Figure 6 ( D ) H & E staining of small intestinal tissues from indicated mice . Two - month - old mice were used and n = 3 fields were taken for each group . Scale bar , 100 μm . Number of crypts were calculated as means ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . ( E ) H & E staining of intestinal tissues from indicated mice at different time points after 8 Gy of radiation damage . Two - month - old mice were used and n = 3 fields were taken for each group . Number of crypts were calculated as means ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . Scale bar , 50 μm . ( F ) Representative organoid images of circBtnl1 + / + ; sgSox9 and circBtnl1 - / - ; sgSox9 mice , sgCtrl served as control . Organoid formation ratios ( n = 3 ) are shown . Organoid formation ratios are shown as mean ± s . d . * P < 0 . 05 by two - tailed Student ' s t - test . Scale bars , 200 μm .

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Figure 6(D) H&E staining of small intestinal tissues from indicated mice. Two-month-old mice were used and n = 3 fields were taken for each group. Scale bar, 100 μm. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel.(E) H&E staining of intestinal tissues from indicated mice at different time points after 8 Gy of radiation damage. Two-month-old mice were used and n = 3 fields were taken for each group. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar, 50 μm.(F) Representative organoid images of circBtnl1+/+; sgSox9 and circBtnl1-/-; sgSox9 mice, sgCtrl served as control. Organoid formation ratios (n = 3) are shown. Organoid formation ratios are shown as mean ± s. d. *P < 0.05 by two-tailed Student's t-test. Scale bars, 200 μm.


Figure 1BATs were isolated from cold - exposed mice ( 4 ℃ , 72 h ) or control mice ( 30 ℃ , 72 h ) , then lysed and subjected to western blotting for analysis of the indicated proteins ( A ) . Relative protein expression levels , normalized to Actin , are shown in ( B ) . mRNA expression levels of the indicated genes were assayed by real - time PCR and normalized to Actb ( C ) . Mitochondrial DNA copy numbers were determined by real - time PCR and normalized to nuclear DNA standards ( D ) . Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted ( E ) . ( F - G ) Representative images showing immunohistochemical detection of FUNDC1 in sections of BATs from mice kept at 30 ℃ or 4 ℃ for 72 h . Scale bar , 50 μm . The expression of FUNDC1 was quantified by analyzing and normalizing the average optical density of FUNDC1 staining .

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Figure 1BATs were isolated from cold-exposed mice (4℃, 72 h) or control mice (30℃, 72 h), then lysed and subjected to western blotting for analysis of the indicated proteins (A). Relative protein expression levels, normalized to Actin, are shown in (B).mRNA expression levels of the indicated genes were assayed by real-time PCR and normalized to Actb (C).Mitochondrial DNA copy numbers were determined by real-time PCR and normalized to nuclear DNA standards (D).Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted (E).(F-G) Representative images showing immunohistochemical detection of FUNDC1 in sections of BATs from mice kept at 30℃ or 4℃ for 72 h. Scale bar, 50 μm. The expression of FUNDC1 was quantified by analyzing and normalizing the average optical density of FUNDC1 staining.


Figure 2Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30 ℃ ( Normal ) or following 72 h at 4 ℃ ( Cold ) ( A ) . Relative protein expression levels were normalized to Actin ( B ) . mRNA expression levels of the indicated genes were assayed by real - time PCR and normalized to Actb ( C ) . Flag - PGC - 1α was introduced into PGC - 1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins ( D ) . Expression levels of the indicated proteins were quantified ( E ) ( mean ± SEM ; n = 3 ) . Scramble , scramble shRNA . Samples were collected for real - time PCR analysis . The expression of Fundc1 was normalized ( F ) . ( G - H ) Scramble and PGC - 1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses ( G ) and quantification of the indicated proteins ( H )

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Figure 2Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30℃ (Normal) or following 72 h at 4℃ (Cold) (A). Relative protein expression levels were normalized to Actin (B).mRNA expression levels of the indicated genes were assayed by real-time PCR and normalized to Actb (C).Flag-PGC-1α was introduced into PGC-1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins (D). Expression levels of the indicated proteins were quantified (E) (mean ± SEM; n=3). Scramble, scramble shRNA.Samples were collected for real-time PCR analysis. The expression of Fundc1 was normalized (F).(G-H) Scramble and PGC-1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses (G) and quantification of the indicated proteins (H)


Figure 3 ( B ) Luciferase activity stimulated by the human Fundc1 promoter . The activity of Fundc1 promoter fragments , either wild type ( WT ) or with a mutation in the potential NRF1 binding site , was assayed using a dual luciferase reporter system . ( C , D ) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 ( F1 probe or mut F1 probe ) , the confirmed NRF1 binding site in the promoter of VSNL1 ( positive probe ) . The probes were incubated with HeLa nuclear extracts , then run on a gel ( C ) . A super - shifted band ( asterisk ) is detected in the presence of an anti - NRF1 antibody ( D ) . Labeled probe : biotin labeled wild type Fundc1 probe ; Cold probe : wild type Fundc1 probe ; Mut probe : NRF1 binding site mutant Fundc1 probe ; asterisk indicates the supershifted band generated with antibody to NRF1 . ( E , F ) ChIP assay to analyze the interaction of NRF1 ( E ) or PGC - 1α ( F ) proteins with the Fundc1 promoter . ( G ) NRF1 - myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies . ( H ) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC - 1α - Flag for 24 h and samples were collected for western blotting with the indicated antibodies . ( I ) ChIP analysis of the interaction of PGC - 1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells .

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Figure 3(B) Luciferase activity stimulated by the human Fundc1 promoter. The activity of Fundc1 promoter fragments, either wild type (WT) or with a mutation in the potential NRF1 binding site, was assayed using a dual luciferase reporter system.(C,D) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 (F1 probe or mut F1 probe) ,the confirmed NRF1 binding site in the promoter of VSNL1 (positive probe). The probes were incubated with HeLa nuclear extracts, then run on a gel (C). A super-shifted band (asterisk) is detected in the presence of an anti-NRF1 antibody (D). Labeled probe: biotin labeled wild type Fundc1 probe; Cold probe: wild type Fundc1 probe; Mut probe: NRF1 binding site mutant Fundc1 probe; asterisk indicates the supershifted band generated with antibody to NRF1.(E,F) ChIP assay to analyze the interaction of NRF1 (E) or PGC-1α (F) proteins with the Fundc1 promoter.(G) NRF1-myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies. (H) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC-1α-Flag for 24 h and samples were collected for western blotting with the indicated antibodies. (I) ChIP analysis of the interaction of PGC-1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells.


Figure 4 ( A , B ) Western blotting analyses of the levels of autophagy - related proteins in BAT from mice at the normal temperature ( 30 ℃ ) or at 4 ℃ for 72 h ( A ) . Expression of the indicated proteins was analyzed before and after 72 h cold exposure ( 4 ℃ ) ( B ) . ( C ) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature ( 30 ℃ ) or 4 ℃ for 72 h . ( D Representative transmission electron microscope ( TEM ) images of BAT cells isolated from mice at the normal temperature ( 30 ℃ ) or 4 ℃ for 72 h . Scale bar , 1 μm ( D ) . The number of mitophagosomes per 100 mitochondria was counted and normalized ( E ) . ( F , G ) BATs from mice exposed to normal conditions ( 30 ℃ ) or cold ( 4 ℃ ) were lysed and immunoprecipitated with antibodies against FUNDC1 , LC3B or IgG , then immunoblotted with the indicated antibodies . The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted ( G ) .

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Figure 4(A,B) Western blotting analyses of the levels of autophagy-related proteins in BAT from mice at the normal temperature (30℃) or at 4℃ for 72 h (A). Expression of the indicated proteins was analyzed before and after 72 h cold exposure (4℃) (B).(C) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature (30 ℃) or 4℃ for 72 h.(D Representative transmission electron microscope (TEM) images of BAT cells isolated from mice at the normal temperature (30℃) or 4℃ for 72 h. Scale bar, 1 μm(D).The number of mitophagosomes per 100 mitochondria was counted and normalized (E).(F,G) BATs from mice exposed to normal conditions (30℃) or cold (4℃) were lysed and immunoprecipitated with antibodies against FUNDC1, LC3B or IgG, then immunoblotted with the indicated antibodies. The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted (G).


Figure 5 ( A , B ) BATs isolated from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) mice at 30 ℃ or following 72 h at 4 ℃ were analyzed by transmission electron microscopy . Representative images are shown , scale bar , 1μm ( A ) . The number of mitophagosomes per 100 mitochondria were counted and normalized ( B ) . ( C , D ) . Representative fluorescence images of BAT sections from Fundc1fl / fl / Adiponectincre - ( Cre - ) and Fundc1fl / fl / Adiponectincre + ( Cre + ) mito - Keima mice at normal conditions ( 30 ℃ ) or 4 ℃ for 72 h . Scale bar , 50 μm ( C ) . Quantification of the ratio between red and green signals ( D ) . ( E ) Fundcfl / fl / Ucp1cre - ( Cre - ) and Fundcfl / fl / Ucpcre + ( Cre + ) mice were maintained at 30 ℃ ( Normal ) or exposed to 4 ℃ for 72 h ( Cold ) . Mitochondrial DNA copy numbers were analyzed by null and normalized to nuclear DNA standards . ( F ) The relative total mito - keima signal ( green signal ) in Fundc1 deficiency and control mito - keima mice under normal condition or cold exposure were quantified and plotted . ( G ) BATs isolated from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) at 30 ℃ or following 72 h at 4 ℃ were analyzed by transmission electron microscopy . The percent of mitochondria without cristae is shown in the histogram . Oxygen consumption rate ( OCR ) ( H ) were measured in BAT from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucpcre + ( Cre + ) mice kept at 30 ℃ or at 4 ℃ for 72 h . mitochondrial superoxide ( I ) were measured in BAT from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucpcre + ( Cre + ) mice kept at 30 ℃ or at 4 ℃ for 72 h .

