Heat map of Egg ChIP - seq within a 10kb window centered on Egg peaks . Note that ChIP peaks was called using ChIPed Input samples Metaplots showing Egg ChIP - seq signals for genomic regions around Egg peaks . Displayed are fold - changes in Egg occupancy normalized to input .

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

Heat map of Egg ChIP-seq within a 10kb window centered on Egg peaks. Note that ChIP peaks was called using the ChIPed and Input samples Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input.


Heatmaps of Egg ChIP - seq signal within 10 kb windows centered on Egg peaks in control ( siEGFP ) Wde - depleted ( siWde ) OSCs .

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

Heatmaps of Egg ChIP-seq signal within 10 kb windows centered on Egg peaks in control (siEGFP) and Wde-depleted (siWde) OSCs.


Metaplots showing Egg ChIP - seq signals for genomic regions around Egg peaks . Displayed are fold - changes in Egg occupancy normalized to input . Metapolts showing Egg ChIP - seq signals for genomic regions around transposon ( TE ) insertions .

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

Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input. Metapolts showing Egg ChIP-seq signals for genomic regions around transposon (TE) insertions.


ChIP - qPCR was performed before ( siEGFP ) after ( siWde ) Wde depletion . qPCR was performed for Piwi target transposons ( Stalker2 , mdg1 , gypsy ) normalized to % input of a PIwi non - target transposon ( roo ) . Error bars represent SD from three independent experiments .

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

ChIP-qPCR was performed before (siEGFP) and after (siWde) Wde depletion. qPCR was performed for Piwi target transposons (Stalker2, mdg1, and gypsy) and normalized to %input of a PIwi non-target transposon (roo). Error bars represent SD from three independent experiments.


Density plots for Egg ChIP - seq signal on Stalker2 , mdg1 , gypsy in control ( siEGFP ) Wde - depleted ( siWde ) OSCs . Input : input was used as a control . RPM : reads per million mapped reads .

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

Density plots for Egg ChIP-seq signal on Stalker2, mdg1, and gypsy in control (siEGFP) and Wde-depleted (siWde) OSCs. Input: input DNA was used as a control. RPM: reads per million mapped reads.


His - tagged GD / 1 / 2019 RBD , GX / P2V / 2017 , SARS - CoV - 2 RBD SARS - CoV - 2 NTD were incubated with BHK 21 expressing eGFP - fused ACE2s . Anti - His / APC antibody was used to detect expressing His - tagged . stained with GD / 1 / 2019 RBD , GX / P2V / 2017 , SARS - CoV - 2 RBD SARS - CoV - 2 NTD are shown in green , orange , marine red , respectively . SARS - CoV - 2 NTD was used as negative control .

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

His-tagged GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins were incubated with BHK 21 cells expressing eGFP-fused ACE2s. Anti-His/APC antibody was used to detect cells expressing His-tagged protein. Cells stained with the GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins are shown in green, orange, marine and red, respectively. The SARS-CoV-2 NTD was used as negative control.


mFc - tagged ACE2s were captured by anti - mIgG Fc antibodies immobilized on CM5 chip , sequentially tested binding with serially diluted SARS - CoV - 2 RBD , GX / P2V / 2017 RBD GD / 1 / 2019 RBD . raw fitted curves are displayed in dotted solid lines , respectively . binding affinity between RBDs ( SARS - CoV - 2 RBD , GX / P2V / 2017 RBD GD / 1 / 2019 RBD ) 13 ACE2s are shown . Mean ± SD represents mean standard deviation of three independent experiments .

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

The mFc-tagged ACE2s were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD. The raw and fitted curves are displayed in dotted and solid lines, respectively. The binding affinity between the RBDs (SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD) and the 13 ACE2s are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.


Hela - hACE2 untransfected HeLa are infected with pseudotyped SARS - CoV - 2 , GX / P2V / 2017 GD / 1 / 2019 , respectively . Green indicates Hela - hACE2 infected with pseudovirus . Untransfected HeLa is used as negative control . scale bar indicates 400 μm . Statistic for infectivity of three pseudoviruses . Data represent results of five replicates . data are presented as mean ± SD . * * * p < 0 . 001 ( Student ' s t - ) . ns , no significant differences .

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

Hela-hACE2 cells and untransfected HeLa cells are infected with the pseudotyped SARS-CoV-2, GX/P2V/2017 and GD/1/2019, respectively. Green fluorescence indicates Hela-hACE2 cells infected with pseudovirus. Untransfected HeLa cell is used as negative control. The scale bar indicates 400 μm. Statistic for the infectivity of the three pseudoviruses. Data represent the results of five replicates. All data are presented as mean ± SD. ***p < 0.001 (Student's t-test). ns, no significant differences.


Flow cytometric of SARS - CoV - 2 RBD its mutants ( K417R , Q498H K417R - Q498H ) binding to human , mouse , rat European hedgehog ACE2s expressed on surface of BHK21 . SARS - CoV - 2 NTD was used as negative control .

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

Flow cytometric assay of the SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H) binding to human, mouse, rat and European hedgehog ACE2s expressed on the cell surface of BHK21 cells. The SARS-CoV-2 NTD was used as negative control.


mFc - tagged ACE2s from human , mouse , rat European hedgehog were captured by anti - mIgG Fc antibodies immobilized on CM5 chip , sequentially tested binding with serially diluted SARS - CoV - 2 RBD its mutants ( K417R , Q498H K417R - Q498H ) .

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

The mFc-tagged ACE2s from human, mouse, rat and European hedgehog were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H).


binding affinities of four ACE2s to SARS - CoV - 2 RBD its mutants are shown . Mean ± SD represents mean standard deviation of three independent experiments .

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

The binding affinities of the four ACE2s to the SARS-CoV-2 RBD and its mutants are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.


B Western blot ( probing with an anti - FLAG mAb ) to detect cleavage of artificial substrate by wild - type ( WT ) / inactive ( S201A ) GlpG encoded on pBAD33 with / without arabinose ( Ara . ) . rhomboid substrates that are uncleaved , by GlpG are marked by black , red , arrows , respectively . Controls , empty pBAD33 ( empty )

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

B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of the artificial substrate by wild-type (WT)/inactive (S201A) GlpG encoded on pBAD33 with/without arabinose (Ara.). rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, empty pBAD33 (empty)


C Western blot to detect cleavage of artificial substrate by wild - type ( WT ) / modified ( S133A or H187A ) Rhom7 encoded on pBAD33 with / without arabinose ( Ara . ) . , rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , blue arrows , respectively . Controls , empty pBAD33 ( empty ) wild - type S . sonnei ( Ss ) .

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

C Western blot analysis to detect cleavage of the artificial substrate by wild-type (WT)/modified (S133A or H187A) Rhom7 encoded on pBAD33 with/without arabinose (Ara.). , rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).


D Activity of Rhom7 with / without its 7th TMD / or C - terminal domain . rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , , blue arrows , respectively . Controls , empty pBAD33 ( empty ) wild - type S . sonnei ( Ss ) .

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

D Activity of Rhom7 with/without its 7th TMD and/or C-terminal domain. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, , and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).


C Western blot of candidate substrates reproducibly by GlpG from screen . Data information : In ( C rhomboid substrates that are uncleaved , by GlpG are marked by black , red , arrows , respectively . Controls , inactive enzymes wild - type S . sonnei ( Ss ) .

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

C Western blot analysis of candidate substrates reproducibly cleaved by GlpG from the screen. Data information: In (C rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).


D Western blot of candidate substrates reproducibly by Rhom7 from screen . Data information : In D ) rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , red , blue arrows , respectively . Controls , inactive enzymes wild - type S . sonnei ( Ss ) .

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

D Western blot analysis of candidate substrates reproducibly cleaved by Rhom7 from the screen. Data information: In D) rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, red, and blue arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).


