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 .

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

Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI , +/- exposure to 400 µM CuCl2 for 30 min. : Rhomboid substrates that are uncleaved, cleaved by GlpG, are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.


F Degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdnH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( S201A , SAHA ) GlpG expressed from pUC19 ( plasmid ) without FdnI ( - ) , , + / - exposure to 400 µM CuCl2 for 30 min . Rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , and blue arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

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

O O O O O O 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 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 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 test .

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

(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 ) 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 .

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

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 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

(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 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 .

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

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

(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 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 .

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

(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 ) 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 .

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

(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 ) 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 test .

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

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


( 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 ) .

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

(B) 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 ) 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 ) .

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

(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 ) 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 ) .

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

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

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

(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 - analysis of footprint size of all 17 inhibitory pairs identified aligning the first codon of the pair in the 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 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 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 ) .

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

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 ) 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 % .

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

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

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 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 .

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

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 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 .

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

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 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 .

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

J, 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 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 ) .

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

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 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 .

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

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 , 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 ) .

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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 , 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 .

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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 , 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 .

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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 , 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 ) .

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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 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 .

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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 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 -

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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 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 .

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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 cells . 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 analysis 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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 and D ) ATG4C and BECN1 protein levels 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 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 ) .

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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 cells . 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 protein degradation following starvation in MCF - 7 cells . 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 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 ) .

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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 cells are dependent on Mec1 . These phosphoproteomic analyses were conducted in the 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 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 .

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

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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 and 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 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 .

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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 assay of wild - type or rev3Δ cells expressing BRCT3 / 4 - SGS1 in the 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 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 .

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

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 assay of sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH .

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

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


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

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

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

(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 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 .

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

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

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

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 O O O O O O 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 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 ) .

O O O O O O O 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 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 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 ) .

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 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 - cell embryos . All samples from one condition were merged for this visualization .

O O O O B-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

(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 and quartiles as dotted line ) .

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

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

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

(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 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 .

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

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 ) 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 .

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

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

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

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

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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).


C ) Z - score distribution per experimental group for all genes within the APPtg - Blue module . Boxplots : center line , median ; box limits , 25th - 75th quartiles ; whiskers , 1 . 5x interquartile range . Empirical p - values are Bonferroni adjusted ( * * * pbonf < 0 . 001 ) and indicate significant shift in z - score

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C) Z-score distribution per experimental group for all genes within the APPtg-Blue module. Boxplots: center line, median; box limits, 25th-75th quartiles; whiskers, 1.5x interquartile range. Empirical p-values are Bonferroni adjusted (***pbonf<0.001) and indicate significant shift in z-score distribution


A ) UMAP clustering of sorted Cd11b + / Cd45 + myeloid cells from mouse hippocampus . ARM : activated response microglia ; CAM : CNS - associated macrophages ; CPM : cycling and proliferating microglia ; HM . 1 : homeostatic microglia cluster 1 ; HM . 2 : homeostatic microglia cluster 2 ; IRM : interferon - response microglia ; MHC . high : high MHC - expressing microglia ; Mnc : monocytes ; TRM : transitioning microglia .

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A) UMAP clustering of sorted Cd11b+/Cd45+ myeloid cells from mouse hippocampus. ARM: activated response microglia; CAM: CNS-associated macrophages; CPM: cycling and proliferating microglia; HM.1: homeostatic microglia cluster 1; HM.2: homeostatic microglia cluster 2; IRM: interferon-response microglia; MHC.high: high MHC-expressing microglia; Mnc: monocytes; TRM: transitioning microglia.


B ) Distribution of cells across the different clusters per experimental group ( n = 2 mice pooled for all experimental groups , except for TAUtg - 11M and TAUwt - 11M with n = 3 mice pooled ) .

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B) Distribution of cells across the different clusters per experimental group (n=2 mice pooled for all experimental groups, except for TAUtg-11M and TAUwt-11M with n=3 mice pooled).


C ) Distribution of cells per experimental group over the different clusters , expressed in percentages as stated on top of each bar . E . g . of all cells within APPtg - 11M , 56 . 7 % of cells are ARM , while 12 . 5 % are HM . 1 cells .

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C) Distribution of cells per experimental group over the different clusters, expressed in percentages as stated on top of each bar. E.g. of all cells within APPtg-11M, 56.7% of cells are ARM, while 12.5% are HM.1 cells.


