HeLa cells were subjected to consecutive rounds of transfection with DNMT1 siRNA targeting the UTR and expression plasmid encoding WT or catalytically inactive ( CI ) GFP - DNMT1 . Cells were then left untreated or incubated with 5 - azadC for 2 h , and SUMOylation of GFP - DNMT1 was analyzed Asterisk denotes unmodified GFP - DNMT1 recognized by the SUMO2 / 3 antibody due to weak cross - reactivity .

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HeLa cells were subjected to consecutive rounds of transfection with DNMT1 siRNA targeting the UTR and expression plasmid encoding WT or catalytically inactive (CI) GFP-DNMT1. Cells were then left untreated or incubated with 5-azadC for 2 h, and SUMOylation of GFP-DNMT1 was analyzed Asterisk denotes unmodified GFP-DNMT1 recognized by the SUMO2/3 antibody due to weak cross-reactivity.


Mass spectrometry analysis of 5 - azadC - induced SUMOylation changes . His10 - SUMO2 conjugates from HeLa / His10 - SUMO2 cells subjected or not to 5 - azadC were purified on Ni - NTA under stringent conditions and analyzed by mass spectrometry . Proteins displaying significantly altered SUMOylation in response to 5 - azadC treatment relative to the control condition were visualized through two - sample t - testing using permutation - based FDR to achieve q - values of < 0 .

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Mass spectrometry analysis of 5-azadC-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to 5-azadC were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. Proteins displaying significantly altered SUMOylation in response to 5-azadC treatment relative to the control condition were visualized through two-sample t-testing using permutation-based FDR to achieve q-values of <0.05


SUMO target proteins displaying altered SUMOylation upon DNMT1 knockdown are shown . Both control ( CTRL ) and DNMT1 siRNA - transfected cells were treated with 5 - azadC .

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SUMO target proteins displaying altered SUMOylation upon DNMT1 knockdown are shown. Both control (CTRL) and DNMT1 siRNA-transfected cells were treated with 5-azadC.


Chromatin - enriched fractions of HeLa cells transfected with indicated siRNAs and subsequently exposed or not to 5 - azadC for 2 h were analyzed by immunoblotting with SUMO2 / 3 and Actin antibodies .

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Chromatin-enriched fractions of HeLa cells transfected with indicated siRNAs and subsequently exposed or not to 5-azadC for 2 h were analyzed by immunoblotting with SUMO2/3 and Actin antibodies.


HeLa cells stably expressing GFP - DNMT1 and transfected with indicated siRNAs were left untreated or exposed to 5 - azadC for 2 h . Cells were then preextracted in stringent preextraction buffer and fixed at the indicated time points after 5 - azadC removal . Mean detergent - resistant GFP signal was determined by quantitative image analysis ( > 6000 cells analyzed per condition ) . Data from a representative experiment are shown .

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HeLa cells stably expressing GFP-DNMT1 and transfected with indicated siRNAs were left untreated or exposed to 5-azadC for 2 h. Cells were then preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal. Mean detergent-resistant GFP signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.


Proliferative capacity of HeLa / GFP - DNMT1 cells transfected with control or UBC9 siRNAs and exposed to the indicated 5 - azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) .

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Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with control or UBC9 siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).


untransfected HeLa cells were exposed or not to 5 - azadC in the presence or absence of a small molecule SUMO E1 enzyme inhibitor ( SUMO - E1i ) .

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untransfected HeLa cells were exposed or not to 5-azadC in the presence or absence of a small molecule SUMO E1 enzyme inhibitor (SUMO-E1i).


HeLa cells treated with 5 - azadC for 2 h in the presence or absence of SUMO - E1i were preextracted in stringent preextraction buffer and fixed at the indicated time points after 5 - azadC removal , and immunostained with DNMT1 antibody . Mean detergent - resistant DNMT1 signal was determined by quantitative image analysis ( > 6000 cells analyzed per condition ) . Data from a representative experiment are shown .

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HeLa cells treated with 5-azadC for 2 h in the presence or absence of SUMO-E1i were preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal, and immunostained with DNMT1 antibody. Mean detergent-resistant DNMT1 signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.


Proliferative capacity of HeLa cells transfected with SPRTN or ZNF451 siRNAs and exposed to indicated 5 - azadC doses for 2 h was

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Proliferative capacity of HeLa cells transfected with SPRTN or ZNF451 siRNAs and exposed to indicated 5-azadC doses for 2 h was assayed


iPOND analysis of 5 - azadC - induced DNMT1 trapping and SUMOylation . HeLa cells were mock - treated or pre - incubated with 5 - azadC for 5 min before addition of EdU for 10 min . Cells were then washed and incubated with thymidine for 0 ( pulse ) or 60 min ( chase ) before performing iPOND .

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iPOND analysis of 5-azadC-induced DNMT1 trapping and SUMOylation. HeLa cells were mock-treated or pre-incubated with 5-azadC for 5 min before addition of EdU for 10 min. Cells were then washed and incubated with thymidine for 0 (pulse) or 60 min (chase) before performing iPOND.


HeLa cells stably expressing GFP - DNMT1 were pre - incubated or not with SUMO - E1i for 30 min , and where indicated 5 - azadC was added to the medium for an additional 60 min . Cells were then processed for GFP immunoprecipitation ( IP ) followed by immunoblotting for the indicated proteins .

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HeLa cells stably expressing GFP-DNMT1 were pre-incubated or not with SUMO-E1i for 30 min, and where indicated 5-azadC was added to the medium for an additional 60 min. Cells were then processed for GFP immunoprecipitation (IP) followed by immunoblotting for the indicated proteins.


Immunoblot analysis of chromatin - enriched fractions of HeLa cells that were synchronized in early S phase by double thymidine block , pulse - labeled with 5 - azadC for 30 min and grown in the presence or absence of proteasome inhibitor ( MG132 ) for the indicated times .

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Immunoblot analysis of chromatin-enriched fractions of HeLa cells that were synchronized in early S phase by double thymidine block, pulse-labeled with 5-azadC for 30 min and grown in the presence or absence of proteasome inhibitor (MG132) for the indicated times.


cells were treated or not with aphidicolin following pulse - labeling with 5 - azadC .

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cells were treated or not with aphidicolin following pulse-labeling with 5-azadC.


Representative images of U2OS cells stably expressing GFP - SPRTN or GFP - ACRC that were transfected with control ( CTRL ) or UBC9 siRNAs , exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme ( UBA1 ) inhibitor TAK - 243 ( Ub - E1i ) as indicated and fixed one h later . Scale bar , 10 μm .

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Representative images of U2OS cells stably expressing GFP-SPRTN or GFP-ACRC that were transfected with control (CTRL) or UBC9 siRNAs, exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme (UBA1) inhibitor TAK-243 (Ub-E1i) as indicated and fixed one h later. Scale bar, 10 μm.


Quantification of data showing proportion of U2OS / GFP - SPRTN cells displaying nuclear GFP - SPRTN foci ( mean ± SEM ; at least 100 cells quantified per condition per experiment ; n = 3 independent experiments ) . As in ( C ) , but showing proportion of U2OS / GFP - ACRC cells displaying nuclear GFP - ACRC foci ( mean ± SEM ; at least 100 cells quantified per condition per experiment ; n = 3 independent experiments ) .