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Figure 5(A,B) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) mice at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. Representative images are shown, scale bar, 1μm (A). The number of mitophagosomes per 100 mitochondria were counted and normalized (B).(C,D).Representative fluorescence images of BAT sections from Fundc1fl/fl/Adiponectincre- (Cre-) and Fundc1fl/fl/Adiponectincre+ (Cre+) mito-Keima mice at normal conditions (30℃) or 4℃ for 72 h. Scale bar, 50 μm (C). Quantification of the ratio between red and green signals (D).(E) Fundcfl/fl/Ucp1cre- (Cre-) and Fundcfl/fl/Ucpcre+ (Cre+) mice were maintained at 30℃ (Normal) or exposed to 4℃ for 72 h (Cold). Mitochondrial DNA copy numbers were analyzed by null and normalized to nuclear DNA standards.(F) The relative total mito-keima signal (green signal ) in Fundc1 deficiency and control mito-keima mice under normal condition or cold exposure were quantified and plotted.(G) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. The percent of mitochondria without cristae is shown in the histogram.Oxygen consumption rate (OCR) (H) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.mitochondrial superoxide (I) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.


Figure 6 ( A ) Body temperature measurements in Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) fasting male mice before and during cold exposure ( 4 ° C ) for the indicated time . Body temperature ( B ) before and after 72 h cold exposure ( 4 ° C ) of Cre - and Cre + male mice . BAT tissue weight ( C ) measurements before and after 72 h cold exposure ( 4 ° C ) of Cre - and Cre + male mice . Fundc1fl / fl / Ucpcre - and Fundc1fl / fl / Ucp1cre + mice were exposed to 4 ℃ for 72 h ( Cold ) , and control mice were kept at 30 ℃ ( Normal ) . Western blotting analysis of BAT protein levels ( D ) . Protein expression levels were normalized to Actin ( E ) . Fundc1fl / fl / Ucpcre - and Fundc1fl / fl / Ucp1cre + mice were exposed to 4 ℃ for 72 h ( Cold ) , and control mice were kept at 30 ℃ ( Normal ) . Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real - time PCR and normalized to Actb ( F ) . ( G ) Representative images of UCP1 immunohistochemistry of BAT sections from Cre - or Cre + mice in normal conditions ( 30 ℃ ) or following 72 h at 4 ℃ . Scale bar , 50 μm .

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Figure 6(A) Body temperature measurements in Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) fasting male mice before and during cold exposure (4°C) for the indicated time. Body temperature (B) before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice.BAT tissue weight (C) measurements before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice.Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Western blotting analysis of BAT protein levels (D). Protein expression levels were normalized to Actin (E).Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real-time PCR and normalized to Actb (F).(G) Representative images of UCP1 immunohistochemistry of BAT sections from Cre- or Cre+ mice in normal conditions (30℃) or following 72 h at 4℃. Scale bar, 50 μm.


Figure 1 ( I ) Normalized death rates in L929 wt cells plotted with pMLKL protein levels measured via immunoblot ( M ) Death rates in TNF - treated cell lines including clonal RelA - knockout population ( mean of three independent experiments ± standard deviation ) .

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Figure 1(I) Normalized death rates in L929 wt cells plotted with pMLKL protein levels measured via immunoblot(M) Death rates in TNF-treated cell lines including clonal RelA-knockout population (mean of three independent experiments ± standard deviation).


Figure 2 ( A ) TNF - induced mRNA expression in indicated L929 cell lines measured via qRT - PCR ( mean of three independent experiments ± standard deviation , two - tailed Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 ) . ( B ) Immunoblot after co - immunoprecipitation ( IP ) of RIPK3 . FT , flow through . ( C ) Relative quantification of A20 in RIPK3 - IP fraction ( means and statistical significance established for 0 , 2 , 4 . 5 and 8 hour time points from three independent experiments ± standard deviation ; two - tailed Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 , n . s . , P > 0 . 05 ) . ( I , J ) ( I ) TNF - induced distributions of death times ( representative data of three independent experiments ) , and ( J ) death rates obtained by live - cell microscopy ( mean of three independent experiments ± standard deviation ) .

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Figure 2(A) TNF-induced mRNA expression in indicated L929 cell lines measured via qRT-PCR (mean of three independent experiments ± standard deviation, two-tailed Student's t-test *P<0.05, **P<0.01).(B) Immunoblot after co-immunoprecipitation (IP) of RIPK3. FT, flow through. (C) Relative quantification of A20 in RIPK3-IP fraction (means and statistical significance established for 0, 2, 4.5 and 8 hour time points from three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05, **P<0.01, n.s., P>0.05). (I,J) (I) TNF-induced distributions of death times (representative data of three independent experiments), and (J) death rates obtained by live-cell microscopy (mean of three independent experiments ± standard deviation).


Figure 3 ( G , H ) ( G ) Distribution of death times ( representative data of three independent experiments ) , or ( H ) death rates in TNF - treated wt , parental A20 KO cells , or A20 KO cells reconstituted with an NFκB - inducible transgene ( fIL8 - A20 ; mean of three independent experiments ± standard deviation ) . ( J ) experimental measurements of 24 - hour fractional survival after varying durations of transient or sustained ( 24h ) TNF stimulation ( mean of three independent experiments ± standard deviation ; two - tailed Student ' s t - test * * * P < 0 . 001 , or no statistically significant difference , n . s . , P > 0 . 05 ) .

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Figure 3(G,H) (G) Distribution of death times (representative data of three independent experiments), or (H) death rates in TNF-treated wt, parental A20 KO cells, or A20 KO cells reconstituted with an NFκB-inducible transgene (fIL8-A20; mean of three independent experiments ± standard deviation).(J) experimental measurements of 24-hour fractional survival after varying durations of transient or sustained (24h) TNF stimulation (mean of three independent experiments ± standard deviation; two-tailed Student's t-test ***P<0.001, or no statistically significant difference, n.s., P>0.05).


Figure 4 ( H ) Death rates in TNF - treated indicated cell lines including isogenic IκBα / IκBε - knockout population ( mean of three independent experiments ± standard deviation ) . ( I ) Death rates in TNF - treated cell lines treated with targeting ( siA20 ) or non - targeting ( siCon ) siRNA ( mean of three independent experiments ± standard deviation ) .

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Figure 4(H) Death rates in TNF-treated indicated cell lines including isogenic IκBα/IκBε-knockout population (mean of three independent experiments ± standard deviation).(I) Death rates in TNF-treated cell lines treated with targeting (siA20) or non-targeting (siCon) siRNA (mean of three independent experiments ± standard deviation).


Figure 2 ( A ‑ B ) BMDCs were plated at different densities and stimulated with poly ( I : C ) for 24h or left unstimulated . DC activation was evaluated based on surface expression of CD86 and CD40 molecules as measured by flow cytometry . ( A ) Representative dot plots showing CD86 and CD40 expression . ( B ) Quantification of CD86 and CD40 upregulation as a fold change in geometric mean fluorescence intensity ( gMFI ) over unstimulated controls . Data are pooled from 3 independent experiments .

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Figure 2(A‑B) BMDCs were plated at different densities and stimulated with poly(I:C) for 24h or left unstimulated. DC activation was evaluated based on surface expression of CD86 and CD40 molecules as measured by flow cytometry. (A) Representative dot plots showing CD86 and CD40 expression. (B) Quantification of CD86 and CD40 upregulation as a fold change in geometric mean fluorescence intensity (gMFI) over unstimulated controls. Data are pooled from 3 independent experiments.


Figure 3 ( A ) IFN ‑ α ( left ) and IFN ‑ β ( right ) production by WT and Irf3 - / - Irf7 - / - BMDCs plated at high density ( 2x105 cells / cm2 ) and stimulated with poly ( I : C ) ( blue dots ) or left unstimulated ( grey dots ) . Dotted lines represent the ELISA threshold of detection . Data are representative of 2 independent experiments . ( B ) CD86 and CD40 expression was quantified on WT ( blue dots ) and Irf3 - / - Irf7 - / - ( orange dots ) BMDCs plated at high density ( 2x105 cells / cm2 ) and stimulated with poly ( I : C ) or left unstimulated . Data are pooled from 2 independent experiments . ( C ) CD86 and CD40 expression on Irf3 - / - Irf7 - / - BMDCs plated at 2x105 cells / cm2 and stimulated with poly ( I : C ) ( dark blue dots ) , IFN - α ( light blue dots ) or a combination of both ( green dots ) . Data are representative of 2 independent experiments .