Western blot ( 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 catalytic serine histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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

Western 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 to detect cleavage of HybA with ( + ) / without ( - ) chromosomal ( native ) or pBAD33 - encoded rhomboids ( plasmid ) . rhomboid substrates that are uncleaved , by GlpG , or by Rhom7 , are marked by black , red , blue arrows , respectively . Wild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of catalytic serine histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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

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 to detect cleavage of HybAG296F by endogenous or pBAD33 - encoded rhomboid . Wild - type S . sonnei , Ss . rhomboid substrates that are uncleaved , by GlpG , or by Rhom7 , re marked by black , red , blue arrows , espectively . ild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of catalytic serine histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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

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 to detect cleavage of HybAP300A by endogenous or pBAD33 - encoded rhomboid . rhomboid substrates that are uncleaved , by GlpG , or by Rhom7 , are marked by black , red , blue arrows , respectively . Wild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of catalytic serine histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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

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.


A two - hybrid with HybA / or HybB fused chromosomally with T25 T18 domains of B . pertussis CyaA , respectively in absence of endogenous CyaA . were grown on LB agar containing 20 µg / ml X - gal at 37 ° C for 14 hours in presence / absence of O2 . Scale bar , 1 cm .

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

A 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 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 by GlpG is marked by black red arrows , respectively .

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

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 of of / uncleaved HybA in strains with ( + ) or without ( - ) HybB . mean ± S . D . of three experiments . * , p < 0 . 05 ; * * * * , p < 0 . 0001 ( One - way ANOVA ) .

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

C Quantification of 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 to detect GlpG - mediated cleavage of native tagged HybA at indicated times after blocking translation by addition of chloramphenicol at T0 in absence of HybB ( - ) . , HybA that is uncleaved or by GlpG is marked by black red arrows , respectively .

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

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 to detect GlpG - mediated cleavage of C - terminally sfCherry - 3xFLAG tagged HybA at times after blocking translation by addition of chloramphenicol at T0 in presence of HybB ( + ) . , HybA that is uncleaved or by GlpG is marked by black red arrows , respectively .

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

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 lacking hydrogenases ( ΔhyaA - F ΔhycE + / - ΔhybO - G ) or GlpG ( ΔglpG ) , or expressing uncleavable HybB ( hybAG296F ) in 5 %

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

F Growth of bacteria lacking hydrogenases (ΔhyaA-F ΔhycE +/- ΔhybO-G) or GlpG (ΔglpG), or expressing uncleavable HybB (hybAG296F) in 5% H2


G H2 consumption by S . sonnei . were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0 . 5 % fumarate , 12 . 5 μg / ml nicotinic acid 0 . 2 % casamino in a sealed glass chromatography vial . headspace was purged with 10 % H2 / 90 % argon cultures were incubated at 37 ° C with shaking at 180 rpm for 9 hours . H2 in headspace was sampled measured by chromatography .

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

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

H S. sonnei growth in 10% H2 with plasmid expressed wild-type or non-functional (pglpGSAHA) GlpG.


B Western blot ( probing with an anti - FLAG mAb ) to detect cleavage of FdoH cleavage in S . sonneiΔrhom7 with ( + ) / without ( - ) FdoI . rhomboid substrates that are uncleaved , by GlpG are marked by black , red arrows , respectively .

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

B 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 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 , by Rhom7 , are marked by black , blue arrows , respectively .

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

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 to detect cleavage of wild - type ( WT ) or uncleavable ( P300A ) HybA by active ( + ) or inactive ( S201A ) chromosomal GlpG in presence ( + ) or absence ( - ) of HybB . rhomboid substrates that are uncleaved , by GlpG are marked by black , red arrows , respectively .

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

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 to detect cleavage of wild - type ( WT ) or modified ( P259A ) FdoH by active ( + ) or inactive ( S201A ) chromosomal GlpG in presence ( + ) or absence ( - ) of FdoI . rhomboid substrates that are uncleaved , 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 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 , by Rhom7 , are marked by black , blue arrows , respectively .

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

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.


A Western blot ( 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 , by GlpG , , are marked by black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

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

A 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 translation at T0 in presence ( + ) or absence ( - ) of HybB . Rhomboid substrates that are uncleaved , by GlpG , black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

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

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 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 , by GlpG are marked by black , red , arrows , respectively .

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

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 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 , by Rhom7 , are marked by black , blue arrows , respectively .

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

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 , by GlpG , are marked by black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

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

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 , by Rhom7 , are marked by black , blue arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

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

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.


A Western blot probing localisation 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 lysate ( Whole . ) , Sol ( Sol . ) , Mem ( detergent - solubilised , Mem . ) , Aggregate ( Agg . ) fractions are shown . HybA that is uncleaved , only by GlpG , further degraded post GlpG cleavage is marked by black , red , green arrows , respectively .

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

A 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.


( A ) Representative flow cytometry plots ( left ) pooled data ( right ) of competition assays with sgControl sgRnf31 B16 - F10 ova after culture with indicated recombinant cytokine ( s ) ( 10 ng / mL ) for 48 hours ; n . s . : no significance , * * * * p < 0 . 0001 , Unpaired t - for n = 3 biological replicates . Pooled data are represented as mean ± .

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

(A) Representative flow cytometry plots (left) and pooled data (right) of competition assays with sgControl and sgRnf31 B16-F10 ova tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( B ) CRISPR / Cas9 screen showing depleted B16 - F10 genes after one week culture with TNF + IFN - γ ( both 10 ng / mL ) ; blue genes indicate overall genes with p < 0 . 001 , black genes indicate genes of interest with p < 0 . 05 . Significance assessed using MAGeCK .

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

(B) CRISPR/Cas9 screen showing depleted B16-F10 genes after one week culture with TNF + IFN-γ (both 10 ng/mL); blue genes indicate overall genes with p < 0.001, black genes indicate genes of interest with p < 0.05. Significance assessed using MAGeCK test.


( H ) Representative flow cytometry plots ( left ) pooled data ( right ) showing PI of sgControl or sgRNF31 A375 after culture with indicated recombinant cytokine ( s ) ( 10 ng / mL ) for 48 hours ; n . s . : no significance , * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - for n = 3 biological replicates . Pooled data are represented as mean ± .

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

(H) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of sgControl or sgRNF31 A375 tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( A ) PI of sgControl or sgRnf31 B16 - F10 treated with combined TNF / IFN - γ ( both 10 ng / mL ) for 24 hours in absence presence of QVD - OPh ( 10 μM ) ; n . s . : no significance , * * * p < 0 . 001 , * * * * p < 0 . 0001 , Unpaired t - for n = 3 biological replicates . Pooled data are represented as mean ± .

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

(A) PI staining of sgControl or sgRnf31 B16-F10 tumor cells treated with combined TNF/IFN-γ (both 10 ng/mL) for 24 hours in the absence and presence of QVD-OPh (10 μM); n.s.: no significance, ***p < 0.001, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( B ) Western blot of indicated on whole lysates for sgControl or sgRnf31 B16 - F10 cultured with indicated cytokine ( s ) ( 10 ng / mL for ) for 0 , 3 , 6 , or 9 hours ; β - actin was used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of the indicated proteins on whole cell lysates for sgControl or sgRnf31 B16-F10 tumor cells cultured with the indicated cytokine(s) (10 ng/mL for all) for 0, 3, 6, or 9 hours; β-actin was used as a loading control.


( D ) CRISPR / Cas9 screen showing enriched genes from sgRnf31 B16 - F10 after successive treatment with combined TNF / IFN - γ , as described in ( C ) ; red genes indicate overall genes with p < 0 . 0001 . Significance assessed using MAGeCK .

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

(D) CRISPR/Cas9 screen showing enriched genes from sgRnf31 B16-F10 tumor cells after successive treatment with combined TNF/IFN-γ, as described in (C); red genes indicate overall genes with p < 0.0001. Significance assessed using MAGeCK test.