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Quantification of data showing proportion of U2OS/GFP-SPRTN cells displaying nuclear GFP-SPRTN foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments). As in (C), but showing proportion of U2OS/GFP-ACRC cells displaying nuclear GFP-ACRC foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).


U2OS cells expressing GFP - ACRC were exposed to the indicated genotoxic agents ( formaldehyde : 600 μM , 1 h ; UV : 20 J / m2 , 6 h recovery ; hydroxyurea ( HU ) : 2mM , 24 h ; mitomycin C ( MMC ) : 40 ng / mL , 24 h ) , preextracted and fixed , and analyzed by microscopy . Representative images are shown . Scale bar , 10 μm .

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U2OS cells expressing GFP-ACRC were exposed to the indicated genotoxic agents (formaldehyde: 600 μM, 1 h; UV: 20 J/m2, 6 h recovery; hydroxyurea (HU): 2mM, 24 h; mitomycin C (MMC): 40 ng/mL, 24 h), preextracted and fixed, and analyzed by microscopy. Representative images are shown. Scale bar, 10 μm.


HeLa cells transfected with plasmids encoding GFP alone or indicated GFP - ACRC alleles were subjected to GFP IP under denaturing conditions . Beads were incubated with recombinant polySUMO22 - 8 chains , washed extensively and processed for immunoblotting with SUMO2 / 3 and GFP antibodies .

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HeLa cells transfected with plasmids encoding GFP alone or indicated GFP-ACRC alleles were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO22-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.


Representative images of U2OS cells expressing indicated GFP - ACRC alleles that were treated with 5 - azadC in the presence or absence of SUMO inhibitor ( SUMO - E1i ) , fixed 2 h later and immunostained with DNMT1 antibody . Scale bar , 10 μm . Quantification of data in ( G ) , showing proportion of cells displaying GFP - ACRC co - localization with DNMT1 foci ( mean ± SEM ; at least 100 cells quantified per condition per experiment ; n = 3 independent experiments ) .

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Representative images of U2OS cells expressing indicated GFP-ACRC alleles that were treated with 5-azadC in the presence or absence of SUMO inhibitor (SUMO-E1i), fixed 2 h later and immunostained with DNMT1 antibody. Scale bar, 10 μm.   Quantification of data in (G), showing proportion of cells displaying GFP-ACRC co-localization with DNMT1 foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).


HeLa cells transfected with plasmids encoding GFP alone , GFP - ACRC or GFP - tagged C . elegans GCNA - 1 were subjected to GFP IP under denaturing conditions . Beads were incubated with recombinant polySUMO23 - 8 chains , washed extensively and processed for immunoblotting with SUMO2 / 3 and GFP antibodies .

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HeLa cells transfected with plasmids encoding GFP alone, GFP-ACRC or GFP-tagged C. elegans GCNA-1 were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO23-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.


GFP expression driven by the gcna - 1 promoter was observed in germ cells , proliferating embryos and young larvae but not in post - mitotic tissues in the head Scale bars , 50 µm .

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GFP expression driven by the gcna-1 promoter was observed in germ cells, proliferating embryos and young larvae but not in post-mitotic tissues in the head Scale bars, 50 µm.


Formaldehyde survival of wild type ( wt ) , dvc - 1 , gcna - 1 loss - of - function ( lof ) and gcna - 1 ; dvc - 1 double mutant C . elegans ( mean ± SEM ; n = 4 independent experiments ) .

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Formaldehyde survival of wild type (wt), dvc-1, gcna-1 loss-of-function (lof) and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=4 independent experiments).


Cisplatin survival of wild type , dvc - 1 , gcna - 1 and gcna - 1 ; dvc - 1 double mutant C . elegans ( mean ± SEM ; n = 3 independent experiments ) .

Cisplatin survival of wild type, dvc-1, gcna-1 and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=3 independent experiments).


Formaldehyde survival of gcna - 1 deletion ( del ) and E364Q mutant C . elegans ( mean ± SEM ; n = 2 independent experiments ) .

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Formaldehyde survival of gcna-1 deletion (del) and E364Q mutant C. elegans (mean±SEM; n=2 independent experiments).


Formaldehyde survival of C . elegans grown on L4440 control ( CTRL ) or smo - 1 RNAi bacteria ( mean ± SEM ; n = 2 independent experiments ) .

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Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).


Formaldehyde survival of C . elegans grown on L4440 control ( CTRL ) or gei - 17 RNAi bacteria ( mean ± SEM ; n = 2 independent experiments ) .

Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or gei-17 RNAi bacteria (mean±SEM; n=2 independent experiments).


Formaldehyde survival of wild type and gcna - 1 deletion ( del ) mutant C . elegans grown on L4440 control ( CTRL ) or smo - 1 RNAi bacteria ( mean ± SEM ; n = 2 independent experiments ) .

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Formaldehyde survival of wild type and gcna-1 deletion (del) mutant C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).


A Detection of the RBPJ / L3MBTL3 interaction using the yeast two - hybrid ( Y2H ) assay . In this Y2H experiment , RBPJ is fused to the GAL4 DNA - binding ( DB ) domain and L3MBTL3 is fused to the GAL4 activation domain ( AD ) . The DB - RBPJ and AD - L3MBTL3 fusion proteins interact with each other , leading to the activation of the ADE2 and HIS3 reporter genes and allowing yeast cells to grow on selective media lacking adenine or histidine . The six Y2H controls were previously described ( Dreze et al , 2010 ) . The experiment was independently replicated thrice .

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A Detection of the RBPJ/L3MBTL3 interaction using the yeast two-hybrid (Y2H) assay. In this Y2H experiment, RBPJ is fused to the GAL4 DNA-binding (DB) domain and L3MBTL3 is fused to the GAL4 activation domain (AD). The DB-RBPJ and AD-L3MBTL3 fusion proteins interact with each other, leading to the activation of the ADE2 and HIS3 reporter genes and allowing yeast cells to grow on selective media lacking adenine or histidine. The six Y2H controls were previously described (Dreze et al, 2010). The experiment was independently replicated thrice.


B Endogenous L3MBTL3 co - purifies specifically with HA - RBPJ but not with HA - EGFP , HA - TBL1X or HA - HEY2 . Immuno - precipitation ( IP ) of HA - tagged RBPJ , EGFP , TBL1X or HEY2 in U87 - MG cells followed by Western blot analyses using HA or L3MBTL3 antibody . The experiment was independently replicated twice .

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B Endogenous L3MBTL3 co-purifies specifically with HA-RBPJ but not with HA-EGFP, HA-TBL1X or HA-HEY2. Immuno-precipitation (IP) of HA-tagged RBPJ, EGFP, TBL1X or HEY2 in U87-MG cells followed by Western blot analyses using HA or L3MBTL3 antibody. The experiment was independently replicated twice.


C Endogenous RBPJ co - purifies specifically with HA - L3MBTL3 but not with HA - EGFP , HA - TBL1X or HA - HEY2 . IPs of HA - tagged L3MBTL3 , EGFP , TBL1X or HEY2 in U87 - MG cells followed by Western blot analyses using HA or RBPJ antibody . The experiment was independently replicated twice . EV : Empty Vector control ; WB : Western blot ; IP : immuno - precipitation .