E ) Differential expression of ARM vs HM . 1 microglia for APPtg and TAUtg mice separately . Genes with a positive log ( fold change ) ( LFC ) are higher expressed in ARMs compared to HM . 1 microglia . As can be seen which genes are more highly expressed in ARMs vs HM . 1 cells are highly similar among APPtg and TAUtg mice ( Spearman correlation r = 0 . 91 , p = 2 . 2e - 16 ) . LFC > | 0 . 2 | with padj < 0 . 05 are considered significant .

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E) Differential expression of ARM vs HM.1 microglia for APPtg and TAUtg mice separately. Genes with a positive log(fold change) (LFC) are higher expressed in ARMs compared to HM.1 microglia. As can be seen which genes are more highly expressed in ARMs vs HM.1 cells are highly similar among APPtg and TAUtg mice (Spearman correlation r=0.91, p=2.2e-16). LFC > |0.2| with padj<0.05 are considered significant.


A , B In - gel kinase assay of OST1 activity in ABA receptor mutants under cold stress . Ten - day - old wild type , ost1 - 3 , pyr1 pyl1 , 4 , pyr1 pyl1 , 2 , 4 , pyr1 pyl4 , 5 , 8 and pyr1 pyl1 , 4 , 5 , 8 mutants were treated at 4 ° C for 2 h . Total protein extracts were prepared and separated on SDS - PAGE gel containing 0 . 1 mg / mL GST - ∆ ABF2 as a substrate , and incubated with 60 μCi of [ γ - 32P ] ATP . Representative pictures are shown in ( A ) and relative kinase activity is shown in ( B ) . Data information : In ( A , Top , gel autoradiograph ; bottom , Coomassie Brilliant Blue ( CBB ) staining of RuBisCO large subunit was used as a loading control . The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In ( B , each bar represents the mean ± SEM of three biological repeats . * P < 0 . 05 ( two - tailed t - test ) .

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A, B In-gel kinase assay of OST1 activity in ABA receptor mutants under cold stress. Ten-day-old wild type, ost1-3, pyr1 pyl1,4, pyr1 pyl1,2,4, pyr1 pyl4,5,8 and pyr1 pyl1,4,5,8 mutants were treated at 4°C for 2 h. Total protein extracts were prepared and separated on SDS-PAGE gel containing 0.1 mg/mL GST-∆ABF2 as a substrate, and incubated with 60 μCi of [γ-32P]ATP. Representative pictures are shown in (A) and relative kinase activity is shown in (B). Data information: In (A , Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (B , each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).


C , D In - gel kinase assay of OST1 activity in ABI1 - OE14 and abi1 abi2 hab1 mutants under cold stress . Representative pictures are shown in ( C ) and relative kinase activity is shown in ( D ) . Data information : In C ) , Top , gel autoradiograph ; bottom , Coomassie Brilliant Blue ( CBB ) staining of RuBisCO large subunit was used as a loading control . The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In D ) , each bar represents the mean ± SEM of three biological repeats . * P < 0 . 05 ( two - tailed t - test ) .

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C, D In-gel kinase assay of OST1 activity in ABI1-OE14 and abi1 abi2 hab1 mutants under cold stress. Representative pictures are shown in (C) and relative kinase activity is shown in (D). Data information: In C), Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In D), each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).


A Interaction of OST1 and EGR2 in yeast . Yeast cells harboring different vector combinations were grown on SC / − Leu / − Trp medium for 3 d or SC / − Leu / − Trp / − His / − Ade medium for 6 d . OST1 - BD / AD and BD / EGR2 - AD were used as negative controls .

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A Interaction of OST1 and EGR2 in yeast. Yeast cells harboring different vector combinations were grown on SC/−Leu/−Trp medium for 3 d or SC/−Leu/−Trp/−His/−Ade medium for 6 d. OST1-BD/AD and BD/EGR2-AD were used as negative controls.


B OST1 interacts with EGR2 in vitro . Purified recombinant MBP - His - EGR2 or MBP - His proteins from E . coli were immunoprecipitated with MBP beads , and then incubated with purified recombinant GST - OST1 . Precipitated proteins were detected with anti - His and anti - GST antibodies .

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B OST1 interacts with EGR2 in vitro. Purified recombinant MBP-His-EGR2 or MBP-His proteins from E. coli were immunoprecipitated with MBP beads, and then incubated with purified recombinant GST-OST1. Precipitated proteins were detected with anti-His and anti-GST antibodies.


