( 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 .

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

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

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 .

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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 .

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(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 ) .

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(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 .

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

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(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 ) .

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(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 ) .

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(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 ) .

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(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 .

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(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 ) .

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(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 O O O O O O O O O O O O O O O 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) 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 .

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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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 genes within APPtg - Blue module . Boxplots : center , median ; box limits , 25th - 75th quartiles ; whiskers , 1 . 5x interquartile range . Empirical p - values are Bonferroni adjusted ( * * * pbonf < 0 . 001 ) indicate significant shift in z - score

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 from mouse hippocampus . ARM : activated response microglia ; CAM : CNS - associated macrophages ; CPM : cycling 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 across different clusters per experimental group ( n = 2 mice pooled for experimental groups , except for TAUtg - 11M TAUwt - 11M with n = 3 mice pooled ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 are highly similar among APPtg TAUtg mice ( Spearman correlation r = 0 . 91 , p = 2 . 2e - 16 ) . LFC > | 0 . 2 | with padj < 0 . 05 are considered significant .

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

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

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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 EGR2 in yeast . Yeast 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 BD / EGR2 - AD were used as negative controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 from E . coli were immunoprecipitated with MBP beads , then incubated with purified recombinant GST - OST1 . Precipitated were detected with anti - His anti - GST antibodies .

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 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 were immunoprecipitated with anti - Myc agarose beads , co - immunoprecipitation products were subjected to immunoblot . EGR2 - Myc HF - OST1 were detected with anti - Myc anti - HA antibodies , respectively .

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

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 . were incubated with 1 μCi [ γ - 32P ] ATP in kinase reaction buffer for 30 min at 30 ° C , then separated by SDS - PAGE . Top , autoradiograph ; bottom , CBB .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 egr2 - 3 mutant egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) 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 ) .

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

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 egr2 - 3 mutant egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) 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 mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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

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 egr2 - 3 mutant egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) 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 mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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 . Two - week - old Super : EGR2 - Myc

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


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

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

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

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

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

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

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

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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).


N - myristoylation of EGR2 EGR2G2A in N . benthamiana leaves . extracts prepared from N . benthamiana leaves expressing EGR2 - Myc or EGR2G2A - Myc were immunoprecipated by anti - Myc agarose beads . Precipitated were detected by anti - Myc anti - myristic acid antibodies .

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N-myristoylation of EGR2 and EGR2G2A in N. benthamiana leaves. Protein extracts prepared from N. benthamiana leaves expressing EGR2-Myc or EGR2G2A-Myc were immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies.


D Immunoblot showing that total level of myristoylated in wild type . Total were prepared from two - week - old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22 ° C for 72 h then subjected to click - reaction with biotin azide . reaction was protected from at room for 3 h . products were detected by anti - streptavidin - HRP antibody .

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D Immunoblot analysis showing that the total level of myristoylated proteins in wild type plants. Total proteins were prepared from two-week-old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22°C for 72 h and then subjected to click-reaction with the biotin azide. The reaction was protected from light at room temperature for 3 h. The products were detected by anti-streptavidin-HRP antibody.


E N - myristoylation of EGR2 in . Total were prepared from two - week - old EGR2 - Myc seedlings products were immunoprecipated by anti - Myc agarose beads detected by anti - streptavidin - HRP antibody . EGR2 - Myc detected by anti - Myc antibody 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

E N-myristoylation of EGR2 in plants. Total proteins were prepared from two-week-old EGR2-Myc seedlings The products were immunoprecipated by anti-Myc agarose beads and detected by anti-streptavidin-HRP antibody. EGR2-Myc detected by anti-Myc antibody was used as a loading control.


F Immunoblot of myristoylated EGR2 under cold stress . Total were prepared form EGR2 - Myc treated with 4 ° C for indicated period , immunoprecipated by anti - Myc agarose beads . Precipitated were detected by anti - Myc anti - myristic acid antibodies . of band detected by anti - myristic acid antibody to anti - Myc antibody without cold treatment was set to 1 . 00 . Representative pictures are shown ( left ) quantitative is shown ( right ) . Each bar represents mean ± of three biological repeats . * * P < 0 . 01 ( two - t - ) .

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

F Immunoblot assay of myristoylated EGR2 under cold stress. Total proteins were prepared form EGR2-Myc plants treated with 4°C for the indicated period, and immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies. The ratio of band intensity detected by anti-myristic acid antibody to anti-Myc antibody without cold treatment was set to 1.00. Representative pictures are shown (left) and quantitative analysis is shown (right). Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


G Expression of EGR2 in egr2 - 3 EGR2 : EGR2G2A - Myc complementation transgenic . Total RNAs were extracted from 2 - week - old seedlings subjected to RT - PCR . EF1α was used as a loading control .