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C Endogenous RBPJ co-purifies specifically with HA-L3MBTL3 but not with HA-EGFP, HA-TBL1X or HA-HEY2. IPs of HA-tagged L3MBTL3, EGFP, TBL1X or HEY2 in U87-MG cells followed by Western blot analyses using HA or RBPJ antibody. The experiment was independently replicated twice. EV: Empty Vector control; WB: Western blot; IP: immuno-precipitation.


B L3MBTL3 - Δ ( 1 - 64 ) does not interact with RBPJ . IP of HA - FLAG - tagged RBPJ in the presence of FLAG - tagged L3MBTL3 ( WT or deletion mutants ) in HEK293T cells followed by Western blotting using FLAG antibody . The experiment was independently replicated twice .

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B L3MBTL3-Δ(1-64) does not interact with RBPJ. IP of HA-FLAG-tagged RBPJ in the presence of FLAG-tagged L3MBTL3 (WT or deletion mutants) in HEK293T cells followed by Western blotting using FLAG antibody. The experiment was independently replicated twice.


D Deletion of the BTD domain impairs the RBPJ / L3MBTL3 interaction . IP of HA - tagged L3MBTL3 in the presence of FLAG - tagged RBPJ ( WT and deletion mutants ) in HEK293T cells followed by Western blotting using HA or FLAG antibody . The experiment was independently replicated twice .

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D Deletion of the BTD domain impairs the RBPJ/L3MBTL3 interaction. IP of HA-tagged L3MBTL3 in the presence of FLAG-tagged RBPJ (WT and deletion mutants) in HEK293T cells followed by Western blotting using HA or FLAG antibody. The experiment was independently replicated twice.


E RBPJF261R point mutant does not interact with L3MBTL3 . IP of HA - tagged L3MBTL3 in the presence of FLAG - tagged RBPJ ( WT and point mutants ) in HEK293T cells followed by Western blotting using HA or FLAG antibody . RBPJV263R and RBPJA284R also show a reduced ability to interact with L3MBTL3 . The experiment was independently replicated twice . WB : Western blot ; IP : immuno - precipitation .

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E RBPJF261R point mutant does not interact with L3MBTL3. IP of HA-tagged L3MBTL3 in the presence of FLAG-tagged RBPJ (WT and point mutants) in HEK293T cells followed by Western blotting using HA or FLAG antibody. RBPJV263R and RBPJA284R also show a reduced ability to interact with L3MBTL3. The experiment was independently replicated twice. WB: Western blot; IP: immuno-precipitation.


A Thermodynamic characterization of the RBPJ / L3MBTL3 interaction . Representative thermograms ( raw heat signal and nonlinear least squares fit to the integrated data ) for L3MBTL3 - ( 31 - 70 ) binding to RBPJ - ( 53 - 474 ) .

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A Thermodynamic characterization of the RBPJ/L3MBTL3 interaction. Representative thermograms (raw heat signal and nonlinear least squares fit to the integrated data) for L3MBTL3-(31-70) binding to RBPJ-(53-474).


B / C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose - dependent manner . IPs were performed in CRISPR / Cas9 - mediated L3MBTL3 knockout ( KO ) HEK293T cells . ( B ) SBP - FLAG - RBPJ and HA - L3MBTL3 - Δ ( SAM ) in the presence of an increasing amount of HA - NOTCH1 ICD . The L3MBTL3 - Δ ( SAM ) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to express HA - L3MBTL3 - Δ ( SAM ) . The experiment was independently replicated thrice . WB : Western blot ; IP : immuno - precipitation .

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

B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (B) SBP-FLAG-RBPJ and HA-L3MBTL3-Δ(SAM) in the presence of an increasing amount of HA-NOTCH1 ICD. The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation.


B / C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose - dependent manner . IPs were performed in CRISPR / Cas9 - mediated L3MBTL3 knockout ( KO ) HEK293T cells . ( C ) SBP - FLAG - RBPJ and HA - NOTCH1 ICD in the presence of an increasing amount of HA - L3MBTL3 - Δ ( SAM ) . The L3MBTL3 - Δ ( SAM ) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to express HA - L3MBTL3 - Δ ( SAM ) . The experiment was independently replicated thrice . WB : Western blot ; IP : immuno - precipitation .

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

B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (C) SBP-FLAG-RBPJ and HA-NOTCH1 ICD in the presence of an increasing amount of HA-L3MBTL3-Δ(SAM). The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation.


A De - repression of Notch target genes upon RBPJ knockdown . Shown are means ± s . d . of quadruplicate experiments . [ * ] P < 0 . 05 , [ * * ] P < 0 . 01 , NS : Not Significant ; one - way ANOVA model on log - transformed data . Inset : Western blot analysis validates the shRNA - mediated depletion of RBPJ .

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

A De-repression of Notch target genes upon RBPJ knockdown. Shown are means ±s.d. of quadruplicate experiments. [*] P<0.05, [**] P<0.01, NS: Not Significant; one-way ANOVA model on log-transformed data. Inset: Western blot analysis validates the shRNA-mediated depletion of RBPJ.


B De - repression of Notch target genes in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of quadruplicate experiments . [ * * ] P < 0 . 01 , NS : Not Significant ; two - sample T - test on log - transformed data . Inset : Western blot analysis validates the CRISPR / Cas9 - mediated KO of L3MBTL3 .

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

B De-repression of Notch target genes in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of quadruplicate experiments. [**] P<0.01, NS: Not Significant; two-sample T-test on log-transformed data. Inset: Western blot analysis validates the CRISPR/Cas9-mediated KO of L3MBTL3.


C RBPJ and L3MBTL3 co - localize at the proximal Notch - responsive elements of Notch target genes . Shown are means ± s . d . of triplicate ChIP experiments . In panels C , D and F : distance in base pairs ( bp ) relative to the transcriptional start site ( TSS ) is indicated below the gene names . Chrom8 was used as negative control ( NEG ) .

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C RBPJ and L3MBTL3 co-localize at the proximal Notch-responsive elements of Notch target genes. Shown are means ±s.d. of triplicate ChIP experiments.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).


D L3MBTL3 occupancy at the proximal Notch - responsive elements of Notch target genes decreases upon RBPJ knockdown . Shown are means ± s . d . of triplicate ChIP experiments . In panels C , D and F : distance in base pairs ( bp ) relative to the transcriptional start site ( TSS ) is indicated below the gene names . Chrom8 was used as negative control ( NEG ) .

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D L3MBTL3 occupancy at the proximal Notch-responsive elements of Notch target genes decreases upon RBPJ knockdown. Shown are means ±s.d. of triplicate ChIP experiments.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).


E The repressive activity of L3MBTL3 at Notch target genes is RBPJ - dependent . Expression analysis of Notch target genes upon RBPJ knockdown and / or overexpression of L3MBTL3 . Shown are means ± s . d . of triplicate experiments . P values were estimated via a one - way ANOVA model on log - transformed data where the difference of differences was tested , which is equivalent to testing the interaction in a two - way ANOVA model . Western blot analysis validates the overexpression of L3MBTL3 and the shRNA - mediated depletion of RBPJ ( Appendix Fig S3E ) . Gene expression analyses of OCT4 was performed as control ( Appendix Fig S3F ) .