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Figure 3(A) IFN‑α (left) and IFN‑β (right) production by WT and Irf3-/-Irf7-/- BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) (blue dots) or left unstimulated (grey dots). Dotted lines represent the ELISA threshold of detection. Data are representative of 2 independent experiments.(B) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3-/-Irf7-/- (orange dots) BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) or left unstimulated. Data are pooled from 2 independent experiments. (C) CD86 and CD40 expression on Irf3-/-Irf7-/- BMDCs plated at 2x105 cells/cm2 and stimulated with poly(I:C) (dark blue dots), IFN-α (light blue dots) or a combination of both (green dots). Data are representative of 2 independent experiments.


Figure 4 ( A ) CD86 and CD40 expression was quantified on WT ( blue dots ) and Irf3 ‑ / ‑ Irf7 ‑ / ‑ ( orange dots ) DCs in non ‑ draining ( ndLN ) and draining ( dLN ) popliteal lymph nodes of WT and Irf3 ‑ / ‑ Irf7 ‑ / ‑ mice injected with poly ( I : C ) or PBS . Data are pooled data from 2 independent experiments . ( D ) Representative dot plots of CD86 and CD40 expression on WT ( blue dots ) and Irf3 ‑ / ‑ Irf7 ‑ / ‑ ( orange dots ) DCs of chimeras injected with PBS or poly ( I : C ) .

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Figure 4(A) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs in non‑draining (ndLN) and draining (dLN) popliteal lymph nodes of WT and Irf3‑/‑Irf7‑/‑ mice injected with poly(I:C) or PBS. Data are pooled data from 2 independent experiments.(D) Representative dot plots of CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs of chimeras injected with PBS or poly(I:C).


Figure 5 ( B ) Representative dot plots showing CD86 and CD40 expression by low ‑ density BMDCs stimulated with poly ( I : C ) for different periods of IFN ‑ α exposure .

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Figure 5(B) Representative dot plots showing CD86 and CD40 expression by low‑density BMDCs stimulated with poly(I:C) for different periods of IFN‑α exposure.


Figure 6Monocyte recruitment in the draining lymph node requires collective DC activation ( A ) Representative dot plots showing the percentage of monocytes ( CD11b + Ly6Chigh cells , black box ) present in popliteal dLN of WT : Irf3 ‑ / ‑ Irf7 ‑ / ‑ chimeric mice injected with poly ( I : C ) ( middle and right ) or with PBS ( left ) . CD11b + Ly6Clow / int cells corresponded to neutrophils and were not included in the analysis . Collective DC activation is essential for the generation of T cell responses . ( G ) Representative dot plots showing the percentage of CD44high tetramer H ‑ 2Kb ‑ OVA + CD8 + T cells in the draining lymph node of chimeric mice injected with PBS or a mixture of OVA + poly ( I : C ) . Collective DC activation is essential for the generation of T cell responses . ( H ) Flow cytometric quantification of CD44high H ‑ 2Kb ‑ OVA tetramer + CD8 + T cells in the draining lymph node of 90 : 10 and 5 : 95 ( WT : Irf3 ‑ / ‑ Irf7 ‑ / ‑ ) chimeric mice injected with PBS or OVA + poly ( I : C ) . Data are pooled from 2 independent experiments .

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Figure 6Monocyte recruitment in the draining lymph node requires collective DC activation (A) Representative dot plots showing the percentage of monocytes (CD11b+ Ly6Chigh cells, black box) present in popliteal dLN of WT:Irf3‑/‑Irf7‑/‑ chimeric mice injected with poly(I:C) (middle and right) or with PBS (left). CD11b+ Ly6Clow/int cells corresponded to neutrophils and were not included in the analysis.Collective DC activation is essential for the generation of T cell responses. (G) Representative dot plots showing the percentage of CD44high tetramer H‑2Kb‑OVA+ CD8+ T cells in the draining lymph node of chimeric mice injected with PBS or a mixture of OVA+poly(I:C).Collective DC activation is essential for the generation of T cell responses. (H) Flow cytometric quantification of CD44high H‑2Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of 90:10 and 5:95 (WT:Irf3‑/‑Irf7‑/‑) chimeric mice injected with PBS or OVA+poly(I:C). Data are pooled from 2 independent experiments.


Figure 1Single - cell expression data in pseudotime of central regulators of naïve pluripotency , epiblast marker genes , glycolytic regulators , and genes with key functions in oxidative metabolism .

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Figure 1Single-cell expression data in pseudotime of central regulators of naïve pluripotency, epiblast marker genes, glycolytic regulators, and genes with key functions in oxidative metabolism.


Figure 2Flow cytometry analysis of Rex1 - GFPd2 cells following addition of 10mM 2 - DG during the 48h EpiLC induction . Representative GFP intensity distributions are depicted . Average proportions of Rex1 - GFPd2 positive ( GFP + ) cells are quantified from two independent biological replicates . Expression analysis by qRT - PCR of naïve pluripotency and epiblast marker genes in bulk 48h cells after 10mM 2 - DG treatment . Relative expression levels , normalized to control culture conditions , are shown . Graphs represent averages from triplicate ( duplicate for Klf4 and Tfcp2l1 ) independent biological experiments . Colony - forming ability following 10mM 2 - DG supplementation during EpiLC stimulation . Representative images of alkaline phosphatase ( AP ) - stained colonies are displayed . Scale bar , 250 um . The average colony formation , normalized to control culture conditions , quantified from two independent biological replicates , is shown .

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Figure 2Flow cytometry analysis of Rex1-GFPd2 cells following addition of 10mM 2-DG during the 48h EpiLC induction. Representative GFP intensity distributions are depicted. Average proportions of Rex1-GFPd2 positive (GFP+) cells are quantified from two independent biological replicates.Expression analysis by qRT-PCR of naïve pluripotency and epiblast marker genes in bulk 48h cells after 10mM 2-DG treatment. Relative expression levels, normalized to control culture conditions, are shown. Graphs represent averages from triplicate (duplicate for Klf4 and Tfcp2l1) independent biological experiments.Colony-forming ability following 10mM 2-DG supplementation during EpiLC stimulation. Representative images of alkaline phosphatase (AP)-stained colonies are displayed. Scale bar, 250 um. The average colony formation, normalized to control culture conditions, quantified from two independent biological replicates, is shown.


Figure 3Pseudotime expression profiles for the αKG - regulating enzymes Idh2 and Dlst during the transition from naïve to primed pluripotency . Tricarboxylic acid ( TCA ) cycle enzymes and metabolites produced within the TCA cycle are illustrated . Representative flow cytometry profiles of Rex1 - GFPd2 cells following 4mM dm - αKG supplementation during the EpiLC induction are depicted . Graphs show average fractions of Rex1 - GFPd2 positive ( GFP + ) cells from six independent biological assays . qRT - PCR analysis of naïve pluripotency regulators and epiblast marker genes following EpiLC stimulation in the presence of 4mM dm - αKG . Expression data are normalized to control culture conditions and represent averages from five biological replicates in bulk 48h cells . Colony forming ability succeeding 4mM dm - αKG treatment during the 48h EpiLC induction . Characteristic images of AP - stained colonies are shown . Scale bar , 250 um . Colony formation is normalized to control - treated cells and quantified from quadruplicate experiments . Representative super - resolution images of TOM - 20 immune - labelled mitochondria in ESCs following 48h culture in 2i / Lif / KSR media , and EpiLC - inducing conditions in the presence of 4mM dm - αKG and DMSO , respectively , are displayed . Scale bar , 3um . 10 - day culture of Rex1 - GFPd2 cells in N2B27 / Lif / KSR with 4mM dm - αKG and DMSO , respectively , with passaging every 2 . 5 - days . ( F ) Characteristic bright field images of Rex1 - GFPd2 cells after 10 days of culture in 2i / Lif / KSR and N2B27 / Lif / KSR , in the presence of dm - αKG and DMSO , respectively . Scale bar , 10um . 10 - day culture of Rex1 - GFPd2 cells in N2B27 / Lif / KSR with 4mM dm - αKG and DMSO , respectively , with passaging every 2 . 5 - days . Flow cytometer - based quantification of Rex1 - GFPd2 positive ( GFP + ) cells . Representative GFP intensity distributions are displayed . The average fractions of GFP + cells are measured from duplicate experiments . 10 - day culture of Rex1 - GFPd2 cells in N2B27 / Lif / KSR with 4mM dm - αKG and DMSO , respectively , with passaging every 2 . 5 - days . qRT - PCR analysis of naïve pluripotency and differentiation markers in bulk cells harvested at 2 . 5 day - intervals during the 10 - day culture in N2B27 / Lif / KSR with dm - αKG or DMSO . Expression data are normalized to time - matched ESCs in 2i / Lif / KSR - culture conditions and are averaged over two independent biological experiments .

