Coomassie brilliant blue staining, showing relative protein loading amount.D Loss of SEC function reduced ATX1 activity in Arabidopsis. Nuclear proteins were extracted and ATX1 was immunoprecipitated with anti-ATX1 antibody from wild-type and sec-5 mutant plants, respectively, and then used for histone H3K4 methyltransferase activity analysis with recombinant H3 as catalyzing substrate. Band intensities were quantified with Image J. The H3 signal was first normalized by input signal and then was used for H3K4me3 signal normalization.E Comparison of ATX1 protein levels in Col-0 and sec-5 plants at stages before and after flowering. Seedlings were collected for protein extraction at 21 days (before flowering) and 35 days (after flowering) after seeds were planted on plates. Anti-ATX1 antibody was used for immunoblot assay.


Figure 5A Mutation of either S947 alone or all 12 serine and threonine residues in the SET domain reduced O - GlcNAc modification level of ATX1ΔN . B Site mutation of S947 or all 12 serine and threonine residues in the ATX1 SET domain inhibited the activation of ATX1 by SEC ; moreover , mutation of five conserved amino acids in the SEC functional domain inhibited SEC activity . C Quantitative analysis of HKMT activity of ATX1ΔN , ATX1ΔN - 12m , and ATX1ΔN - m and the effect of SECΔN and ΔSEC5m in activating those proteins . Experiments were repeated three times . Error bars are SD . His - ΔSEC5m and His - SECΔN were used as the control .

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

Figure 5A Mutation of either S947 alone or all 12 serine and threonine residues in the SET domain reduced O-GlcNAc modification level of ATX1ΔN.B Site mutation of S947 or all 12 serine and threonine residues in the ATX1 SET domain inhibited the activation of ATX1 by SEC; moreover, mutation of five conserved amino acids in the SEC functional domain inhibited SEC activity.C Quantitative analysis of HKMT activity of ATX1ΔN, ATX1ΔN-12m, and ATX1ΔN-m and the effect of SECΔN and ΔSEC5m in activating those proteins. Experiments were repeated three times. Error bars are SD. His-ΔSEC5m and His-SECΔN were used as the control.


Figure 6A Flowering phenotypes of Col - 0 , atx1 - 2 , atx1 - 2 35S : : ATX1 - FLAG , and atx1 - 2 35S : : ATX1m - FLAG ( with Ser947 replaced with alanine ) plants under LD conditions . Scale bar : 1 cm . Two independent lines are shown for each transformation . B Quantitative real - time PCR analysis of ATX1 and FLC transcript levels in Col - 0 , atx1 - 2 , atx1 - 2 35S : : ATX1 - FLAG , and atx1 - 2 35S : : ATX1m - FLAG plants . The transcript levels were normalized to that of TUBULIN . Data are mean ± s . d . of three independent biological replicates , n = 3 . Flowering time phenotype analysis of lines Three independent biological repeats were conducted , and data from a total of 71 plants were used for statistical analysis . Data are mean ± s . d . , ( * * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 01 . Flowering time phenotype analysis of lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination , respectively . Three independent biological repeats were conducted , and data from a total of 71 plants were used for statistical analysis . Data are mean ± s . d . , ( * * ) , Statistical significance ( two - tailed t - test ) with P < 0 . 01 .

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

Figure 6A Flowering phenotypes of Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG (with Ser947 replaced with alanine) plants under LD conditions. Scale bar: 1 cm. Two independent lines are shown for each transformation.B Quantitative real-time PCR analysis of ATX1 and FLC transcript levels in Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG plants. The transcript levels were normalized to that of TUBULIN. Data are mean ± s.d. of three independent biological replicates, n=3.Flowering time phenotype analysis of lines Three independent biological repeats were conducted, and data from a total of 71 plants were used for statistical analysis. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01.Flowering time phenotype analysis of lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Three independent biological repeats were conducted, and data from a total of 71 plants were used for statistical analysis. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01.