( G ) PI of sgControl , sgRnf31 , sgRnf31 / Stat1 , sgRnf31 / Casp8 , sgRnf31 / Bid , or sgRnf31 / Rela B16 - F10 treated with combined TNF / IFN - γ for 24 hours ; * p < 0 . 05 , * * * * p < 0 . 0001 , asterisk ( s ) indicated group vs TNF / IFN - γ - treated sgRnf31 group , Unpaired t - for n > 3 biological replicates . Pooled data are represented as mean ± .

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

(G) PI staining of sgControl, sgRnf31, sgRnf31/Stat1, sgRnf31/Casp8, sgRnf31/Bid, or sgRnf31/Rela B16-F10 tumor cells treated with combined TNF/IFN-γ for 24 hours; *p < 0.05, ****p < 0.0001, asterisk(s) indicated group vs TNF/IFN-γ-treated sgRnf31 group, Unpaired t-test for n > 3 biological replicates. Pooled data are represented as mean ± SEM.


( B ) Representative flow cytometry plots ( left ) pooled data ( right ) showing PI of parental B16 - F10 left untreated or treated with supernatant ( Sup ) ( derived from a previous overnight OT - I / B16 - F10 ova co - culture at indicated E : T for 48 hours , following a 4 hour pre - treatment with vehicle or HOIPIN - 1 ( 50 μM ) ( of vehicle / HOIPIN - 1 was halved upon addition of Sup ) ; * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - for n = 3 biological replicates . Pooled data are represented as mean ± .

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

(B) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental B16-F10 tumor cells left untreated or treated with supernatant (Sup) (derived from a previous overnight OT-I/B16-F10 ova co-culture at the indicated E:T ratio for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of Sup); *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( C ) Representative flow cytometry plots ( left ) pooled data ( right ) showing PI of parental A375 left untreated or treated with TNF / IFN - γ ( both 10 ng / mL ) for 48 hours , following a 4 hour pre - treatment with vehicle or HOIPIN - 1 ( 50 μM ) ( of vehicle / HOIPIN - 1 was halved upon addition of cytokines ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , Unpaired t - for n = 3 biological replicates . Pooled data are represented as mean ± .

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

(C) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental A375 tumor cells left untreated or treated with TNF/IFN-γ (both 10 ng/mL) for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of cytokines); *p < 0.05, **p < 0.01, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( B ) ( left ) average ( right ) in vivo growth of subcutaneous wild - type or sgRnf31 B16 - F10 ova tumors left untreated or treated with adoptively transferred OT - I CD8 + T ( on days indicated by black arrows ) ; * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - for OT - I - treated wild - type vs OT - I - treated sgRnf31 with n = 5 / 6 mice per group . Pooled data are represented as mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Individual (left) and average (right) in vivo growth of subcutaneous wild-type or sgRnf31 B16-F10 ova tumors left untreated or treated with adoptively transferred OT-I CD8+ T cells (on days indicated by black arrows); *p < 0.05, **p < 0.01, Unpaired t-test for OT-I-treated wild-type vs OT-I-treated sgRnf31 with n = 5/6 mice per group. Pooled data are represented as mean ± SEM.


( C ) Overall survival of mice from ( B ) ; * p < 0 . 05 , * * p < 0 . 01 , Log - rank .

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

(C) Overall survival of mice from (B); *p < 0.05, **p < 0.01, Log-rank test.


( B ) Representative eTLCs ( left ) corresponding elongation kinetics ( right ) for CGA - CGA inhibitory pair ( red ) CGC optimal pair ( green ) . Product formation is normalized to fraction of Met ICs that form Met - Puro when reacted with puromycin ( Fig EV1A ) .

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

(B) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CGA inhibitory pair (red) and the CGC optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).


( C ) Representative eTLCs ( left ) corresponding elongation kinetics ( right ) for CGA - CCG inhibitory pair ( red ) CGC - CCA optimal pair ( green ) . Product formation is normalized to fraction of Met ICs that form Met - Puro when reacted with puromycin ( Fig EV1A ) .

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

(C) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CCG inhibitory pair (red) and the CGC-CCA optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).


( A ) Comparison of observed rates of elongation for inhibitory pairs ( red ) their optimal controls ( green ) at limiting tRNA concentrations ( hatched bars , 15 to 25 nM aa - tRNA ) saturating tRNA concentrations ( solid bars , 150 to 250 nM aa - tRNA ) .

(A) Comparison of observed rates of elongation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).


( B ) Comparison of total peptide formation for inhibitory pairs ( red ) their optimal controls ( green ) at limiting tRNA concentrations ( hatched bars , 15 to 25 nM aa - tRNA ) saturating tRNA concentrations ( solid bars , 150 to 250 nM aa - tRNA ) .

(B) Comparison of total peptide formation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).


( C ) Elongation kinetics for CGA - CGA inhibitory codon pair with native arginine ICGtRNA ( red ) or non - native arginine UCGtRNA ( pink ) for CGC - CGC optimal control pair with native arginine ICGtRNA ( green ) .

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

(C) Elongation kinetics for the CGA-CGA inhibitory codon pair with the native arginine ICGtRNA (red) or the non-native arginine UCGtRNA (pink) and for the CGC-CGC optimal control pair with the native arginine ICGtRNA (green).


( A ) Meta - of footprint of 17 inhibitory pairs identified aligning first codon of pair in ribosomal P site .

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

(A) Meta-analysis of footprint size of all 17 inhibitory pairs identified aligning the first codon of the pair in the ribosomal P site.


( B ) Metacodon of 21 nt RPFs centered at first codon of each inhibitory pair ( red ) compared to their corresponding optimal pair ( green ) . P - values were determined by Student ' s t - between each inhibitory corresponding optimal pair ( * * p < 0 . 01 , n = 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Metacodon analysis of 21 nt RPFs centered at the first codon of each inhibitory pair (red) compared to their corresponding optimal pair (green). P-values were determined by Student's t-test between each inhibitory and the corresponding optimal pair (** p < 0.01, n=2).


( C ) Comparison of 21 nt RPFs aligned at 17 inhibitory codons from libraries made with CHX / ANS ( blue ) CHX / TIG ( black ) ( left ) to their corresponding optimal pairs with same antibiotic combination ( right ) . No significant difference found ( n . s . : p = 0 . 9961 , n = 2 ) between CHX - TIG CHX - ANS .

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

(C) Comparison of 21 nt RPFs aligned at all 17 inhibitory codons from libraries made with CHX/ANS (blue) and CHX/TIG (black) (left) to their corresponding optimal pairs with the same antibiotic combination (right). No significant difference found (n.s.: p=0.9961, n=2) between CHX-TIG and CHX-ANS.


E - F , Candidate gene regions with neurogenesis - associated de novo methylation ( E ) demethylation ( F ) . Depicted are mean methylation per group at CpGs within specified genomic regions . Stars indicate significantly differentially methylated CpGs with q < 0 . 05 ( logistic regression with multiple testing correction using SLIM method ) methylation difference greater than 20 % .

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

E-F, Candidate gene regions with neurogenesis-associated de novo methylation (E) and demethylation (F). Depicted are mean methylation levels per group at individual CpGs within the specified genomic regions. Stars indicate significantly differentially methylated CpGs with q < 0.05 (logistic regression with multiple testing correction using SLIM method) and methylation difference greater than 20 %.


D , Dnmt3a / b - KO did not affect NPC proliferation . Depicted are data points for every sample with genotype means ± standard errors of mean ( ) . E , Reduced of dead ( Annexin V / propidium iodide ( PI ) double - positive ) in KO cultures at 46 h after start of differentiation . Depicted are data points for every sample with genotype means ± .