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Figure 3Pseudotime expression profiles for the αKG-regulating enzymes Idh2 and Dlst during the transition from naïve to primed pluripotency. Tricarboxylic acid (TCA) cycle enzymes and metabolites produced within the TCA cycle are illustrated.Representative flow cytometry profiles of Rex1-GFPd2 cells following 4mM dm-αKG supplementation during the EpiLC induction are depicted. Graphs show average fractions of Rex1-GFPd2 positive (GFP+) cells from six independent biological assays.qRT-PCR analysis of naïve pluripotency regulators and epiblast marker genes following EpiLC stimulation in the presence of 4mM dm-αKG. Expression data are normalized to control culture conditions and represent averages from five biological replicates in bulk 48h cells.Colony forming ability succeeding 4mM dm-αKG treatment during the 48h EpiLC induction. Characteristic images of AP-stained colonies are shown. Scale bar, 250 um. Colony formation is normalized to control-treated cells and quantified from quadruplicate experiments.Representative super-resolution images of TOM-20 immune-labelled mitochondria in ESCs following 48h culture in 2i/Lif/KSR media, and EpiLC-inducing conditions in the presence of 4mM dm-αKG and DMSO, respectively, are displayed. Scale bar, 3um.10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. (F) Characteristic bright field images of Rex1-GFPd2 cells after 10 days of culture in 2i/Lif/KSR and N2B27/Lif/KSR, in the presence of dm-αKG and DMSO, respectively. Scale bar, 10um.10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. Flow cytometer-based quantification of Rex1-GFPd2 positive (GFP+) cells. Representative GFP intensity distributions are displayed. The average fractions of GFP+ cells are measured from duplicate experiments.10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. qRT-PCR analysis of naïve pluripotency and differentiation markers in bulk cells harvested at 2.5 day-intervals during the 10-day culture in N2B27/Lif/KSR with dm-αKG or DMSO. Expression data are normalized to time-matched ESCs in 2i/Lif/KSR-culture conditions and are averaged over two independent biological experiments.


Figure 4FACS analysis of Prdm1 - GFP positive ( GFP + ) cells in day - 4 embryoids specified in the presence of 4mM dm - αKG and PGC cytokines . Representative flow cytometer profiles are depicted . Graphs show the average fractions of GFP + cells from duplicate experiments . P1 - GFP , Prdm1 - GFP . FACS analysis of Prdm1 - GFP positive ( GFP + ) cells in day - 2 embryoids aggregated under addition of LIF ( 10ng ml - 1 ) and BMP4 ( 500ng ml - 1 ) , dm - αKG ( 4mM ) , or DMSO . Representative flow cytometer profiles are displayed . Average fractions of GFP + cells are calculated from duplicate assays . P1 - GFP , Prdm1 - GFP . FACS analysis of Prdm1 - GFP positive ( GFP + ) cells in day - 4 PGCLC aggregates specified from 4mM dm - αKG - treated ( t = 48h to t = 72h ) EpiLCs . Representative flow cytometer profiles are depicted . The average fractions of GFP + cells , quantified from triplicate experiments , are shown . P1 - GFP , Prdm1 - GFP . Transcript analysis by qRT - PCR of PGC specifiers , demethylating enzymes , mesoderm , endoderm , and ESC regulators in FACS - sorted day - 4 Prdm1 - GFP embryoids induced from 4mM dm - αKG - treated 72h EpiLCs . Expression levels are normalized to Prdm1 - GFP negative cells from control embryoids . Graphs represent averages from triplicate experiments . + , Prdm1 - GFP positive cells ; - , Prdm1 - GFP negative cells . Western blot analysis for H3K9me2 , H3K27me3 , and DNMT3b in 4mM dm - αKG - treated EpiLCs . H3 is used as a loading control . 48h , dm - αKG and DMSO , respectively , supplementation from t = 24h to t = 48h during the EpiLC differentiation . 72h , dm - αKG and DMSO , respectively , supplementation from t = 48h to t = 72h during the EpiLC differentiation . ChIP - qPCR analysis of H3K9me2 and H3K27me3 in putative enhancer regions of genes associated with the naïve pluripotent state ( Esrrb , Arid5b ) and PGC fate ( Tfap2c ) , respectively , in naïve ESCs and at t = 48h following EpiLC induction in the presence of 4mM dm - αKG and DMSO , respectively . Graphs show enrichment of H3K9me2 , H3K27me3 , and IgG control , respectively , relative to DMSO - treated EpiLCs .

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Figure 4FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 embryoids specified in the presence of 4mM dm-αKG and PGC cytokines. Representative flow cytometer profiles are depicted. Graphs show the average fractions of GFP+ cells from duplicate experiments. P1-GFP, Prdm1-GFP.FACS analysis of Prdm1-GFP positive (GFP+) cells in day-2 embryoids aggregated under addition of LIF (10ng ml-1) and BMP4 (500ng ml-1), dm-αKG (4mM), or DMSO. Representative flow cytometer profiles are displayed. Average fractions of GFP+ cells are calculated from duplicate assays. P1-GFP, Prdm1-GFP.FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 PGCLC aggregates specified from 4mM dm-αKG-treated (t=48h to t=72h) EpiLCs. Representative flow cytometer profiles are depicted. The average fractions of GFP+ cells, quantified from triplicate experiments, are shown. P1-GFP, Prdm1-GFP.Transcript analysis by qRT-PCR of PGC specifiers, demethylating enzymes, mesoderm, endoderm, and ESC regulators in FACS-sorted day-4 Prdm1-GFP embryoids induced from 4mM dm-αKG-treated 72h EpiLCs. Expression levels are normalized to Prdm1-GFP negative cells from control embryoids. Graphs represent averages from triplicate experiments. +, Prdm1-GFP positive cells; -, Prdm1-GFP negative cells.Western blot analysis for H3K9me2, H3K27me3, and DNMT3b in 4mM dm-αKG-treated EpiLCs. H3 is used as a loading control. 48h, dm-αKG and DMSO, respectively, supplementation from t=24h to t=48h during the EpiLC differentiation. 72h, dm-αKG and DMSO, respectively, supplementation from t=48h to t=72h during the EpiLC differentiation.ChIP-qPCR analysis of H3K9me2 and H3K27me3 in putative enhancer regions of genes associated with the naïve pluripotent state (Esrrb, Arid5b) and PGC fate (Tfap2c), respectively, in naïve ESCs and at t=48h following EpiLC induction in the presence of 4mM dm-αKG and DMSO, respectively. Graphs show enrichment of H3K9me2, H3K27me3, and IgG control, respectively, relative to DMSO-treated EpiLCs.


Figure 1A , B . Western blot showing the absence of HUWE1 protein in 293T ( A ) and HeLa ( B ) cells subjected to CRISPR / Cas9 - mediated HUWE1 - deletion . C , D . HUWE1 - knockout 293T ( C ) and HeLa ( D ) cells show increased DNA breaks in the absence of exogenous DNA damage treatment . Results from the Alkaline Comet Assay are shown . The " n " numbers of comet tails analyzed ( pooled from two independent experiments ) , as well as the mean , are indicated on the graphs . HUWE1 - knockout HeLa cells did not show increases breakage in the Neutral Comet assay ( Fig EV1B ) , indicating that the majority of breaks observed in cycling HUWE1 - knockout cells are not double strand breaks , but rather single strand breaks and other types of lesions . E , F . Increased S - phase arrest in HUWE1 - knockout 293T ( E ) and HeLa ( F ) cells . Cycling cells were incubated with BrdU and subjected to BrdU / PI bi - dimensional flow cytometry . Representative flow cytometry profiles are presented in Fig EV1D - F . Bars represent the fold increase in the percentage of cells with S - phase DNA content ( between 2N and 4N ) but negative for BrdU staining . Bars represent the average of three independent experiments , with error bars showing SD . The p - values are 0 . 0091 ( E ) and 0 . 0007 ( F ) . G , H . The DNA fiber assay shows reduced replication tract length in HUWE1 - knockout 293T ( G ) and HeLa ( H ) cells in the absence of exogenous DNA damage treatment . The " n " numbers of fibers analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . P - values are 9 . 8x10 - 22 ( G ) and 1 . 0x10 - 10 ( H ) .

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Figure 1A, B. Western blot showing the absence of HUWE1 protein in 293T (A) and HeLa (B) cells subjected to CRISPR/Cas9-mediated HUWE1-deletion.C, D. HUWE1-knockout 293T (C) and HeLa (D) cells show increased DNA breaks in the absence of exogenous DNA damage treatment. Results from the Alkaline Comet Assay are shown. The "n" numbers of comet tails analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs. HUWE1-knockout HeLa cells did not show increases breakage in the Neutral Comet assay (Fig EV1B), indicating that the majority of breaks observed in cycling HUWE1-knockout cells are not double strand breaks, but rather single strand breaks and other types of lesions.E, F. Increased S-phase arrest in HUWE1-knockout 293T (E) and HeLa (F) cells. Cycling cells were incubated with BrdU and subjected to BrdU/PI bi-dimensional flow cytometry. Representative flow cytometry profiles are presented in Fig EV1D-F. Bars represent the fold increase in the percentage of cells with S-phase DNA content (between 2N and 4N) but negative for BrdU staining. Bars represent the average of three independent experiments, with error bars showing SD. The p-values are 0.0091 (E) and 0.0007 (F).G, H. The DNA fiber assay shows reduced replication tract length in HUWE1-knockout 293T (G) and HeLa (H) cells in the absence of exogenous DNA damage treatment. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-values are 9.8x10-22 (G) and 1.0x10-10 (H).