C Co - IP assay of OST1 with EGR2 in vivo . 35S : HF - OST1 / Super : EGR2 - Myc or 35S : HF - OST1 / Super : Myc was expressed in N . benthamiana leaves . Total proteins were immunoprecipitated with anti - Myc agarose beads , and the co - immunoprecipitation products were subjected to immunoblot analysis . EGR2 - Myc and HF - OST1 were detected with anti - Myc and anti - HA antibodies , respectively .

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C Co-IP assay of OST1 with EGR2 in vivo. 35S:HF-OST1/Super:EGR2-Myc or 35S:HF-OST1/Super:Myc was expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti-Myc agarose beads, and the co-immunoprecipitation products were subjected to immunoblot analysis. EGR2-Myc and HF-OST1 were detected with anti-Myc and anti-HA antibodies, respectively.


D Bimolecular fluorescence complementation ( BiFC ) assay . Wild - type Arabidopsis protoplasts transformed with OST1 - YFPC and EGR2 - YFPN proteins were incubated for 18 h . The interaction signal was detected by confocal microcopy . The combinations of OST1 - YFPC / GUS - YFPN and GUS - YFPC / EGR2 - YFPN were used as negative controls . Scale bars : 100 μm .

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D Bimolecular fluorescence complementation (BiFC) assay. Wild-type Arabidopsis protoplasts transformed with OST1-YFPC and EGR2-YFPN proteins were incubated for 18 h. The interaction signal was detected by confocal microcopy. The combinations of OST1-YFPC/GUS-YFPN and GUS-YFPC/EGR2-YFPN were used as negative controls. Scale bars: 100 μm.


E EGR2 inhibits OST1 kinase activity in vitro . Proteins were incubated with 1 μCi [ γ - 32P ] ATP in kinase reaction buffer for 30 min at 30 ° C , and then separated by SDS - PAGE . Top , autoradiograph ; bottom , CBB staining .

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E EGR2 inhibits OST1 kinase activity in vitro. Proteins were incubated with 1 μCi [γ-32P]ATP in kinase reaction buffer for 30 min at 30°C, and then separated by SDS-PAGE. Top, autoradiograph; bottom, CBB staining.


F , G In - gel assay of OST1 activity in 10 - d - old wild type , egr1 and egr2 single mutants , and the egr1 egr2 double mutant under cold stress . Data information : the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In ( F representative pictures are shown : Top , autoradiograph ; bottom , CBB staining . In ( G relative kinase activity is shown . Each bar represents the mean ± SEM of three biological repeats . * * P < 0 . 01 ( two - tailed t - test ) .

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F, G In-gel assay of OST1 activity in 10-d-old wild type, egr1 and egr2 single mutants, and the egr1 egr2 double mutant under cold stress. Data information: the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (F representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In (G relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


H , I In - gel assay of OST1 activity in 10 - d - old wild - type and EGR2 - overexpressing plants under cold stress . Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In H ) , representative pictures are shown : Top , autoradiograph ; bottom , CBB staining . In I ) , relative kinase activity is shown . Each bar represents the mean ± SEM of three biological repeats . * * P < 0 . 01 ( two - tailed t - test ) .

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H, I In-gel assay of OST1 activity in 10-d-old wild-type and EGR2-overexpressing plants under cold stress. Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In H), representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In I), relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


Freezing phenotype ( A ) of the egr2 - 3 mutant and egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) and acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) .

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Freezing phenotype (A) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA).


survival rate ( B ) of the egr2 - 3 mutant and egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) and acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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survival rate (B) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA). Data information each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


ion leakage ( C ) of the egr2 - 3 mutant and egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) and acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

ion leakage (C) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA). Data information each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Freezing phenotype of EGR2 - overexpressing plants . Two - week - old Super : EGR2 - Myc

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Freezing phenotype of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants


survival rate ( E ) of EGR2 - overexpressing plants . Two - week - old Super : EGR2 - Myc plants Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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survival rate (E) of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


ion leakage ( F ) of EGR2 - overexpressing plants . Two - week - old Super : EGR2 - Myc plants Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

ion leakage (F) of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Expression of CBFs ( G in egr2 - 3 mutant plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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Expression of CBFs (G in egr2-3 mutant plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Expression of CBFs target genes ( H in egr2 - 3 mutant plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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Expression of CBFs target genes (H in egr2-3 mutant plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Expression of CBFs in EGR2 - overexpressing plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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

Expression of CBFs in EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Expression of CBFs target genes J ) in EGR2 - overexpressing plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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

Expression of CBFs target genes J) in EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).