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G Expression of EGR2 in egr2-3 EGR2:EGR2G2A-Myc complementation transgenic plants. Total RNAs were extracted from 2-week-old seedlings and subjected to RT-PCR analysis. EF1α was used as a loading control.


Freezing tolerance assays of egr2 - 3 mutant EGR2 : EGR2G2A - Myc transgenic in egr2 - 3 mutant . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar at 22 ° C were treated at − 5 ° C for 0 - 1 . 5 h ( NA ) or − 8 ° C for 0 - 1 . 5 h ( CA ) . Representative ( - 5oC , 0 . 5 h ; - 8oC , 0 . 5 h ) are shown in ( H )

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Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). Representative photographs (-5oC, 0.5 h; -8oC, 0.5 h) are shown in (H)


Freezing tolerance assays of egr2 - 3 mutant EGR2 : EGR2G2A - Myc transgenic in egr2 - 3 mutant . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar at 22 ° C were treated at − 5 ° C for 0 - 1 . 5 h ( NA ) or − 8 ° C for 0 - 1 . 5 h ( CA ) . survival rate is shown in ( I ) . Data shown in ( I ) are mean values ± of three biological replicates , each of which has three technical replicates . Asterisks indicate significant differences compared with wild type for same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) ; n . s . , not significant .

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Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). survival rate is shown in (I). Data shown in (I) are mean values ± SEM of three biological replicates, each of which has three technical replicates. Asterisks indicate significant differences compared with wild type for the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test); n. s., not significant.


A In vitro pull - down showing interaction of NMT1 EGR2 . Recombinant GST - NMT1 was incubated with immunoprecipated MBP - His - EGR2 or MBP - His at 4 ° C for 2 h . Anti - GST anti - MBP were used to detect NMT1 , EGR2 MBP - His , respectively .

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A In vitro pull-down assay showing the interaction of NMT1 and EGR2. Recombinant GST-NMT1 was incubated with immunoprecipated MBP-His-EGR2 or MBP-His at 4°C for 2 h. Anti-GST and anti-MBP were used to detect NMT1, EGR2 and MBP-His, respectively.


Co - localization of NMT1 EGR2 in N . benthamiana leaves . Scale bar : 50

Co-localization of NMT1 and EGR2 in N. benthamiana leaves. Scale bar: 50 μm


C , D Effect of cold stress on interaction of EGR2 NMT1 determined by co - IP . Arabidopsis protoplasts expressing HF / NMT1 - Myc HF - EGR2 / NMT1 - Myc were treated with or without 4 ° C for 2 h . Total were immunoprecipated by anti - HA agarose beads . Anti - Myc anti - HA were used to detect NMT1 - Myc HF - EGR2 , respectively . In ( C ) , relative band is shown below co - IP blot . In ( D ) , each bar represents mean ± of three biological experiments ( * * P < 0 . 01 , two - t - ) .

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C, D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co-IP assay. Arabidopsis protoplasts expressing HF/NMT1-Myc and HF-EGR2/NMT1-Myc were treated with or without 4°C for 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc and anti-HA were used to detect NMT1-Myc and HF-EGR2, respectively. In (C), relative band intensity is shown below the co-IP blot. In (D), each bar represents the mean ± SEM of three biological experiments (**P < 0.01, two-tailed t-test).


E In vitro pull - down showing decreased interaction of EGR2 ( 4 ° C ) NMT1 compared with that of EGR2 ( 22 ° C ) NMT1 . Total prepared from two - week - old EGR2 - Myc grown at 4 ° C for 0 h 6 h were immunoprecipated by anti - Myc agarose beads incubated with GST - NMT1 in pull down buffer for 2 h at 4 ° C . Anti - Myc anti - GST antibodies were used to detect EGR2 NMT1 . Relative band is shown below output blot .

E In vitro pull-down assay showing decreased interaction intensity of EGR2 (4°C) and NMT1 compared with that of EGR2 (22°C) and NMT1. Total proteins prepared from two-week-old EGR2-Myc plants grown at 4°C for 0 h and 6 h were immunoprecipated by anti-Myc agarose beads and incubated with GST-NMT1 in pull down buffer for 2 h at 4°C. Anti-Myc and anti-GST antibodies were used to detect EGR2 and NMT1 proteins. Relative band intensity is shown below the output blot.


