Representative of U2OS expressing indicated GFP - ACRC alleles that were treated with 5 - azadC in presence or absence of SUMO inhibitor ( SUMO - E1i ) , fixed 2 h later immunostained with DNMT1 antibody . Scale bar , 10 μm . of data in ( G ) , showing proportion of displaying GFP - ACRC co - localization with DNMT1 foci ( mean ± ; at least 100 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 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 processed for immunoblotting with SUMO2 / 3 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 gcna - 1 promoter was observed in germ , proliferating young larvae but not in post - mitotic tissues in 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 ) gcna - 1 ; dvc - 1 double mutant C . elegans ( mean ± ; 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 gcna - 1 ; dvc - 1 double mutant C . elegans ( mean ± ; 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 ) E364Q mutant C . elegans ( mean ± ; 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 ( mean ± ; 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 ( mean ± ; 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 gcna - 1 deletion ( del ) mutant C . elegans grown on L4440 control ( CTRL ) or smo - 1 RNAi ( mean ± ; 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 RBPJ / L3MBTL3 interaction using yeast two - hybrid ( Y2H ) . In this Y2H experiment , RBPJ is fused to GAL4 - binding ( DB ) domain L3MBTL3 is fused to GAL4 activation domain ( AD ) . DB - RBPJ AD - L3MBTL3 fusion interact with each other , leading to activation of ADE2 HIS3 reporter genes allowing yeast to grow on selective media lacking adenine or histidine . six Y2H controls were previously described ( Dreze et al , 2010 ) . 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 followed by Western blot analyses using HA or L3MBTL3 antibody . 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 followed by Western blot analyses using HA or RBPJ antibody . 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 presence of FLAG - tagged L3MBTL3 ( WT or deletion mutants ) in HEK293T followed by Western blotting using FLAG antibody . 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 BTD domain impairs RBPJ / L3MBTL3 interaction . IP of HA - tagged L3MBTL3 in presence of FLAG - tagged RBPJ ( WT deletion mutants ) in HEK293T followed by Western blotting using HA or FLAG antibody . 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 presence of FLAG - tagged RBPJ ( WT point mutants ) in HEK293T followed by Western blotting using HA or FLAG antibody . RBPJV263R RBPJA284R also show a reduced ability to interact with L3MBTL3 . 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 RBPJ / L3MBTL3 interaction . Representative thermograms ( raw heat signal nonlinear least squares fit to 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 . ( B ) SBP - FLAG - RBPJ HA - L3MBTL3 - Δ ( SAM ) in presence of an increasing amount of HA - NOTCH1 ICD . L3MBTL3 - Δ ( SAM ) mutant construct was used instead of L3MBTL3 WT construct in order to allow of both NOTCH1 ICD L3MBTL3 in same Western blot . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to express HA - L3MBTL3 - Δ ( SAM ) . experiment was independently replicated thrice . WB : Western blot ; IP : immuno - precipitation .

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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 . ( C ) SBP - FLAG - RBPJ HA - NOTCH1 ICD in presence of an increasing amount of HA - L3MBTL3 - Δ ( SAM ) . L3MBTL3 - Δ ( SAM ) mutant construct was used instead of L3MBTL3 WT construct in order to allow of both NOTCH1 ICD L3MBTL3 in same Western blot . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to express HA - L3MBTL3 - Δ ( SAM ) . experiment was independently replicated thrice . WB : Western blot ; IP : immuno - precipitation .

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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 validates shRNA - mediated depletion of RBPJ .

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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 . Shown are means ± s . d . of quadruplicate experiments . [ * * ] P < 0 . 01 , NS : Not Significant ; two - sample T - on log - transformed data . Inset : Western blot validates CRISPR / Cas9 - mediated KO of L3MBTL3 .