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

D, Dnmt3a/b-KO did not affect NPC proliferation. Depicted are data points for every sample with genotype means ± standard errors of the mean (SEM). E, Reduced percentage of dead cells (Annexin V/propidium iodide (PI) double-positive) in KO cultures at 46 h after start of differentiation. Depicted are data points for every sample with genotype means ± SEM.


F - G , NPCs from WT KO mice differentiated into astrocytes ( GFAP - positive ) neurons ( Tubb3 - positive ) . Increased numbers of both neurons astrocytes were generated from KO NPCs . Depicted are data points for every sample with genotype means ± . Scale bar : 100 µm .

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

F-G, NPCs from WT and KO mice differentiated into astrocytes (GFAP-positive) and neurons (Tubb3-positive). Increased numbers of both neurons and astrocytes were generated from KO NPCs. Depicted are data points for every sample with genotype means ± SEM. Scale bar: 100 µm.


H , High magnification image of differentiated neurons . Scale bar : 20 µm . I , Sholl of Tubb3 - labeled WT KO neurons ( n = 29 neurons , WT ; n = 30 neurons , KO ) . Reported p - value corresponds to genotype effect from two - way ANOVA . Asterisks highlight data points with p < 0 . 05 after multiple testing adjustment ( Holm method ) of repeated t - . Depicted are means ± .

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

H, High magnification fluorescent image of differentiated neurons. Scale bar: 20 µm. I, Sholl analysis of Tubb3-labeled WT and KO neurons (n = 29 neurons, WT; n = 30 neurons, KO). Reported p-value corresponds to genotype effect from two-way ANOVA. Asterisks highlight data points with p < 0.05 after multiple testing adjustment (Holm method) of repeated t-tests. Depicted are means ± SEM.


J , High magnification image of differentiated astrocytes . Scale bar : 20 µm . K , Astrocytes from KO cultures covered a larger than WT astrocytes . Data points represent means of cultures ( n > 30 astrocytes per culture ) . Depicted are data points per sample with genotype means ± .

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J, High magnification fluorescent image of differentiated astrocytes. Scale bar: 20 µm. K, Astrocytes from KO cultures covered a larger area than WT astrocytes. Data points represent means of individual cultures (n > 30 astrocytes per culture). Depicted are data points per sample with genotype means ± SEM.


H , Dnmt3a / b - dependent loss of methylation at neuronal gene candidates was associated with reduced transcription in neurons . Depicted are mean CpG methylation at specified genomic regions as determined by RRBS ( left ) expression fold changes in KO versus WT neurons as determined by qRT - PCR ( right ; n = 4 cultures per genotype ; p - values from Mann - Whitney ; depicted are data points for every sample with genotype means ± ) .

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H, Dnmt3a/b-dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons. Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS (left) and expression fold changes in KO versus WT neurons as determined by qRT-PCR (right; n = 4 cultures per genotype; p-values from Mann-Whitney test; depicted are data points for every sample with genotype means ± SEM).


A , No difference in numbers of Ki67 - positive , proliferating was observed between subgranular zone ( SGZ ) of Dnmt3a / b - WT Dnmt3a / b - KO mice one week after tamoxifen administration . Depicted are data points for every with genotype means ± . B , Bright field image of Ki67 - positive stained using diaminobenzidine method . Scale bar : 100 µm .

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A, No difference in numbers of Ki67-positive, proliferating cells was observed between the subgranular zone (SGZ) of Dnmt3a/b-WT and Dnmt3a/b-KO mice one week after tamoxifen administration. Depicted are data points for every animal with genotype means ± SEM. B, Bright field image of Ki67-positive cells stained using diaminobenzidine method. Scale bar: 100 µm.


C , KO of Dnmt3a / b did not affect numbers of new - born neurons in dentate gyrus . Mice were administered with tamoxifen analyzed 5 after first injection . BrdU was injected 3 . 5 before . Depicted are data points for every with genotype means ± . D , image of BrdU / NeuN - positive new - born neurons . Scale bar : 25 µm .

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C, KO of Dnmt3a/b did not affect numbers of new-born neurons in the dentate gyrus. Mice were administered with tamoxifen and analyzed 5 weeks after the first injection. BrdU was injected 3.5 weeks before analysis. Depicted are data points for every animal with genotype means ± SEM. D, Fluorescence image of BrdU/NeuN-positive new-born neurons. Scale bar: 25 µm.


B , Representative image of mature immature neurons . Scale bar : 50 µm . C , KO mice exhibit reduced total numbers of Calb2 - positive relative to Dcx - positive in dentate gyrus .

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B, Representative fluorescence image of mature and immature neurons. Scale bar: 50 µm. C, KO mice exhibit reduced total numbers of Calb2-positive cells relative to Dcx-positive cells in the dentate gyrus.


F , Representative image of immunostaining used for detection of mature neurons . Scale bar : 100 µm . G , Sholl of Gfp - positive revealed reduced dendritic outgrowth of adult - born neurons in KO mice ( n = 20 neurons , WT ; n = 23 neurons , KO ) . Depicted p - value reports genotype effect from two - way ANOVA ( to soma : p < 0 . 001 ) . Asterisks highlight data points with p < 0 . 05 after multiple testing correction ( Holm method ) of repeated t - . Data points are means ± . Experimental scheme as depicted in Fig . 4E .

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F, Representative fluorescence image of the immunostaining used for detection of mature neurons. Scale bar: 100 µm. G, Sholl analysis of Gfp-positive cells revealed reduced dendritic outgrowth of adult-born neurons in KO mice (n = 20 neurons, WT; n = 23 neurons, KO). Depicted p-value reports genotype effect from two-way ANOVA (distance to soma: p < 0.001). Asterisks highlight data points with p < 0.05 after multiple testing correction (Holm method) of repeated t-tests. Data points are means ± SEM. Experimental scheme as depicted in Fig. 4E.


I , Adult - born neurons of KO mice exhibit reduced numbers of dendritic spines in outer molecular layer ( n = 32 neurons from 3 mice , WT ; n = 46 neurons from 4 mice , KO ) . Depicted are data points for every neuron with genotype means ± ( p - value from unpaired t - ) . J , Mean spine per was reduced in KO mice ( n = 50 spines per mouse ) . K , Mean of mushroom spines ( diameter of spine head ≥ 0 . 45 µm ) per was lower in KO mice compared to WT mice . L , Representative image of dendritic spines in WT KO mice . Scale bar : 5 µm .

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I, Adult-born neurons of KO mice exhibit reduced numbers of dendritic spines in the outer molecular layer (n = 32 neurons from 3 mice, WT; n = 46 neurons from 4 mice, KO). Depicted are individual data points for every neuron with genotype means ± SEM (p-value from unpaired t-test). J, Mean spine size per animal was reduced in KO mice (n = 50 spines per mouse). K, Mean percentage of mushroom spines (diameter of spine head ≥ 0.45 µm) per animal was lower in KO mice compared to WT mice. L, Representative fluorescence image of dendritic spines in WT and KO mice. Scale bar: 5 µm.


A - B , of adult - born ( BrdU - positive ) in dentage gyrus ( DG ) of Dnmt3a / b - KO WT mice after five or three months of environmental enrichment ( ENR ) or standard housing ( STD ) . BrdU was injected 4 . 5 before . Depicted p - values correspond to genotype effects from two - way ANOVA . Influences of ENR were significant with p < 0 . 01 . Depicted are data points for every with group means ± .

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A-B, Quantification of adult-born (BrdU-positive) cells in the dentage gyrus (DG) of Dnmt3a/b-KO and WT mice after five weeks or three months of environmental enrichment (ENR) or standard housing (STD). BrdU was injected 4.5 weeks before analysis. Depicted p-values correspond to genotype effects from two-way ANOVA. Influences of ENR were significant with p < 0.01. Depicted are data points for every animal with group means ± SEM.