F Expression of NMT1 in NMT1 - Myc overexpression . EF1α was used as a loading control .

F Expression of NMT1 in NMT1-Myc overexpression plants. EF1α was used as a loading control.


Freezing phenotype ( G ) of NMT1 - Myc overexpression transgenic . Two - week - old Super : NMT1 - Myc were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) .

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Freezing phenotype (G) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA).


survival rate ( H ) of NMT1 - Myc overexpression transgenic . Two - week - old Super : NMT1 - Myc were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . In ( H ) , each bar represents mean ± of three biological experiments , asterisks indicate significant differences compared with wild type for same treatment ( * P < 0 . 05 , two t - ) .

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survival rate (H) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA). In (H), each bar represents the mean ± SEM of three biological experiments, and asterisks indicate significant differences compared with the wild type for the same treatment (*P < 0.05, two tailed t-test).


OST1 kinase activity in NMT1 - Myc overexpression under cold stress . Top , autoradiograph ; bottom , CBB . Representative pictures are shown in ( I ) In ( I ) : of band of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1 . 00 .

OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. Top, autoradiograph; bottom, CBB staining. Representative pictures are shown in (I) In (I): the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1.00.


OST1 kinase activity in NMT1 - Myc overexpression under cold stress . relative kinase activity is shown in ( J ) . In ( J ) : each bar represents mean ± of three independent experiments ( * * P < 0 . 01 , two t - ) .

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OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. the relative kinase activity is shown in (J). In (J): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two tailed t-test).


A , B Pull - down . Total prepared from EGR2 - Myc or EGR2G2A - Myc transgenic was immunoprecipated with anti - Myc agarose beads , then incubated with GST - OST1 . Anti - Myc anti - GST antibody were used to detect GST - OST1 EGE2 - Myc . Representative pictures are shown in ( A ) . Relative interaction is shown in ( B ) . Data information : In ( A Relative band intensities are shown . In ( B each bar represents mean ± of three independent experiments ( * * P < 0 . 01 , two - t - ) .

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A, B Pull-down assay. Total proteins prepared from EGR2-Myc or EGR2G2A-Myc transgenic plants was immunoprecipated with anti-Myc agarose beads, and then incubated with GST-OST1. Anti-Myc and anti-GST antibody were used to detect GST-OST1 and EGE2-Myc. Representative pictures are shown in (A). Relative interaction is shown in (B). Data information: In (A Relative band intensities are shown. In (B each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


C LCI of EGR2 EGR2G2A with OST1 in N . benthamiana leaves . EGR2 - cLuc / OST1 - nLuc EGR2G2A - cLuc / OST1 - nLuc were expressed in N . benthamiana leaves for 48 h . EGR2 - cLuc / nLuc , EGR2G2A - cLuc / nLuc , cLuc / OST1 - nLuc were used as negative controls . Scale bar : 2 cm .

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C LCI of EGR2 and EGR2G2A with OST1 in N. benthamiana leaves. EGR2-cLuc/OST1-nLuc and EGR2G2A-cLuc/OST1-nLuc were expressed in N. benthamiana leaves for 48 h. EGR2-cLuc/nLuc, EGR2G2A-cLuc/nLuc, and cLuc/OST1-nLuc were used as negative controls. Scale bar: 2 cm.


D , E Co - IP . EGR2 - Myc / HF - OST1 , EGR2G2A - Myc / HF - OST1 , HF / EGR2 - Myc HF / EGR2G2A - Myc were expressed in N . benthamiana leaves . Total were immunoprecipated by anti - HA agarose beads . Anti - HA anti - Myc antibodies were used to detect HF - OST1 , EGR2 - Myc EGR2G2A - Myc , respectively . Representative pictures are shown in ( D ) , relative interaction is shown in ( E ) . Data information : In : Relative band intensities are shown . In each bar represents mean ± of three independent experiments ( * * P < 0 . 01 , two - t - ) .

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D, E Co-IP assay. EGR2-Myc/HF-OST1, EGR2G2A-Myc/HF-OST1, HF/EGR2-Myc and HF/EGR2G2A-Myc were expressed in N. benthamiana leaves. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-HA and anti-Myc antibodies were used to detect HF-OST1, EGR2-Myc and EGR2G2A-Myc, respectively. Representative pictures are shown in (D), and relative interaction is shown in (E). Data information: In : Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


F , G Effect of EGR2G2A on interaction of OST1 EGR2 in Arabidopsis protoplasts expressing HF - OST1 / EGR2 - GFP / EGR2 - Myc , HF - OST1 / EGR2 - GFP / EGR2G2A - Myc , or HF - OST1 / EGR2 - GFP . Total were immunoprecipated by anti - HA agarose beads . Anti - Myc , anti - GFP , anti - HA antibodies were used to detect EGR2 - Myc , EGR2 - GFP , HF - OST1 , respectively . Representative pictures are shown in ( F ) , relative interaction is shown in ( G ) . Data information In Relative band intensities are shown . In each bar represents mean ± of three independent experiments ( * * P < 0 . 01 , two - t - ) .