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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 L3MBTL3 co - localize at proximal Notch - responsive elements of Notch target genes . Shown are means ± s . d . of triplicate ChIP experiments . In panels C , D F : in base pairs ( bp ) relative to transcriptional start site ( TSS ) is indicated below 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 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 F : in base pairs ( bp ) relative to transcriptional start site ( TSS ) is indicated below 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 repressive activity of L3MBTL3 at Notch target genes is RBPJ - dependent . Expression of Notch target genes upon RBPJ knockdown / 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 difference of differences was tested , which is equivalent to testing interaction in a two - way ANOVA model . Western blot validates overexpression of L3MBTL3 shRNA - mediated depletion of RBPJ ( Appendix Fig S3E ) . Gene expression analyses of OCT4 was performed as control ( Appendix Fig S3F ) .

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

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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 L3MBTL3 - ( 1 - 64 ) domain is required for downregulation of HES1 HEY2 in U87 - MG . Expression 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 .

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

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

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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 proximal Notch - responsive elements of Notch target genes is dependent on L3MBTL3 both its RBPJ interaction KDM1A interaction domains . ChIP of endogenous KDM1A in L3MBTL3 KO U87 - MG 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 D : in bp relative to TSS is indicated below gene names . Chrom8 was used as negative control ( NEG ) .

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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 proximal Notch - responsive element of HES1 . ChIP of H3K4me2 at 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 . 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 D : in bp relative to TSS is indicated below gene names . Chrom8 was used as negative control ( NEG ) .

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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 of HES1 upon overexpression of LacZ control ( Control ) , L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) mutants in L3MBTL3 KO U87 - MG . 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 context of this experiment , i . e . , in absence of endogenous L3MBTL3 , overexpression of L3MBTL3 - Δ ( 1 - 64 ) does not result in increased expression of HES1 , contrasting with result obtained in Fig 4G , i . e . , in presence of endogenous L3MBTL3 . Indeed , as expected , dominant negative effect of L3MBTL3 - ( 1 - 64 ) on endogenous WT L3MBTL3 ' s ability to repress expression of Notch target genes can only be observed when WT L3MBTL3 is expressed .

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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 , Drosophila homolog of L3MBTL3 , directly interacts with Su ( H ) , Drosophila homolog of RBPJ . In vitro transcribed translated dL ( 3 ) mbt or dNotch ICD ( dNotch ICD fragment containing RAM domain ANK repeats ) , as positive control , was incubated with bacterially purified GST - Su ( H ) or GST alone pre - bound to GSH beads . were resolved via SDS - PAGE signals were acquired via X - ray exposure . 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 co - localization of dL ( 3 ) mbt Su ( H ) at dNotch ( N ) locus .

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


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

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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 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 proportions via a two - proportion Z - . 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 lin - 61 / L3MBTL3 during C . elegans vulva development . Proportion of ( 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 .

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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 distribution of GFP - tagged Hxt2 changes during division . In small medium budded ( arrowheads ) , Hxt2 is mainly present in of mother . In large budded , Hxt2 is distributed over entire plasma ( 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 . FISH with an HXT2 - specific probe . Early in cycle mRNA is predominantly found in mother ( arrowheads ) while it is equally distributed after meta - to - anaphase transition ( MAT ) ( yellow arrowheads ) . C of ( B ) . intensities from mother bud were independently measured of signal from mother over signal from bud was calculated . Values > 1 indicate a stronger signal in mother , < 1 a stronger signal in bud around 1 equal distribution . Three independent experiments with 50 mother 50 buds per experiment were analyzed . Boxes represent interquartile range from 25th to 75th percentile with median . Whiskers represent 10th 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 cytoskeleton . were either treated for 15 min with 30 mg / ml latrunculin A or with 30 mg / ml benomyl . arrowheads point to small - budded yellow arrowheads to large budded . E of ( D ) of three in dependent experiments with at least 50 each . Values > 1 indicate a stronger signal in mother , < 1 a stronger signal in bud around 1 equal distribution . Boxes represent interquartile range from 25th to 75th percentile with median . Whiskers represent 10th 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 retention of HXT2 mRNA in mother 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 . 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 in comparison to prt1 - 1 mutant . data represents 3 independent experiments 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 cycle . HXT2 mRNA level bound to either di - / trisomes polysomes ( ≥ 4 ribosomes ) over monosomes was determined by extraction of mRNA from elutriated harvested at G1 / S or M - phase followed by qPCR . At G1 / S similar of HXT2 mRNA were found in 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 localization of HXT2 mRNA is independent of Signal Recognition Particle ( SRP ) . Deletion of SRP54 does not affect retention in mother or 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 bud . FISH experiment with deletions in various binding .

