C , D . HUWE1 - knockout 293T ( C ) HeLa ( D ) show increased breaks in absence of exogenous damage treatment . Results from Alkaline Comet are shown . " n " numbers of comet tails analyzed ( pooled from two independent experiments ) , as well as mean , are indicated on graphs . HUWE1 - knockout HeLa did not show increases breakage in Neutral Comet ( Fig EV1B ) , indicating that majority of breaks observed in cycling HUWE1 - knockout are not double strand breaks , but rather single strand breaks other types of lesions .

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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 ) HeLa ( F ) . Cycling were incubated with BrdU subjected to BrdU / PI bi - dimensional flow cytometry . Representative flow cytometry profiles are presented in Fig EV1D - F . Bars represent fold increase in of with S - phase content ( between 2N 4N ) but negative for BrdU . Bars represent average of three independent experiments , with error bars showing SD . p - values are 0 . 0091 ( E ) 0 . 0007 ( F ) .

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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 . fiber shows reduced replication tract in HUWE1 - knockout 293T ( G ) HeLa ( H ) in absence of exogenous damage treatment . " n " numbers of analyzed ( pooled from three independent experiments ) , as well as mean + / - , are indicated on graphs . P - values are 9 . 8x10 - 22 ( G ) 1 . 0x10 - 10 ( H ) .

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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).


A . HUWE1 are efficiently downregulated by two different HUWE1 siRNA oligonucleotides in 8988T . B . of HUWE1 expression following siRNA treatment . Band was quantified using ImageJ software normalized to Tubulin loading control . average of three experiments is shown . Error bars indicate SD .

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A. 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 . cycle analyses by flow cytometry using BrdU / PI double show increased replication arrest in HUWE1 - depleted 8988T in absence of exogenous damage treatment . C . Representative flow cytometry profiles of control HUWE1 - knockdown . R1 - labeled region indicates mid late S - phase ( BrdU - positive , > 2N content ) , while R2 - labeled region indicates S - phase arrested ( BrdU - negative , content between 2N 4N ) .

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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 . cycle analyses by flow cytometry using BrdU / PI double show increased replication arrest in HUWE1 - depleted 8988T in absence of exogenous damage treatment . C . Representative flow cytometry profiles of control HUWE1 - knockdown . R1 - labeled region indicates mid late S - phase ( BrdU - positive , > 2N content ) , while R2 - labeled region indicates S - phase arrested ( BrdU - negative , content between 2N 4N ) . D . of S - phase . of in R1 region is shown . Bars represent average of three independent experiments . Error bars indicate SD . P - value is 0 . 0014 . E . of S - phase arrested . Bars represent fold increase in of in R2 region , normalized to siControl - treated . average of three independent experiments is shown . Error bars indicate SD . P - value is 0 . 0135 .

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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 . fiber experiment showing reduced replication tract in HUWE1 - depleted HeLa in absence of exogenous damage treatment . " n " numbers of analyzed ( pooled from two independent experiments ) , as well as mean , are indicated on graphs .

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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 showing that HUWE1 knockdown in 8988T are sensitive to UV ( G ) , HU ( H ) . As controls , both transfected with non - targeting siRNA ( siControl ) , non - transfected , were used . Note that siHUWE1 # 2 oligonucleotide shows a stronger downregulation of HUWE1 , confers increased sensitivity compared to siHUWE1 # 1 . average of nine experiments is shown . Error bars represent . P - values ( representing statistical difference between samples at highest dose treatment ) are : 4 . 55x10 - 9 ( G ) 1 . 8x10 - 8 ( H ) .

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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 . fiber experiments showing decreased replication tract following UV - B treatment ( 30J / m2 ) in HUWE1 - depleted HeLa . " n " numbers of analyzed ( pooled from three independent experiments ) , as well as mean + / - , are indicated on graphs . P - value is 2 . 11x10 - 20 .

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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.


A . iPOND experiment showing that HUWE1 localizes to replication forks in 293T . Binding to nascent was increased after UV exposure . Because of difficulties in removing crosslinks in high molecular weightproteins , HUWE1 is detected as a high molecular smear . A control experiment using HUWE1 - knockout ( shown in right side panel ) confirmed specificity of HUWE1 signal .

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A. 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 foci in HeLa exposed to replication fork stalling agents HU ( 2mM for 16h ) UV ( 40J / m2 , analyzed 2h later ) .

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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 PCNA in foci . U2OS were analyzed 2h after exposure to UV ( 40J / m2 ) . Quantifications of co - localization , additional are presented in Fig EV2A - D .

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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.


B . Anti - PCNA immunoprecipitation , showing that HUWE1 co - precipitates .

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B. Anti-PCNA immunoprecipitation, showing that HUWE1 co-precipitates.


C . Reciprocal experiment showing that PCNA co - precipitates with HUWE1 in anti - HUWE1 immunoprecipitation .

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C. Reciprocal experiment showing that PCNA co-precipitates with HUWE1 in anti-HUWE1 immunoprecipitation.


D . As control , endogenous HUWE1 was depleted by siRNA treatment , extracts were subjected to anti - HUWE1 immunoprecipitation . HUWE1 blot shows that much less HUWE1 is precipitated by anti - HUWE1 antibodies from HUWE1 - knockdown than control , as expected . PCNA is co - precipitated by anti - HUWE1 antibodies from control , but not HUWE1 - depleted , showing that interaction is specific not due to unspecific binding of PCNA to anti - HUWE1

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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 ) 8988T ( F ) , showing that HUWE1 - PCNA interaction can be detected in different lines .

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. 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 RAD18 - knockout 293T , showing that HUWE1 - PCNA interaction is not affected by loss of PCNA ubiquitination . RAD18 - knockout were obtained by CRISPR / Cas9 technology . RAD18 PCNA blots show loss of RAD18 , of PCNA ubiquitination respectively .

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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 . Anti - PCNA immunoprecipitation showed that C - terminal HUWE1 fragment interacts with PCNA .

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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 of recombinant GST - PCNA expressed purified from .

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I. Coomassie staining of recombinant GST-PCNA expressed and purified from bacteria.


J . GST - pulldowns using GST - PCNA or GST - empty as control , extracts of 293T transfected with Myc - HUWE1C - ter , showing interaction of this fragment with recombinant PCNA .