Figure 2A . HUWE1 levels are efficiently downregulated by two different HUWE1 siRNA oligonucleotides in 8988T cells . B . Quantification of HUWE1 expression following siRNA treatment . Band intensity was quantified using ImageJ software and normalized to Tubulin loading control . The average of three experiments is shown . Error bars indicate SD . C - E . Cell cycle analyses by flow cytometry using BrdU / PI double staining show increased replication arrest in HUWE1 - depleted 8988T cells in the absence of exogenous DNA damage treatment . C . Representative flow cytometry profiles of control and HUWE1 - knockdown cells . R1 - labeled region indicates mid and late S - phase cells ( BrdU - positive , > 2N DNA content ) , while R2 - labeled region indicates S - phase arrested cells ( BrdU - negative , DNA content between 2N and 4N ) . C - E . Cell cycle analyses by flow cytometry using BrdU / PI double staining show increased replication arrest in HUWE1 - depleted 8988T cells in the absence of exogenous DNA damage treatment . C . Representative flow cytometry profiles of control and HUWE1 - knockdown cells . R1 - labeled region indicates mid and late S - phase cells ( BrdU - positive , > 2N DNA content ) , while R2 - labeled region indicates S - phase arrested cells ( BrdU - negative , DNA content between 2N and 4N ) . D . Quantification of S - phase cells . Percentage of cells in R1 region is shown . Bars represent the average of three independent experiments . Error bars indicate SD . P - value is 0 . 0014 . E . Quantification of S - phase arrested cells . Bars represent the fold increase in the percentage of cells in R2 region , normalized to siControl - treated cells . The average of three independent experiments is shown . Error bars indicate SD . P - value is 0 . 0135 . F . DNA fiber experiment showing reduced replication tract length in HUWE1 - depleted HeLa cells in the absence of exogenous DNA damage treatment . The " n " numbers of fibers analyzed ( pooled from two independent experiments ) , as well as the mean , are indicated on the graphs . G , H . Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV ( G ) , and HU ( H ) . As controls , both cells transfected with non - targeting siRNA ( siControl ) , and non - transfected cells , were used . Note that the siHUWE1 # 2 oligonucleotide shows a stronger downregulation of HUWE1 , and confers increased sensitivity compared to siHUWE1 # 1 . The average of nine experiments is shown . Error bars represent SEM . P - values ( representing the statistical difference between the samples at the highest dose treatment ) are : 4 . 55x10 - 9 ( G ) and 1 . 8x10 - 8 ( H ) . I . DNA fiber experiments showing decreased replication tract length following UV - B treatment ( 30J / m2 ) in HUWE1 - depleted HeLa cells . The " n " numbers of fibers analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . P - value is 2 . 11x10 - 20 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. HUWE1 levels are efficiently downregulated by two different HUWE1 siRNA oligonucleotides in 8988T cells. B. Quantification of HUWE1 expression following siRNA treatment. Band intensity was quantified using ImageJ software and normalized to Tubulin loading control. The average of three experiments is shown. Error bars indicate SD.C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N).C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N). D. Quantification of S-phase cells. Percentage of cells in R1 region is shown. Bars represent the average of three independent experiments. Error bars indicate SD. P-value is 0.0014. E. Quantification of S-phase arrested cells. Bars represent the fold increase in the percentage of cells in R2 region, normalized to siControl-treated cells. The average of three independent experiments is shown. Error bars indicate SD. P-value is 0.0135.F. DNA fiber experiment showing reduced replication tract length in HUWE1-depleted HeLa cells in the absence of exogenous DNA damage treatment. The "n" numbers of fibers analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs.G, H. Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV (G), and HU (H). As controls, both cells transfected with non-targeting siRNA (siControl), and non- transfected cells, were used. Note that the siHUWE1#2 oligonucleotide shows a stronger downregulation of HUWE1, and confers increased sensitivity compared to siHUWE1#1. The average of nine experiments is shown. Error bars represent SEM. P-values (representing the statistical difference between the samples at the highest dose treatment) are: 4.55x10-9 (G) and 1.8x10-8 (H).I. DNA fiber experiments showing decreased replication tract length following UV-B treatment (30J/m2) in HUWE1-depleted HeLa cells. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-value is 2.11x10-20.


Figure 3A . iPOND experiment showing that HUWE1 localizes to replication forks in 293T cells . Binding to nascent DNA was increased after UV exposure . Because of the difficulties in removing all the crosslinks in high molecular weightproteins , HUWE1 is detected as a high molecular weight smear . A control experiment using the HUWE1 - knockout cells ( shown in the right side panel ) confirmed the specificity of the HUWE1 signal . B . Immunofluorescence experiment showing that HUWE1 localizes to chromatin foci in HeLa cells exposed to replication fork stalling agents HU ( 2mM for 16h ) and UV ( 40J / m2 , analyzed 2h later ) . C . Co - immunofluorescence experiments showing co - localization of HUWE1 and PCNA in chromatin foci . U2OS cells were analyzed 2h after exposure to UV ( 40J / m2 ) . Quantifications of co - localization , and additional micrographs are presented in Fig EV2A - D .

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Figure 3A. iPOND experiment showing that HUWE1 localizes to replication forks in 293T cells. Binding to nascent DNA was increased after UV exposure. Because of the difficulties in removing all the crosslinks in high molecular weightproteins, HUWE1 is detected as a high molecular weight smear. A control experiment using the HUWE1-knockout cells (shown in the right side panel) confirmed the specificity of the HUWE1 signal.B. Immunofluorescence experiment showing that HUWE1 localizes to chromatin foci in HeLa cells exposed to replication fork stalling agents HU (2mM for 16h) and UV (40J/m2, analyzed 2h later).C. Co-immunofluorescence experiments showing co-localization of HUWE1 and PCNA in chromatin foci. U2OS cells were analyzed 2h after exposure to UV (40J/m2). Quantifications of co-localization, and additional micrographs are presented in Fig EV2A-D.


Figure 4B . Anti - PCNA immunoprecipitation , showing that HUWE1 co - precipitates . C . Reciprocal experiment showing that PCNA co - precipitates with HUWE1 in anti - HUWE1 immunoprecipitation . D . As control , endogenous HUWE1 was depleted by siRNA treatment , and extracts were subjected to anti - HUWE1 immunoprecipitation . The HUWE1 blot shows that much less HUWE1 is precipitated by anti - HUWE1 antibodies from HUWE1 - knockdown cells than control , as expected . PCNA is co - precipitated by anti - HUWE1 antibodies from control , but not HUWE1 - depleted cells , showing that the interaction is specific and not due to unspecific binding of PCNA to anti - HUWE1 antibodies . E , F . HUWE1 co - precipitates with PCNA in anti - PCNA immunoprecipitation from extracts of MCF7 ( E ) and 8988T ( F ) cells , showing that the HUWE1 - PCNA interaction can be detected in different cell lines . G . Co - immunoprecipitation experiment from control and RAD18 - knockout 293T cells , showing that the HUWE1 - PCNA interaction is not affected by loss of PCNA ubiquitination . RAD18 - knockout cells were obtained by CRISPR / Cas9 technology . The RAD18 and PCNA blots show loss of RAD18 , and of PCNA ubiquitination respectively . H . Myc - HUWE1C - ter fragment was transfected in 293T cells . Anti - PCNA immunoprecipitation showed that the C - terminal HUWE1 fragment interacts with PCNA . I . Coomassie staining of recombinant GST - PCNA expressed and purified from bacteria . J . GST - pulldowns using GST - PCNA or GST - empty as control , and extracts of 293T cells transfected with Myc - HUWE1C - ter , showing interaction of this fragment with recombinant PCNA .

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Figure 4B. Anti-PCNA immunoprecipitation, showing that HUWE1 co-precipitates.C. Reciprocal experiment showing that PCNA co-precipitates with HUWE1 in anti-HUWE1 immunoprecipitation.D. As control, endogenous HUWE1 was depleted by siRNA treatment, and extracts were subjected to anti-HUWE1 immunoprecipitation. The HUWE1 blot shows that much less HUWE1 is precipitated by anti-HUWE1 antibodies from HUWE1-knockdown cells than control, as expected. PCNA is co-precipitated by anti-HUWE1 antibodies from control, but not HUWE1-depleted cells, showing that the interaction is specific and not due to unspecific binding of PCNA to anti-HUWE1 antibodies. E, F. HUWE1 co-precipitates with PCNA in anti-PCNA immunoprecipitation from extracts of MCF7 (E) and 8988T (F) cells, showing that the HUWE1-PCNA interaction can be detected in different cell lines.G. Co-immunoprecipitation experiment from control and RAD18-knockout 293T cells, showing that the HUWE1-PCNA interaction is not affected by loss of PCNA ubiquitination. RAD18-knockout cells were obtained by CRISPR/Cas9 technology. The RAD18 and PCNA blots show loss of RAD18, and of PCNA ubiquitination respectively.H. Myc-HUWE1C-ter fragment was transfected in 293T cells. Anti-PCNA immunoprecipitation showed that the C-terminal HUWE1 fragment interacts with PCNA.I. Coomassie staining of recombinant GST-PCNA expressed and purified from bacteria.J. GST-pulldowns using GST-PCNA or GST-empty as control, and extracts of 293T cells transfected with Myc-HUWE1C-ter, showing interaction of this fragment with recombinant PCNA.