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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 effect of ∆ scp160 on HXT2 mRNA localization is independent of its increased ploidy . Scp160 truncations that lack either last two ( ∆ C2 ) or last four ( ∆ C4 ) KH domains or a Tet - off SCP160 construct , in which expression is blocked by 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 bud . Treatment of with forskolin ( + Forsko ) or using a ∆ bcy1 , 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 glucose affinity of four most important hexose transporters . B FISH of HXT1 , 3 4 under glucose rich conditions . arrowheads depict before , yellow arrowheads after MAT . C 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 HXTs after MAT .

D Hyperactive PKA promotes enrichment of all HXTs after MAT.


E HXT2 mRNA is only HXT mRNA showing enrichment in 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 upon refeeding . Daughters mothers were separated through elutriation . qPCR was performed to determine relative of different HXTs 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 bud leads to higher Hxt2 - GFP transporter at plasma of daughter . Daughter were enriched through elutriation . of of plasma from life imaging from three independent experiments in which at least 50 were analyzed per condition experiment . Western blot confirms higher amount of Hxt2 - GFP transporter present in daughter 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 degradation is not essential for asymmetric HXT2 mRNA localization . FISH of wild - type ∆ xrn1 .

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


B cytoskeleton contributes to HXT2 mRNA enrichment in bud . 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 envelope enrichment in bud requires Kar9 but not Bim1 function . HXT2 mRNA FISH with ∆ bim1 ∆ kar9 strains . Arrows point to a nucleus with HXT2 signal . D of daughter enrichment from data displayed in ( C ) . E of HXT2 mRNA signal in with two nuclei in mother from experiment depicted in ( C ) . of with asymmetric HXT2 mRNA 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 MLP1 / 2 reduced HXT2 mRNA enrichment in bud . FISH under glucose shift conditions with strains in which different NPC components were deleted . B 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 envelope after MAT upon glucose shift . FISH - IF experiment with a probe against HXT2 mRNA an antibody against HA tag of pore component Nup84 - HA . arrows point to HXT2 mRNA signals on 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 envelope into bud during mitosis after glucose shift . Stills of time lapse movies . HXT2 mRNA was visualized using MS2 - GFP system . Under glucose shift conditions HXT2 mRNA is mostly localized to bud together with nucleus . Under glucose rich conditions HXT2 mRNA moves mostly independent of nucleus . Cyan : Nup84 - mCherry ; 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 other HXT only . 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 of competition . 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 HXT2 mRNA with U1A loops leads to its artificial enrichment in bud in 96 % of independently of cycle . Arrows point to examples of HXT2 - U1A mRNA in bud early late in 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 that always enrich HXT2 mRNA in bud have a growth advantage in first hours when coming from quiescence compared to WT or 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 do not retain a memory of previous stress situation encountered by their mothers . were re - fed after starvation 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 presence of enterocytes ( E - cad ) , Goblet ( Muc - 2 ) 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 are shown . Error bars indicate standard deviation . Statistics are from unpaired t - .

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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 , HAstV1 infected 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 are shown . Error bars indicate standard deviation . Statistics are from unpaired t - .

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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 presence of HAstV1 infected ( green ) were visualized by indirect immunofluorescence . Apical basolateral membranes were immunostained for actin ( magenta ) 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 are shown . Error bars indicate standard deviation . Statistics are from unpaired t - .

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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 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 presence of IFNλ in media was tested by ELISA . Dotted indicates detection limit of .

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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 induced 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 course of infection 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 . of ( OLFM4 ) goblet ( FGCBP ) , mature enterocytes ( APOA4 ) of enterocyte lineage ( FGCBP ) from total organoid population ( black bar ) in mock - treated organoids after HAstV1 infection . of infected ( red bars ) bystander ( blue bars ) within each type is shown . data was obtained from multiplex in situ hybridization in 2D organoids . n = 10 , mean ± . Statistics show comparison of infected between mock - treated , 4h pi 16h pi organoids . An Ordinary one - way ANOVA Tukey ' s multiple comparisons 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 multiplex in situ FISH . A representative region of was chosen cropped in order to have a zoom - in of a specific for better visualization . Shown are enterocyte lineage marker FABP6 ( green ) , HAstV1 infection ( ) , CCL2 gene expression ( red ) of mock - treated infected organoids . DAPI is in blue . arrows show co - localization of FABP6 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 . ( arbitrary units ) of innate immune marker expression in ( OLFM4 positive ) , goblet ( FCGBP positive ) , enterocyte lineage ( FABP6 positive ) , mature enterocytes ( APOA4 positive ) . Bystander are in blue infected in red . 10 - 90 percentile box plots , each dot represents one ( 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 between lineages red statistics show comparison of infected between lineages . Ordinary one - way ANOVA Tukey ' s multiple comparisons were used . Grey statistics show comparison between infected bystander within each lineage . Unpaired t - with Welch ' s correction was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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

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(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 enzymatic activity of RNF26 . 293 ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) HA - Ub ( 1 µg ) together with RNF26 or its mutants ( 1 µg each ) . IP under denatured conditions ubiquitination was performed as described in ( A ) .

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(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 . RNF26 MITA were obtained by in vitro transcription translation , then incubated with biotin - Ub , E1 indicated E2s . Polyubiquitination of MITA was examined by immunoblot with HRP - streptavidin ( top panel ) . inputs of RNF26 MITA were analyzed by immunoblots with anti - MITA 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 its mutants were obtained by in vitro transcription translation . Biotin - Ub , E1 , UbcH5 MITA were incubated with RNF26 or its mutants , followed by ubiquitination immunoblot as described in ( C ) . experiments were repeated for at least three times with similar results .

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(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 . 293 ( 5 × 106 ) were transfected with indicated plasmids ( 5 µg each ) . Twenty - four hours later , were lysed lysates were immunoprecipitated with anti - Flag or control IgG . immunoprecipitates whole lysates were analyzed by immunoblots with anti - HA or anti - Flag .

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(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 ( 2 × 107 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected . were lysed immunoprecipitation immunoblot was performed with antibodies against indicated . 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 ER mitochondria . HeLa ( 2 × 105 ) were transfected with indicated plasmids ( 0 . 5 µg each ) . Twenty - four hours after transfection , were stained with Mito - Tracker Red for 15 minutes or left untreated . were fixed with 4 % paraformaldehyde subjected for confocal microscopy .

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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 . 293 ( 5 × 107 ) were infected with SeV for indicated time points or left uninfected . Subcellular fractionation assays were performed fractions were analyzed by immunoblots with antibodies against indicated .

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

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

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(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 its mutants were obtained by in vitro transcription translation . Biotin - Ub , E1 , UbcH5 RNF26 were incubated with MITA or its mutants . ubiquitination of MITA was examined by immunoblot with HRP - streptavidin ( top panel ) . inputs of RNF26 MITA were analyzed by immunoblots with anti - MITA anti - RNF26 ( bottom panels ) . 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 B ) RNF26 targeted MITA for K11 - linked polyubiquitination . 293 ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Twenty - four hours after transfection , 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 . immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . whole lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated . Ub - AKR , lysine residues of ubiquitin were mutated to arginine .

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(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 expression of RNF26 . In upper panels , 293 ( 1 × 106 ) were transfected with expression plasmids for RNF26 - Flag ( 0 . 5 µg ) HA - β - actin ( 0 . 1 µg ) together with indicated RNAi plasmids ( 1 µg each ) . Twenty - four hours after transfection , whole lysates were analyzed by immunoblots with anti - Flag or anti - HA . In lower panels , 293 were transduced with a control or RNF26 - RNAi by retrovirus mediated gene transfer . ( 1 × 106 ) were lysed whole 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 of Flag - tagged ubiquitin expression in THP - 1 stably transfected with Flag - Ub - K11O plasmid . Whole lysates of THP - 1 - Flag - Ub - K11O - RNF26 - RNAi control ( 1 × 106 ) were analyzed by immunoblots with antibodies against indicated . RNF26 - RNAi # 1 was used here in 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 F ) Effects of RNF26 knockdown on - induced K11 - linked polyubiquitination of endogenous MITA . In ( E ) , THP - 1 - Flag - Ub - K11O - RNF26 - RNAi or control ( 2 × 107 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected followed by IP under denatured conditions with anti - MITA . immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panels ) or anti - MITA ( lower panels ) . whole lysates were analyzed by immunoblots with antibodies against indicated or viral . In ( F ) , 293 - HA - Ub - K11O ( 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 culture medium . were selected for twenty - four hours infected with SeV or left uninfected for indicated time points followed by IP under denatured conditions immunoblot as in ( E ) . experiments were repeated for at least three times with similar results .

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

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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.


( B ) Effects of RNF5 knockdown on - induced K11 - linked polyubiquitination of endogenous MITA . 293 - HA - Ub - K11O ( 2 × 107 ) were transfected with indicated RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , were selected with puromycin ( 1 µg / mL ) for twenty - four hours infected with SeV for indicated time points or left uninfected . lysates were subjected to IP under denatured conditions with anti - MITA immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - MITA ( lower panel ) . whole lysates were analyzed by immunoblots with antibodies against indicated antibodies .

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(B) Effects of RNF5 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. The 293-HA-Ub-K11O cells (2×107) were transfected with the indicated RNAi plasmid (10 µg each). Twelve hours after transfection, the cells were selected with puromycin (1 µg/mL) for twenty-four hours and infected with SeV 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-HA (upper panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies.


( C ) RNF26 RNF5 competed with each other on MITA polyubiquitination . 293 ( 5 × 106 ) were transfected with MITA ( 5 µg ) , Flag - Ub - K48O HA - Ub - K11O ( 1 µg each ) together with indicated amount of RNF26 RNF5 . Twenty - four hours after transfection , lysates were subjected to IP under denatured conditions with anti - MITA immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panel ) , anti - HA ( middle panel ) or anti - MITA ( lower panel ) . whole lysates were analyzed by immunoblots with antibodies against indicated .

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(C) RNF26 and RNF5 competed with each other on MITA polyubiquitination. The 293 cells (5×106) were transfected with MITA (5 µg), Flag-Ub-K48O and HA-Ub-K11O (1 µg each) together with indicated amount of RNF26 and RNF5. Twenty-four hours after transfection, cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panel), anti-HA (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.


( D ) Effects of RNF26 knockdown on - triggered MITA degradation . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected . were lysed whole lysates were analyzed by immunoblots with antibodies against indicated ( upper immunoblots ) . relative of MITA in reference to β - actin were analyzed by Quantity One program data shown are mean ± S . D . of three independent experiments ( lower histographs ) .

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(D) Effects of RNF26 knockdown on virus-triggered MITA degradation. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins (upper immunoblots). The relative protein levels of MITA in reference to β-actin were analyzed by Quantity One program and data shown are mean ± S.D. of three independent experiments (lower histographs).


( A ) Effects of RNF26 knockdown on SeV - triggered activation of IFN - β promoter . 293 ( 2 × 105 ) were transfected with IFN - β promoter reporter ( 0 . 1 µg ) indicated RNAi plasmid ( 0 . 5 µg each ) . Thirty hours after transfection , were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

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(A) Effects of RNF26 knockdown on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and the indicated RNAi plasmid (0.5 µg each). Thirty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( B C ) Effects of RNF26 knockdown on - triggered induction of IFN - β in THP - 1 . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected followed by quantitative real - time PCR ( B )

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(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B)


( B C ) Effects of RNF26 knockdown on - triggered induction of IFN - β in THP - 1 . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected followed by quantitative real - time PCR ( B ) or ELISA (

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(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C


( D ) Effects of RNF26 knockdown on - triggered induction of IFNB1 gene in THP - 1 . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with VSV , EMCV or ECTV for indicated time points or left uninfected before quantitative real - time PCR was performed as in ( B ) .

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(D) Effects of RNF26 knockdown on virus-triggered induction of IFNB1 gene in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with VSV, EMCV or ECTV for the indicated time points or left uninfected before quantitative real-time PCR analysis was performed as in (B).


( E F ) Effects of RNF26 knockdown on - triggered induction of TNFα in THP - 1 . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected followed by quantitative real - time PCR ( E ) ELISA ( F ) .

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(E and F) Effects of RNF26 knockdown on virus-triggered induction of TNFα in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (E) and ELISA analysis (F).


( G ) Effects of RNF26 knockdown on - triggered phosphorylation of TBK1 , IRF3 IκBα . THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were infected with SeV or HSV - 1 for indicated time points or left uninfected , whole lysates were analyzed by immunoblots with anti - p - TBK1 , anti - TBK1 , anti - p - IRF3 , anti - IRF3 , anti - p - IκBα , anti - IκBα , anti - RNF26 or anti - β - actin as indicated . experiments were repeated for at least three times with similar results . bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

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(G) Effects of RNF26 knockdown on virus-triggered phosphorylation of TBK1, IRF3 and IκBα. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected, whole cell lysates were analyzed by immunoblots with anti-p-TBK1, anti-TBK1, anti-p-IRF3, anti-IRF3, anti-p-IκBα, anti-IκBα, anti-RNF26 or anti-β-actin as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


A ) Effects of RNF26 knockdown on IRF3 level . In left panel , whole lysates of THP - 1 - RNF26 - RNAi or control ( 1 × 106 ) were analyzed by immunoblots with indicated antibodies . In right panel , ( 1 × 106 ) were subjected to quantitative real - time PCR .

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A) Effects of RNF26 knockdown on IRF3 level. In the left panel, whole cell lysates of THP-1-RNF26-RNAi or control cells (1×106) were analyzed by immunoblots with the indicated antibodies. In the right panel, cells (1×106) were subjected to quantitative real-time PCR analysis.


B ) Effects of RNF26 on IRF3 stability . 293 ( 1 × 106 ) were transfected with HA - IRF3 ( 0 . 5 µg ) HA - β - actin ( 0 . 1 µg ) together with RNF26 or its mutant ( 0 . 3 µg each ) . Twenty - four hours later , whole lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

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B) Effects of RNF26 on IRF3 stability. The 293 cells (1×106) were transfected with HA-IRF3 (0.5 µg) and HA-β-actin (0.1 µg) together with RNF26 or its mutant (0.3 µg each). Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( C ) Effects of RNF26 its mutant on SeV - triggered activation of IFN - β promoter . 293 ( 2 × 105 ) were transfected with IFN - β promoter reporter ( 0 . 1 µg ) RNF26 or its mutant ( 0 . 1 µg each ) . Twenty hours after transfection , were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

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(C) Effects of RNF26 and its mutant on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and RNF26 or its mutant (0.1 µg each). Twenty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( D ) Effects of RNF26 on IRF3 ubiquitination . 293 ( 5 × 106 ) were transfected with Flag - IRF3 ( 5 µg ) RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during experiment . lysates were subjected to IP under denatured conditions with anti - Flag immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panels ) or anti - Flag ( lower panels ) . whole lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated .

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(D) Effects of RNF26 on IRF3 ubiquitination. The 293 cells (5×106) were transfected with Flag-IRF3 (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Eighteen hours after transfection, 3-MA (500 ng/mL) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment. The cell lysates were subjected to IP under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panels) or anti-Flag (lower panels). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.