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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.


A , 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 VFF - indicated residues , critical for PIP - box , were mutated to A ) , were purified from ( see Coomassiestaining ) employed for GST - pulldowns using extracts of 293T . PCNA co - precipitated with wild - type , but not PIP - box mutants . B . Reciprocal GST - pulldown using recombinant GST - PCNA extracts of 293T overexpressing Myc - HUWE1C - ter . Wild - type , but not PIP - box mutant ( VFF ) HUWE1 fragment bound to GST - PCNA .

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A, 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 were transfected with Myc - HUWE1C - ter variants , 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 .

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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 PIP - box in full - HUWE1 also blocks PCNA interaction . Full - Myc - tagged HUWE1 ( wild - type or PIP - mutant ) was transfected in 293T . Following Myc - immunoprecipitation , endogenous PCNA was detected in wild - type , but not PIP - box mutant ( FF ) complexes .

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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.


A . Stable expression of Myc - tagged wild - type , PIP - box mutant , catalytic inactive HUWE1 variants in HUWE1 - knockout 293T . Western blot shows that expression of Myc - tagged variants stably transfected in knockout lines , is similar to endogenous HUWE1 expression .

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A. 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 corrected lines described above . As expected , PCNA co - precipitates with wild - type , but not PIP - box mutant HUWE1 ( FF ) .

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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 , corrected with wild - type or PIP - mutant HUWE1 . PIP - mutant shows reduced crosslinking to nascent , indicated defective recruitment to replication forks .

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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 showing that stable expression of wild - type HUWE1 , but not of PIP box mutant or catalytic mutant , corrects HU sensitivity of HUWE1 - knockout 293T . Shown is average of 3 independent experiments - / + SD . If not indicated otherwise , p - values shown specify statistical significance relative to 293T ( for 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 ) .

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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 . S - phase arrest phenotype is also rescued by wild - type , but not PIP - mutant or catalytic mutant HUWE1 . S - phase arrested were quantified using BrdU / PI bi - dimensional flow cytometry . Shown is average of 3 independent experiments - / + SD . p - values shown specify statistical significance relative to 293T ( 0 . 018 , 0 . 957 , 0 . 015 , 0 . 024 respectively ) . A of same data , showing of arrested in S - phase , is shown in Fig EV3A .

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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 , quantified using fiber , is restored by expressing wild - type , but not PIP - mutant HUWE1 , in HUWE1 - knockout 293T . " n " numbers of analyzed ( pooled from three independent experiments ) , as well as mean + / - , are indicated on graphs . If not indicated otherwise , p - values shown specify 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 ) .

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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 showing that wild - type , but not FF mutant can correct breakage phenotype of HUWE1 - knockout 293T . " n " numbers of comet tails analyzed ( pooled from three independent experiments ) , as well as mean + / - , are indicated on graphs . p - values indicated are : 4 . 18x10 - 43 ( 293T vs HUWE - / - samples ) , 6 . 89x10 - 41 ( HUWE1 - / - + WT vs FF samples ) , 5 . 97x10 - 50 ( HUWE1 - / - + WT vs C4341A samples ) .

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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).


A , B . impact of HUWE1 on H2AX phosphorylation 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 were analyzed .

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A, 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 showing that recombinant HUWE1C - ter can mono - ubiquitinate H2AX .

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C. In vitro ubiquitination assay showing that recombinant HUWE1C-ter can mono-ubiquitinate H2AX.


D . Wild - type , but not PIP - mutant can correct defective H2AX ubiquitination in HUWE1 - knockout 293T .

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D. Wild-type, but not the PIP-mutant can correct the defective H2AX ubiquitination in HUWE1-knockout 293T cells.


. E . immunoprecipitation showing that H2AX binding to common fragile site FRA3B is reduced in HUWE1 - knockout 8988T . were treated with 600nM aphidicolin for 24h . Binding was quantified relative to input material . Shown is average of 4 experiments - / + SD . P - value is 4 . 4x10 - 6 .

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. 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 , BMI1 were knocked - down in HeLa . efficiency of knockdown is shown in Fig EV4 . were treated with 600nM aphidicolin for 24h , 2mM HU for 24h , or analyzed 2h after exposure to 40J / m2 UV .

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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 . immunoprecipitation from HeLa , showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B . were treated with 600nM aphidicolin for 24h . Binding was quantified relative to input material . Shown is average of 3 experiments - / + SD . p - values shown indicate statistical significance relative to siControl ( 0 . 0016 , 0 . 0005 , 0 . 0009 respectively ) .

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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 . fractionation experiments showing that HUWE1 - knockout HeLa , treated with 2mM HU for 24h , fail to efficiently recruit BRCA1 BRCA2 to . In contrast , 53BP1 association is not induced by HU treatment , not affected by HUWE1 knockout , thus can serve as loading control . Shown are blots of whole extract ( WCE ) samples , representing input material , of pellet samples .

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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.


B Recombinant MAVS - Region III - dimer ( lanes 1 - 4 ) , trimer ( lanes 5 - 8 ) or tetramer ( lanes 9 - 12 ) were subjected to IRF3 dimerization respectively in vitro . Increasing doses of recombinant MAVS - Region III were used shown in Fig EV1C . Immunoblotting was performed following Native - PAGE for reaction mixture .

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B 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 - constructs expressing MAVS - Region III - dimer , trimer tetramer were transfected into HEK293T respectively . ISG54 induction were measured thirty - six hours after transfection by quantitative PCR ( qPCR ) .

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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 following an experimental procedure outlined in Fig EV1G . IP products were resolved in SDS - PAGE a specific band pulled - down with MAVS - Region III - tetramer was indicated by an arrow . TRAF3IP3 was identified in specific band by mass spectrometry .

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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 samples as described in ( D ) were subjected to immunoblotting . TRAF3IP3 band was indicated by an arrow asterisk indicated nonspecific

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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 bands


F pcDNA3 - FLAG - TRAF3IP3 ( full - truncated forms ) pcDNA3 - HA - MAVS were transfected into HEK293T , which were harvested thirty - six hours post transfection . Co - IP experiments were performed with anti - HA antibody , followed by immunoblotting .