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

E The repressive activity of L3MBTL3 at Notch target genes is RBPJ-dependent. Expression analysis of Notch target genes upon RBPJ knockdown and/or overexpression of L3MBTL3. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA model on log-transformed data where the difference of differences was tested, which is equivalent to testing the interaction in a two-way ANOVA model. Western blot analysis validates the overexpression of L3MBTL3 and the shRNA-mediated depletion of RBPJ (Appendix Fig S3E). Gene expression analyses of OCT4 was performed as control (Appendix Fig S3F).


F L3MBTL3 occupancy at the proximal Notch - responsive elements of Notch target genes is dependent on its RBPJ interaction domain . ChIP analyses of HA - L3MBTL3 WT and HA - L3MBTL3 - Δ ( 1 - 64 ) occupancy at the proximal Notch - responsive elements of Notch target genes . Shown are means ± s . d . of duplicate experiments measured twice each . In panels C , D and F : distance in base pairs ( bp ) relative to the transcriptional start site ( TSS ) is indicated below the gene names . Chrom8 was used as negative control ( NEG ) .

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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 L3MBTL3 occupancy at the proximal Notch-responsive elements of Notch target genes is dependent on its RBPJ interaction domain. ChIP analyses of HA-L3MBTL3 WT and HA-L3MBTL3-Δ(1-64) occupancy at the proximal Notch-responsive elements of Notch target genes. Shown are means ±s.d. of duplicate experiments measured twice each.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).


G The L3MBTL3 - ( 1 - 64 ) domain is required for the downregulation of HES1 and HEY2 in U87 - MG cells . Expression analysis of Notch target genes upon overexpression of L3MBTL3 WT , L3MBTL3 - Δ ( 1 - 64 ) or LacZ control ( Control ) . Shown are means ± s . d . of triplicate experiments . [ * ] P < 0 . 05 , [ * * ] P < 0 . 01 , NS : Not Significant ; one - way ANOVA model on log - transformed data .

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

G The L3MBTL3-(1-64) domain is required for the downregulation of HES1 and HEY2 in U87-MG cells. Expression analysis of Notch target genes upon overexpression of L3MBTL3 WT, L3MBTL3-Δ(1-64) or LacZ control (Control). Shown are means ±s.d. of triplicate experiments. [*] P<0.05, [**] P<0.01, NS: Not Significant; one-way ANOVA model on log-transformed data.


A The RBPJ / KDM1A interaction is indirect and occurs via L3MBTL3 . IP of HA - KDM1A in the presence of overexpressed V5 - L3MBTL3 or V5 - L3MBTL3 - Δ ( 1 - 64 ) in L3MBTL3 KO U87 - MG cells . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to overexpress the L3MBTL3 proteins . The experiment was independently replicated twice .

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

A The RBPJ/KDM1A interaction is indirect and occurs via L3MBTL3. IP of HA-KDM1A in the presence of overexpressed V5-L3MBTL3 or V5-L3MBTL3-Δ(1-64) in L3MBTL3 KO U87-MG cells. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to overexpress the L3MBTL3 proteins. The experiment was independently replicated twice.


B KDM1A occupancy at the proximal Notch - responsive elements of Notch target genes is L3MBTL3 - dependent . ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of duplicate experiments measured twice each . Panels B , C and D : distance in bp relative to the TSS is indicated below the gene names . Chrom8 was used as negative control ( NEG ) .

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

B KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is L3MBTL3-dependent. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).


C KDM1A occupancy at the proximal Notch - responsive elements of Notch target genes is dependent on L3MBTL3 and both its RBPJ interaction and KDM1A interaction domains . ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87 - MG cells upon overexpression of L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) . Control : empty vector . Shown are means ± s . d . of duplicate experiments measured twice each . Panels B , C and D : distance in bp relative to the TSS is indicated below the gene names . Chrom8 was used as negative control ( NEG ) .

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

C KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is dependent on L3MBTL3 and both its RBPJ interaction and KDM1A interaction domains. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells upon overexpression of L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM). Control: empty vector. Shown are means ±s.d. of duplicate experiments measured twice each.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).


D L3MBTL3 , but neither L3MBTL3 - Δ ( 1 - 64 ) nor L3MBTL3 - Δ ( SAM ) , leads to decreasing H3K4me2 at the proximal Notch - responsive element of HES1 . ChIP analysis of H3K4me2 at the proximal Notch - responsive element of HES1 upon overexpression of LacZ control ( Control ) , L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of duplicate experiments measured twice each . P values were estimated via a one - way ANOVA on log - transformed data . Panels B , C and D : distance in bp relative to the TSS is indicated below the gene names . Chrom8 was used as negative control ( NEG ) .

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

D L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), leads to decreasing H3K4me2 at the proximal Notch-responsive element of HES1. ChIP analysis of H3K4me2 at the proximal Notch-responsive element of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each. P values were estimated via a one-way ANOVA on log-transformed data.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).


E L3MBTL3 , but neither L3MBTL3 - Δ ( 1 - 64 ) nor L3MBTL3 - Δ ( SAM ) , represses HES1 . Expression analysis of HES1 upon overexpression of LacZ control ( Control ) , L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) mutants in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of triplicate experiments . P values were estimated via a one - way ANOVA on log - transformed data . NS : Not Significant . WB : Western blot ; IP : immuno - precipitation . We note that in the context of this experiment , i . e . , in the absence of endogenous L3MBTL3 , the overexpression of L3MBTL3 - Δ ( 1 - 64 ) does not result in the increased expression of HES1 , contrasting with the result obtained in Fig 4G , i . e . , in the presence of endogenous L3MBTL3 . Indeed , as expected , the dominant negative effect of L3MBTL3 - ( 1 - 64 ) on endogenous WT L3MBTL3 ' s ability to repress the expression of Notch target genes can only be observed when WT L3MBTL3 is expressed .

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

E L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), represses HES1. Expression analysis of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) mutants in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA on log-transformed data. NS: Not Significant. WB: Western blot; IP: immuno-precipitation. We note that in the context of this experiment, i.e., in the absence of endogenous L3MBTL3, the overexpression of L3MBTL3-Δ(1-64) does not result in the increased expression of HES1, contrasting with the result obtained in Fig 4G, i.e., in the presence of endogenous L3MBTL3. Indeed, as expected, the dominant negative effect of L3MBTL3-(1-64) on endogenous WT L3MBTL3's ability to repress the expression of Notch target genes can only be observed when WT L3MBTL3 is expressed.


A GST pulldown showing that dL ( 3 ) mbt , the Drosophila homolog of L3MBTL3 , directly interacts with Su ( H ) , the Drosophila homolog of RBPJ . In vitro transcribed and translated dL ( 3 ) mbt or dNotch ICD ( dNotch ICD fragment containing the RAM domain and ANK repeats ) , as positive control , was incubated with bacterially purified GST - Su ( H ) or GST alone pre - bound to GSH beads . Proteins were resolved via SDS - PAGE and signals were acquired via X - ray exposure . The experiment was independently replicated four times .