Figure 1A : Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures . Wild type , mps1 - 3 and mps1 - 1 mutant cells were synchronised in G1 with α - factor at permissive temperature ( 25 ° C ) and then released at restrictive temperature ( 34 ° C , t = 0 ) . Cells were collected at the indicated time points for FACS analysis of DNA contents ( C ) Wild type , mps1 - 3 and mps1 - 1 mutant cells were synchronised in G1 with α - factor at permissive temperature ( 25 ° C ) and then released at restrictive temperature ( 34 ° C , t = 0 ) . Cells were collected at the indicated time points for immunofluorescence using anti - tubulin antibodies in order to score metaphase and anaphase spindles ( D ) . Budding and nuclear division were scored on the FACS samples . F : Wild type and mps1 mutant cells bearing the TetO / TetR - GFP system to mark the centromere of chromosome V [ 43 ] and expressing the SPBs marker Spc97 - mCherry were synchronised as in ( C - D ) and arrested in anaphase through the temperature - sensitive cdc15 - 2 allele . At 120 and 150 minutes , cells were fixed for scoring chromosome V segregation ( n ≥ 172 ) . Arrowheads indicate the sister chromatids of chromosome V . Error bars : SD . N = 3 . Representative images of cells for each genotype are shown on the right . Scale bar : 5 μm . G : Wild type and mps1 - 3 mutant cells carrying the TetO / TetR - GFP markers for CEN5 labelling and expressing mCherry - Tub1 were grown at 25 ° C and then shifted to 34 ° C for 1 hour before filming . Cells were filmed at 34 ° C every 2 minutes by time lapse fluorescence microscopy . DIC : differential interference contrast . Scale bar : 5 μm .

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

Figure 1A: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents (C)Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for immunofluorescence using anti-tubulin antibodies in order to score metaphase and anaphase spindles (D). Budding and nuclear division were scored on the FACS samples.F: Wild type and mps1 mutant cells bearing the TetO/TetR-GFP system to mark the centromere of chromosome V [43] and expressing the SPBs marker Spc97-mCherry were synchronised as in (C-D) and arrested in anaphase through the temperature-sensitive cdc15-2 allele. At 120 and 150 minutes, cells were fixed for scoring chromosome V segregation (n≥172). Arrowheads indicate the sister chromatids of chromosome V. Error bars: SD. N=3. Representative images of cells for each genotype are shown on the right. Scale bar: 5 μm.G: Wild type and mps1-3 mutant cells carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for 1 hour before filming. Cells were filmed at 34°C every 2 minutes by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm.


Figure 2A : Wild type and mps1 mutant cells were synchronised in G1 with α - factor at 25 ° C and then released at 34 ° C in the presence of nocodazole ( t = 0 ) . Cells were collected at the indicated time points for FACS analysis of DNA contents . B : Cells were treated as in ( A ) and after 2 hours from the release α - factor was re - added to prevent cells from undergoing a second cell cycle . Cell samples were collected at the indicated time points for western blot analysis of the indicated proteins . Pgk1 was used as loading control . Cyc : cycling cells . C : Top : in vitro kinase assays with recombinant Mps1 and Mps1 - 3 purified from E . coli and incubated at 34 ° C for the indicated times in the presence of radioactive ATP and a recombinant Spc105 N - terminal fragment ( aa 1 - 320 ) as substrate . Bottom left : kinase activity was quantified on the autoradiographs by Image J and normalized to the levels of the full length protein on the Coomassie Blue - stained gel ( right ) . Bottom right : 1μg of the recombinant Mps1 kinases used for kinase assays was loaded on SDS page and stained with Coomassie Blue for normalisation of kinase assays . D : Wild type and mps1 - 3 cells were synchronised in G1 with α - factor at 25 ° C and then released at 34 ° C in the presence of nocodazole . Cells were collected after 90 minutes and fixed with formaldehyde for ChIP - seq analysis . ChIP sequence reads were normalised against sequence reads from corresponding input samples , and relative enrichment is plotted for chromosome III around the centromere ( see the centromeric regions of all 16 yeast chromosomes in Fig . S2 ) . Y ‐ axis shows enrichment values ( linear scale , range is 0 - 10 ) . Values below 1 . 5 are shown in grey , and values above 1 . 5 ( i . e . sequences enriched in ChIP samples ) are red coloured . E : Cells with the indicated genotypes were synchronised in G1 with α - factor at 25 ° C and then released in fresh medium at 34 ° C ( t = 0 ) . Cells were collected at the indicated time points for FACS analysis of DNA contents .