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F 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 , including THP1 , HeLa , HEK293 , HEK293T as well as Traf3ip3 - / - HEK293T , were lysed subjected to immunoblotting to examine TRAF3IP3 expression .

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G Human cells, including THP1, HeLa, HEK293, HEK293T as well as Traf3ip3-/- HEK293T, were lysed and subjected to immunoblotting to examine TRAF3IP3 expression.


A Three pairs of small interfering against TRAF3IP3 were transfected into HEK293T respectively immunoblotting were then employed to examine knockdown efficiency . A pair of scrambled oligoes was transfected signified as con .

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A 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 . Twenty - four hours after transfection , were infected with ( SeV ) or without ( mock ) Sendai , which were harvested six hours post infection to measure luciferase activity .

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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 , which were then treated with mock , VSV , SeV , poly ( I : C ) or HSV - 1 . After various treatments , IFNB inductions were measured by qPCR .

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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 Traf3ip3 - / - HEK293T .

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D Immunoblotting to detect TRAF3IP3 in wild type and Traf3ip3-/- HEK293T cells.


E Wild type Traf3ip3 - / - HEK293T were treated with or without VSV , SeV or poly ( I : C ) as indicated for twelve hours . IFNB induction was measured by qPCR .

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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 Traf3ip3 - / - HEK293T were infected with GFP - VSV . 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 ( right ) .

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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 Traf3ip3 - / - HEK293T . Thirty - six hours after transfection , IFNB induction was measured by qPCR .

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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 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 , were infected with SeV for twelve hours . IFNB induction was measured by qPCR .

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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.


A experiment was performed as described in Fig 2C . Instead of IFNB , ISG54 IL6 inductions were measured by qPCR .

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A The experiment was performed as described in Fig 2C. Instead of IFNB, ISG54 and IL6 inductions were measured by qPCR.


B HEK293 were transfected with si - TRAF3IP3 oligos then treated with various stimuli . Following subcellular fractionation , S5 fractions were obtained subjected to native PAGE for detecting IRF3 dimer by immunoblotting . whole lysates were also subjected to SDS - PAGE for anti - IRF3 tubulin immunoblotting .

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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 experiment was performed as described in Fig 2E . ISG54 IL6 induction was measured by qPCR .

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C The experiment was performed as described in Fig 2E. ISG54 and IL6 induction was measured by qPCR.


D Wild type Traf3ip3 - / - HEK293T were infected with or without VSV . At indicated time points , were collected analyzed by immunoblotting . Following subcellular fractionation , S5 fractions were subjected to native PAGE to examine IRF3 dimer , P5 fractions contained mitochondria were subjected to SDD - to examine MAVS aggregation . mitochondrial outer Prohibitin was immunoblotted as an internal control .

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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.


A pcDNA - FLAG - TRADD pcDNA - FLAG - TRAF3IP3 were transfected into HEK293T at increasing amount respectively . ISG54 IL6 induction were measured thirty - six hours post - transfection by qPCR . Expression were shown in Fig EV4B . data are presented as mean values on three independent experiments , error bars indicate s . d . P values were determined by unpaired two - Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 NS indicates no statistically significant difference .

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A 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 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 , were collected anti - HA immunoprecipitations were performed . IP products were subjected to immunoblotting .

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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 . Twenty - four hours after transduction , were treated with puromycin ( 2 μg / mL ) for forty - eight hours then transfected with pcDNA3 - FLAG - MAVS - ( Region III only ) . Twenty - four hours after transfection , were infected with or without VSV for twelve hours . were then collected subjected to immunoprecipitation immunoblotting .

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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 Traf3ip3 - / - HEK293T were transfected with pcDNA3 - FLAG - MAVS - ( Region III only ) . Twenty - four hours post - transfection , were infected with or without VSV . Twelve hours post - infection , were collected subjected to immunoprecipitation immunoblotting .

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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.


A Subcellular fractionation was performed for HEK293T infected with or without SeV to get three fractions , i . e . , cytoplasm , nuclei extracts , which were then subjected to Immunoblotting as indicated .

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A 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 ER extracts as described in methods , followed by immunoblotting .

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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 were transfected with pcDNA3 - FLAG - TRAF3IP3 . Twenty - four hours after transfection , were infected with or without SeV for twelve hours . Anti - FLAG M2 ( FITC ) was used for immunofluorescence of FLAG - TRAF3IP3 . Nuclei were stained with DAPI . Mitochondria was stained with Mitotracker Red ( C ) . Calnexin ( an ER ) was stained for ER ( D ) . GM130 ( a Golgi ) was stained for Golgi ( E ) . Scale bar represents 5 micrometers . F Quantitative of yellow color as shown in Fig 5C , D E , indicating TRAF3IP3 localization on various organelles with or without infection . was performed with ImageJ . data are presented as mean values on three independent experiments , error bars indicate s . d . P values were determined by unpaired two - Student ' s t - . * * * P < 0 . 001 . NS indicates no statistically significant difference .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SUBCELLULAR O 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-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-E 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.


BMDMs from wild type , Traf3ip3 - / - 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 . data are presented as mean values on three independent experiments , error bars indicate s . d . P values were determined by unpaired two - Student ' s t - . * P < 0 . 05 * * P < 0 . 01 . NS indicates no statistically significant difference .

B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O B-ORGANISM O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. 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 - / - 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 ) , Cxcl10 ( E ) induction was measured respectively by qPCR . data are presented as mean values on three independent experiments , error bars indicate s . d . P values were determined by unpaired two - Student ' s t - . * P < 0 . 05 * * P < 0 . 01 . NS indicates no statistically significant difference .

B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O B-ORGANISM O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 - / - Mavs - / - mice were infected with or without VSV for six hours . were then collected following subcellular fractionation , S5 fractions were subjected to native PAGE to examine IRF3 dimer , P5 fractions contained mitochondria were subjected to SDD - to examine MAVS aggregation .

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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.