D , of c - Fos - positive in DG CA3 of WT KO mice . Scale bar : 200 µm . E , Numbers of c - Fos - positive in DG without ( STD ) with acute stimulation in ENR for 1 h ( n = 2 , WT STD ; n = 2 , KO STD ; n = 5 , WT ENR ; n = 5 , KO ENR ) . F , Numbers of c - Fos - positive in suprapyramidal infrapyramidal blades of DG after acute stimulation in ENR ( n = 5 mice , WT ; n = 5 mice , KO ) .

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D, Fluorescence images of c-Fos-positive cells in the DG and CA3 of WT and KO mice. Scale bar: 200 µm. E, Numbers of c-Fos-positive cells in the DG without (STD) and with acute stimulation in ENR for 1 h (n = 2, WT STD; n = 2, KO STD; n = 5, WT ENR; n = 5, KO ENR). F, Numbers of c-Fos-positive cells in suprapyramidal and infrapyramidal blades of the DG after acute stimulation in ENR (n = 5 mice, WT; n = 5 mice, KO).


G , of new - born neurons ( BrdU / NeuN - positive ) that expressed c - Fos after ENR ( n = 5 mice , WT ; n = 5 mice , KO ) . H , Representative image of c - Fos expression in BrdU / NeuN - positive in DG . Scale bar : 25 µm .

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G, Percentage of new-born neurons (BrdU/NeuN-positive) that expressed c-Fos after ENR (n = 5 mice, WT; n = 5 mice, KO). H, Representative fluorescent image of c-Fos expression in BrdU/NeuN-positive cells in the DG. Scale bar: 25 µm.


I , of c - Fos - positive that expressed Dcx in WT KO mice ( n = 5 mice , WT ; n = 4 mice , KO ) . J , Representative image of c - Fos - positive Dcx - positive in DG . Scale bar : 100 µm .

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I, Percentage of c-Fos-positive cells that expressed Dcx in WT and KO mice (n = 5 mice, WT; n = 4 mice, KO). J, Representative fluorescent image of c-Fos-positive cells and Dcx-positive cells in the DG. Scale bar: 100 µm.


L , image of c - Fos expression in Parvalbumin ( Pv ) - positive interneurons in CA3 . Scale bar : 50 µm . M , A higher of Pv - positive interneurons in CA3 expressed c - Fos in KO mice ( n = 4 mice , WT ; n = 4 mice , KO ) .

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L, Fluorescent image of c-Fos expression in Parvalbumin (Pv)-positive interneurons in CA3. Scale bar: 50 µm. M, A higher percentage of Pv-positive interneurons in CA3 expressed c-Fos in KO mice (n = 4 mice, WT; n = 4 mice, KO).


D , Average time needed to reach platform per day . Note that KO mice showed increased path latency on days 4 5 . E , Time mice spent on day 5 in quadrant in which goal was located during acquisition phase .

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D, Average time animals needed to reach the platform per day. Note that KO mice showed increased path length and latency on days 4 and 5. E, Time mice spent on day 5 in the quadrant in which the goal was located during the acquisition phase.


( A ) MCF - 7 were co - transfected with either MIR - CNT ( control plasmid ) or MIR376A GFP - LC3 plasmid , GFP - LC3 dot formation was analyzed . arrows indicate clusters of GFP - LC3 dots in . ( B ) of experiments in A . MIR376A overexpression , but not MIR - CNT expression , blocked starvation ( STV ) - induced autophagy ( mean ± S . D . of independent experiments , n = 4 , * * * p < 0 , 01 ) . NON STV , non -

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(A) MCF-7 cells were co-transfected with either MIR-CNT (control plasmid) or MIR376A and GFP-LC3 plasmid, and GFP-LC3 dot formation was analyzed. White arrows indicate clusters of the GFP-LC3 dots in cells. (B) Quantification of the experiments in A. MIR376A overexpression, but not MIR-CNT expression, blocked starvation (STV)-induced autophagy (mean ± S.D. of independent experiments, n = 4, ***p<0,01). NON STV, non-starved


( C ) Overexpression of MIR376A resulted in a decrease in autophagic activity of MCF - 7 . Starvation - induced conversion of LC3 - I to LC3 - II in MCF - 7 was analyzed . were performed in presence or absence of E64d ( 10 µg / ml ) Pepstatin A ( 10 µg / ml ) ( E + P ) . LC3 - II / LC3 - I densitometric ratios were marked . ACTB was used as a loading control .

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(C) Overexpression of MIR376A resulted in a decrease in the autophagic activity of MCF-7 cells. Starvation-induced conversion of LC3-I to LC3-II in MCF-7 cells was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.


( D ) MIR376A blocked starvation induced SQSTM1 degradation in MCF - 7 . ACTB was used as a loading control . SQSTM1 / ACTIN densitometric ratios were marked .

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(D) MIR376A blocked starvation induced SQSTM1 degradation in MCF-7 cells. ACTB was used as a loading control. SQSTM1/ACTIN densitometric ratios were marked.


( A ) MIR376A blocked GFP - LC3 dot formation under starvation condition .

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(A) MIR376A blocked GFP-LC3 dot formation under starvation condition.


( A ) MIR376A blocked GFP - LC3 dot formation under starvation condition . ( B ) Quantitative of experiments in A ( mean ± S . D . of independent experiments , n = 3 , * * * p < 0 , 01 ) .

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(A) MIR376A blocked GFP-LC3 dot formation under starvation condition. (B) Quantitative analysis of experiments in A (mean ± S.D. of independent experiments, n = 3, ***p<0,01).


( C ) Overexpression of MIR376A resulted in decreased autophagic flux in Huh - 7 . Starvation - induced conversion of LC3 - I to LC3 - II was analyzed . were performed in presence or absence of E64d ( 10 µg / ml ) Pepstatin A ( 10 µg / ml ) ( E + P ) . LC3 - II / LC3 - I densitometric ratios were marked . ACTB was used as a loading control .

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(C) Overexpression of MIR376A resulted in decreased autophagic flux in Huh-7 cells. Starvation-induced conversion of LC3-I to LC3-II was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.


( C D ) qPCR of ATG4C BECN1 mRNA expression in control miRNA ( MIR - CONT ) or MIR376A overexpressing under non - starved ( NON STV ) or starved ( STV ) condition ( mean ± S . D . of independent experiments , n = 3 , * p < 0 , 05 ) .

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(C and D) qPCR analysis of ATG4C and BECN1 mRNA expression in control miRNA (MIR-CONT) or MIR376A overexpressing cells under non-starved (NON STV) or starved (STV) condition (mean ± S.D. of independent experiments, n = 3, *p<0,05).


( E F ) Immunoblots showing ATG4C BECN1 following control miRNA ( MIR - CONT ) or MIR376A overexpression . ACTB was used as a loading control . ATG4C / ACTB or BECN1 / ACTB densitometric ratios were marked .

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(E and F) Immunoblots showing ATG4C and BECN1 protein levels following control miRNA (MIR-CONT) or MIR376A overexpression. ACTB was used as a loading control. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.


( A B ) qPCR of ATG4C BECN1 mRNA in MCF - 7 transfected with control antagomirs ( CNT - Ant ) or antagomir - 376a ( Ant - 376a ) ( mean ± S . D . of independent experiments , n = 3 , * * p < 0 , 03 for ATG4C , n = 3 , * * p < 0 , 03 for BECN1 ) . Results were expressed as fold changes against GAPDH mRNA .

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(A and B) qPCR analysis of ATG4C and BECN1 mRNA levels in MCF-7 cells transfected with control antagomirs (CNT-Ant) or antagomir-376a (Ant-376a) (mean ± S.D. of independent experiments, n = 3, **p<0,03 for ATG4C, and n = 3, **p<0,03 for BECN1). Results were expressed as fold changes against GAPDH mRNA levels.


( C D ) ATG4C BECN1 were increased following antagomir - 376a ( Ant - 376a ) transfection . ATG4C / ACTB or BECN1 / ACTB densitometric ratios were marked .