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

Figure 2A: Wild type and mps1 mutant cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.B: Cells were treated as in (A) and after 2 hours from the release α-factor was re-added to prevent cells from undergoing a second cell cycle. Cell samples were collected at the indicated time points for western blot analysis of the indicated proteins. Pgk1 was used as loading control. Cyc: cycling cells.C: Top: in vitro kinase assays with recombinant Mps1 and Mps1-3 purified from E. coli and incubated at 34°C for the indicated times in the presence of radioactive ATP and a recombinant Spc105 N-terminal fragment (aa 1-320) as substrate. Bottom left: kinase activity was quantified on the autoradiographs by Image J and normalized to the levels of the full length protein on the Coomassie Blue-stained gel (right). Bottom right: 1μg of the recombinant Mps1 kinases used for kinase assays was loaded on SDS page and stained with Coomassie Blue for normalisation of kinase assays.D: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole. Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Fig. S2). Y‐axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.E: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.


Figure 3A : Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at 25 ° C and 34 ° C . C : Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures . D : Cells with the indicated genotypes were synchronised in G1 with α - factor at 25 ° C and then released in fresh medium at 34 ° C in presence of nocodazole ( t = 0 ) . Cells were collected at the indicated time points for FACS analysis of DNA contents . E : Cells were treated as in ( D ) and collected at the indicated time points for western blot analysis of Mad1 - 3HA . Cyc : cycling cells . F : Cells with the indicated genotypes were synchronised at 25 ° C and then released in fresh medium at 34 ° C ( t = 0 ) . Cells were collected at the indicated time points for FACS analysis of DNA contents . G - H : Cells with the indicated genotypes carrying the TetO / TetR - GFP markers for CEN5 labelling and expressing mCherry - Tub1 were grown at 25 ° C and then shifted to 34 ° C for one hour before filming . Cells were filmed at 34 ° C every 2 or 4 minutes by time lapse fluorescence microscopy . Chromosome V segregation errors are reported in the table ( H ) . Representative cells are shown as examples in the montages ( G ) . Representative montages for wild type and mps1 - 3 cells are shown in Fig . 1F . DIC : differential interference contrast . Scale bar : 5 μm . I : Wild type , mps1 - 3 , mps1 - 3 spc105 - 18 , and mps1 - 3 GLC7 - 24 cells were synchronised in G1 with α - factor at 25 ° C and then released at 32 ° C in the presence of nocodazole ( note that the presence of 3HA tags at the C - terminus of Mps1 - 3 slightly decreases the maximal temperature of suppression ) . Cells were collected after 90 minutes and fixed with formaldehyde for ChIP - seq analysis . ChIP sequence reads were normalised against sequence reads from corresponding input samples , and relative enrichment is plotted for chromosome III around the centromere ( see the centromeric regions of all 16 yeast chromosomes in Fig . S5 ) . Y ‐ axis shows enrichment values ( linear scale , range is 0 - 10 ) . Values below 1 . 5 are shown in grey , and values above 1 . 5 ( i . e . sequences enriched in ChIP samples ) are red coloured .

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

Figure 3A: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at 25°C and 34°C.C: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.D: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C in presence of nocodazole (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.E: Cells were treated as in (D) and collected at the indicated time points for western blot analysis of Mad1-3HA. Cyc: cycling cells.F: Cells with the indicated genotypes were synchronised at 25°C and then released in fresh medium at 34°C (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.G-H: Cells with the indicated genotypes carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for one hour before filming. Cells were filmed at 34°C every 2 or 4 minutes by time lapse fluorescence microscopy. Chromosome V segregation errors are reported in the table (H). Representative cells are shown as examples in the montages (G). Representative montages for wild type and mps1-3 cells are shown in Fig. 1F. DIC: differential interference contrast. Scale bar: 5 μm.I: Wild type, mps1-3, mps1-3 spc105-18, and mps1-3 GLC7-24 cells were synchronised in G1 with α-factor at 25°C and then released at 32°C in the presence of nocodazole (note that the presence of 3HA tags at the C-terminus of Mps1-3 slightly decreases the maximal temperature of suppression). Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Fig. S5). Y‐axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.