Figure 5A , B . GST - pulldowns showing that HUWE1 PIP - box mutants do not interact with PCNA . A . Recombinant GST - tagged HUWE1C - ter , either wild - type or PIP - box mutant variants ( QVFF and VFF - the indicated residues , critical for the PIP - box , were mutated to A ) , were purified from bacteria ( see Coomassiestaining ) and employed for GST - pulldowns using extracts of 293T cells . PCNA co - precipitated with wild - type , but not PIP - box mutants . B . Reciprocal GST - pulldown using recombinant GST - PCNA and extracts of 293T cells overexpressing Myc - HUWE1C - ter . Wild - type , but not the PIP - box mutant ( VFF ) HUWE1 fragment bound to GST - PCNA . C , D . Reciprocal co - immunoprecipitationexperiments showing that PIP - box mutation abolishes PCNA interaction . C . 293T cells were transfected with Myc - HUWE1C - ter variants , and subjected to anti - PCNA ( C ) or anti - Myc ( D ) immunoprecipitation . Only wild - type , but not PIP - box mutant ( VFF ) HUWE1 interacted with PCNA in both experimental setups . E . Mutation of the PIP - box in full - length HUWE1 also blocks PCNA interaction . Full - length Myc - tagged HUWE1 ( wild - type or PIP - mutant ) was transfected in 293T cells . Following Myc - immunoprecipitation , endogenous PCNA was detected in wild - type , but not PIP - box mutant ( FF ) complexes .

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Figure 5A, B. GST-pulldowns showing that HUWE1 PIP-box mutants do not interact with PCNA. A. Recombinant GST-tagged HUWE1C-ter, either wild-type or PIP-box mutant variants (QVFF and VFF -the indicated residues, critical for the PIP-box, were mutated to A), were purified from bacteria (see Coomassiestaining) and employed for GST-pulldowns using extracts of 293T cells. PCNA co-precipitated with wild-type, but not PIP-box mutants. B. Reciprocal GST-pulldown using recombinant GST-PCNA and extracts of 293T cells overexpressing Myc-HUWE1C-ter. Wild-type, but not the PIP-box mutant (VFF) HUWE1 fragment bound to GST-PCNA.C, D. Reciprocal co-immunoprecipitationexperiments showing that PIP-box mutation abolishes PCNA interaction. C. 293T cells were transfected with Myc-HUWE1C-ter variants, and subjected to anti-PCNA (C) or anti-Myc (D) immunoprecipitation. Only wild-type, but not PIP-box mutant (VFF) HUWE1 interacted with PCNA in both experimental setups.E. Mutation of the PIP-box in full-length HUWE1 also blocks PCNA interaction. Full-length Myc-tagged HUWE1 (wild-type or PIP-mutant) was transfected in 293T cells. Following Myc-immunoprecipitation, endogenous PCNA was detected in wild-type, but not PIP-box mutant (FF) complexes.


Figure 6A . Stable expression of Myc - tagged wild - type , PIP - box mutant , and catalytic inactive HUWE1 variants in HUWE1 - knockout 293T cells . The Western blot shows that expression of the Myc - tagged variants stably transfected in the knockout cell lines , is similar to the endogenous HUWE1 expression . B . Myc - tag immunoprecipitation using extracts of the corrected cell lines described above . As expected , PCNA co - precipitates with wild - type , but not PIP - box mutant HUWE1 ( FF ) . C . iPOND experiment using HUWE1 - knockout 293T cells , corrected with wild - type or PIP - mutant HUWE1 . The PIP - mutant shows reduced crosslinking to nascent DNA , indicated defective recruitment to replication forks . D . Clonogenic assay showing that stable expression of wild - type HUWE1 , but not of the PIP box mutant or the catalytic mutant , corrects the HU sensitivity of the HUWE1 - knockout 293T cells . Shown is the average of 3 independent experiments - / + SD . If not indicated otherwise , the p - values shown specify the statistical significance relative to 293T ( for the 0 . 5mM HU condition ) . P - values are : 0 . 0003 ( 293T vs HUWE1 - / - ; 0 . 157 ( 293T vs HUWE1 - / - + WT ) ; 0 . 0045 ( 293T vs HUWE1 - / - + FF ) ; 0 . 0006 ( 293T vs HUWE1 - / - + C4341A ; 0 . 047 ( HUWE1 - / - + WT vs HUWE1 - / - + FF ) . E . The S - phase arrest phenotype is also rescued by wild - type , but not PIP - mutant or catalytic mutant HUWE1 . S - phase arrested cells were quantified using the BrdU / PI bi - dimensional flow cytometry assay . Shown is the average of 3 independent experiments - / + SD . The p - values shown specify the statistical significance relative to 293T ( 0 . 018 , 0 . 957 , 0 . 015 , and 0 . 024 respectively ) . A quantification of the same data , showing the percentage of cells arrested in S - phase , is shown in Fig EV3A . F . Normal replication tract length , quantified using the DNA fiber assay , is restored by expressing wild - type , but not PIP - mutant HUWE1 , in the HUWE1 - knockout 293T cells . The " n " numbers of fibers analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . If not indicated otherwise , the p - values shown specify the statistical significance relative to 293T . P - values are : 1 . 47x10 - 11 ( 293T vs HUWE - / - samples ) , 0 . 094 ( 293T vs HUWE - / - + WT samples ) , 1 . 40x10 - 7 ( 293T vs HUWE - / - + FF samples ) , 1 . 22x10 - 7 ( HUWE - / - vs HUWE - / - + WT samples ) , 0 . 679 ( HUWE - / - vs HUWE - / - + FF samples ) . G . Alkaline Comet Assay showing that wild - type , but not the FF mutant can correct the breakage phenotype of HUWE1 - knockout 293T cells . The " n " numbers of comet tails analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . The p - values indicated are : 4 . 18x10 - 43 ( 293T vs HUWE - / - samples ) , 6 . 89x10 - 41 ( HUWE1 - / - + WT vs FF samples ) , and 5 . 97x10 - 50 ( HUWE1 - / - + WT vs C4341A samples ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Stable expression of Myc-tagged wild-type, PIP-box mutant, and catalytic inactive HUWE1 variants in HUWE1-knockout 293T cells. The Western blot shows that expression of the Myc-tagged variants stably transfected in the knockout cell lines, is similar to the endogenous HUWE1 expression.B. Myc-tag immunoprecipitation using extracts of the corrected cell lines described above. As expected, PCNA co-precipitates with wild-type, but not PIP-box mutant HUWE1 (FF).C. iPOND experiment using HUWE1-knockout 293T cells, corrected with wild-type or PIP-mutant HUWE1. The PIP-mutant shows reduced crosslinking to nascent DNA, indicated defective recruitment to replication forks.D. Clonogenic assay showing that stable expression of wild-type HUWE1, but not of the PIP box mutant or the catalytic mutant, corrects the HU sensitivity of the HUWE1-knockout 293T cells. Shown is the average of 3 independent experiments -/+SD. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T (for the 0.5mM HU condition). P-values are: 0.0003 (293T vs HUWE1-/-; 0.157 (293T vs HUWE1-/- +WT); 0.0045 (293T vs HUWE1-/- +FF); 0.0006 (293T vs HUWE1-/- +C4341A; 0.047 (HUWE1-/- +WT vs HUWE1-/- +FF).E. The S-phase arrest phenotype is also rescued by wild-type, but not PIP-mutant or catalytic mutant HUWE1. S-phase arrested cells were quantified using the BrdU/PI bi-dimensional flow cytometry assay. Shown is the average of 3 independent experiments -/+SD. The p-values shown specify the statistical significance relative to 293T (0.018, 0.957, 0.015, and 0.024 respectively). A quantification of the same data, showing the percentage of cells arrested in S-phase, is shown in Fig EV3A.F. Normal replication tract length, quantified using the DNA fiber assay, is restored by expressing wild-type, but not PIP-mutant HUWE1, in the HUWE1-knockout 293T cells. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T. P-values are: 1.47x10-11 (293T vs HUWE-/- samples), 0.094 (293T vs HUWE-/- +WT samples), 1.40x10-7 (293T vs HUWE-/- +FF samples), 1.22x10-7 (HUWE-/- vs HUWE-/- +WT samples), 0.679 (HUWE-/- vs HUWE-/- +FF samples).G. Alkaline Comet Assay showing that wild-type, but not the FF mutant can correct the breakage phenotype of HUWE1-knockout 293T cells. The "n" numbers of comet tails analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. The p-values indicated are: 4.18x10-43 (293T vs HUWE-/- samples), 6.89x10-41 (HUWE1-/- +WT vs FF samples), and 5.97x10-50 (HUWE1-/- +WT vs C4341A samples).