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A GST pulldown showing that dL(3)mbt, the Drosophila homolog of L3MBTL3, directly interacts with Su(H), the Drosophila homolog of RBPJ. In vitro transcribed and translated dL(3)mbt or dNotch ICD (dNotch ICD fragment containing the RAM domain and ANK repeats), as positive control, was incubated with bacterially purified GST-Su(H) or GST alone pre-bound to GSH beads. Proteins were resolved via SDS-PAGE and signals were acquired via X-ray exposure. The experiment was independently replicated four times.


C Snapshot showing the co - localization of dL ( 3 ) mbt and Su ( H ) at the dNotch ( N ) locus .

C Snapshot showing the co-localization of dL(3)mbt and Su(H) at the dNotch (N) locus.


D In the wing imaginal disc , dL ( 3 ) mbt overexpression in the dorso - ventral ( D - V ) boundary results in the downregulation of the Notch target gene cut . Wing discs expressing UAS - GFP ( top panels ) or UAS - HA - dL ( 3 ) mbt ; UAS - GFP ( bottom panels ) under the vg - Gal4 driver at 25 ° C were stained for cut and HA . GFP marks the vg - Gal4 expression domain . Insets below each panel show a closer view of the D - V boundary with yellow arrows marking the regions where HA - dL ( 3 ) mbt is expressed and cut is downregulated . At least 20 discs for each genotype were analyzed . Representative images are shown . Scale bars : 100μm .

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

D In the wing imaginal disc, dL(3)mbt overexpression in the dorso-ventral (D-V) boundary results in the downregulation of the Notch target gene cut. Wing discs expressing UAS-GFP (top panels) or UAS-HA-dL(3)mbt;UAS-GFP (bottom panels) under the vg-Gal4 driver at 25°C were stained for cut and HA. GFP marks the vg-Gal4 expression domain. Insets below each panel show a closer view of the D-V boundary with yellow arrows marking the regions where HA-dL(3)mbt is expressed and cut is downregulated. At least 20 discs for each genotype were analyzed. Representative images are shown. Scale bars: 100μm.


E The vg - Gal4 - driven HA - dL ( 3 ) mbt overexpression causes a serrated wing ( wing notching ) phenotype . Flies expressing either UAS - GFP or UAS - HA - dL ( 3 ) mbt ; UAS - GFP under vg - Gal4 were reared to adulthood at 25 ° C . P values were estimated by comparing the proportions via a two - proportion Z - test . Scale bars : 200μm .

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E The vg-Gal4-driven HA-dL(3)mbt overexpression causes a serrated wing (wing notching) phenotype. Flies expressing either UAS-GFP or UAS-HA-dL(3)mbt;UAS-GFP under vg-Gal4 were reared to adulthood at 25°C. P values were estimated by comparing the proportions via a two-proportion Z-test. Scale bars: 200μm.


F Functional interaction between lag - 1 / RBPJ and lin - 61 / L3MBTL3 during C . elegans vulva development . Proportion of animals ( n ≥ 100 ) displaying a protruding vulva ( Pvl ) phenotype after RNAi treatment for two generations . Worms were grown at 25 ° C . Shown are means ± s . d of duplicate experiments . EV : Empty Vector control .

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

F Functional interaction between lag-1/RBPJ and lin-61/L3MBTL3 during C. elegans vulva development. Proportion of animals (n ≥100) displaying a protruding vulva (Pvl) phenotype after RNAi treatment for two generations. Worms were grown at 25°C. Shown are means ±s.d of duplicate experiments. EV: Empty Vector control.


A The distribution of the GFP - tagged Hxt2 changes during the cell division . In small and medium budded cells ( white arrowheads ) , Hxt2 is mainly present in the membrane of the mother cell . In large budded cells , Hxt2 is distributed over the entire plasma membrane ( yellow arrowheads ) .

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A The distribution of the GFP-tagged Hxt2 changes during the cell division. In small and medium budded cells (white arrowheads), Hxt2 is mainly present in the membrane of the mother cell. In large budded cells, Hxt2 is distributed over the entire plasma membrane (yellow arrowheads).


B HXT2 mRNA localization correlate with that of the protein . FISH with an HXT2 - specific probe . Early in the cell cycle the mRNA is predominantly found in the mother cell ( white arrowheads ) while it is equally distributed after meta - to - anaphase transition ( MAT ) ( yellow arrowheads ) . C Quantification of ( B ) . The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated . Values > 1 indicate a stronger signal in the mother , < 1 a stronger signal in the bud and around 1 equal distribution . Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed . Boxes represent the interquartile range from the 25th to the 75th percentile with the median . Whiskers represent the 10th and 90th percentile , respectively .

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B HXT2 mRNA localization correlate with that of the protein. FISH with an HXT2-specific probe. Early in the cell cycle the mRNA is predominantly found in the mother cell (white arrowheads) while it is equally distributed after meta-to-anaphase transition (MAT) (yellow arrowheads). C Quantification of (B). The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.


D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton . Cells were either treated for 15 min with 30 mg / ml latrunculin A or with 30 mg / ml benomyl . White arrowheads point to small - budded and yellow arrowheads to large budded cells . E Quantification of ( D ) of three in dependent experiments with at least 50 cells each . Values > 1 indicate a stronger signal in the mother , < 1 a stronger signal in the bud and around 1 equal distribution . Boxes represent the interquartile range from the 25th to the 75th percentile with the median . Whiskers represent the 10th and 90th percentile , respectively .

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D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton. Cells were either treated for 15 min with 30 mg/ml latrunculin A or with 30 mg/ml benomyl. White arrowheads point to small-budded and yellow arrowheads to large budded cells. E Quantification of (D) of three in dependent experiments with at least 50 cells each. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.


A , Inhibiting translation by either applying Cycloheximide ( CHX ) or Verrucarin A ( VerruA ) leads to the retention of HXT2 mRNA in the mother cell after MAT . Chloro : chloroform ; solvent control for VerruA ; NT : not treated ; control for CHX .

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A, Inhibiting translation by either applying Cycloheximide (CHX) or Verrucarin A (VerruA) leads to the retention of HXT2 mRNA in the mother cell after MAT. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.


FISH of CHX or VerruA - treated cells . Chloro : chloroform ; solvent control for VerruA ; NT : not treated ; control for CHX .

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FISH of CHX or VerruA-treated cells. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.


C CHX treatment leads to increased transcript stability . Quantitative PCR of HXT2 mRNA in CHX - treated cells in comparison to prt1 - 1 mutant cells . The data represents 3 independent experiments and the standard deviation is given .

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C CHX treatment leads to increased transcript stability. Quantitative PCR of HXT2 mRNA in CHX-treated cells in comparison to prt1-1 mutant cells. The data represents 3 independent experiments and the standard deviation is given.


D Translation of HXT2 mRNA is more active late in the cell cycle . The HXT2 mRNA level bound to either di - / trisomes and polysomes ( ≥ 4 ribosomes ) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1 / S or M - phase followed by qPCR . At G1 / S similar levels of the HXT2 mRNA were found in the different ribosome fractions , whereas in M - phase more mRNA was bound to polysomes , suggesting a more active translation in M - phase .

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D Translation of HXT2 mRNA is more active late in the cell cycle. The HXT2 mRNA level bound to either di-/trisomes and polysomes (≥ 4 ribosomes) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1/S or M-phase followed by qPCR. At G1/S similar levels of the HXT2 mRNA were found in the different ribosome fractions, whereas in M-phase more mRNA was bound to polysomes, suggesting a more active translation in M-phase.