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(C and D) ATG4C and BECN1 protein levels were increased following antagomir-376a (Ant-376a) transfection. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.


( C D ) Normalized luciferase activity measuremets of lysates from 293T that were co - transfected with either MIR - CNT or MIR376A plasmids together with wild - type or mutant luciferase constructs ( mean ± S . D . of independent experiments , n = 3 for ATG4C , * p < 0 , 05 n = 5 for BECN1 , * * * p < 0 , 01 ) .

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(C and D) Normalized luciferase activity measuremets of lysates from 293T cells that were co-transfected with either MIR-CNT or MIR376A plasmids together with the wild-type or mutant luciferase constructs (mean ± S.D. of independent experiments, n = 3 for ATG4C tests, *p<0,05 and n = 5 for BECN1 tests, ***p<0,01).


( A ) Blockage of endogenous MIR376A by Ant - 376a , but not CNT - Ant further stimulated starvation ( STV ) - activated LC3 - I to LC3 - II conversion in MCF - 7 . ACTB was used as a loading control . LC3 - II / LC3 - I densitometric ratios were marked .

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(A) Blockage of endogenous MIR376A by Ant-376a, but not CNT-Ant further stimulated starvation (STV)-activated LC3-I to LC3-II conversion in MCF-7 cells. ACTB was used as a loading control. LC3-II/LC3-I densitometric ratios were marked.


( B ) Ant - 376a , but not CNT - Ant resulted in further activation of SQSTM1 degradation following starvation in MCF - 7 . SQSTM1 / ACTB ratios were marked .

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(B) Ant-376a, but not CNT-Ant resulted in further activation of SQSTM1 protein degradation following starvation in MCF-7 cells. SQSTM1/ACTB ratios were marked.


MIR376A MIR376B level changes in response to starvation stress . ( A B ) TaqMan quantitative PCR ( qPCR ) of endogenous MIR376A ( A ) MIR376B ( B ) under control ( CNT , no starvation ) or starvation ( STV , 1 to 8 hours ) conditions . TaqMan qPCR data was normalized using U6 small 1 ( RNU6 - 1 ) ( U6 ) mRNA ( mean ± S . D . of independent experiments , n = 5 for both A B , * * * p < 0 , 01 ) .

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MIR376A and MIR376B level changes in response to starvation stress. (A and B) TaqMan quantitative PCR (qPCR) analysis of endogenous MIR376A (A) and MIR376B (B) levels under control (CNT, no starvation) or starvation (STV, 1 to 8 hours) conditions. TaqMan qPCR data was normalized using U6 small nuclear 1 (RNU6-1) (U6) mRNA levels (mean ± S.D. of independent experiments, n = 5 for both A and B, ***p<0,01).


Quantitative phosphoproteomics dataset showing that most phosphorylation events induced in rad9Δ are dependent on Mec1 . These phosphoproteomic analyses were conducted in presence of 0 . 02 % MMS for 2 hours .

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Quantitative phosphoproteomics dataset showing that most phosphorylation events induced in rad9Δ cells are dependent on Mec1. These phosphoproteomic analyses were conducted in the presence of 0.02% MMS for 2 hours.


Immunoblot of Sgs1 N - terminus ( 1 - 647 ) from treated with increasing doses of alkylating drug MMS in absence of RAD9 / or MEC1 . This truncated Sgs1 was expressed from its native promoter in this subsequent experiments .

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Immunoblot analysis of Sgs1 N-terminus (amino acids 1-647) from cells treated with increasing doses of the DNA alkylating drug MMS in the absence of RAD9 and/or MEC1. This truncated Sgs1 protein was expressed from its native promoter in this and all subsequent experiments.


Rmi1 , a component of Sgs1 - Top3 - Rmi1 ( STR ) , is hyper - phosphorylated on S / T - Q motifs in rad9Δ .

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Rmi1, a component of the Sgs1-Top3-Rmi1 (STR) complex, is hyper-phosphorylated on S/T-Q motifs in rad9Δ cells.


Immunoblot showing co - immunoprecipitation of Dpb11 with Sgs1 . Both DPB11 - 3xHA SGS11 - 647 - 5xFLAG were expressed at their endogenous loci .

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Immunoblot showing co-immunoprecipitation of Dpb11 with Sgs1. Both DPB11-3xHA and SGS11-647-5xFLAG were expressed at their endogenous loci.


Immunoblots showing co - immunoprecipitation between Dpb11 Sgs1 in presence of 0 . 04 % MMS in either wildtype , mec1Δ , tel1Δ or mec1Δ tel1Δ lines . DPB11 - 3HA was tagged at its endogenous locus co - immunoprecipitated with SGS11 - 647 - FLAG tagged truncated at its endogenous locus .

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Immunoblots showing co-immunoprecipitation between Dpb11 and Sgs1 in the presence of 0.04% MMS in either wildtype, mec1Δ, tel1Δ or mec1Δ tel1Δ cell lines. DPB11-3HA was tagged at its endogenous locus and co-immunoprecipitated with SGS11-647-FLAG tagged and truncated at its endogenous locus.


Dilution of wild - type or rev3Δ expressing BRCT3 / 4 - SGS1 in presence of 0 . 0025 % MMS .

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Dilution assay of wild-type or rev3Δ cells expressing BRCT3/4-SGS1 in the presence of 0.0025% MMS.


Measurement of BIR efficiency in carrying an empty vector or expressing DPB11BRCT3 / 4 fused to wild - type or mutant versions of SGS1 . Mutants include a helicase deficient allele of SGS1 ( SGS1hd ) or an allele lacking Top3 Interacting Motif ( TIM ; SGS1TIM ) . Error bars represent of at least 3 replicate experiments . P value was calculated with a two - , unpaired t - . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 . C . Measurement of BIR efficiency in carrying an empty vector or expressing DPB11BRCT3 / 4 fused to either SGS1 , TOP3 , or RMI1 . Error bars represent of at least 3 replicate experiments . P value was calculated with a two - , unpaired t - . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 . D . Measurement of BIR efficiency in carrying an empty vector or DPB11BRCT3 / 4 fused to either SGS1 , hBLM , PIF1 , or RRM3 . Error bars represent of at least 3 replicate experiments . P value was calculated with a two - , unpaired t - . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 .

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

Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3/4 fused to wild-type or mutant versions of SGS1. Mutants include a helicase deficient allele of SGS1 (SGS1hd) or an allele lacking the Top3 Interacting Motif (TIM; SGS1TIM). Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001. C. Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3/4 fused to either SGS1, TOP3, or RMI1. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001. D. Measurement of BIR efficiency in cells carrying an empty vector or DPB11BRCT3/4 fused to either SGS1, hBLM, PIF1, or RRM3. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.


Serial dilution of sgs1Δ lines expressing either SGS1 or sgs1ΔACIDICPATCH .

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

Serial dilution assay of sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH.


BIR in rad9Δ sgs1Δ lines expressing either SGS1 or sgs1ΔACIDICPATCH . Error bars represent of 3 independent experiments . Error bars represent of at least 3 replicate experiments . P value was calculated with a two - , unpaired t - . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 .

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

BIR assay in rad9Δ sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH. Error bars represent SEM of 3 independent experiments. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.


( B - C ) Representative time - lapse of ( B ) Tet1 - 3 triple KD ( Tet - TKD ) , ( C ) Tet3 - KD control at 3 . 5 days post fertilization . Scale bar = 100 µm .

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

(B-C) Representative time-lapse images of (B) Tet1-3 triple KD (Tet-TKD), (C) Tet3-KD and control embryos at 3.5 days post fertilization. Scale bar = 100 µm.