Figure 7A , B . The impact of HUWE1 on H2AX phosphorylation and ubiquitination , under normal conditions ( A ) or upon induction of replication stress ( 2mM HU for 18h , or 2h after exposure to 40J / m2 UV ) . Control or HUWE1 - knockout HeLa cells were analyzed . C . In vitro ubiquitination assay showing that recombinant HUWE1C - ter can mono - ubiquitinate H2AX . D . Wild - type , but not the PIP - mutant can correct the defective H2AX ubiquitination in HUWE1 - knockout 293T cells . . E . Chromatin immunoprecipitation showing that H2AX binding to the common fragile site FRA3B is reduced in HUWE1 - knockout 8988T cells . Cells were treated with 600nM aphidicolin for 24h . Binding was quantified relative to input material . Shown is the average of 4 experiments - / + SD . P - value is 4 . 4x10 - 6 . F . Western blots showing that several H2AX ubiquitin ligases participate in H2AX ubiquitination following replication stress . HUWE1 , RNF168 , and BMI1 were knocked - down in HeLa cells . The efficiency of the knockdown is shown in Fig EV4 . Cells were treated with 600nM aphidicolin for 24h , 2mM HU for 24h , or analyzed 2h after exposure to 40J / m2 UV . G . Chromatin immunoprecipitation from HeLa cells , showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B . Cells were treated with 600nM aphidicolin for 24h . Binding was quantified relative to input material . Shown is the average of 3 experiments - / + SD . The p - values shown indicate the statistical significance relative to siControl ( 0 . 0016 , 0 . 0005 , and 0 . 0009 respectively ) . H . Chromatin fractionation experiments showing that HUWE1 - knockout HeLa cells , treated with 2mM HU for 24h , fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA . In contrast , 53BP1 chromatin association is not induced by HU treatment , and not affected by HUWE1 knockout , and thus can serve as loading control . Shown are blots of whole cell extract ( WCE ) samples , representing input material , and of chromatin pellet samples .

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Figure 7A, B. The impact of HUWE1 on H2AX phosphorylation and ubiquitination, under normal conditions (A) or upon induction of replication stress (2mM HU for 18h, or 2h after exposure to 40J/m2 UV). Control or HUWE1-knockout HeLa cells were analyzed.C. In vitro ubiquitination assay showing that recombinant HUWE1C-ter can mono-ubiquitinate H2AX.D. Wild-type, but not the PIP-mutant can correct the defective H2AX ubiquitination in HUWE1-knockout 293T cells.. E. Chromatin immunoprecipitation showing that H2AX binding to the common fragile site FRA3B is reduced in HUWE1-knockout 8988T cells. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 4 experiments -/+ SD. P-value is 4.4x10-6.F. Western blots showing that several H2AX ubiquitin ligases participate in H2AX ubiquitination following replication stress. HUWE1, RNF168, and BMI1 were knocked-down in HeLa cells. The efficiency of the knockdown is shown in Fig EV4. Cells were treated with 600nM aphidicolin for 24h, 2mM HU for 24h, or analyzed 2h after exposure to 40J/m2 UV.G. Chromatin immunoprecipitation from HeLa cells, showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 3 experiments -/+ SD. The p-values shown indicate the statistical significance relative to siControl (0.0016, 0.0005, and 0.0009 respectively).H. Chromatin fractionation experiments showing that HUWE1-knockout HeLa cells, treated with 2mM HU for 24h, fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA. In contrast, 53BP1 chromatin association is not induced by HU treatment, and not affected by HUWE1 knockout, and thus can serve as loading control. Shown are blots of whole cell extract (WCE) samples, representing input material, and of chromatin pellet samples.


Figure 1B Recombinant MAVS - Region III - dimer ( lanes 1 - 4 ) , trimer ( lanes 5 - 8 ) or tetramer ( lanes 9 - 12 ) were subjected to IRF3 dimerization assay respectively in vitro . Increasing doses of recombinant MAVS - Region III were used and shown in Fig EV1C . Immunoblotting analysis was performed following Native - PAGE for the reaction mixture . C pcDNA3 - based constructs expressing MAVS - Region III - dimer , trimer and tetramer were transfected into HEK293T cells respectively . ISG54 induction were measured thirty - six hours after transfection by quantitative PCR ( qPCR ) . D Recombinant MAVS - Region III - dimer or tetramer were immunoprecipitated with anti - FLAG M2 beads with or without a preincubation with S100 from HEK293T cells following an experimental procedure outlined in Fig EV1G . The IP products were resolved in SDS - PAGE and a specific protein band pulled - down with MAVS - Region III - tetramer was indicated by an arrow . TRAF3IP3 was identified in the specific band by mass spectrometry . E Protein samples as described in ( D ) were subjected to immunoblotting analysis . The TRAF3IP3 band was indicated by an arrow and the asterisk indicated nonspecific bandsF pcDNA3 - FLAG - TRAF3IP3 ( full - length and truncated forms ) and pcDNA3 - HA - MAVS were transfected into HEK293T cells , which were harvested thirty - six hours post transfection . Co - IP experiments were performed with anti - HA antibody , followed by immunoblotting . G Human cells , including THP1 , HeLa , HEK293 , HEK293T as well as Traf3ip3 - / - HEK293T , were lysed and subjected to immunoblotting to examine TRAF3IP3 expression .

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Figure 1B Recombinant MAVS-Region III-dimer (lanes 1-4), trimer (lanes 5-8) or tetramer (lanes 9-12) were subjected to IRF3 dimerization assay respectively in vitro. Increasing doses of recombinant MAVS-Region III were used and shown in Fig EV1C. Immunoblotting analysis was performed following Native-PAGE for the reaction mixture.C pcDNA3-based constructs expressing MAVS-Region III-dimer, trimer and tetramer were transfected into HEK293T cells respectively. ISG54 induction were measured thirty-six hours after transfection by quantitative PCR (qPCR).D Recombinant MAVS-Region III-dimer or tetramer were immunoprecipitated with anti-FLAG M2 beads with or without a preincubation with S100 from HEK293T cells following an experimental procedure outlined in Fig EV1G. The IP products were resolved in SDS-PAGE and a specific protein band pulled-down with MAVS-Region III-tetramer was indicated by an arrow. TRAF3IP3 was identified in the specific band by mass spectrometry.E Protein samples as described in (D) were subjected to immunoblotting analysis. The TRAF3IP3 band was indicated by an arrow and the asterisk indicated nonspecific bandsF pcDNA3-FLAG-TRAF3IP3 (full-length and truncated forms) and pcDNA3-HA-MAVS were transfected into HEK293T cells, which were harvested thirty-six hours post transfection. Co-IP experiments were performed with anti-HA antibody, followed by immunoblotting.G Human cells, including THP1, HeLa, HEK293, HEK293T as well as Traf3ip3-/- HEK293T, were lysed and subjected to immunoblotting to examine TRAF3IP3 expression.


Figure 2A Three pairs of small interfering RNA against TRAF3IP3 were transfected into HEK293T cells respectively and immunoblotting were then employed to examine the knockdown efficiency . A pair of scrambled RNA oligoes was transfected and signified as con . B Three pairs of si - TRAF3IP3 together with IFNβ - luciferase reporter were transfected into HEK 293T cells . Twenty - four hours after transfection , the cells were infected with ( SeV ) or without ( mock ) Sendai virus , which were harvested six hours post infection to measure the luciferase activity . C Si - TRAF3IP3 ( oligo # 1 ) were transfected into HEK 293 cells , which were then treated with mock , VSV , SeV , poly ( I : C ) or HSV - 1 . After various treatments , IFNB inductions were measured by qPCR . D Immunoblotting to detect TRAF3IP3 in wild type and Traf3ip3 - / - HEK293T cells . E Wild type and Traf3ip3 - / - HEK293T cells were treated with or without VSV , SeV or poly ( I : C ) as indicated for twelve hours . IFNB induction was measured by qPCR . F Wild type and Traf3ip3 - / - HEK293T cells were infected with GFP - VSV . Fluorescent images were taken to examine GFP - VSV proliferation at eight hours post infection ( left ) . Scale bar represents 10 micrometers . GFP - VSV titers were quantitated by plaque assay ( right ) . G Various expression constructs were transfected into WT and Traf3ip3 - / - HEK293T cells . Thirty - six hours after transfection , IFNB induction was measured by qPCR . H Mavs - / - HEK293T cells were transfected with empty vector , pcDNA - FLAG - MAVS , pcDNA - FLAG - MAVS - ( Region III only ) , or pcDNA - FLAG - MAVS - ( ΔRegion III ) , together with pcDNA3 - FLAG - TRAF3IP3 or not as indicated . Twenty - four hours after transfection , cells were infected with SeV for twelve hours . IFNB induction was measured by qPCR .