Figure 4 : Wild type and mps1 - 3 cells were synchronised in G1 with α - factor at 25 ° C and then released at 34 ° C in the presence ( A ) of nocodazole ( t = 0 ) . Cells were collected at the indicated time points for western blot analysis of the indicated proteins . Equal amounts of protein extracts were loaded on two different gels , for western blot of Spc105 - 3PK and Clb2 / Pgk1 respectively . Clb2 was used as mitotic marker and Pgk1 as loading control . Cyc : cycling cells . Wild type and mps1 - 3 cells were synchronised in G1 with α - factor at 25 ° C and then released at 34 ° C in the absence ( B ) of nocodazole ( t = 0 ) . Cells were collected at the indicated time points for western blot analysis of the indicated proteins . Equal amounts of protein extracts were loaded on two different gels , for western blot of Spc105 - 3PK and Clb2 / Pgk1 respectively . Clb2 was used as mitotic marker and Pgk1 as loading control . Cyc : cycling cells . Cells with the indicated genotypes expressing Bub1 - GFP and the kinetochore marker Mtw1 - Tomato were grown in SD glu 2 % and then shifted to 34 ° C for 1 hour before filming ; they were then filmed every 4 minutes by time lapse fluorescence microscopy at 34 ° C . Note that BUB1 - GFP is synthetic lethal with mps1 - 3 . Thus , we used mps1 - 3 GALs - MPS1 BUB1 - GFP cells that were grown in - His RG medium at 30 ° C ; glucose was added to the culture for 30 minutes to shut off GALs - MPS1 , followed by shifting cells to SD medium at 34 ° C for 1 before imaging in the same medium . Montages show representative cells ( C ) . Arrowheads indicate Bub1 - GFP signals at kinetochores . DIC : differential interference contrast . Scale bar : 5 μm . Wild type and mps1 - 3 cells were synchronised in G1 with α - factor at 25 ° C and then released at 34 ° C in the presence ( E - F ) of nocodazole ( t = 0 ) . Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents Equal amounts of protein extracts were loaded on two different gels , for western blot of Bub1 - 3HA / Cdc5 and Clb2 / Pgk1 respectively . A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation . Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control . Cyc : cycling cells . Wild type and mps1 - 3 cells were synchronised in G1 with α - factor at 25 ° C and then released at 34 ° C in the absence ( G - H ) of nocodazole ( t = 0 ) . Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents ( F , H ) . Equal amounts of protein extracts were loaded on two different gels , for western blot of Bub1 - 3HA / Cdc5 and Clb2 / Pgk1 respectively . A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation . Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control . Cyc : cycling cells .

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

Figure 4: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (A) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for western blot of Spc105-3PK and Clb2/Pgk1 respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the absence (B) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for western blot of Spc105-3PK and Clb2/Pgk1 respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.Cells with the indicated genotypes expressing Bub1-GFP and the kinetochore marker Mtw1-Tomato were grown in SD glu 2% and then shifted to 34°C for 1 hour before filming; they were then filmed every 4 minutes by time lapse fluorescence microscopy at 34°C. Note that BUB1-GFP is synthetic lethal with mps1-3. Thus, we used mps1-3 GALs-MPS1 BUB1-GFP cells that were grown in -His RG medium at 30°C; glucose was added to the culture for 30 minutes to shut off GALs-MPS1, followed by shifting cells to SD medium at 34°C for 1 before imaging in the same medium. Montages show representative cells (C). Arrowheads indicate Bub1-GFP signals at kinetochores. DIC: differential interference contrast. Scale bar: 5 μm.Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (E-F) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents Equal amounts of protein extracts were loaded on two different gels, for western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1 respectively. A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the absence (G-H) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents (F, H). Equal amounts of protein extracts were loaded on two different gels, for western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1 respectively. A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.