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F, G Effect of EGR2G2A on the interaction of OST1 and EGR2 in Arabidopsis protoplasts expressing HF-OST1/EGR2-GFP/EGR2-Myc, HF-OST1/EGR2-GFP/EGR2G2A-Myc, or HF-OST1/EGR2-GFP. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc, anti-GFP, and anti-HA antibodies were used to detect EGR2-Myc, EGR2-GFP, and HF-OST1, respectively. Representative pictures are shown in (F), and relative interaction is shown in (G). Data information In Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


H , I Effect of cold stress on interaction of OST1 EGR2 . Arabidopsis protoplasts expressing EGR2 - Myc / HF - OST1 EGR2 - Myc / HF were treated at 4 ° C for 0 2 h . Total were immunoprecipated by anti - HA agarose beads . co - immunoprecipitated products were detected by anti - HA anti - Myc antibodies , respectively . Representative pictures are shown in ( H ) , relative interaction is shown in ( I ) . Data information : In H ) Relative band intensities are shown . In I ) : each bar represents mean ± of three independent experiments ( * * P < 0 . 01 , two - t - ) .

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H, I Effect of cold stress on the interaction of OST1 and EGR2. Arabidopsis protoplasts expressing EGR2-Myc/HF-OST1 and EGR2-Myc/HF were treated at 4°C for 0 and 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. The co-immunoprecipitated products were detected by anti-HA and anti-Myc antibodies, respectively. Representative pictures are shown in (H), and relative interaction is shown in (I). Data information: In H) Relative band intensities are shown. In I): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


Freezing phenotype ( A ) of wild type , ost1 - 3 , egr2 - 3 , ost1 egr2 mutants . Two - week - old seedlings grown on MS medium were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) .

Freezing phenotype (A) of wild type, ost1-3, egr2-3, and ost1 egr2 mutants. Two-week-old seedlings grown on MS medium were treated at −5°C (NA) or −8°C for 0.5 h (CA).


survival rate ion leakage ( B ) of wild type , ost1 - 3 , egr2 - 3 , ost1 egr2 mutants . Two - week - old seedlings grown on MS medium were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information : each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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survival rate and ion leakage (B) of wild type, ost1-3, egr2-3, and ost1 egr2 mutants. Two-week-old seedlings grown on MS medium were treated at −5°C (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).


C Expression of CBFs in wild type , ost1 - 3 , egr2 - 3 , ost1 egr2 - 3 under cold stress . Two - week - old seedlings grown at 22 ° C were treated at 4 ° C for 0 h 3 h . Data information : each bar represents mean ± of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to wild type with same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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C Expression of CBFs in wild type, ost1-3, egr2-3, and ost1 egr2-3 plants under cold stress. Two-week-old seedlings grown at 22°C were treated at 4°C for 0 h and 3 h. 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).


( C ) 293T were transfected with empty vector ( EV ) , a vector encoding FLAG - tagged full CYLD ( wt ) , or constructs encoding FLAG - tagged CYLD fragments 1 - 581 ( F1 ) lacking C - terminus , or 581 - 956 ( F2 ) lacking N - terminus , as indicated , followed by FLAG - IP . blot was probed with antibodies recognizing SPATA2 , FLAG tubulin . Endogenous SPATA2 was co - immunoprecipitated to comparable by CYLD C - terminal CYLD fragment , containing USP domain .

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(C) 293T cells were transfected with empty vector (EV), a vector encoding FLAG-tagged full length CYLD (wt), or constructs encoding FLAG-tagged CYLD fragments 1-581 (F1) lacking the C-terminus, or 581-956 (F2) lacking the N-terminus, as indicated, followed by FLAG-IP. The blot was probed with antibodies recognizing SPATA2, FLAG and tubulin. Endogenous SPATA2 was co-immunoprecipitated to comparable levels by CYLD and the C-terminal CYLD protein fragment, containing the USP domain.


( D ) CYLD - / - MEF were infected with retrovirus encoding FLAG - CYLD as indicated CYLD was purified by FLAG - IP . blot was probed with antibodies recognizing CYLD , SPATA2 actin .

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(D) CYLD-/- MEF were infected with retrovirus encoding FLAG-CYLD as indicated and CYLD was purified by FLAG-IP. The blot was probed with antibodies recognizing CYLD, SPATA2 and actin.


( E ) 293T were transfected with empty vector ( EV ) , a vector encoding FLAG - tagged full SPATA2 or a construct encoding FLAG - tagged N - terminal part of SPATA2 ( NT ) , as indicated , followed by FLAG - IP . blot was probed with antibodies recognizing CYLD , SPATA2 tubulin . M , marker lane .

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(E) 293T cells were transfected with empty vector (EV), a vector encoding FLAG-tagged full length SPATA2 or a construct encoding the FLAG-tagged N-terminal part of SPATA2 (NT), as indicated, followed by FLAG-IP. The blot was probed with antibodies recognizing CYLD, SPATA2 and tubulin. M, marker lane.


( A ) 293T were transfected with a vector encoding V5 - tagged full SPATA2 , along with empty vector ( EV ) a vector encoding FLAG - tagged HOIP wild - type ( wt ) or a vector encoding a FLAG - tagged PUB domain mutant of HOIP ( N102A ) . After FLAG - IP , blot was probed with antibodies recognizing HOIP , CYLD , V5 actin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were transfected with a vector encoding V5-tagged full length SPATA2, along with empty vector (EV) a vector encoding FLAG-tagged HOIP wild-type (wt) or a vector encoding a FLAG-tagged PUB domain mutant of HOIP (N102A). After FLAG-IP, the blot was probed with antibodies recognizing HOIP, CYLD, V5 and actin.


( B ) 293T were transfected with empty vector or a vector encoding FLAG - tagged HOIP along with either V5 - tagged SPATA2 ( wt ) or a V5 - tagged PIM mutant ( PIMmut ) of SPATA2 . After V5 - IP , blot was probed with antibodies recognizing FLAG , CYLD , V5 tubulin .

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(B) 293T cells were transfected with empty vector or a vector encoding FLAG-tagged HOIP along with either V5-tagged SPATA2 (wt) or a V5-tagged PIM mutant (PIMmut) of SPATA2. After V5-IP, the blot was probed with antibodies recognizing FLAG, CYLD, V5 and tubulin.


( C ) 293T were transfected with a vector encoding FLAG - tagged HOIP increasing amounts of V5 - tagged SPATA2 . After FLAG - IP , blot was sequentially probed with antibodies recognizing OTULIN , HOIP , V5 tubulin .

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(C) 293T cells were transfected with a vector encoding FLAG-tagged HOIP and increasing amounts of V5-tagged SPATA2. After FLAG-IP, the blot was sequentially probed with antibodies recognizing OTULIN, HOIP, V5 and tubulin.


( D ) 293T were transfected with empty vector , FLAG - SPATA2 or FLAG - HOIP . After FLAG - IP , blot was probed with antibodies recognizing OTULIN , CYLD , FLAG actin .

O O O O O O O O O O O O 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) 293T cells were transfected with empty vector, FLAG-SPATA2 or FLAG-HOIP. After FLAG-IP, the blot was probed with antibodies recognizing OTULIN, CYLD, FLAG and actin.


( E ) 293T were transfected with empty vector , V5 - tagged SPATA2 ( wt ) or a V5 - tagged PUB domain mutant PUBmut of SPATA2 . After V5 - IP , blot was probed with antibodies recognizing CYLD , FLAG , V5 tubulin .

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(E) 293T cells were transfected with empty vector, V5-tagged SPATA2 (wt) or a V5-tagged PUB domain mutant PUBmut of SPATA2. After V5-IP, the blot was probed with antibodies recognizing CYLD, FLAG, V5 and tubulin.


( F ) A549 were treated with FLAG - TNF ( 2µg / ml ) for indicated time . purified TNF - RSC was sequentially probed with antibodies recognizing SPATA2 , CYLD , RIPK1 , TRADD , SHARPIN , TNF tubulin as indicated .

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(F) A549 cells were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, RIPK1, TRADD, SHARPIN, TNF and tubulin as indicated.


( A ) A549 were infected to express a CRISPR / Cas9 system targeting luciferase ( as control , indicated wt ) or spata2 gene ( ko ) . Mixed cultures were treated with FLAG - huTNF ( 2µg / ml ) for indicated time . purified TNF - RSC was sequentially probed with antibodies recognizing SPATA2 , CYLD , HOIP , SHARPIN , TRADD , TNF tubulin as indicated .