E The localization of HXT2 mRNA is independent of the Signal Recognition Particle ( SRP ) . Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA .

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E The localization of HXT2 mRNA is independent of the Signal Recognition Particle (SRP). Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA.


A Deletion of SCP160 , ASC1 , or BFR1 increases HXT2 mRNA signal in the bud . FISH experiment with deletions in various RNA binding proteins .

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A Deletion of SCP160, ASC1, or BFR1 increases HXT2 mRNA signal in the bud. FISH experiment with deletions in various RNA binding proteins.


B The effect of ∆ scp160 on HXT2 mRNA localization is independent of its increased ploidy . Scp160 truncations that lack either the last two ( ∆ C2 ) or the last four ( ∆ C4 ) KH domains or a Tet - off SCP160 construct , in which expression is blocked by the addition of doxycycline ( Doxy )

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B The effect of ∆scp160 on HXT2 mRNA localization is independent of its increased ploidy. Scp160 truncations that lack either the last two (∆C2) or the last four (∆C4) KH domains or a Tet-off SCP160 construct, in which expression is blocked by the addition of doxycycline (Doxy)


D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud . Treatment of cells with forskolin ( + Forsko ) or using a ∆ bcy1 strain , causes HXT2 mRNA to be bud - enriched .

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D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud. Treatment of cells with forskolin (+Forsko) or using a ∆bcy1 strain, causes HXT2 mRNA to be bud-enriched.


E HXT2 mRNA enrichment upon refeeding is Ras2 - dependent .

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E HXT2 mRNA enrichment upon refeeding is Ras2-dependent.


A Table of the glucose affinity of the four most important hexose transporters . B FISH of HXT1 , 3 and 4 under glucose rich conditions . White arrowheads depict cells before , yellow arrowheads cells after MAT . C Quantification of ( B ) .

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A Table of the glucose affinity of the four most important hexose transporters. B FISH of HXT1, 3 and 4 under glucose rich conditions. White arrowheads depict cells before, yellow arrowheads cells after MAT. C Quantification of (B).


D Hyperactive PKA promotes enrichment of all HXTs after MAT .

D Hyperactive PKA promotes enrichment of all HXTs after MAT.


E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation .

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E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation.


F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding . Daughters and mothers were separated through elutriation . qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions .

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F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding. Daughters and mothers were separated through elutriation. qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions.


G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2 - GFP transporter levels at the plasma membrane of the daughter cells . Daughter cells were enriched through elutriation . Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment . Western blot analysis confirms the higher amount of Hxt2 - GFP transporter protein present in daughter cells under glucose - shift

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G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2-GFP transporter levels at the plasma membrane of the daughter cells. Daughter cells were enriched through elutriation. Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment. Western blot analysis confirms the higher amount of Hxt2-GFP transporter protein present in daughter cells under glucose-shift conditions


A RNA degradation is not essential for asymmetric HXT2 mRNA localization . FISH of wild - type and ∆ xrn1 cells .

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A RNA degradation is not essential for asymmetric HXT2 mRNA localization. FISH of wild-type and ∆xrn1 cells.


B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud . Cells were treated upon glucose addition with either 120 mg / ml benomyl for 15 min , 30 mg / ml latrunculin A for 30 min or 200 µM CK - 666 for 30 min . HXT2 mRNA was detect by FISH .

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B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud. Cells were treated upon glucose addition with either 120 mg/ml benomyl for 15 min, 30 mg/ml latrunculin A for 30 min or 200 µM CK-666 for 30 min. HXT2 mRNA was detect by FISH.


C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function . HXT2 mRNA FISH with ∆ bim1 and ∆ kar9 strains . Arrows point to a nucleus with HXT2 signal . D Quantification of the daughter cell enrichment from data displayed in ( C ) . E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in ( C ) . Percentage of cells with asymmetric HXT2 mRNA staining is displayed .

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C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function. HXT2 mRNA FISH with ∆bim1 and ∆kar9 strains. Arrows point to a nucleus with HXT2 signal. D Quantification of the daughter cell enrichment from data displayed in (C). E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in (C). Percentage of cells with asymmetric HXT2 mRNA staining is displayed.


A Deletion of NUP2 and MLP1 / 2 reduced HXT2 mRNA enrichment in the bud . FISH under glucose shift conditions with strains in which different NPC components were deleted . B Quantification of ( A ) .

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A Deletion of NUP2 and MLP1/2 reduced HXT2 mRNA enrichment in the bud. FISH under glucose shift conditions with strains in which different NPC components were deleted. B Quantification of (A).


C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift . FISH - IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84 - HA . White arrows point to HXT2 mRNA signals on the nuclear envelope .

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C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift. FISH-IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84-HA. White arrows point to HXT2 mRNA signals on the nuclear envelope.


D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift . Stills of time lapse movies . HXT2 mRNA was visualized using the MS2 - GFP system . Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus . Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus . Cyan : Nup84 - mCherry ; white arrowhead : HXT2 mRNA - MS2 + MCP - GFP .

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D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift. Stills of time lapse movies. HXT2 mRNA was visualized using the MS2-GFP system. Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus. Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus. Cyan: Nup84-mCherry; white arrowhead: HXT2 mRNA-MS2 + MCP-GFP.


HXT2 only strains grow faster than the other HXT only strain . Growth was assessed 2 - 3 days after incubation at 30 ° C .

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HXT2 only strains grow faster than the other HXT only strain. Growth was assessed 2-3 days after incubation at 30°C.


D Quantification of competition assay . qPCR with primers against either HXT1 , 2 , 3 or 4 was performed . HXT gene abundance was normalized against actin .

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D Quantification of competition assay. qPCR with primers against either HXT1, 2, 3 or 4 was performed. HXT gene abundance was normalized against actin.


E Strains that lack HXT2 grow slower when coming from quiescence compared to WT .

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E Strains that lack HXT2 grow slower when coming from quiescence compared to WT.


F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96 % of cells independently of the cell cycle . Arrows point to examples of HXT2 - U1A mRNA in the bud early and late in the cell cycle .

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F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96% of cells independently of the cell cycle. Arrows point to examples of HXT2-U1A mRNA in the bud early and late in the cell cycle.


G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2 .

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G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2.


H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers . Cells were re - fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points .

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H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers. Cells were re-fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points.


A . Cryo - sections of human organoids were analyzed for the presence of enterocytes ( E - cad ) , Goblet cells ( Muc - 2 ) and tight junctions ( ZO - 1 ) by indirect immunofluorescence . Nuclei are stained with DAPI . Scale bar 25 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test .

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A. Cryo-sections of human organoids were analyzed for the presence of enterocytes (E-cad), Goblet cells (Muc-2) and tight junctions (ZO-1) by indirect immunofluorescence. Nuclei are stained with DAPI. Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.


B . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . 16hpi organoids were frozen , cryo - sectioned , and HAstV1 infected cells were visualized by indirect immunofluorescence ( HAstV1 ( red ) , nuclei ( DAPI , blue ) . Scale bar 25 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test .

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B. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. 16hpi organoids were frozen, cryo-sectioned, and HAstV1 infected cells were visualized by indirect immunofluorescence (HAstV1 (red), nuclei (DAPI, blue). Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.


C . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells ( green ) were visualized by indirect immunofluorescence . Apical and basolateral membranes were immunostained for actin ( magenta ) and Laminin ( red ) respectively . Nuclei are stained with DAPI ( blue ) . Scale bar is 20 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test .

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C. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells (green) were visualized by indirect immunofluorescence. Apical and basolateral membranes were immunostained for actin (magenta) and Laminin (red) respectively. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.


E . Human intestinal organoids were infected with HAstV1 . At indicated time post - infection the increase in viral copy number was determined by q - RT - PCR .

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E. Human intestinal organoids were infected with HAstV1. At indicated time post-infection the increase in viral copy number was determined by q-RT-PCR.


F . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . At 24 hpi the presence of IFNλ in the media was tested by ELISA . Dotted line indicates detection limit of the assay .

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F. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. At 24 hpi the presence of IFNλ in the media was tested by ELISA. Dotted line indicates detection limit of the assay.


G . Same as E but for the induced levels of either type I IFN ( IFNβ1 ) or type III IFN ( IFNλ ) .

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G. Same as E but for the induced levels of either type I IFN (IFNβ1) or type III IFN (IFNλ).


H . Human intestinal organoids were pre - treated for 24h with 2000IU / mL of IFNβ1 or 300ng / mL of IFNλ1 - 3 . Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q - RT - PCR .

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H. Human intestinal organoids were pre-treated for 24h with 2000IU/mL of IFNβ1 or 300ng/mL of IFNλ1-3. Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q-RT-PCR.


J - M . Percentage of stem cells ( OLFM4 ) goblet cell ( FGCBP ) , mature enterocytes ( APOA4 ) and cells of the enterocyte lineage ( FGCBP ) from the total organoid cells population ( black bar ) in mock - treated organoids and after HAstV1 infection . The percentage of infected cells ( red bars ) and bystander cells ( blue bars ) within each cell type is shown . The data was obtained from multiplex in situ RNA hybridization in 2D organoids . n = 10 , mean ± SEM . Statistics show comparison of infected cells between mock - treated , 4h pi and 16h pi organoids . An Ordinary one - way ANOVA and Tukey ' s multiple comparisons test was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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J-M. Percentage of stem cells (OLFM4) goblet cell (FGCBP), mature enterocytes (APOA4) and cells of the enterocyte lineage (FGCBP) from the total organoid cells population (black bar) in mock-treated organoids and after HAstV1 infection. The percentage of infected cells (red bars) and bystander cells (blue bars) within each cell type is shown. The data was obtained from multiplex in situ RNA hybridization in 2D organoids. n=10, mean ± SEM. Statistics show comparison of infected cells between mock-treated, 4h pi and 16h pi organoids. An Ordinary one-way ANOVA and Tukey's multiple comparisons test was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001.


A . One image per condition was used to show the multiplex in situ RNA FISH . A representative region of the images was chosen and cropped in order to have a zoom - in of a specific area for better visualization . Shown are the enterocyte lineage marker FABP6 ( green ) , HAstV1 infection ( white ) , and CCL2 gene expression ( red ) of mock - treated and infected organoids . DAPI is in blue . White arrows show co - localization of FABP6 and CCL2 with HAstV1 signal . Scale bar 100µm .

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A. One image per condition was used to show the multiplex in situ RNA FISH. A representative region of the images was chosen and cropped in order to have a zoom-in of a specific area for better visualization. Shown are the enterocyte lineage marker FABP6 (green), HAstV1 infection (white), and CCL2 gene expression (red) of mock-treated and infected organoids. DAPI is in blue. White arrows show co-localization of FABP6 and CCL2 with HAstV1 signal. Scale bar 100µm.


B - D . Fluorescence intensity ( arbitrary units ) of innate immune marker expression in stem cells ( OLFM4 positive ) , goblet cells ( FCGBP positive ) , enterocyte lineage cells ( FABP6 positive ) , and mature enterocytes ( APOA4 positive ) . Bystander cells are in blue and infected cells in red . 10 - 90 percentile box plots , each dot represents one cell ( mock N = 2274 , 4h N = 3682 , 16h N = 1510 . C . mock N = 2693 , 4h N = 3706 , 16h N = 1921 . D . mock N = 2274 , 4h N = 3706 , 16h N = 1514 . ) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages . Ordinary one - way ANOVA and Tukey ' s multiple comparisons test were used . Grey statistics show the comparison between infected and bystander cells within each cell lineage . Unpaired t - test with Welch ' s correction was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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

B-D. Fluorescence intensity (arbitrary units) of innate immune marker expression in stem cells (OLFM4 positive), goblet cells (FCGBP positive), enterocyte lineage cells (FABP6 positive), and mature enterocytes (APOA4 positive). Bystander cells are in blue and infected cells in red. 10-90 percentile box plots, each dot represents one cell (mock N=2274, 4h N=3682, 16h N=1510. C. mock N=2693, 4h N=3706, 16h N=1921. D. mock N=2274, 4h N=3706, 16h N=1514.) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages. Ordinary one-way ANOVA and Tukey's multiple comparisons test were used. Grey statistics show the comparison between infected and bystander cells within each cell lineage. Unpaired t-test with Welch's correction was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001


( A ) Overexpression of RNF26 promoted polyubiquitination of MITA . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with a control or RNF26 expression plasmid ( 0 . 5 , 1 , 2 µg ) . Twenty - four hours after transfection , cells were subjected to immunoprecipitation ( IP ) under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - HA ( upper panel ) , anti - Flag ( middle panel ) and anti - RNF26 ( bottom panel ) . Ub , ubiquitin .

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

(A) Overexpression of RNF26 promoted polyubiquitination of MITA. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with a control or RNF26 expression plasmid (0.5, 1, 2 µg). Twenty-four hours after transfection, cells were subjected to immunoprecipitation (IP) under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-HA (upper panel), anti-Flag (middle panel) and anti-RNF26 (bottom panel). Ub, ubiquitin.


( B ) RNF26 - mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26 . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with RNF26 or its mutants ( 1 µg each ) . IP under denatured conditions and ubiquitination assay was performed as described in ( A ) .

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

(B) RNF26-mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with RNF26 or its mutants (1 µg each). IP under denatured conditions and ubiquitination assay was performed as described in (A).


( C ) UbcH5 mediated polyubiquitination of MITA by RNF26 . The RNF26 and MITA proteins were obtained by in vitro transcription and translation , then incubated with biotin - Ub , E1 and the indicated E2s . Polyubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) .

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(C) UbcH5 mediated polyubiquitination of MITA by RNF26. The RNF26 and MITA proteins were obtained by in vitro transcription and translation, then incubated with biotin-Ub, E1 and the indicated E2s. Polyubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels).


( D ) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro . MITA , RNF26 and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and MITA were incubated with RNF26 or its mutants , followed by ubiquitination and immunoblot analysis as described in ( C ) . All experiments were repeated for at least three times with similar results .

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

(D) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro. MITA, RNF26 and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and MITA were incubated with RNF26 or its mutants, followed by ubiquitination and immunoblot analysis as described in (C). All experiments were repeated for at least three times with similar results.