Figure 5A : SPC105 - GBD cells expressing Bub1 - GFP were grown at 30 ° C and filmed every 4 minutes at 30 ° C by time lapse fluorescence microscopy . DIC : differential interference contrast . Scale bar : 5 μm . B : Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures . C : cells with the indicated genotypes were grown at 25 ° C and then shifted at 34 ° C ( t = 0 ) . Every hour after the temperature shift the same number of cells was plated on YEPD for each strain and incubated at 25 ° C to determine the number of colony - forming units . Percentages of viable cells have been calculated for each strain relative to t = 0 . Error bars : SD . N = 3 . D : Cells with the indicated genotypes were synchronised in G1 with α - factor at 25 ° C and then released in fresh medium at 34 ° C . Cells were collected at the indicated time points for FACS analysis of DNA contents .

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

Figure 5A: SPC105-GBD cells expressing Bub1-GFP were grown at 30°C and filmed every 4 minutes at 30°C by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm.B: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.C: cells with the indicated genotypes were grown at 25°C and then shifted at 34°C (t=0). Every hour after the temperature shift the same number of cells was plated on YEPD for each strain and incubated at 25°C to determine the number of colony-forming units. Percentages of viable cells have been calculated for each strain relative to t=0. Error bars: SD. N=3.D: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C. Cells were collected at the indicated time points for FACS analysis of DNA contents.


Figure 1B . 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 tD . 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 μmE . 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 tF . 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 ≥ 3G . 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 tH . Representative images of tdTomato + and tdTomato - embryos derived from ICSI or SCNT , after 4 . 5 days of culturing in vitro . Scale bar , 100 μmI . 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 .

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

Figure 1B. 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
 tD. 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 μmE. 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
 tF. 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 ≥ 3G. 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
 tH. Representative images of tdTomato+ and tdTomato- embryos derived from ICSI or SCNT, after 4.5 days of culturing in vitro. Scale bar, 100 μmI. 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.


Figure 2B . 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 μmC . 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 μmF . 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 μmG . 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 tH . 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 tI . RT - qPCR analysis for somatic cell genes in SCNT 1 - cell embryos , 2 - cell tdTomato + , and 2 - cell tdTomato - embryos . Results were normalized based on the geometric mean of the expression levels of two reference genes ( Ywhaz and Gapdh ) . Error bars , s . e . m . , n = 3 . * * * P < 0 . 001 by two - tailed Student " s

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

Figure 2B. 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 μmC. 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 μmF. 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 μmG. 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
 tH. 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
 tI. RT-qPCR analysis for somatic cell genes in SCNT 1-cell embryos, 2-cell tdTomato+, and 2-cell tdTomato- embryos. Results were normalized based on the geometric mean of the expression levels of two reference genes (Ywhaz and Gapdh). Error bars, s.e.m., n = 3. ***P < 0.001 by two-tailed Student"s
 t


Figure 3A . 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 tC . Western blot results showing H3K27me3 levels in SCNT and ICSI embryos . α - Tubulin ( TUBB ) was blotted as a loading controlD . 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 μmE . 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 μmF . 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 μmG . 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 .

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

Figure 3A. 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
 tC. Western blot results showing H3K27me3 levels in SCNT and ICSI embryos. α-Tubulin (TUBB) was blotted as a loading controlD. 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 μmE. 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 μmF. 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 μmG. 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.


Figure 4A , 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 tC . 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 tagD . 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 μmF . Representative images of SCNT embryos at 115 h after injection of different mRNA as indicated . Scale bar , 50 μmG . 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 signifI . 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 embryosI . 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 embryosK . 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 .

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

Figure 4A, 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 tC. 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 tagD. 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 μmF. Representative images of SCNT embryos at 115 h after injection of different mRNA as indicated. Scale bar, 50 μmG. 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 signifI. 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 embryosI. 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 embryosK. 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.


Figure 5A . RT - qPCR analysis of KDM6A / B in SCNT embryos injected with siRNA - 6A or siRNA - 6B . The SCNT embryos were subject to injection of 10 μM or 20 μM siRNA as indicated . Data are mean expression relative to Gapdh with siRNA - control normalized to 1 . Error bars , s . e . m . , n = 3 . * * P < 0 . 01 , * * * P < 0 . 001 according to two - tailed Student " s t - test . n . s . , not significant . B . The bar chart shows the KDM6A / B transcript levels in SCNT embryo with 20 μM siRNA - 6A / B . All data are mean expression relative to Gapdh with control siRNA injected SCNT embryos normalized to 1 . Error bars , s . e . m . , n = 3 , * * P < 0 . 01 by Student & quoC . Immunofluorescence staining results showing H3K27me3 of 2 - stage SCNT embryos . Embryo was injected with siRNA as indicated . The H3K27me3 levels between control and double injected with siRNA - 6A - 6B cannot observe any difference , but a marked decrease was observed when injected with either siRNA - 6A or siRNA - 6B . Scale bar , 20 μmD . Western blot showing the expression of KDM6A and KDM6B in SCNT embryos after injection of different siRNA as indicated . Protein lysates from 1 , 500 embryos were loaded in each lane . The results of one representative of two independent experiments are presentedF . Representative images of SCNT embryos at 115 h after injection of different siRNA as indicated . These images are representative examples of the quantification shown in Fig 5G . n ≥ 3 . Scale bar , 50 μm . G . Injection of siRNA - 6B improved the preimplantation development rate of SCNT embryos . Both cumulus cells , Sertoli cells , and C57 MEF cells were used as donor cells . The bovine intraspecies SCNT embryos derived from bovine ear fibroblast cells . Shown is the percentage of embryos that reached the indicated stages . ♂ and ♀ indicated the male and female , respectively . Error bars , s . d . , n ≥ 3 . H RT - qPCR analysis for select ZGA genes in ICSI and SCNT embryos . The SCNT embryo was injected with siRNA as indicated . Results were normalized based on the geometric mean of the expression levels of two reference genes ( Ywhaz and Gapdh ) . Error bars , s . e . m . , n = 3 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 according to two - tailed Student " s t - test . n . s . , not

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

Figure 5A. RT-qPCR analysis of KDM6A/B in SCNT embryos injected with siRNA-6A or siRNA-6B. The SCNT embryos were subject to injection of 10 μM or 20 μM
 siRNA as indicated. Data are mean expression relative to Gapdh with siRNA-control normalized to 1. Error bars, s.e.m., n = 3. **P < 0.01, ***P < 0.001 according to two-tailed Student"s t-test. n.s., not significant. B. The bar chart shows the KDM6A/B transcript levels in SCNT embryo with 20 μM siRNA-6A/B. All data are mean expression relative to Gapdh with control siRNA injected SCNT embryos normalized to 1.
 Error bars, s.e.m., n = 3, **P < 0.01 by Student&quoC. Immunofluorescence staining results showing H3K27me3 of 2-stage SCNT embryos. Embryo was injected with siRNA as indicated. The H3K27me3 levels between control and double injected with siRNA-6A-6B cannot observe any difference, but a marked decrease was observed when injected with either siRNA-6A or siRNA-6B. Scale bar, 20 μmD. Western blot showing the expression of KDM6A and KDM6B in SCNT embryos after injection of different siRNA as indicated. Protein lysates from 1,500 embryos were loaded in each lane. The results of one representative of two independent experiments are presentedF. Representative images of SCNT embryos at 115 h after injection of different siRNA as indicated. These images are representative examples of the quantification shown in Fig 5G. n ≥ 3. Scale bar, 50 μm. G. Injection of siRNA-6B improved the preimplantation development rate of SCNT embryos. Both cumulus cells, Sertoli cells, and C57 MEF cells were used as donor cells. The bovine intraspecies SCNT embryos derived from bovine ear fibroblast cells. Shown is the percentage of embryos that reached the indicated stages. ♂ and ♀ indicated the male and female, respectively. Error bars, s.d., n ≥ 3. H RT-qPCR analysis for select ZGA genes in ICSI and SCNT embryos. The SCNT embryo was injected with siRNA as indicated. Results were normalized based on the geometric mean of the expression levels of two reference genes (Ywhaz and Gapdh). Error bars, s.e.m., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 according to two-tailed Student"s t-test. n.s., not signif