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Figure 2A Three pairs of small interfering RNA against TRAF3IP3 were transfected into HEK293T cells respectively and immunoblotting were then employed to examine the knockdown efficiency. A pair of scrambled RNA oligoes was transfected and signified as con.B Three pairs of si-TRAF3IP3 together with IFNβ-luciferase reporter were transfected into HEK 293T cells. Twenty-four hours after transfection, the cells were infected with (SeV) or without (mock) Sendai virus, which were harvested six hours post infection to measure the luciferase activity.C Si-TRAF3IP3 (oligo #1) were transfected into HEK 293 cells, which were then treated with mock, VSV, SeV, poly(I:C) or HSV-1. After various treatments, IFNB inductions were measured by qPCR.D Immunoblotting to detect TRAF3IP3 in wild type and Traf3ip3-/- HEK293T cells.E Wild type and Traf3ip3-/- HEK293T cells were treated with or without VSV, SeV or poly (I:C) as indicated for twelve hours. IFNB induction was measured by qPCR.F Wild type and Traf3ip3-/- HEK293T cells were infected with GFP-VSV. Fluorescent images were taken to examine GFP-VSV proliferation at eight hours post infection (left). Scale bar represents 10 micrometers. GFP-VSV titers were quantitated by plaque assay (right).G Various expression constructs were transfected into WT and Traf3ip3-/- HEK293T cells. Thirty-six hours after transfection, IFNB induction was measured by qPCR.H Mavs-/- HEK293T cells were transfected with empty vector, pcDNA-FLAG-MAVS, pcDNA-FLAG-MAVS-(Region III only), or pcDNA-FLAG-MAVS-(ΔRegion III), together with pcDNA3-FLAG-TRAF3IP3 or not as indicated. Twenty-four hours after transfection, cells were infected with SeV for twelve hours. IFNB induction was measured by qPCR.


Figure 3A The experiment was performed as described in Fig 2C . Instead of IFNB , ISG54 and IL6 inductions were measured by qPCR . B HEK293 cells were transfected with si - TRAF3IP3 oligos and then treated with various stimuli . Following subcellular fractionation , S5 fractions were obtained and subjected to native PAGE for detecting IRF3 dimer by immunoblotting . The whole cell lysates were also subjected to SDS - PAGE for anti - IRF3 and tubulin immunoblotting . C The experiment was performed as described in Fig 2E . ISG54 and IL6 induction was measured by qPCR . D Wild type and Traf3ip3 - / - HEK293T cells were infected with or without VSV . At indicated time points , Cells were collected and analyzed by immunoblotting . Following subcellular fractionation , S5 fractions were subjected to native PAGE to examine IRF3 dimer , and P5 fractions contained mitochondria were subjected to SDD - AGE to examine MAVS aggregation . The mitochondrial outer membrane protein Prohibitin was immunoblotted as an internal control .

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Figure 3A The experiment was performed as described in Fig 2C. Instead of IFNB, ISG54 and IL6 inductions were measured by qPCR.B HEK293 cells were transfected with si-TRAF3IP3 oligos and then treated with various stimuli. Following subcellular fractionation, S5 fractions were obtained and subjected to native PAGE for detecting IRF3 dimer by immunoblotting. The whole cell lysates were also subjected to SDS-PAGE for anti- IRF3 and tubulin immunoblotting.C The experiment was performed as described in Fig 2E. ISG54 and IL6 induction was measured by qPCR.D Wild type and Traf3ip3-/- HEK293T cells were infected with or without VSV. At indicated time points, Cells were collected and analyzed by immunoblotting. Following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation. The mitochondrial outer membrane protein Prohibitin was immunoblotted as an internal control.


Figure 4A pcDNA - FLAG - TRADD and pcDNA - FLAG - TRAF3IP3 were transfected into HEK293T cells at increasing amount respectively . ISG54 and IL6 induction were measured thirty - six hours post - transfection by qPCR . Expression levels were shown in Fig EV4B . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 NS indicates no statistically significant difference . B HEK293T cells were transfected with pcDNA3 - HA - TRAF3IP3 , in combination with pcDNA3 - FLAG - RIG - I , MAVS , TBK1 , IRF3 , TRAF2 , TRAF3 , TRAF5 , or TRAF6 as indicated . Thirty - six hours after transfection , cells were collected and anti - HA immunoprecipitations were performed . IP products were subjected to immunoblotting . C shRNA targeting TRAF3IP3 ( sh - TRAF3IP3 ) was transduced into Mavs - / - HEK293T cells . Twenty - four hours after transduction , cells were treated with puromycin ( 2 μg / mL ) for forty - eight hours and then transfected with pcDNA3 - FLAG - MAVS - ( Region III only ) . Twenty - four hours after transfection , cells were infected with or without VSV for twelve hours . Cells were then collected and subjected to immunoprecipitation assay and immunoblotting . D Wild type and Traf3ip3 - / - HEK293T cells were transfected with pcDNA3 - FLAG - MAVS - ( Region III only ) . Twenty - four hours post - transfection , cells were infected with or without VSV . Twelve hours post - infection , cells were collected and subjected to immunoprecipitation assay and immunoblotting .

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Figure 4A pcDNA-FLAG-TRADD and pcDNA-FLAG-TRAF3IP3 were transfected into HEK293T cells at increasing amount respectively. ISG54 and IL6 induction were measured thirty-six hours post-transfection by qPCR. Expression levels were shown in Fig EV4B. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05, **P<0.01 and ***P<0.001 NS indicates no statistically significant difference.B HEK293T cells were transfected with pcDNA3-HA-TRAF3IP3, in combination with pcDNA3-FLAG- RIG-I, MAVS, TBK1, IRF3, TRAF2, TRAF3, TRAF5, or TRAF6 as indicated. Thirty-six hours after transfection, cells were collected and anti-HA immunoprecipitations were performed. IP products were subjected to immunoblotting.C shRNA targeting TRAF3IP3 (sh-TRAF3IP3) was transduced into Mavs-/- HEK293T cells. Twenty-four hours after transduction, cells were treated with puromycin (2 μg/mL) for forty-eight hours and then transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours after transfection, cells were infected with or without VSV for twelve hours. Cells were then collected and subjected to immunoprecipitation assay and immunoblotting.D Wild type and Traf3ip3-/- HEK293T cells were transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours post-transfection, cells were infected with or without VSV. Twelve hours post-infection, cells were collected and subjected to immunoprecipitation assay and immunoblotting.


Figure 5A Subcellular fractionation was performed for HEK293T cells infected with or without SeV to get three fractions , i . e . , cytoplasm , membrane and nuclei extracts , which were then subjected to Immunoblotting analysis as indicated . B Membranous fraction obtained in Fig 5A were further separated into mitochondria , Golgi and ER extracts as described in methods , followed by immunoblotting analysis . C - E Immunofluorescent microscopic imaging for TRAF3IP3 . HeLa cells were transfected with pcDNA3 - FLAG - TRAF3IP3 . Twenty - four hours after transfection , cells were infected with or without SeV for twelve hours . Anti - FLAG M2 ( FITC ) was used for immunofluorescence staining of FLAG - TRAF3IP3 . Nuclei were stained with DAPI . Mitochondria was stained with Mitotracker Red ( C ) . Calnexin ( an ER protein ) was stained for ER ( D ) . GM130 ( a Golgi protein ) was stained for Golgi ( E ) . Scale bar represents 5 micrometers . F Quantitative analysis of yellow color as shown in Fig 5C , D and E , indicating TRAF3IP3 localization on various organelles with or without virus infection . The quantification was performed with ImageJ . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * * * P < 0 . 001 . NS indicates no statistically significant difference .

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Figure 5A Subcellular fractionation was performed for HEK293T cells infected with or without SeV to get three fractions, i.e., cytoplasm, membrane and nuclei extracts, which were then subjected to Immunoblotting analysis as indicated.B Membranous fraction obtained in Fig 5A were further separated into mitochondria, Golgi and ER extracts as described in methods, followed by immunoblotting analysis.C-E Immunofluorescent microscopic imaging for TRAF3IP3. HeLa cells were transfected with pcDNA3-FLAG-TRAF3IP3. Twenty-four hours after transfection, cells were infected with or without SeV for twelve hours. Anti-FLAG M2 (FITC) was used for immunofluorescence staining of FLAG-TRAF3IP3. Nuclei were stained with DAPI. Mitochondria was stained with Mitotracker Red (C). Calnexin (an ER protein) was stained for ER (D). GM130 (a Golgi protein) was stained for Golgi (E). Scale bar represents 5 micrometers. F Quantitative analysis of yellow color as shown in Fig 5C, D and E, indicating TRAF3IP3 localization on various organelles with or without virus infection. The quantification was performed with ImageJ. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. ***P<0.001. NS indicates no statistically significant difference.


Figure 6BMDMs from wild type , Traf3ip3 - / - and Mavs - / - mice were treated with or without VSV , SeV , poly ( I : C ) or HSV - 1 respectively as indicated for six hours . Ifnb ( A ) , Ifna4 ( B ) , Il6 induction was measured respectively by qPCR . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . BMDMs from wild type , Traf3ip3 - / - and Mavs - / - mice were treated with or without VSV , SeV , poly ( I : C ) or HSV - 1 respectively as indicated for six hours . Il6 ( C ) , Isg54 ( D ) , and Cxcl10 ( E ) induction was measured respectively by qPCR . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . F BMDMs from wild type , Traf3ip3 - / - and Mavs - / - mice were infected with or without VSV for six hours . Cells were then collected and following subcellular fractionation , S5 fractions were subjected to native PAGE to examine IRF3 dimer , and P5 fractions contained mitochondria were subjected to SDD - AGE to examine MAVS aggregation .

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Figure 6BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Ifnb (A), Ifna4 (B), Il6 induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Il6 (C), Isg54 (D), and Cxcl10 (E) induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.F BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were infected with or without VSV for six hours. Cells were then collected and following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation.