Figure 1A . Immunoblot analysis of nuclear and cytoplasmic extracts prepared from parasites synchronized at ring ( R ) , trophozoite ( T ) and schizont ( S ) stages with anti - histone H3 C - terminus antibodies identifies three bands : full - length histone H3 , an intermediate form ( H3 - int ) and a fully processed form ( H3p ) . Anti - histone H4 and anti - PfAldolase antibodies served as loading controls for the nuclear and cytoplasmic fraction , respectively . B . Immunoblot assays of mononucleosomes with anti - histone H3 C - terminus or anti - histone H4 antibodies . The right panel shows a mononoucleosome preparation separated on a denaturing polyacrylamide gel and stained with Coomassie brilliant blue ( C . B . ) . C . Immunoblot analysis of a schizont - stage histone preparation with antibodies against the indicated post - translational modification ( PTM ) in the N - terminal tail of histone H3 ( upper panel ) .

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Figure 1A. Immunoblot analysis of nuclear and cytoplasmic extracts prepared from parasites synchronized at ring (R), trophozoite (T) and schizont (S) stages with anti-histone H3 C-terminus antibodies identifies three bands: full-length histone H3, an intermediate form (H3-int) and a fully processed form (H3p). Anti-histone H4 and anti-PfAldolase antibodies served as loading controls for the nuclear and cytoplasmic fraction, respectively.B. Immunoblot assays of mononucleosomes with anti-histone H3 C-terminus or anti-histone H4 antibodies. The right panel shows a mononoucleosome preparation separated on a denaturing polyacrylamide gel and stained with Coomassie brilliant blue (C.B.).C. Immunoblot analysis of a schizont-stage histone preparation with antibodies against the indicated post-translational modification (PTM) in the N-terminal tail of histone H3 (upper panel).


Figure 2B . GST - PfH3 or GST - PfH2A were incubated with increasing amounts of nuclear extracts , the reactions resolved using denaturing gel electrophoresis and analyzed by immunoblotting with anti - GST antibodies . C . GST - PfH3 was incubated with 25 μg of nuclear extracts and the indicated protease inhibitor . The resulting products were analyzed by denaturing gel electrophoresis and immunoblotting with anti - GST antibodies . ( - ) indicates a reaction without nuclear extract and protease inhibitor .

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Figure 2B. GST-PfH3 or GST-PfH2A were incubated with increasing amounts of nuclear extracts, the reactions resolved using denaturing gel electrophoresis and analyzed by immunoblotting with anti-GST antibodies.C. GST-PfH3 was incubated with 25 μg of nuclear extracts and the indicated protease inhibitor. The resulting products were analyzed by denaturing gel electrophoresis and immunoblotting with anti-GST antibodies. (-) indicates a reaction without nuclear extract and protease inhibitor.


Figure 3A . Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p - HA in ring ( R ) , trophozoite ( T ) and schizont ( S ) stages of P . falciparum asexual growth . PfH3p - HA was detected using anti - HA antibodies ( green ) and endogenous histone H3 with anti - histone H3 N - terminal antibodies ( red ) . DAPI ( blue ) was used to stain the nucleus . Scale bar = 5 μm . B . Nuclei isolated from wild - type ( WT ) or PfH3p - HA - expressing ( WT + PfH3p - HA ) schizont stage parasites were treated with 4 U / ml of micrococcal nuclease ( MNase ) for the indicated amounts of time , the DNA purified and migrated on a 2 % agarose gel , and stained with ethidium bromide . Mononucleosomes purified after 10 minutes of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant blue ( C . B . ) or visualized by immunoblotting with anti - HA ( α - HA ) or anti - C - terminal histone H3 ( α - H3c ) antibodies . C . Co - immunoprecipitation ( IP ) experiments of purified mononucleosomes obtained from wild - type ( WT ) or transfected ( WT + PfH3p - HA ) schizont stage parasites were performed with either anti - HA antibodies or mouse IgG . Immunoprecipitated products ( right panel ) were analyzed by immunoblotting using anti - HA or anti - histone H4 antibodies .

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Figure 3A. Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p-HA in ring (R), trophozoite (T) and schizont (S) stages of P. falciparum asexual growth. PfH3p-HA was detected using anti-HA antibodies (green) and endogenous histone H3 with anti-histone H3 N-terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm.B. Nuclei isolated from wild-type (WT) or PfH3p-HA-expressing (WT + PfH3p-HA) schizont stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 minutes of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant blue (C.B.) or visualized by immunoblotting with anti-HA (α-HA) or anti-C-terminal histone H3 (α-H3c) antibodies.C. Co-immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild-type (WT) or transfected (WT + PfH3p-HA) schizont stage parasites were performed with either anti-HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti-HA or anti-histone H4 antibodies.


Figure 4A . Genome - wide distribution of ectopically expressed PfH3p - HA in P . falciparum schizont stages is represented as fold - enrichment of PfH3p - HA ChIP - seq signal over Input ( PfH3p - HA / Input in blue ; y - axis scale 1 - 10 ) or over PfH3n ( PfH3p - HA / Input in teal ; y - axis scale 1 - 10 ) calculated using MACS2 . The coverage of PfH3p - HA ( red ; y - axis scale 0 - 50 ) , Input ( black ; y - axis scale 0 - 30 ) and PfH3n ( green ; y - axis scale 0 - 30 ) is also shown and represents average reads per million over 1000 nt bins of the genome . The x - axis represents chromosome size in Mb . Major MACS2 - derived peaks identified in all replicates are indicated using an orange arrowhead . B . Principal Component Analysis of the different ChIP - seq replicates ( bigwig files derived from deduplicated bam files ) was performed using the plotPCA function of deepTools on a multibigwig summary file , over 150 nt bins . The Eigen values of the top two principal components PC1 and PC2 are shown and meaningful clustering of replicates highlighted . Rep = Biological Replicate . C . Summary of the PfH3p - enriched peaks - and corresponding genes - identified in the PfH3p - HA versus Input or PfH3p - HA versus PfH3n comparisons using MACS2 peak - calling analysis . The overlap between the two comparisons is indicated using a Venn diagram . FE = Fold Enrichment ; q - value represents the Benjamini and Hochberg false discovery rate . D . The genomic context of the six peaks identified in all three PfH3p - HA ChIP - seq replicates is shown . The fold enrichment of PfH3p - HA ChIP - seq signal over Input ( PfH3p - HA / Input ; blue ) or PfH3n ( PfH3p - HA / PfH3n ; teal ) is shown , as are the coverage plots of PfH3p - HA ( red ) , PfH3n ( green ) and Input ( black ) , which are represented as average reads per million over 1000 nt bins of the genome . DNA replication genes : Replication Protein A 1 ( RPA1 ) , Proliferating Cell Nuclear Antigen 1 ( PCNA1 ) , Single Stranded DNA binding protein ( SSB ) , Topoisomerase I ( TOPO - I ) , DNA polymerase alpha subunit ( DNA pol a ) , and Topoisomerase II ( TOPO - II ) are highlighted and their directionality indicated . Note that the data in part D correspond to Biological Replicate 1 .

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Figure 4A. Genome-wide distribution of ectopically expressed PfH3p-HA in P. falciparum schizont stages is represented as fold-enrichment of PfH3p-HA ChIP-seq signal over Input (PfH3p-HA / Input in blue; y-axis scale 1-10) or over PfH3n (PfH3p-HA / Input in teal; y-axis scale 1-10) calculated using MACS2. The coverage of PfH3p-HA (red; y-axis scale 0-50), Input (black; y-axis scale 0-30) and PfH3n (green; y-axis scale 0-30) is also shown and represents average reads per million over 1000 nt bins of the genome. The x-axis represents chromosome size in Mb. Major MACS2-derived peaks identified in all replicates are indicated using an orange arrowhead. B. Principal Component Analysis of the different ChIP-seq replicates (bigwig files derived from deduplicated bam files) was performed using the plotPCA function of deepTools on a multibigwig summary file, over 150 nt bins. The Eigen values of the top two principal components PC1 and PC2 are shown and meaningful clustering of replicates highlighted. Rep = Biological Replicate. C. Summary of the PfH3p-enriched peaks - and corresponding genes - identified in the PfH3p-HA versus Input or PfH3p-HA versus PfH3n comparisons using MACS2 peak-calling analysis. The overlap between the two comparisons is indicated using a Venn diagram. FE = Fold Enrichment; q-value represents the Benjamini and Hochberg false discovery rate. D. The genomic context of the six peaks identified in all three PfH3p-HA ChIP-seq replicates is shown. The fold enrichment of PfH3p-HA ChIP-seq signal over Input (PfH3p-HA / Input; blue) or PfH3n (PfH3p-HA / PfH3n; teal) is shown, as are the coverage plots of PfH3p-HA (red), PfH3n (green) and Input (black), which are represented as average reads per million over 1000 nt bins of the genome. DNA replication genes: Replication Protein A 1 (RPA1), Proliferating Cell Nuclear Antigen 1 (PCNA1), Single Stranded DNA binding protein (SSB), Topoisomerase I (TOPO-I), DNA polymerase alpha subunit (DNA pol a), and Topoisomerase II (TOPO-II) are highlighted and their directionality indicated. Note that the data in part D correspond to Biological Replicate 1.


Figure 1 ( C ) Co - immunoprecipitation between VP30 and the indicated PPxPxY containing host factors . Empty expression plasmid ( Vector ) or FLAG - tagged host protein expression plasmids were co - transfected with an HA - VP30 expression plasmid in HEK293T cells . An anti - FLAG immunoprecipitation ( IP : Anti - FLAG ) was performed . Western blots of IP and whole cell lysates ( WCL ) are shown . Anti - β - tubulin was used as a loading control for the WCLs . ( D ) Cells were transfected with empty vector or HA - VP30 expression plasmid and IPs were performed with anti - HA antibody ( IP : Anti - HA ) . Western blotting with anti - HA tag or antibodies to the indicated host proteins was performed on IP and on whole cell lysates ( WCL ) .

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Figure 1(C) Co-immunoprecipitation between VP30 and the indicated PPxPxY containing host factors. Empty expression plasmid (Vector) or FLAG-tagged host protein expression plasmids were co-transfected with an HA-VP30 expression plasmid in HEK293T cells. An anti-FLAG immunoprecipitation (IP: Anti-FLAG) was performed. Western blots of IP and whole cell lysates (WCL) are shown. Anti-β-tubulin was used as a loading control for the WCLs.(D) Cells were transfected with empty vector or HA-VP30 expression plasmid and IPs were performed with anti-HA antibody (IP: Anti-HA). Western blotting with anti-HA tag or antibodies to the indicated host proteins was performed on IP and on whole cell lysates (WCL).


Figure 2 ( B ) Co - immunoprecipitation between HA - VP30 and either GFP or GFP - fused to peptides derived from the host proteins was performed . Western blots of the co - immunoprecipitation and whole - cell lysate ( WCL ) are shown . ( C ) Equilibrium dissociation curves of FITC - RBBP6 from eVP30130 - 272 in the presence of increasing concentrations ( 0 . 13 - 500 μM ) of the PPxPxY containing peptides . Fluorescence polarization was determined with constant concentrations of FITC - RBBP6 and eVP30130 - 272 , at 0 . 50 μM and 3 . 8 μM respectively . Experiments were performed in two independent replicates . Error bars represent standard deviation .

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Figure 2(B) Co-immunoprecipitation between HA-VP30 and either GFP or GFP-fused to peptides derived from the host proteins was performed. Western blots of the co-immunoprecipitation and whole-cell lysate (WCL) are shown.(C) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 in the presence of increasing concentrations (0.13-500 μM) of the PPxPxY containing peptides. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272, at 0.50 μM and 3.8 μM respectively. Experiments were performed in two independent replicates. Error bars represent standard deviation.


Figure 3 ( A ) HDX kinetics data for VP30 alone and for the indicated host peptides incubated with VP30 are shown . VP30 peptide amino acid residue positions and sequences are indicated at the top of each curve . The error bars indicate mean ± S . D . ( N = 3 ) .

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Figure 3(A) HDX kinetics data for VP30 alone and for the indicated host peptides incubated with VP30 are shown. VP30 peptide amino acid residue positions and sequences are indicated at the top of each curve. The error bars indicate mean ± S.D. (N=3).


Figure 4 ( A ) Co - immunoprecipitation experiment demonstrating that NP interferes with VP30 - PPxPxY binding . VP30 was co - expressed with the indicated HA - tagged PPxPxY proteins and either empty vector ( - ) or increasing amounts of FLAG - tagged NP expression plasmid . IPs were performed with anti - HA antibodies followed by Western blots of IPs or WCL by using anti - HA , anti - FLAG and anti - VP30 antibodies . ( C ) Co - immunoprecipitations between VP30 and the indicated host proteins upon titration of RBBP6 . VP30 was co - expressed with FLAG - tagged PPxPxY containing host proteins and either a 5 - fold or 10 - fold excess of RBBP6 plasmid . IP was performed using anti - FLAG beads followed by Western blotting using the indicated antibodies .

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Figure 4(A) Co-immunoprecipitation experiment demonstrating that NP interferes with VP30-PPxPxY binding. VP30 was co-expressed with the indicated HA-tagged PPxPxY proteins and either empty vector (-) or increasing amounts of FLAG-tagged NP expression plasmid. IPs were performed with anti-HA antibodies followed by Western blots of IPs or WCL by using anti-HA, anti-FLAG and anti-VP30 antibodies.(C) Co-immunoprecipitations between VP30 and the indicated host proteins upon titration of RBBP6. VP30 was co-expressed with FLAG-tagged PPxPxY containing host proteins and either a 5-fold or 10-fold excess of RBBP6 plasmid. IP was performed using anti-FLAG beads followed by Western blotting using the indicated antibodies.


Figure 5 ( B ) Equilibrium dissociation curves of FITC - RBBP6 from eVP30130 - 272 as it is outcompeted by increasing concentrations ( 0 . 13 - 500 μM ) of RBBP6 alanine mutants . Fluorescence polarization was determined with constant concentrations of FITC - RBBP6 and eVP30130 - 272 at 0 . 50 µM and 3 . 8 µM respectively . Experiments were performed in two independent replicates . Error bars represent the standard deviation . ( D ) Minigenome ( MG ) activity upon titration of plasmids encoding GFP fused to wildtype or single point mutant - RBBP6 peptides . GFP alone or GFP - RBBP6 peptide plasmids were transfected at 12 . 5 and 25 ng amounts along with the plasmids of the MG system . The data represent the mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . The experiment was reproduced in two additional , independent experiments ( see Appendix Figure S2 ) . Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons test . * * * * p < 0 . 00005 ; * * * p < 0 .

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Figure 5(B) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 as it is outcompeted by increasing concentrations (0.13-500 μM) of RBBP6 alanine mutants. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272 at 0.50 µM and 3.8 µM respectively. Experiments were performed in two independent replicates. Error bars represent the standard deviation. (D) Minigenome (MG) activity upon titration of plasmids encoding GFP fused to wildtype or single point mutant-RBBP6 peptides. GFP alone or GFP-RBBP6 peptide plasmids were transfected at 12.5 and 25 ng amounts along with the plasmids of the MG system. The data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments (see Appendix Figure S2). Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005


Figure 6 ( C ) MG activity upon knockdown of host genes . HEK293T cells were transfected with scrambled siRNA or siRNA targeting RBBP6 , hnRNP L , hnRNPUL1 or PEG10 . Twenty - four hours post - transfection , cells were transfected with MG assay plasmids including VP30 at 12 . 5 and 25ng doses . Data information : , the data represent the mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . The experiment was reproduced in two additional , independent experiments Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons test . * * * * p < 0 . 00005 ; * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

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Figure 6(C) MG activity upon knockdown of host genes. HEK293T cells were transfected with scrambled siRNA or siRNA targeting RBBP6, hnRNP L, hnRNPUL1 or PEG10. Twenty-four hours post-transfection, cells were transfected with MG assay plasmids including VP30 at 12.5 and 25ng doses. Data information: , the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05.


Figure 7 ( A ) HeLa cells were transfected with scrambled siRNA ( SCR si ) or siRNAs targeting hnRNP L , hnRNPUL1 or PEG10 , 48 h post - transfection , cells were challenged with EBOV - EGFP at an MOI of 0 . 5 . 24 h post - infection , cells were fixed , stained for nuclei with Hoechst dye and imaged . Scale bar is 200 µm . The graph ( right ) depicts relative number of infected cells as determined by calculating the ratio of number of infected cells to nuclei and shown relative to SCR siRNA . The data shows a representative result of two independent experiments with the mean ± S . D . calculated from three independently treated wells . Statistical significance was calculated relative to the values obtained in SCR siRNA - treated cells using ANOVA with Dunnett ' s multiple comparisons test . * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 ( B ) Levels of transcription and replication in the MG assay upon over - expression of the indicated host factors . MG assays were performed with 50 and 500 ng of host protein expression plasmid . RNA was isolated and analyzed by qRT - PCR to measure the levels of viral mRNA / cRNA and vRNA . The sample with vector control was set to 100 % and mean ± S . D . values from two independent experiments are shown ( each experiment was performed in duplicate , n = 2 ) . * * p < 0 . 005 , * p < 0 . 05 . Statistical significance was calculated relative to vector control using ANOVA with Tukey ' s multiple comparisons test . ( C ) Immunoblots to assess VP30 and pVP30 levels in lysates from MG assay upon knockdown of RBBP6 , hnRNP L or hnRNPUL1 . ( D ) Immunoblots to assess the levels of VP30 , pVP30 and NP in EBOV infected lysates upon knockdown of NPC1 or RBBP6 . HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6 . 48 h post - transfection cells were infected with EBOV at MOI = 0 . 1 . At 24 h post - infection , cells were lysed in TRIzol . Protein was extracted from the TRIzol reagent and analyzed by Western blotting to determine levels of VP30 , pSer29 VP30 and NP . ( E ) Western blots detecting pVP30 , VP30 and NP levels in EBOV infected HeLa cells upon hnRNP L , hnRNPUL1 or PEG10 knockdown .