Wild type , Traf3ip3 - / - Mavs - / - mice ( three per group ) were infected intravenously with VSV at a MOI of 2 × 108 . spleens were collected 4 hours or 8 hours after infection Ifna4 ( A ) , Ifnb ( B ) , Isg54 ( C ) Il6 ( D ) inductions were measured respectively by qPCR . data are presented as mean values on three independent experiments , error bars indicate s . d . P values were determined by unpaired two - Student ' s t - . * P < 0 . 05 * * P < 0 . 01 . NS indicates no statistically significant difference .

O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and Ifna4 (A), Ifnb (B), Isg54 (C) and Il6 (D) inductions were 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.


Wild type , Traf3ip3 - / - Mavs - / - mice ( three per group ) were infected intravenously with VSV at a MOI of 2 × 108 . spleens were collected 4 hours or 8 hours after infection VSV were measured by qPCR ( E ) . data are presented as mean values on three independent experiments , error bars indicate s . d . P values were determined by unpaired two - Student ' s t - . * P < 0 . 05 * * P < 0 . 01 . NS indicates no statistically significant difference .

O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and VSV RNA levels were measured by qPCR (E). 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.


Wild type , Traf3ip3 - / - Mavs - / - mice ( three per group ) were infected intravenously with VSV at a MOI of 2 × 108 . sera were collected from treated mice used for measurement of IFNβ production by ELISA ( F ) . data are presented as mean values on three independent experiments , error bars indicate s . d . P values were determined by unpaired two - Student ' s t - . * P < 0 . 05 * * P < 0 . 01 . NS indicates no statistically significant difference .

O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The sera were collected from the treated mice and used for the measurement of IFNβ production by ELISA (F). 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.


G Wild type , Traf3ip3 - / - Mavs - / - mice ( n = 6 each ) were injected with VSV via tail vein injection at 5 × 107 pfu per mouse , survival rates were monitored for 15 days . P values were determined by unpaired two - Student ' s t - . * P < 0 .

O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-ORGANISM O B-TISSUE I-TISSUE O O O O O O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

G Wild type, Traf3ip3-/- and Mavs-/- mice (n=6 each) were injected with VSV via tail vein injection at 5×107 pfu per mouse, and the survival rates were monitored for 15 days. P values were determined by unpaired two-tailed Student's t-test. *P<0.05


| Representative of B220 IgM surface expression in bone marrow ( left ) spleen ( right ) of mice from indicated genotypes .

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| Representative analysis of B220 and IgM surface expression in bone marrow (left) and spleen cells (right) of mice from the indicated genotypes.


Representative flow cytometric of splenocytes from mice of indicated genotypes ( pre - gated on B : B220 + / CD19 + ) for surface expression of IgM 3 - 83 idiotype ( 54 . 1 ) . Shown data are representative 11 - 35 mice per genotype .

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Representative flow cytometric analysis of splenocytes from mice of the indicated genotypes (pre-gated on B cells: B220+/CD19+) for surface expression of IgM and the 3-83 idiotype (54.1). Shown data are representative 11 - 35 individual mice per genotype.


PCR fragments amplified with specific primers for Vκ Jκ5 from genomic of purified splenic B from Ptenf / f x 3 - 83ki Ptenf / f x mb1 - cre x 3 - 83ki mice on respective backgrounds . Genomic tail from a 3 - 83ki mouse from purified splenic B of a control ( ctrl ) mouse were used as controls . PCR for splicing factor Srp20 served as a loading control . Kb Kd indicate respective background of mice , ( H2 - Kb : + Ag ) .

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PCR fragments amplified with specific primers for Vκ and Jκ5 from genomic DNA of purified splenic B cells from Ptenf/f x 3-83ki and Ptenf/f x mb1-cre x 3-83ki mice on the respective backgrounds. Genomic tail DNA from a 3-83ki mouse and DNA from purified splenic B cells of a control (ctrl) mouse were used as controls. PCR for the splicing factor Srp20 served as a loading control. Kb and Kd indicate the respective background of the mice, (H2-Kb: +Ag).


A | Intracellular expression of IgM ( Ig - μHC ic ) in bone marrow ( left ) spleen ( right ) was determined by flow cytometry compared between populations of B ( green , identified by B220 CD19 expression ) non - B ( gray ) . Numbers in histograms indicate percentages of positive populations . Figures are representative of 11 - 35 mice per genotype .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O

A | Intracellular expression of IgM (Ig-μHC ic) in bone marrow (left) and spleen cells (right) was determined by flow cytometry and compared between the populations of B cells (green, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Figures are representative of 11 - 35 individual mice per genotype.


B | Intracellular Ca2 + influx was measured in CD90 . 2 / Thy1 . 2 - splenocytes derived from mice of indicated genotypes following stimulation with 10 μg / ml α - κLC antibody . Figures are representative of at least 3 mice per genotype .

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B | Intracellular Ca2+ influx was measured in CD90.2/Thy1.2- splenocytes derived from mice of the indicated genotypes following stimulation with 10 μg/ml α-κLC antibody. Figures are representative of at least 3 individual mice per genotype.


C | Serum IgM concentrations measured in mice of indicated genotypes . Mean ± SD , symbols represent IgM concentrations from mice ( n ) . Statistical significance was calculated by using Kruskal - Wallis ( see also Table S1 ) , n . s . = not significant ; * * * * p ≤ 0 . 0001 .

O O B-TISSUE B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C | Serum IgM concentrations measured in mice of the indicated genotypes. Mean ±SD, symbols represent IgM concentrations from individual mice (n). Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S1), n. s. = not significant; **** p ≤ 0.0001.


D , E | Absolute numbers of B in bone marrow ( D ) spleens ( E ) from mice of respective genotypes , determined by B220 / CD19 surface expression . Mean ± SD , symbols indicate numbers of B in mice ( n ) . Statistical significance was calculated by using Kruskal - Wallis ( see also Tables S2 - 3 ) , n . s . = not significant ; * p ≤ 0 . 05 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-TISSUE I-TISSUE O O O B-TISSUE O O O O B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E | Absolute numbers of B cells in bone marrow (D) and spleens (E) from mice of the respective genotypes, determined by B220/CD19 surface expression. Mean ±SD, symbols indicate the numbers of B cells in individual mice (n). Statistical significance was calculated by using the Kruskal-Wallis test (see also Tables S2-3), n. s. = not significant; * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001.


Splenocytes from mice of indicated genotypes , ( A ) Ptenf / f , Ptenf / f x mb1 - cre Ptenf / f x mb1 - cre 3 - 83ki mice on respective backgrounds were analyzed by flow cytometry . B were pre - gated by B220 CD19 expression IgM / IgD surface expression ( dot plots , left ) was determined . Histograms ( right ) show a comparison of intracellular IgD expression ( Ig - δHC ic ) between splenic populations of B ( orange , identified by B220 CD19 expression ) non - B ( gray ) . Numbers in histograms indicate percentages of positive populations . Data are representative of 11 - 35 mice per genotype .

B-CELL_TYPE O B-ORGANISM O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O

Splenocytes from mice of the indicated genotypes, (A) Ptenf/f, Ptenf/f x mb1-cre and Ptenf/f x mb1-cre 3-83ki mice on the respective backgrounds were analyzed by flow cytometry. B cells were pre-gated by B220 and CD19 expression and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig-δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11 - 35 individual mice per genotype.


( B ) Ptenf / f x MD4tg Ptenf / f x mb1 - cre x MD4tg mice in presence absence of antigen ( ML5tg ) , were analyzed by flow cytometry . B were pre - gated by B220 CD19 expression IgM / IgD surface expression ( dot plots , left ) was determined . Histograms ( right ) show a comparison of intracellular IgD expression ( Ig - δHC ic ) between splenic populations of B ( orange , identified by B220 CD19 expression ) non - B ( gray ) . Numbers in histograms indicate percentages of positive populations . Data are representative of 11 - 35 mice per genotype .

O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-TISSUE O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O

(B) Ptenf/f x MD4tg and Ptenf/f x mb1-cre x MD4tg mice in presence and absence of antigen (ML5tg), were analyzed by flow cytometry. B cells were pre-gated by B220 and CD19 expression and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig-δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11 - 35 individual mice per genotype.


C | of surface IgD mean ( MFI ) in Ptenf / f x MD4 - trangenic mice in presence ( mb1 - cre + / + ) absence ( mb1 - cre + / ki ) of Pten . Single dots represent average MFI of 4 independent measurements per mouse ( n ) , mean ± SD . Statistical significance was calculated by using an unpaired two - t - .

O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

C | Quantification of surface IgD mean fluorescence intensity (MFI) in Ptenf/f x MD4-trangenic mice in presence (mb1-cre +/+) and absence (mb1-cre +/ki) of Pten. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. Statistical significance was calculated by using an unpaired two-tailed t-test.


D | Serum anti - HEL titers . Sera from MD4tg mice of indicated genotypes were adjusted to an IgM of 500 μg / ml applied in triplicates to HEL - coated plates in dilution steps of 1 : 3 , mean ± . Statistical significance was calculated by using Kruskal - Wallis ( see also Table S4 ) . Statements of significance in figure refer to comparison between Ptenf / f x MD4tg ( filled red squares ) Ptenf / f x MD4tg x MD5tg sera ( filled orange triangles ) , * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 .

O O B-TISSUE O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O B-GENEPROD B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

D | Serum anti-HEL titers. Sera from MD4tg mice of the indicated genotypes were adjusted to an IgM concentration of 500 μg/ml and applied in triplicates to HEL-coated plates in dilution steps of 1 : 3, mean ±SEM. Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S4). Statements of significance in the figure refer to the comparison between Ptenf/f x MD4tg (filled red squares) and Ptenf/f x MD4tg x MD5tg sera (filled orange triangles), ** p ≤ 0.01; *** p ≤ 0.001.


E | Immunohistochemistry of sections from spleens of Ptenf / f , Ptenf / f x mb1 - cre , Ptenf / f x mb1 - cre x 3 - 83ki Ptenf / f x mb1 - cre x MD4tg mice for CD169 ( green ) , CD5 ( red ) IgM ( cyan ) at 10x magnification . Pictures in second row show enlarged areas indicated by squares , respectively . Double - arrows indicate between T ( red ) marginal zone ( green ) . Shown pictures are representative of 2 - 3 mice per

O O B-EXP_ASSAY O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O 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-CELL_TYPE O O O B-TISSUE I-TISSUE O O O O O O O O O O O O B-ORGANISM O

E | Immunohistochemistry of sections from spleens of Ptenf/f, Ptenf/f x mb1-cre, Ptenf/f x mb1-cre x 3-83ki and Ptenf/f x mb1-cre x MD4tg mice for CD169 (green), CD5 (red) and IgM (cyan) at 10x magnification. Pictures in the second row show enlarged areas indicated by the white squares, respectively. Double-arrows indicate the distance between T cell (red) and marginal zone (green). Shown pictures are representative of 2 - 3 mice per genotype


B | 3 days post transduction surface expression of IgM / IgD ( dot plots , top ) , IgD CD23 ( histograms , bottom ) was measured by flow cytometry compared between EV - FoxO1 - A3 - transduced . minus one ( FMO ) stainings , lacking α - IgD or α - CD23 antibodies respectively , served as controls ( ctrl ) . Bar diagrams below flow cytometry data display percentages of IgD + ( n = 12 ; left ) MFI of CD23 ( as relative units , RU ) , normalized to MFI measured in respective EV transduced ; n = 12 ; middle ) . Fcer2 transcript were measured by qRT - PCR ( right , n = 4 ) . Mean ± SD , symbols indicate data from mice . Statistical significance of IgD expression was determined by applying Wilcoxon matched - pairs signed rank for CD23 by applying one sample two - t - for Fcer2 expression .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD 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-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY O

B | 3 days post transduction the surface expression of IgM/IgD (dot plots, top), IgD and CD23 (histograms, bottom) was measured by flow cytometry and compared between EV- and FoxO1-A3-transduced cells. Fluorescence minus one (FMO) stainings, lacking α-IgD or α-CD23 antibodies respectively, served as controls (ctrl). Bar diagrams below flow cytometry data display the percentages of IgD+ cells (n = 12; left) and MFI of CD23 (as relative units, RU), normalized to the MFI measured in the respective EV transduced cells; n = 12; middle). Fcer2 transcript levels were measured by qRT-PCR (right, n = 4). Mean ±SD, symbols indicate data from individual mice. Statistical significance of IgD expression was determined by applying the Wilcoxon matched-pairs signed rank test for CD23 and by applying the one sample two-tailed t-test for Fcer2 expression.


C | Cstf64 ( n = 8 ) , Ell2 ( n = 8 ) Zfp318 ( n = 11 ) expression were measured at 3 days post transduction by qRT - PCR . FoxO1 - A3 - EV - transduced splenic B were FACS - purified according to gating strategy Mean ± SD , symbols indicate expression in mice . Statistical significance was calculated by using one - sample two - t - .

O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-TISSUE B-CELL_TYPE O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O

C | Cstf64 (n = 8), Ell2 (n = 8) and Zfp318 (n = 11) expression levels were measured at 3 days post transduction by qRT-PCR. FoxO1-A3- and EV-transduced splenic B cells were FACS-purified according to the gating strategy Mean ±SD, symbols indicate the expression in individual mice. Statistical significance was calculated by using the one-sample two-tailed t-test.


O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O

E: IgD (n = 6), CD23 (n = 4) and Fcer2 (n = 4).


F: Cstf64 (n = 4), Ell2 (n = 4) and Zfp318 (n = 4).


B | Representative flow cytometric of Ramos reconstituted with HH10 - mu IgM , HH10 - mu IgD . were stained with α - murine μHC - , δHC - κLC - antibodies , respectively . EV - transduced HL - KO , expressing GFP only , were used as negative control ( gray ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-ORGANISM B-GENEPROD O O O B-ORGANISM B-GENEPROD O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

B | Representative flow cytometric analysis of Ramos cells reconstituted with HH10-mu IgM, HH10-mu IgD. Cells were stained with α-murine μHC-, δHC- and κLC-antibodies, respectively. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray).


C | Representative flow cytometric of HEL - binding in reconstituted Ramos after with fluorescently - labeled HEL . EV - transduced HL - KO , expressing GFP only , were used as negative control ( gray ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O

C | Representative flow cytometric analysis of HEL-binding in reconstituted Ramos cells after staining with fluorescently-labeled HEL. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray).


D | Representative intracellular Ca2 + influx in HH10 - mu IgM - HH10 - mu IgD - expressing upon stimulation with multivalent HEL ( cHEL ) , monovalent ( soluble sHEL ) ( both at a of 1 μg / ml ) , or 10 μg / ml α - mouse κLC antibody , respectively .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

D | Representative intracellular Ca2+ influx in HH10-mu IgM- and HH10-mu IgD-expressing cells upon stimulation with multivalent HEL (complex cHEL), monovalent (soluble sHEL) (both at a concentration of 1 μg/ml), or 10 μg/ml α-mouse κLC antibody, respectively.


E | Representative intracellular Ca2 + influx of HH10 - mu IgM - ( top ) HH10 - mu IgD - ( bottom ) expressing upon stimulation with indicated ratios of 1 μg / ml cHEL sHEL , a 1 : 25 mixture of cHEL with bovine serum albumin ( BSA ) , where 1 = 1 μg / ml .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM B-GENEPROD O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-ORGANISM B-TISSUE B-GENEPROD O B-GENEPROD O O O O O O O O O O

E | Representative intracellular Ca2+ influx of HH10-mu IgM- (top) and HH10-mu IgD- (bottom) expressing cells upon stimulation with indicated ratios of 1 μg/ml cHEL and sHEL, and a 1 : 25 mixture of cHEL with bovine serum albumin (BSA), where 1 = 1 μg/ml.


F | Schematic overview of reconstitution of BCR - deficient Ramos ( HL - KO ) with HH10 - specific human IgM ( HH10 - hu IgM ) IgD ( HH10 - hu IgD ) BCR isotype . G - J | using HH10 - hu IgM - HH10 - hu IgD - expressing instead of HH10 - mu IgM - HH10 - mu IgD - expressing , respectively . Data shown in G to J are representative of minimum three independent experiments .

O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-ORGANISM B-GENEPROD O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O B-ORGANISM B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O B-ORGANISM B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O

F | Schematic overview of reconstitution of BCR-deficient Ramos (HL-KO) cells with HH10-specific human IgM (HH10-hu IgM) and IgD (HH10-hu IgD) BCR isotype. G - J | using HH10-hu IgM- and HH10-hu IgD-expressing cells instead of HH10-mu IgM- and HH10-mu IgD-expressing cells, respectively. Data shown in G to J are representative of minimum three independent experiments.


A | of surface IgM ( top ) IgD ( bottom ) MFI in B from mice of indicated genotypes . Single dots represent average MFI of 4 independent measurements per mouse ( n ) , mean ± SD . Statistical significance was calculated by using Kruskal - Wallis n . s . = not significant ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A | Quantification of surface IgM (top) and IgD (bottom) MFI in B cells from mice of the indicated genotypes. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. Statistical significance was calculated by using the Kruskal-Wallis test n. s. = not significant; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


| Representative intracellular Ca2 + influx measured in Thy1 . 2 - splenocytes , derived from mice of indicated genotypes , upon stimulation with cHEL , monovalent sHEL ( both at a of 1 μg / ml ) or 10 μg / ml α - mouse κLC antibody , respectively . diagrams below dot plots show overlayed MFI of Ca2 + influx kinetics displayed in plots above .

O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O B-ORGANISM O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

| Representative intracellular Ca2+ influx measured in Thy1.2- splenocytes, derived from mice of the indicated genotypes, upon stimulation with cHEL, monovalent sHEL (both at a concentration of 1 μg/ml) or 10 μg/ml α-mouse κLC antibody, respectively. Line diagrams below the dot plots show the overlayed MFI of the Ca2+ influx kinetics displayed in the plots above.


C | of intracellular Ca2 + influx median under curve of Ca2 + influx kinetics was calculated , single dots represent data from mice , mean ± SD . Statistical significance was calculated by applying Mann - Whitney - U n . s . = not significant , * * p ≤ 0 . 01 .

O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C | Quantification of intracellular Ca2+ influx The median area under the curve of the Ca2+ influx kinetics was calculated, single dots represent data from individual mice, mean ±SD. Statistical significance was calculated by applying the Mann-Whitney-U test n. s. = not significant, ** p ≤ 0.01.


B | of GC B in splenic B220 + B from wild type ( WT , top ) IgD - / - ( bottom ) after 4 , 7 10 days ( left to right ) post immunization . Percentages of follicular B ( Fo . B ; CD38 + / CD95 - ) GC B ( CD38 - / CD95 + ) are depicted in plots . GC B ( CD38 - / CD95 + ) were further analyzed for GL - 7 expression , compared to Fo . B quantified .

O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O

B | Analysis of GC B cells in splenic B220+ B cells from wild type (WT, top) and IgD-/- (bottom) animals after 4, 7 and 10 days (left to right) post immunization. Percentages of follicular B cells (Fo.B; CD38+/CD95-) and GC B cells (CD38-/CD95+) are depicted in the plots. GC B cells (CD38-/CD95+) were further analyzed for GL-7 expression, compared to Fo.B cells and quantified.


C | of Fo . B ( top , percentages ) CD38 - / CD95 + / GL - 7 + GC ( percentages absolute numbers , bottom panels ) in WT ( black ) IgD - / - ( green ) after 4 , 7 , 10 14 days post immunization , mean ± SD ( numbers of indicated in top plot ) . Statistical significance was analyzed by a two - unpaired t - .

O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

C | Quantification of Fo.B cells (top, percentages) and CD38-/CD95+/GL-7+ GC cells (percentages and absolute numbers, bottom panels) in WT (black) and IgD-/- (green) animals after 4, 7, 10 and 14 days post immunization, mean ±SD (numbers of animals indicated in the top plot). Statistical significance was analyzed by a two-tailed unpaired t-test.


D | of GC in spleen sections from WT IgD - / - after 4 , 7 10 days post immunization . Representative of 2 x 2 mm sections stained with DAPI ( nuclei ) peanut agglutinin ( PNA ) are shown for indicated time points . Scale bar represents 500 μm . Dashed or yellow lines mark distinct GC foci in merged . E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within of D are shown as enlarged single GCs . As no distinct GC foci were observed in IgD - / - samples from days 4 7 , an identical 800 x 800 μm region is shown in each case . F | of number of GCs per mm2 of spleen sections from WT ( black ) IgD - / - ( green ) after 4 , 7 10 days post immunization . Data represent mean ± SD of two sections ( from 3 per group time point ) were analyzed by two - unpaired t - .

O O O B-CELL_TYPE O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O 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-CELL_TYPE O O O O B-CELL_TYPE O O O O B-GENEPROD O 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-EXP_ASSAY O B-CELL_TYPE O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D | Analysis of GC in spleen sections from WT and IgD-/- animals after 4, 7 and 10 days post immunization. Representative images of 2 x 2 mm sections stained with DAPI (nuclei) and peanut agglutinin (PNA) are shown for the indicated time points. Scale bar represents 500 μm. Dashed white or yellow lines mark distinct GC foci in the merged images. E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within the images of D are shown as enlarged single GCs. As no distinct GC foci were observed in IgD-/- samples from days 4 and 7, an identical 800 x 800 μm region is shown in each case. F | Quantification of number of GCs per mm2 area of spleen sections from WT (black) and IgD-/- (green) animals after 4, 7 and 10 days post immunization. Data represent mean ±SD of two sections (from 3 animals per group and time point) and were analyzed by two-tailed unpaired t-test.


G | Total serum α - TNP IgG . Sera from PBS - or TNP - Ova - immunized WT ( left ) IgD - / - ( middle ) mice , collected at day 7 following immunization , were adjusted to an IgG of 1 μg / ml applied in duplicates dilution steps of 1 : 3 to TNP - coated plates , respectively , mean ± . Right : Overlay of α - TNP IgG titers from TNP - Ova - immunized WT IgD - / - mice . Statistical significance was calculated by using Mann - Whitney - U ( see also Table S7 ) , n . s . = not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

O O O B-TISSUE O O B-SMALL_MOLECULE B-GENEPROD O B-TISSUE O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G | Total serum α-TNP IgG. Sera from PBS- or TNP-Ova-immunized WT (left) and IgD-/- (middle) mice, collected at day 7 following immunization, were adjusted to an IgG concentration of 1 μg/ml and applied in duplicates and dilution steps of 1 : 3 to TNP-coated plates, respectively, mean ±SEM. Right: Overlay of α-TNP IgG titers from TNP-Ova-immunized WT and IgD-/- mice. Statistical significance was calculated by using the Mann-Whitney-U test (see also Table S7), n. s. = not significant, * p ≤ 0.05, ** p ≤ 0.01.


( a ) TbMCU - GFP co - localization with MitoTracker ( MT ) in mitochondria of PCF trypanosomes as detected by immunofluorescence analyses with antibodies against GFP ( Pearson ' s correlation coefficient ( PCC ) : 0 . 912 ) . ( b , c ) TbMCU co - localization with MitoTracker in mitochondria of PCF ( b ) or BSF ( c ) trypanosomes , as detected by immunofluorescence analyses with antibodies against TbMCU . PCC : 0 . 930 0 . 920 , respectively . Merge show co - localization in yellow . DIC , differential interference contrast microscopy . Scale bar , 10 μm ( a - c ) .

O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-ORGANISM I-ORGANISM O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-ORGANISM O O O O B-ORGANISM O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

(a) TbMCU-GFP co-localization with MitoTracker (MT) in mitochondria of PCF trypanosomes as detected by immunofluorescence analyses with antibodies against GFP (Pearson's correlation coefficient (PCC): 0.912). (b,c) TbMCU co-localization with MitoTracker in the mitochondria of PCF (b) or BSF (c) trypanosomes, as detected by immunofluorescence analyses with antibodies against TbMCU. PCC: 0.930 and 0.920, respectively. Merge images show the co-localization in yellow. DIC, differential interference contrast microscopy. Scale bar, 10 μm (a-c).


( d , e ) Growth of BSF ( d ) PCF ( e ) trypanosomes in absence ( black lines , − Tet ) or presence ( red lines , + Tet ) of 1 μg ml − 1 tetracycline for indicated number of days . Values are mean ± s . d . ( n = 3 ) .

O O O O O B-EXP_ASSAY O B-ORGANISM O O O B-ORGANISM O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(d,e) Growth of BSF (d) and PCF (e) trypanosomes in the absence (black lines, −Tet) or presence (red lines, +Tet) of 1 μg ml−1 tetracycline for the indicated number of days. Values are mean±s.d. (n=3).


( f , g ) Northern blot analyses of TbMCU RNAi of BSF ( f ) PCF ( g ) trypanosomes grown in absence ( 0 ) presence ( 2 - 8 ; 2 - 10 ) of tetracycline ( top ) . Tubulin is shown as a loading control ( bottom ) . Markers are shown on right . transcript of TbMCU , including 5 ′ - 3 ′ - UTR was ~ 1 . 5 kb in .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-ORGANISM O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(f,g) Northern blot analyses of TbMCU RNAi of BSF (f) and PCF (g) trypanosomes grown in the absence (0) and presence (2-8; 2-10) of tetracycline (top). Tubulin is shown as a loading control (bottom). Markers are shown on the right. The transcript of TbMCU, including the 5′- and 3′-UTR was ~1.5 kb in length.


( h , i ) Western blot analyses of TbMCU RNAi of BSF ( h ) PCF ( i ) trypanosomes grown in absence ( 0 ) or presence ( 2 - 8 ; 2 - 10 ) of tetracycline . Total lysates ( 30 μg ) were subjected to 10 % SDS - polyacrylamide gel electrophoresis before transfer to a nitrocellulose then stained with antibodies against TbMCU ( top ) . One band of ~ 30 kDa was detected in T . brucei homogenates . Membranes were stripped re - incubated with antibody against voltage - dependent anion channel ( TbVDAC ) as a loading control ( bottom ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-ORGANISM O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

(h,i) Western blot analyses of TbMCU RNAi of BSF (h) and PCF (i) trypanosomes grown in the absence (0) or presence (2-8; 2-10) of tetracycline. Total lysates (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against TbMCU (top). One band of ~30 kDa was detected in T. brucei homogenates. Membranes were stripped and re-incubated with antibody against the voltage-dependent anion channel (TbVDAC) as a loading control (bottom).


( a ) reaction buffer contained 2 mM succinate 1 μM Calcium Green 5 - N . After several pulses of Ca2 + ( 8 μM final ) ( 5 × 107 trypanosomes grown in absence ( black tracing ) or presence ( green tracing ) of tetracycline ) were added to reaction medium ( 2 . 45 ml ) reaction was started adding 50 μM digitonin . Ruthenium red ( RR , 40 μM , yellow red tracing ) , FCCP ( 5 μM ) were added where indicated . A decrease in indicates decreasing medium Ca2 + or increasing vesicular Ca2 + . ( b ) Relative Ca2 + uptake at 700 s as compared with that of control trypanosomes grown in absence of tetracycline considered as 1 . 0 ( − Tet ) ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - ) .

O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) The reaction buffer contained 2 mM succinate and 1 μM Calcium Green 5-N. After several pulses of Ca2+ (8 μM final concentration) cells (5 × 107 trypanosomes grown in the absence (black tracing) or presence (green tracing) of tetracycline) were added to the reaction medium (2.45 ml) and the reaction was started adding 50 μM digitonin. Ruthenium red (RR, 40 μM, yellow and red tracing), and FCCP (5 μM) were added where indicated. A decrease in fluorescence indicates decreasing medium Ca2+ or increasing vesicular Ca2+. (b) Relative Ca2+ uptake at 700 s as compared with that of control trypanosomes grown in the absence of tetracycline considered as 1.0 (−Tet) (means±s.d., n=3, **P0.001, Student's t-test).


( c ) Similar conditions as in a except that further CaCl2 additions ( 8 μM each time ) were added to − Tet trypanosomes to show high mitochondrial capacity to take up Ca2 + . EGTA ( 8 μM ) CaCl2 ( Ca2 + ) were added where indicated to show presence of medium Ca2 + . ( d ) Relative initial rate of Ca2 + uptake by + Tet as compared with − Tet trypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - ) .

O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE B-ORGANISM O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Similar conditions as in a except that further CaCl2 additions (8 μM each time) were added to −Tet trypanosomes to show the high mitochondrial capacity to take up Ca2+. EGTA (8 μM) and CaCl2 (Ca2+) were added where indicated to show the presence of medium Ca2+. (d) Relative initial rate of Ca2+ uptake by +Tet as compared with −Tet trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).


( e ) Representative traces of Ca2 + uptake kinetics in digitonin - permeabilized TbMCU BSF trypanosomes cultured in absence ( − Tet ) or presence ( + Tet ) of tetracycline . reactions were started adding 40 μM digitonin in presence of 1 mM ATP 500 μM sodium orthovanadate . Ca2 + uptake was monitored over time using 1 μM Calcium Green - 5N . Ruthenium red ( RR , 40 μM , yellow red tracings ) , oligomycin ( Oligo , 2 . 5 μg ml − 1 ) , EGTA ( 0 . 4 μM ) CaCl2 ( pulses of 0 . 4 μM ) were added where indicated . ( f ) Relative Ca2 + uptake at 200 s as compared with that of control BSF trypanosomes grown in absence of tetracycline considered as 1 ( − Tet ) ( means ± s . d . , n = 3 , * ( + Tet ± RR ) , * * ( − Tet ± RR ) , * * * ( − Tet / + Tet ) , P0 . 001 , Student ' s t - ) .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD B-ORGANISM I-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(e) Representative traces of Ca2+ uptake kinetics in digitonin-permeabilized TbMCU BSF trypanosomes cultured in the absence (−Tet) or presence (+Tet) of tetracycline. The reactions were started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate. Ca2+ uptake was monitored over time using 1 μM Calcium Green-5N. Ruthenium red (RR, 40 μM, yellow and red tracings), oligomycin (Oligo, 2.5 μg ml−1), EGTA (0.4 μM) and CaCl2 (pulses of 0.4 μM) were added where indicated. (f) Relative Ca2+ uptake at 200 s as compared with that of control BSF trypanosomes grown in the absence of tetracycline considered as 1 (−Tet) (means±s.d., n=3, *(+Tet±RR), **(−Tet±RR), ***(−Tet/+Tet) , P0.001, Student's t-test).