(A) A549 cells were infected to express a CRISPR/Cas9 system targeting luciferase (as control, indicated wt) or the spata2 gene (ko). Mixed cell cultures were treated with FLAG-huTNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, HOIP, SHARPIN, TRADD, TNF and tubulin as indicated.


( B ) MEF were infected to express a CRISPR / Cas9 system targeting luciferase ( wt ) or spata2 gene ( ko ) . Single clones were generated , which were treated with FLAG - TNF ( 2µg / ml ) for indicated time . purified TNF - RSC was sequentially probed with antibodies recognizing M1 - linked ubiquitin , CYLD , RIPK1 , tubulin as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MEF were infected to express a CRISPR/Cas9 system targeting luciferase (wt) or the spata2 gene (ko). Single clones were generated, which were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing M1-linked ubiquitin, CYLD, RIPK1, and tubulin as indicated.


FLAG - tagged CYLD or catalytically inactive FLAG - CYLD ( C598A ) , respectively , were stably expressed in CYLD - / - MEF , followed by infection with control retrovirus , or retrovirus encoding SPATA2 , purified by FLAG - IP . Purified CYLD or inactive mutant were added to recombinant di - ubiquitin for 0 , 20 or 40 min at 37 ° C as indicated . After electrophoresis , gel was cut lower part was silver stained to visualize mono - di - ubiquitin , while upper part was transferred to a probed for FLAG V5 . In ( A ) , experiment was done with M1 - linked di - ubiquitin substrate , in ( B ) K63 - linked di - ubiquitin substrate was subjected to , in ( C ) K48 - linked di - ubiquitin substrate was analyzed .

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FLAG-tagged CYLD or catalytically inactive FLAG-CYLD (C598A), respectively, were stably expressed in CYLD-/-MEF, followed by infection with control retrovirus, or retrovirus encoding SPATA2, and purified by FLAG-IP. Purified CYLD or the inactive mutant were added to recombinant di-ubiquitin for 0, 20 or 40 min at 37°C as indicated. After electrophoresis, the gel was cut and the lower part was silver stained to visualize mono-and di-ubiquitin, while the upper part was transferred to a membrane and probed for FLAG and V5. In (A), the experiment was done with M1-linked di-ubiquitin substrate, in (B) K63-linked di-ubiquitin substrate was subjected to the assay, and in (C) K48-linked di-ubiquitin substrate was analyzed.


( A ) MEF expressing CRISPR / Cas9 targeting luciferase ( wt ) or single clones generated from MEF expressing CRISPR / Cas9 targeting spata2 gene ( ko ) were treated with mTNF ( 10 ng / ml ) as indicated . blots were probed with antibodies recognizing P - IkBαIkBα , P - p38 , p38 , P - JNK , JNK , P - p65 , p65 tubulin .

(A) MEF expressing CRISPR/Cas9 targeting luciferase (wt) or single clones generated from MEF expressing CRISPR/Cas9 targeting the spata2 gene (ko) were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBαIkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin.


( B ) 15P - 1 Sertoli expressing CRISPR / Cas9 targeting luciferase ( as control ) or single clones generated from 15P - 1 Sertoli expressing CRISPR / Cas9 targeting spata2 gene were treated with mTNF ( 10 ng / ml ) as indicated . blots were probed with antibodies recognizing P - IkBα , IkBα , P - p38 , p38 , P - JNK , JNK , P - p65 , p65 tubulin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting luciferase (as control) or single clones generated from 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting the spata2 gene were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBα, IkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin.


( C ) TAK1 - / - MEF were infected to express a CRISPR / Cas9 system targeting luciferase or spata2 gene , from which single clones were generated . were treated with mTNF ( 10 ng / ml ) or mTNF along with Necrostatin - 1 ( Nec - 1 ) for 1 - 3 h as indicated . blot was sequentially probed with antibodies recognizing PARP , caspase - 3 tubulin .

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(C) TAK1-/-MEF were infected to express a CRISPR/Cas9 system targeting luciferase or the spata2 gene, from which single clones were generated. The cells were treated with mTNF (10 ng/ml) or mTNF along with Necrostatin-1 (Nec-1) for 1-3 h as indicated. The blot was sequentially probed with antibodies recognizing PARP, cleaved caspase-3 and tubulin.


( D ) . same as used in ( C ) were treated as before with TNF ( 10 ng / ml ) or TNF along with Necrostatin - 1 ( Nec - 1 ) for one 2 h as indicated . Caspase activity was determined using fluorogenic substrate DEVD - AMC .