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Figure 7(A) HeLa cells were transfected with scrambled siRNA (SCR si) or siRNAs targeting hnRNP L, hnRNPUL1 or PEG10, 48 h post-transfection, cells were challenged with EBOV-EGFP at an MOI of 0.5. 24 h post-infection, cells were fixed, stained for nuclei with Hoechst dye and imaged. Scale bar is 200 µm.The graph (right) depicts relative number of infected cells as determined by calculating the ratio of number of infected cells to nuclei and shown relative to SCR siRNA. The data shows a representative result of two independent experiments with the mean ± S.D. calculated from three independently treated wells. Statistical significance was calculated relative to the values obtained in SCR siRNA-treated cells using ANOVA with Dunnett's multiple comparisons test. ***p<0.0001, ***p<0.001, **p<0.01(B) Levels of transcription and replication in the MG assay upon over-expression of the indicated host factors. MG assays were performed with 50 and 500 ng of host protein expression plasmid. RNA was isolated and analyzed by qRT-PCR to measure the levels of viral mRNA/cRNA and vRNA. The sample with vector control was set to 100% and mean ± S.D. values from two independent experiments are shown (each experiment was performed in duplicate, n=2). **p<0.005, *p<0.05. Statistical significance was calculated relative to vector control using ANOVA with Tukey's multiple comparisons test.(C) Immunoblots to assess VP30 and pVP30 levels in lysates from MG assay upon knockdown of RBBP6, hnRNP L or hnRNPUL1.(D) Immunoblots to assess the levels of VP30, pVP30 and NP in EBOV infected lysates upon knockdown of NPC1 or RBBP6. HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6. 48 h post-transfection cells were infected with EBOV at MOI=0.1. At 24 h post-infection, cells were lysed in TRIzol. Protein was extracted from the TRIzol reagent and analyzed by Western blotting to determine levels of VP30, pSer29 VP30 and NP.(E) Western blots detecting pVP30, VP30 and NP levels in EBOV infected HeLa cells upon hnRNP L, hnRNPUL1 or PEG10 knockdown.


Figure 1B . The in vitro synthesized [ 35S ] Met - labeled single - cysteine Bax proteins were activated and targeted to the mitochondria that were pretreated with NEM to block the sulfhydryls of mitochondrial proteins . The resulting mitochondria were isolated and oxidized by CuPhe for 30 min . NEM and EDTA were then added to stop the oxidation . For the " 0 min " controls , NEM and EDTA were added before the addition of CuPhe . The resulting samples were analyzed by phosphor - imaging after non - reducing or reducing SDS - PAGE ( see Appendix Figure S2A ) . C . Oxidized mitochondria with the radioactive single - cysteine Bax protein pair or double - cysteine Bax protein were prepared and analyzed as in ( B ) . D . Oxidized mitochondria with the radioactive single - cysteine Bax protein pair or double - cysteine Bax protein were prepared as in ( B ) . After an aliquot was withdrawn as input , another aliquot was extracted by Na2CO3 ( pH 11 . 5 ) and centrifuged through a sucrose cushion to separate the integral proteins in the membrane pellet from the soluble and peripheral proteins in the supernatant . The input , pellet and supernatant were analyzed by non - reducing SDS - PAGE and phosphor - imaging . In a parallel control experiment , the pellet and supernatant were analyzed by reducing SDS - PAGE and immunoblotting with an antibody specific to PDHE1α , a soluble mitochondrial matrix protein .