( E ) Effects of inhibitors on RNF26 - mediated destabilization of IRF3 . 293 ( 1 × 106 ) were transfected with indicated plasmids as described in ( B ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) , NH4Cl ( 25 mM ) or MG132 ( 100 µM ) was added into culture medium for four hours . Whole lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

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(E) Effects of inhibitors on RNF26-mediated destabilization of IRF3. The 293 cells (1×106) were transfected with the indicated plasmids as described in (B). Eighteen hours after transfection, 3-MA (500 ng/mL), NH4Cl (25 mM) or MG132 (100 µM) was added into culture medium for four hours. Whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( F ) Effects of ATG12 knockdown on RNF26 - mediated destabilization of IRF3 . 293 - ATG12 - RNAi or control ( 1 × 106 ) were transfected with indicated plasmids . Twenty - four hours later , whole lysates were analyzed by immunoblots with anti - HA , anti - RNF26 or anti - ATG12 as indicated . experiments were repeated for at least three times with similar results . bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

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(F) Effects of ATG12 knockdown on RNF26-mediated destabilization of IRF3. The 293-ATG12-RNAi or control cells (1×106) were transfected with the indicated plasmids. Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA, anti-RNF26 or anti-ATG12 as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


( A , B ) Expression of UVRAG under stress . HL60 HCT116 were treated with doxorubicin ( 1 μg / ml ) , cisplatin ( 10 μm ) UV irradiation ( 5 min after 50 mJ / cm2 ) for indicated time , then apoptosis , autophagy of UVRAG was assayed . Scale bar , 10 μm .

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(A,B) Expression of UVRAG under cell stress. HL60 and HCT116 cells were treated with doxorubicin (1 μg/ml), cisplatin (10 μm) and UV irradiation (5 min after 50 mJ/cm2) for the indicated time, and then apoptosis, autophagy and fluorescence intensity of UVRAG was assayed. Scale bar, 10 μm.


( C ) In parallel , total extracts were used for western blot .

(C) In parallel, the total protein extracts were used for western blot analysis.


( D , E ) Pharmacological inhibition ( for example , 3 ‐ methyladenine ( 3 ‐ MA ) , 10 mM ) or genetic deletion ( for example , ATG5 − / − ) of autophagy pathway decreases mRNA expression of UVRAG increases apoptosis at 12 h in HL60 treated with doxorubicin ( Doxo ) UV irradiation ( n = 3 , * P0 . 05 ) . AU , arbitrary units ; C ‐ PARP , poly ‐ ADP ribose polymerase ; LC3 , chain 3 ; mRNA , messenger ; PI , propidium iodide ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance ‐ associated gene .

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(D,E) Pharmacological inhibition (for example, 3‐methyladenine (3‐MA), 10 mM) or genetic deletion (for example, ATG5−/−) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin (Doxo) and UV irradiation (n=3, *P0.05). AU, arbitrary units; C‐PARP, cleaved poly‐ADP ribose polymerase; LC3, light chain 3; mRNA, messenger RNA; PI, propidium iodide; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance‐associated gene.


( A - C ) After transfection with UVRAG shRNA or control shRNA for 48 h , HL60 ( A ) , HCT116 ( B ) HeLa ( C ) were treated with doxorubicin ( Doxo , 1 μg / ml ) , cisplatin ( Cis , 10 μm ) UV irradiation ( 5 min after 50 mJ / cm2 ) for 12 h or starvation ( HBSS ) for 3 h , then apoptosis autophagy were assayed ( n = 3 , * P0 . 05 ) .

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(A-C) After transfection with UVRAG shRNA or control shRNA for 48 h, HL60 (A), HCT116 (B) and HeLa (C) cells were treated with doxorubicin (Doxo, 1 μg/ml), cisplatin (Cis, 10 μm) and UV irradiation (5 min after 50 mJ/cm2) for 12 h or starvation (HBSS) for 3 h, and then apoptosis and autophagy were assayed (n=3, *P0.05).