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(D). The same cells as used in (C) were treated as before with TNF (10 ng/ml) or TNF along with Necrostatin-1 (Nec-1) for one and 2 h as indicated. Caspase activity was determined using the fluorogenic substrate DEVD-AMC.


( E ) from experiment as shown in ( D ) were analyzed for apoptosis by Annexin V after 2 h . In ( D ) ( E ) , a representative experiment is shown with error bars referring to technical replicates .

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(E) Cells from the experiment as shown in (D) were analyzed for apoptosis by Annexin V staining after 2 h. In (D) and (E), a representative experiment is shown with error bars referring to technical replicates.


( F ) as described before were treated with mTNF ( 10 ng / ml ) for indicated time lysates were subjected to immunoprecipitation with an anti - FADD antibody . immunoprecipitates were subjected to Western blotting probed for RIPK1 , FADD actin .

(F) Cells as described before were treated with mTNF (10 ng/ml) for the indicated time and lysates were subjected to immunoprecipitation with an anti-FADD antibody. The immunoprecipitates were subjected to Western blotting and probed for RIPK1, FADD and actin.


skin fibroblasts from a normal , SLOS , ZS , X - ALD , NPC incubated for 24 h without serum were immunostained with anti - pericentrin ( red ) anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( green ) . Arrows indicate cilia . Scale bar , 5 μm . of Filipin III signal at cilia from ( a ) was remarkably reduced in SLOS ZS ( * * p < 0 . 01 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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Primary skin fibroblasts from a normal individual, SLOS patient, ZS patients, X-ALD patient, and NPC patient incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 5 μm. The intensity of Filipin III signal at primary cilia from (a) was remarkably reduced in SLOS and ZS patient cells (**p<0.01: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Immunofluorescent of skin fibroblasts in quiescent G0 phase treated with 50 nM Shh - N for 24 h using anti - Smo ( green ) , anti - acetylated - tubulin ( blue ) , anti - γ - tubulin ( red ) antibodies . Arrows indicate cilia . Scale bar , 2 . 5 μm . of proportion of Smo - positive ciliated upon Shh - N stimulation from ( C ) . populations of Smo - positive ciliated fibroblasts from SLOS ZS were significantly reduced ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 100 per experiment ) . Smo at cilia in fibroblasts from ( C ) . ciliary localization of Smo upon Shh - N stimulation was significantly impaired in fibroblasts from SLOS ZS ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 90 - 100 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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

Immunofluorescent staining of primary skin fibroblasts in quiescent G0 phase treated with 50 nM Shh-N for 24 h using anti-Smo (green), anti-acetylated-tubulin (blue), and anti-γ-tubulin (red) antibodies. Arrows indicate primary cilia. Scale bar, 2.5 μm. Quantification of proportion of Smo-positive ciliated cells upon Shh-N stimulation from (C). The populations of Smo-positive ciliated fibroblasts from the SLOS and ZS patients were significantly reduced (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 100 cells per experiment). The Smo intensity at primary cilia in the fibroblasts from (C). The ciliary localization of Smo upon Shh-N stimulation was significantly impaired in fibroblasts from the SLOS and ZS patients (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 90-100 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


fibroblasts from a normal , SLOS ZS were treated with 1 . 5 % methyl - β - cyclodextrin for 45 min to remove cholesterol , then incubated with or without cholesterol ( cholesterol / methyl - β - cyclodextrin ) for 1 h . After washing out exogenous cholesterol , fibroblasts were stimulated with 50 nM Shh - N for 24 h in presence of pravastatin , then immunostained with anti - Smo ( green ) , anti - acetylated - tubulin ( blue ) , anti - pericentrin ( red ) antibodies . For alternative cholesterol complementation , LDL ( 0 . 06 mg / ml ) was co - incubated with Shh - N pravastatin for 24 h after methyl - β - cyclodextrin - mediated cholesterol depletion . Scale bar , 2 . 5 μm . of Smo at cilia in fibroblasts from ( F ) . ciliary accumulation of Smo upon Shh - N stimulation of fibroblasts from an SLO was restored by complementation of both exogenous cholesterol / methyl - β - cyclodextrin LDL , while that of from ZS was efficiently rescued by not LDL but cholesterol / methyl - β - cyclodextrin ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 90 - 100 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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

Primary fibroblasts from a normal individual, and SLOS and ZS patients were treated with 1.5% methyl-β-cyclodextrin for 45 min to remove cellular cholesterol, and then incubated with or without cholesterol (cholesterol/methyl-β-cyclodextrin complex) for 1 h. After washing out the exogenous cholesterol, the fibroblasts were stimulated with 50 nM Shh-N for 24 h in the presence of pravastatin, and then immunostained with anti-Smo (green), anti-acetylated-tubulin (blue), and anti-pericentrin (red) antibodies. For the alternative cholesterol complementation, LDL (0.06 mg/ml) was co-incubated with Shh-N and pravastatin for 24 h after methyl-β-cyclodextrin-mediated cholesterol depletion. Scale bar, 2.5 μm. Quantification of the Smo intensity at primary cilia in the fibroblasts from (F). The ciliary accumulation of Smo upon the Shh-N stimulation of fibroblasts from an SLO patient was restored by the complementation of both exogenous cholesterol/methyl-β-cyclodextrin complex and LDL, while that of cells from ZS patients was efficiently rescued by not LDL but cholesterol/methyl-β-cyclodextrin complex (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 90-100 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Western blot showing depletion of PEX1 PEX14 normal processing of Shh in PEX1 − / − PEX14 − / − hTERT - RPE1 clones . GAPDH served as a loading control .

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Western blot analysis showing the depletion of PEX1 and PEX14 and the normal processing of Shh protein in the PEX1−/− and PEX14−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.


Immunostaining with anti - PEX1 ( red ) or PEX14 ( red ) , anti - PMP70 ( green ) , anti - ninein ( blue ) , anti - acetylated tubulin ( ) in wild - type , PEX1 − / − , PEX14 − / − hTERT - RPE1 in quiescent G0 phase . Arrowheads indicate cilia . Scale bar , 10 μm . of number of PMP70 - positive peroxisomes per from ( B ) . Peroxisome formation in PEX1 − / − PEX14 − / − hTERT - RPE1 was significantly impaired compared with that of parental ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 20 - 25 per experiment ) . of proportion of ciliated from ( B ) . Ciliogenesis in PEX1 − / − PEX14 − / − hTERT - RPE1 was not significantly altered compared with that of parental ( mean ± s . d . : one - way ANOVA with Tukey ' s HSD , n = 3 : 190 - 200 per experiment ) .

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Immunostaining with anti-PEX1 (red) or PEX14 (red), anti-PMP70 (green), anti-ninein (blue), and anti-acetylated tubulin (white) in wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells in quiescent G0 phase. Arrowheads indicate primary cilia. Scale bar, 10 μm. Quantification of the number of PMP70-positive peroxisomes per cell from (B). Peroxisome formation in PEX1−/− and PEX14−/− hTERT-RPE1 cells was significantly impaired compared with that of the parental cells (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 20-25 cells per experiment). Quantification of proportion of ciliated cells from (B). Ciliogenesis in PEX1−/− and PEX14−/− hTERT-RPE1 cells was not significantly altered compared with that of the parental cells (mean ± s.d.: one-way ANOVA with Tukey's HSD, n=3: 190-200 cells per experiment).


Quiescent G0 - phase wild - type , PEX1 − / − , PEX14 − / − hTERT - RPE1 transfected with AcGFP1 - tagged D4 as a cholesterol probe were immunostained with anti - pericentrin ( ) anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( red ) . Arrows arrowheads indicate cilia accumulations of AcGFP1 - tagged D4 , respectively . Scale bar , 5 μm . of Filipin III at cilia from ( E ) . PEX1 − / − PEX14 − / − hTERT - RPE1 had significant reductions in ciliary signal of Filipin III ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively . of AcGFP1 - tagged D4 at cilia from ( E ) . ciliary signal of AcGFP1 - tagged D4 in PEX1 − / − PEX14 − / − hTERT - RPE1 was significantly diminished ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 25 - 30 per experiment ) . In boxplot , medians , 25th / 75th percentile min / max were represented by central lines , box limits , whiskers / error bars , representatively .

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

Quiescent G0-phase wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells transfected with AcGFP1-tagged D4 as a cholesterol probe were immunostained with anti-pericentrin (white) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (red). Arrows and arrowheads indicate primary cilia and cytosolic accumulations of AcGFP1-tagged D4, respectively. Scale bar, 5 μm. Quantification of the Filipin III intensity at primary cilia from (E). PEX1−/− and PEX14−/− hTERT-RPE1 cells had significant reductions in the ciliary signal of Filipin III (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively. Quantification of the AcGFP1-tagged D4 intensity at primary cilia from (E). The ciliary signal of AcGFP1-tagged D4 in PEX1−/− and PEX14−/− hTERT-RPE1 cells was significantly diminished (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 25-30 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.