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 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 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

Figure 1B. The in vitro synthesized [35S]Met-labeled single-cysteine Bax proteins were activated and targeted to the mitochondria that were pretreated with NEM to block the sulfhydryls of mitochondrial proteins. The resulting mitochondria were isolated and oxidized by CuPhe for 30 min. NEM and EDTA were then added to stop the oxidation. For the "0 min" controls, NEM and EDTA were added before the addition of CuPhe. The resulting samples were analyzed by phosphor-imaging after non-reducing or reducing SDS-PAGE (see Appendix Figure S2A).C. Oxidized mitochondria with the radioactive single-cysteine Bax protein pair or double-cysteine Bax protein were prepared and analyzed as in (B).D. Oxidized mitochondria with the radioactive single-cysteine Bax protein pair or double-cysteine Bax protein were prepared as in (B). After an aliquot was withdrawn as input, another aliquot was extracted by Na2CO3 (pH 11.5) and centrifuged through a sucrose cushion to separate the integral proteins in the membrane pellet from the soluble and peripheral proteins in the supernatant. The input, pellet and supernatant were analyzed by non-reducing SDS-PAGE and phosphor-imaging. In a parallel control experiment, the pellet and supernatant were analyzed by reducing SDS-PAGE and immunoblotting with an antibody specific to PDHE1α, a soluble mitochondrial matrix protein.


Figure 2B . The in vitro synthesized radioactive Bax proteins , each with a single cysteine positioned in helix α2 , α3 , α4 or α5 , were activated and targeted to the mitochondria . The resulting mitochondria were isolated and treated with IASD in the absence or presence of CHAPS , urea , or both . After 30 min , the labeling reactions were stopped by mercaptoethanol . For the " 0 min " controls , the samples were pretreated with mercaptoethanol before addition of IASD . The IASD - labeled radioactive Bax proteins were resolved from the unlabeled ones using either isoelectric focusing ( IEF ; as indicated ) or gradient SDS - PAGE and detected by phosphor - imaging . Triangles and arrows indicate the unlabeled and IASD - labeled Bax proteins , respectively . n = 3 for V111C , and K119C ; 4 for W107C , and A117C ; 2 for other mutants . C - D . The phosphor - imaging data for IASD labeling of Bax mutants in ( B ) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices as described and shown in Appendix Figure S3 . The membrane burial indices were normalized by that of G179C in α9 , a reference for the membrane - buried residues . The resulting relative membrane burial indices are shown in ( C ) . The residues with the relative membrane burial index ≥ 40 % of that of Gly179 are considered as buried in the MOM . Similarly , the relative protein burial indices shown in ( D ) were obtained by using Y115C in α5 as a reference for the protein - buried residues . The residues with the relative protein burial index ≥ 40 % of that of Tyr115 are considered as buried in the protein or its complex .

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Figure 2B. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in helix α2, α3, α4 or α5, were activated and targeted to the mitochondria. The resulting mitochondria were isolated and treated with IASD in the absence or presence of CHAPS, urea, or both. After 30 min, the labeling reactions were stopped by mercaptoethanol. For the "0 min" controls, the samples were pretreated with mercaptoethanol before addition of IASD. The IASD-labeled radioactive Bax proteins were resolved from the unlabeled ones using either isoelectric focusing (IEF; as indicated) or gradient SDS-PAGE and detected by phosphor-imaging. Triangles and arrows indicate the unlabeled and IASD-labeled Bax proteins, respectively. n = 3 for V111C, and K119C; 4 for W107C, and A117C; 2 for other mutants.C-D. The phosphor-imaging data for IASD labeling of Bax mutants in (B) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices as described and shown in Appendix Figure S3. The membrane burial indices were normalized by that of G179C in α9, a reference for the membrane-buried residues. The resulting relative membrane burial indices are shown in (C). The residues with the relative membrane burial index ≥ 40% of that of Gly179 are considered as buried in the MOM. Similarly, the relative protein burial indices shown in (D) were obtained by using Y115C in α5 as a reference for the protein-buried residues. The residues with the relative protein burial index ≥ 40% of that of Tyr115 are considered as buried in the protein or its complex.


Figure 3A - B . Oxidized mitochondria with the radioactive single - or double - cysteine Bax proteins , or the single - cysteine Bax protein pairs were prepared and analyzed as in Figure 1B . C . Oxidized mitochondria with the radioactive double - cysteine Bax protein were prepared , subjected to Na2CO3 extraction and analyzed as in Figure 1D .