( D - E ) Developmental rates of ( D ) control ( control - MO injected ) , Tet1 - , Tet2 - Tet3 - single KD Tet - TKD ( E ) control ( control - MO + Tet2 mRNA co - injected ) , Tet2 - KD2 ( Tet2 MO2 injected ) , Tet2 - KD2 + GFP mRNA Tet2 - KD2 + Tet2 mRNA until blastocyst stage . Indicated p - values were calculated using log rank ( Mantel - Cox ) against ( D ) control or ( E ) Tet2 - KD2 or as indicated ( ns = non - significant , * = p < 0 . 05 , * * * * = p < 0 . 0001 ; numbers of analyzed for each condition are indicated in parenthesis ) .

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

(D-E) Developmental rates of (D) control (control-MO injected), Tet1-, Tet2- and Tet3-single KD and Tet-TKD embryos and (E) control (control-MO + Tet2 mRNA co-injected), Tet2-KD2 (Tet2 MO2 injected), Tet2-KD2 + GFP mRNA and Tet2-KD2 + Tet2 mRNA embryos until blastocyst stage. Indicated p-values were calculated using log rank (Mantel-Cox) test against (D) control embryos or (E) Tet2-KD2 embryos or as indicated (ns = non-significant, * = p < 0.05, **** = p < 0.0001; numbers of analyzed embryos for each condition are indicated in parenthesis).


( A - B ) Representative of ( A ) 5hmC - ( B ) 5mC 5caC - IF of control ( control - MO injected ) , Tet1 + 2 - DKD Tet - TKD zygotes at 12 hpf . Paternal maternal pronuclei are indicated , Pb = polar ; Sp = sperm ; Scale bar = 20 μm .

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

(A-B) Representative images of (A) 5hmC- and (B) 5mC and 5caC-IF of control (control-MO injected), Tet1+2-DKD and Tet-TKD zygotes at 12 hpf. Paternal and maternal pronuclei are indicated, Pb = polar body; Sp = sperm; Scale bar = 20 μm.


( C - E ) of paternal / maternal ( C ) 5mC - , ( D ) 5hmC - , ( E ) 5caC - signal ratios of 12 hpf derived zygotes normalized against signal . A total of 5 - 16 zygotes from two to three experiments per condition were analyzed . Significance to control zygotes was calculated using ordinary one - way ANOVA with Tukey ' s multiple comparisons ( C , E ) or Welch ANOVA with Dunnett ' s T3 multiple comparisons ( D ) significant differences are indicated ( * = p < 0 . 05 , * * = < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 , data are represented as mean ± SD , with dots representing single zygotes ) .

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

(C-E) Quantification of paternal/maternal (C) 5mC-, (D) 5hmC-, and (E) 5caC-signal ratios of 12 hpf derived zygotes normalized against DNA signal. A total of 5-16 zygotes from two to three experiments per condition were analyzed. Significance to control zygotes was calculated using ordinary one-way ANOVA with Tukey's multiple comparisons test (C, E) or Welch ANOVA with Dunnett's T3 multiple comparisons test (D) and significant differences are indicated (* = p < 0.05, ** = < 0.01, *** = p < 0.001, **** = p < 0.0001, data are represented as mean ± SD, with dots representing single zygotes).


( B ) MA plots of - seq data from Tet3 - KD / control ( left ) Tet - TKD / control ( right ) 2 - . Significant differentially expressed genes are colored . amount of up - downregulated genes is indicated . Differentially expressed genes were determined using DEseq2 ( padj < 0 . 05 , | log2FC | > 0 . 7 ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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) MA plots of RNA-seq data from Tet3-KD/control (left) and Tet-TKD/control (right) 2-cell embryos. Significant differentially expressed genes are colored. The amount of up- and downregulated genes is indicated. Differentially expressed genes were determined using DEseq2 (padj<0.05, |log2FC|>0.7).


( A ) Expression changes of different classes of TEs in Tet3 - KD Tet - TKD . Shown are log2 - fold changes of KD compared to control 2 - of different repetitive elements subdivided in classes of TEs . Indicated p - values were calculated using a paired two - t - ( * * * = p < 0 . 001 , * * * * = p < 0 . 0001 , data are represented as medium smoothed violin plots with indicated median quartiles as dotted lines ) .

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

(A) Expression changes of different classes of TEs in Tet3-KD and Tet-TKD. Shown are log2-fold changes of KD embryos compared to control 2-cell embryos of different repetitive elements subdivided in classes of TEs. Indicated p-values were calculated using a paired two-tailed t-test (*** = p < 0.001, **** = p <0.0001, data are represented as medium smoothed violin plots with indicated median and quartiles as dotted lines).


( B - C ) Expression of ( B ) maternally expressed ERV3 - LTRs ( C ) 2 - specific ERV3 - LTRs in control , Tet3 - KD Tet - TKD 2 - ( p - values were calculated using DEseq2 ; * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 , data are represented as mean ± SD , dots represent single ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Expression levels of (B) maternally expressed ERV3-LTRs and (C) 2-cell embryo specific ERV3-LTRs in control, Tet3-KD and Tet-TKD 2-cell embryos (p-values were calculated using DEseq2; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, data are represented as mean ± SD, dots represent single embryos).


( E ) Expression of important candidate factors in control , Tet3 - KD Tet - TKD 2 - , implicated in generation of 2C - like as important regulators of EGA ( p - values were calculated using DEseq2 ; * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 , data are represented as mean ± SD , dots represent single ) .

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

(E) Expression levels of important candidate factors in control, Tet3-KD and Tet-TKD 2-cell embryos, implicated in the generation of 2C-like cells and as important regulators of EGA (p-values were calculated using DEseq2; * = p < 0.05, ** = p < 0.01, **** = p < 0.0001, data are represented as mean ± SD, dots represent single embryos).


( A ) % CpG methylation of 20 kb tiles in 5 % bins in control or Tet - TKD 2 - . samples from one condition were merged for this visualization .

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

(A) % CpG methylation of 20 kb tiles in 5% bins in control or Tet-TKD 2-cell embryos. All samples from one condition were merged for this visualization.


( F ) Significant methylation changes of specific TEs subdivided in different classes of TEs ( calculated using logistic regression function of Seqmonk , padj < 0 . 05 ; data are represented as medium smoothed violin plot with indicated median quartiles as dotted ) .

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

(F) Significant methylation changes of specific TEs subdivided in different classes of TEs (calculated using logistic regression function of Seqmonk, padj < 0.05; data are represented as medium smoothed violin plot with indicated median and quartiles as dotted line).


( G ) Correlation of significant methylation changes in TEs ( calculated with logistic regression function of Seqmonk , padj < 0 . 05 , methylation difference > 5 % ) , which are also significant differentially expressed ( DEseq2 , padj < 0 . 05 , | log2FC | > 0 . 7 ) .

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

(G) Correlation of significant DNA methylation changes in TEs (calculated with logistic regression function of Seqmonk, padj < 0.05, methylation difference >5%), which are also significant differentially expressed (DEseq2, padj < 0.05, |log2FC|>0.7).


( H ) Developmental rate of derived from control , Tet2 - MO2 , Tet2 - MO2 + Tet2 full - mRNA ( as shown in Fig . 1E ) Tet2 - MO2 + Tet2 truncated mRNA ( no catalytical domain ) injected oocytes . Indicated p - values were calculated using log rank ( Mantel - Cox ) against Tet2 - MO2 KD or as indicated ( * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 , numbers of analyzed for each condition is indicated in parenthesis ) .

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

(H) Developmental rate of embryos derived from control, Tet2-MO2, Tet2-MO2 + Tet2 full-length mRNA (as shown in Fig. 1E) and Tet2-MO2 + Tet2 truncated mRNA (no catalytical domain) injected oocytes. Indicated p-values were calculated using the log rank (Mantel-Cox) test against Tet2-MO2 KD or as indicated (* = p < 0.05, *** = p < 0.001, **** = p < 0.0001, numbers of analyzed embryos for each condition is indicated in parenthesis).