Figure 6B . Representative fluorescence images of siRNA - control or siRNA - 6B injected SCNT embryos . Scale bar , 50 μmC . Quantification of embryos that expression tdTomato after injection with siRNA - control or siRNA - 6B . Numbers of observed embryos are indicated . n ≥ 3 , Error bars , s . e . m . , * * * P < 0 . 001 according to two - tailed Student " s tD . Representative images of green fluorescence in Oct4 : : EGFP reconstructed blastocysts derived from injected siRNA - control or siRNA - 6B SCNT embryos . Representative images from ≥ 25 embryos analyzed in three independent micromanipulations are shown . Green fluorescence indicates that the Oct4 : : EGFP transgene has been expressed . Scale bar , 50 μmE . Oct4 : : EGFP was up - regulated significantly in ICSI embryos compared with SCNT embryos at blastocyst stages , as determined by RT - qPCR . Data shown are mean expression values relative to Gapdh . The value in ICSI embryos was set as 1 . Error bars , s . e . m . , n ≥ 3 . * P < 0 . 05 , * * * P < 0 . 001 by two - tailed Student " s tF . Representative image of cloned mice derived by siRNA - 6B injected SCNT embryosG . Bar graph showing the efficiency of attachment to the feeder cells ( left ) and si6B - mdx - ntES derivation ( right ) . The mdx sick mice tail - tip fibroblasts as nuclear donors . N , total number of embryos analyzed for each condition . Results are from four replicate experimentsH . Immunostaining images of si6B - mdx - ntES expressed pluripotency markers . Scale bar , 50 μmI . The si6B - mdx - ntES possessed multiple - differentiation potential , as shown in embryoid body . Scale bar , 100 μm . J . An image of a chimeric mouse derived from si6B - mdx -

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

Figure 6B. Representative fluorescence images of siRNA-control or siRNA-6B injected SCNT embryos. Scale bar, 50 μmC. Quantification of embryos that expression tdTomato after injection with siRNA-control or siRNA-6B. Numbers of observed embryos are indicated. n ≥ 3, Error bars, s.e.m., ***P < 0.001 according to two-tailed Student"s tD. Representative images of green fluorescence in Oct4::EGFP reconstructed blastocysts derived from injected siRNA-control or siRNA-6B SCNT embryos. Representative images from ≥ 25 embryos analyzed in three independent micromanipulations are shown. Green fluorescence indicates that the Oct4::EGFP transgene has been expressed. Scale bar, 50 μmE. Oct4::EGFP was up-regulated significantly in ICSI embryos compared with SCNT embryos at blastocyst stages, as determined by RT-qPCR. Data shown are mean expression values relative to Gapdh. The value in ICSI embryos was set as 1. Error bars, s.e.m., n ≥ 3. *P < 0.05, ***P < 0.001 by two-tailed Student"s
 tF. Representative image of cloned mice derived by siRNA-6B injected SCNT embryosG. Bar graph showing the efficiency of attachment to the feeder cells (left) and si6B-mdx-ntES derivation (right). The mdx sick mice tail-tip fibroblasts as nuclear donors. N, total number of embryos analyzed for each condition. Results are from four replicate experimentsH. Immunostaining images of si6B-mdx-ntES expressed pluripotency markers. Scale bar, 50 μmI. The si6B-mdx-ntES possessed multiple-differentiation potential, as shown in embryoid body.
 Scale bar, 100 μm.J. An image of a chimeric mouse derived from si6B-mdx-ntES