( A ) RNF26 interacted with MITA . The 293 cells ( 5 × 106 ) were transfected with the indicated plasmids ( 5 µg each ) . Twenty - four hours later , cells were lysed and cell lysates were immunoprecipitated with anti - Flag or control IgG . The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti - HA or anti - Flag .

O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNF26 interacted with MITA. The 293 cells (5×106) were transfected with the indicated plasmids (5 µg each). Twenty-four hours later, cells were lysed and cell lysates were immunoprecipitated with anti-Flag or control IgG. The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti-HA or anti-Flag.


( B ) RNF26 was physiologically associated with MITA . THP - 1 cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins . NC , negative control .

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(B) RNF26 was physiologically associated with MITA. THP-1 cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins. NC, negative control.


( C ) RNF26 is localized to the ER and mitochondria . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Twenty - four hours after transfection , the cells were stained with Mito - Tracker Red for 15 minutes or left untreated . The cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis .

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(C) RNF26 is localized to the ER and mitochondria. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Twenty-four hours after transfection, the cells were stained with Mito-Tracker Red for 15 minutes or left untreated. The cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis.


D ) Subcellular distribution of RNF26 . The 293 cells ( 5 × 107 ) were infected with SeV for the indicated time points or left uninfected . Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins .

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D) Subcellular distribution of RNF26. The 293 cells (5×107) were infected with SeV for the indicated time points or left uninfected. Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins.


( E ) RNF26 and MITA were colocalized to the ER . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Eighteen hours after transfection , cells were infected with SeV or HSV - 1 for 6 hours or left uninfected , followed by ER - Tracker Blue / White staining for 30 minutes . Cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis . All experiments were repeated for at least three times with similar results .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNF26 and MITA were colocalized to the ER. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Eighteen hours after transfection, cells were infected with SeV or HSV-1 for 6 hours or left uninfected, followed by ER-Tracker Blue/White staining for 30 minutes. Cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis. All experiments were repeated for at least three times with similar results.


( A ) RNF26 mediated polyubiquitination of MITA at K150 . The 293 cells ( 5 × 106 ) were transfected with HA - Ub ( 1 µg ) and RNF26 ( 1 µg ) together with Flag - MITA or the indicated mutants ( 5 µg each ) . Twenty - four hours after transfection , cells were subjected IP under denatured conditions with anti - Flag and immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated .

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

(A) RNF26 mediated polyubiquitination of MITA at K150. The 293 cells (5×106) were transfected with HA-Ub (1 µg) and RNF26 (1 µg) together with Flag-MITA or the indicated mutants (5 µg each). Twenty-four hours after transfection, cells were subjected IP under denatured conditions with anti-Flag and immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.


B ) RNF26 targeted MITA for polyubiquitination at K150 in vitro . RNF26 , MITA and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and RNF26 were incubated with MITA or its mutants . The ubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) . All experiments were repeated for at least three times with similar results .

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B) RNF26 targeted MITA for polyubiquitination at K150 in vitro. RNF26, MITA and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and RNF26 were incubated with MITA or its mutants. The ubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels). All experiments were repeated for at least three times with similar results.


( A and B ) RNF26 targeted MITA for K11 - linked polyubiquitination . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Twenty - four hours after transfection , cells were subjected for IP under denatured conditions with limited amount of anti - Flag ( 0 . 5 µg ) so that equal amount of Flag - MITA is pulled down . The immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated . Ub - AKR , all lysine residues of ubiquitin were mutated to arginine .

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

(A and B) RNF26 targeted MITA for K11-linked polyubiquitination. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Twenty-four hours after transfection, cells were subjected for IP under denatured conditions with limited amount of anti-Flag (0.5 µg) so that equal amount of Flag-MITA is pulled down. The immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated. Ub-AKR, all lysine residues of ubiquitin were mutated to arginine.


( C ) Effects of RNF26 - RNAi plasmids on the expression of RNF26 . In the upper panels , the 293 cells ( 1 × 106 ) were transfected with the expression plasmids for RNF26 - Flag ( 0 . 5 µg ) and HA - β - actin ( 0 . 1 µg ) together with the indicated RNAi plasmids ( 1 µg each ) . Twenty - four hours after transfection , whole cell lysates were analyzed by immunoblots with anti - Flag or anti - HA . In the lower panels , 293 cells were transduced with a control or RNF26 - RNAi by retrovirus mediated gene transfer . Cells ( 1 × 106 ) were lysed and whole cell lysates were analyzed by immunoblots with anti - RNF26 or anti - β - actin .

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(C) Effects of RNF26-RNAi plasmids on the expression of RNF26. In the upper panels, the 293 cells (1×106) were transfected with the expression plasmids for RNF26-Flag (0.5 µg) and HA-β-actin (0.1 µg) together with the indicated RNAi plasmids (1 µg each). Twenty-four hours after transfection, whole cell lysates were analyzed by immunoblots with anti-Flag or anti-HA. In the lower panels, 293 cells were transduced with a control or RNF26-RNAi by retrovirus mediated gene transfer. Cells (1×106) were lysed and whole cell lysates were analyzed by immunoblots with anti-RNF26 or anti-β-actin.


( D ) Immunoblot analysis of Flag - tagged ubiquitin expression in THP - 1 cells stably transfected with Flag - Ub - K11O plasmid . Whole cell lysates of THP - 1 - Flag - Ub - K11O - RNF26 - RNAi and control cells ( 1 × 106 ) were analyzed by immunoblots with antibodies against the indicated proteins . RNF26 - RNAi # 1 was used here and in the following experiments if not noted .

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(D) Immunoblot analysis of Flag-tagged ubiquitin expression in THP-1 cells stably transfected with Flag-Ub-K11O plasmid. Whole cell lysates of THP-1-Flag-Ub-K11O-RNF26-RNAi and control cells (1×106) were analyzed by immunoblots with antibodies against the indicated proteins. RNF26-RNAi #1 was used here and in the following experiments if not noted.


( E and F ) Effects of RNF26 knockdown on virus - induced K11 - linked polyubiquitination of endogenous MITA . In ( E ) , THP - 1 - Flag - Ub - K11O - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti - MITA . The immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panels ) or anti - MITA ( lower panels ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins . In ( F ) , 293 - HA - Ub - K11O cells ( 2 × 107 ) were transfected with a control or RNF26 - RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , puromycin ( 1 µg / mL ) was added into the culture medium . The cells were selected for twenty - four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in ( E ) . All experiments were repeated for at least three times with similar results .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F) Effects of RNF26 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. In (E), THP-1-Flag-Ub-K11O-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti-MITA. The immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panels) or anti-MITA (lower panels). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins. In (F), 293-HA-Ub-K11O cells (2×107) were transfected with a control or RNF26-RNAi plasmid (10 µg each). Twelve hours after transfection, puromycin (1 µg/mL) was added into the culture medium. The cells were selected for twenty-four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in (E). All experiments were repeated for at least three times with similar results.


A ) Effects of RNF26 knockdown on virus - induced polyubiquitination of endogenous MITA . The THP - 1 - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - Ub ( K48 ) ( upper panel ) , anti - Ub ( K63 ) ( middle panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins .

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

A) Effects of RNF26 knockdown on virus-induced polyubiquitination of endogenous MITA. The THP-1-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Ub(K48) (upper panel), anti-Ub(K63) (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.