A ) Visualisation of pathology load at 9M of of TAUwt , TAUtg APPtg mice at 9M of . Immunofluorescent for X34 ( a derivative of Congo Red ) , Iba1 ( microglia ) TO - PRO - 3 ( nuclei ) has been pseudocolored . Scale bar = 100μm .

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

A) Visualisation of the pathology load at 9M of age of TAUwt, TAUtg and APPtg mice at 9M of age. Immunofluorescent staining for X34 (a fluorescent derivative of Congo Red), Iba1 (microglia) and TO-PRO-3 (nuclei) has been pseudocolored. Scale bar = 100μm.


Log2 fold change ( LFC ) in TAUtg ( x - axis ) APPtg mice ( y - axis ) after differential expression . Upregulated genes are on right part ( TAUtg mice ) or upper part ( APPtg mice ) of graph ; downregulated genes are on left part ( TAUtg ) or lower part ( APPtg ) of graph . Colored dots represent significantly differentially expressed genes ( Benjamini - Yuketieli - adjusted p - values ( Padj ) < 0 . 05 ) for APPtg ( green dots ) , TAUtg ( yellow dots ) or for both ( red dots ) . Spearman correlation assesses correlation between APPtg TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up - to most down - regulated on a combined score of LFC padj ( i . e . signed log10 ( p - value ) , where sign is determined by LFC ) . A ) Genes differentially expressed as a function of , i . e . 4M vs 10M , independently of genotype . Thus , genes with a positive LFC are more highly expressed in 10M mice over 4M mice .

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

Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis. Upregulated genes are on the right part (TAUtg mice) or upper part (APPtg mice) of the graph; downregulated genes are on the left part (TAUtg) or lower part (APPtg) of the graph. Colored dots represent significantly differentially expressed genes (Benjamini-Yuketieli-adjusted p-values (Padj) <0.05) for APPtg (green dots), TAUtg (yellow dots) or for both (red dots). Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up- to most down-regulated on a combined score of LFC and padj (i.e. signed log10(p-value), where the sign is determined by the LFC). A) Genes differentially expressed as a function of age, i.e. 4M vs 10M, independently of genotype. Thus, genes with a positive LFC are more highly expressed in 10M mice over 4M mice.


Log2 fold change ( LFC ) in TAUtg ( x - axis ) APPtg mice ( y - axis ) after differential expression . Upregulated genes are on right part ( TAUtg mice ) or upper part ( APPtg mice ) of graph ; downregulated genes are on left part ( TAUtg ) or lower part ( APPtg ) of graph . Colored dots represent significantly differentially expressed genes ( Benjamini - Yuketieli - adjusted p - values ( Padj ) < 0 . 05 ) for APPtg ( green dots ) , TAUtg ( yellow dots ) or for both ( red dots ) . Spearman correlation assesses correlation between APPtg TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up - to most down - regulated on a combined score of LFC padj ( i . e . signed log10 ( p - value ) , where sign is determined by LFC ) . B ) Genes differentially expressed due to genotype , i . e . WT vs TG , independently of . Thus , genes with a positive LFC are more highly expressed in TG mice than in WT mice .

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

Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis. Upregulated genes are on the right part (TAUtg mice) or upper part (APPtg mice) of the graph; downregulated genes are on the left part (TAUtg) or lower part (APPtg) of the graph. Colored dots represent significantly differentially expressed genes (Benjamini-Yuketieli-adjusted p-values (Padj) <0.05) for APPtg (green dots), TAUtg (yellow dots) or for both (red dots). Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up- to most down-regulated on a combined score of LFC and padj (i.e. signed log10(p-value), where the sign is determined by the LFC). B) Genes differentially expressed due to genotype, i.e. WT vs TG, independently of age. Thus, genes with a positive LFC are more highly expressed in TG mice than in WT mice.


Log2 fold change ( LFC ) in TAUtg ( x - axis ) APPtg mice ( y - axis ) after differential expression . Upregulated genes are on right part ( TAUtg mice ) or upper part ( APPtg mice ) of graph ; downregulated genes are on left part ( TAUtg ) or lower part ( APPtg ) of graph . Colored dots represent significantly differentially expressed genes ( Benjamini - Yuketieli - adjusted p - values ( Padj ) < 0 . 05 ) for APPtg ( green dots ) , TAUtg ( yellow dots ) or for both ( red dots ) . Spearman correlation assesses correlation between APPtg TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up - to most down - regulated on a combined score of LFC padj ( i . e . signed log10 ( p - value ) , where sign is determined by LFC ) . C ) Genes differentially expressed in * genotype interaction comparison , i . e . comparing TG - 10M mice to other experimental groups ( see Fig . 1C ) . Thus , genes with a positive LFC are more highly expressed in TG - 10M mice compared to other experimental groups .

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

Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis. Upregulated genes are on the right part (TAUtg mice) or upper part (APPtg mice) of the graph; downregulated genes are on the left part (TAUtg) or lower part (APPtg) of the graph. Colored dots represent significantly differentially expressed genes (Benjamini-Yuketieli-adjusted p-values (Padj) <0.05) for APPtg (green dots), TAUtg (yellow dots) or for both (red dots). Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up- to most down-regulated on a combined score of LFC and padj (i.e. signed log10(p-value), where the sign is determined by the LFC). C) Genes differentially expressed in the age*genotype interaction comparison, i.e. comparing the TG-10M mice to all other experimental groups (see Fig.1C). Thus, genes with a positive LFC are more highly expressed in the TG-10M mice compared to all other experimental groups.


Log2 fold change ( LFC ) in TAUtg ( x - axis ) APPtg mice ( y - axis ) after differential expression . Upregulated genes are on right part ( TAUtg mice ) or upper part ( APPtg mice ) of graph ; downregulated genes are on left part ( TAUtg ) or lower part ( APPtg ) of graph . Colored dots represent significantly differentially expressed genes ( Benjamini - Yuketieli - adjusted p - values ( Padj ) < 0 . 05 ) for APPtg ( green dots ) , TAUtg ( yellow dots ) or for both ( red dots ) . Spearman correlation assesses correlation between APPtg TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up - to most down - regulated on a combined score of LFC padj ( i . e . signed log10 ( p - value ) , where sign is determined by LFC ) . D ) Depicts 314 Marioni - GWAS genes at p < 0 . 001 onto LFC / LFC plot of * genotype comparison ( panel C ) . Green dots are significantly changed .

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

Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis. Upregulated genes are on the right part (TAUtg mice) or upper part (APPtg mice) of the graph; downregulated genes are on the left part (TAUtg) or lower part (APPtg) of the graph. Colored dots represent significantly differentially expressed genes (Benjamini-Yuketieli-adjusted p-values (Padj) <0.05) for APPtg (green dots), TAUtg (yellow dots) or for both (red dots). Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up- to most down-regulated on a combined score of LFC and padj (i.e. signed log10(p-value), where the sign is determined by the LFC). D) Depicts the 314 Marioni-based GWAS genes at p<0.001 onto the LFC/LFC plot of the age*genotype comparison (panel C). Green dots are significantly changed.


B ) Log2 fold change ( LFC ) in TAUtg ( x - axis ) APPtg mice ( y - axis ) after differential expression , assessing effects of * genotype interaction . Color code for dots / numbers : grey = genes in hippocampus ( n = 15824 ) ; black = genes in APPtg - Blue ( n = 4236 ) ; green = genes significantly differentially expressed in APPtg mice ( n = 493 ) ; yellow = significantly differentially expressed genes in TAUtg mice ( n = 9 ) ; red = significantly differentially expressed genes in both ( n = 9 ) .

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B) Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis, assessing the effects of age*genotype interaction. Color code for dots/numbers: grey = genes in hippocampus (n=15824); black = genes in APPtg-Blue (n=4236); green = genes significantly differentially expressed in APPtg mice (n=493); yellow = significantly differentially expressed genes in TAUtg mice (n=9); red = significantly differentially expressed genes in both (n=9).