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Figure 3A-B. Oxidized mitochondria with the radioactive single- or double-cysteine Bax proteins, or the single-cysteine Bax protein pairs were prepared and analyzed as in Figure 1B.C. Oxidized mitochondria with the radioactive double-cysteine Bax protein were prepared, subjected to Na2CO3 extraction and analyzed as in Figure 1D.


Figure 4B and D . The radioactive single - or double - cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated ( mito ) or pretreated with NEM ( NEM - mito ) , then oxidized and analyzed as in Figure 1B . Protein standards , and monomers and disulfide - linked dimers of the Bax proteins are indicated as in Figure 1B . n = 2 for all mutants . C . The in vitro synthesized [ 35S ] Met - labeled Bax E69C protein with or without the additional mutations ( G108E and / or S184V ) were activated by the in vitro synthesized [ 35S ] Met - labeled tBid L105C protein and targeted to the Bax - / - / Bak - / - mitochondria that were pretreated with NEM . The resulting mitochondria were isolated and oxidized by CuPhe for 0 or 30 min . The resulting " 0 min " samples ( 1 equivalent each ) and " 30 min " samples ( 1 equivalent each ) were analyzed by non - reducing SDS - PAGE and phosphor - imaging . The remaining " 30 min " samples ( 4 equivalent each ) were immunoprecipitated ( IP ) by either Bax - or tBid - specific antibody , and then analyzed by non - reducing SDS - PAGE and phosphor - imaging . The identities of the four major products , indicated on the right side of the image , were based on their Mr and recognition by the respective antibody . n = 2 . B and D . The radioactive single - or double - cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated ( mito ) or pretreated with NEM ( NEM - mito ) , then oxidized and analyzed as in Figure 1B . Protein standards , and monomers and disulfide - linked dimers of the Bax proteins are indicated as in Figure 1B . n = 2 for all mutants .

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 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 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

Figure 4B and D. The radioactive single- or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated (mito) or pretreated with NEM (NEM-mito), then oxidized and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.C. The in vitro synthesized [35S]Met-labeled Bax E69C protein with or without the additional mutations (G108E and/or S184V) were activated by the in vitro synthesized [35S]Met-labeled tBid L105C protein and targeted to the Bax-/-/Bak-/- mitochondria that were pretreated with NEM. The resulting mitochondria were isolated and oxidized by CuPhe for 0 or 30 min. The resulting "0 min" samples (1 equivalent each) and "30 min" samples (1 equivalent each) were analyzed by non-reducing SDS-PAGE and phosphor-imaging. The remaining "30 min" samples (4 equivalent each) were immunoprecipitated (IP) by either Bax- or tBid-specific antibody, and then analyzed by non-reducing SDS-PAGE and phosphor-imaging. The identities of the four major products, indicated on the right side of the image, were based on their Mr and recognition by the respective antibody. n = 2.B and D. The radioactive single- or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated (mito) or pretreated with NEM (NEM-mito), then oxidized and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.


Figure 5A . The in vitro synthesized radioactive Bax proteins , each with a single cysteine positioned in α9 , were activated and targeted to the mitochondria , and labeled with IASD as in Figure 2B . The IASD - labeled and unlabeled Bax proteins were resolved using gradient SDS - PAGE , detected by phosphor - imaging and indicated by triangles and arrows , respectively . For all mutants , n = 2 , except for A183C , n = 3 . B - C . The IASD labeling data in ( A ) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices shown in Appendix Figure S5 , from which the relative membrane ( B ) or protein ( C ) burial indices were obtained as described in Figure 2C - D , and shown . D . The structure of α9 ( extracted from the NMR structure of Bax monomer , PDB entry 1F16 ) is shown with the residues that are buried in the MOM or exposed to the aqueous milieu , as the IASD labeling data in ( A - C ) suggested , presented in yellow or cyan stick form , respectively .

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Figure 5A. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in α9, were activated and targeted to the mitochondria, and labeled with IASD as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using gradient SDS-PAGE, detected by phosphor-imaging and indicated by triangles and arrows, respectively. For all mutants, n = 2, except for A183C, n = 3.B-C. The IASD labeling data in (A) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices shown in Appendix Figure S5, from which the relative membrane (B) or protein (C) burial indices were obtained as described in Figure 2C-D, and shown.D. The structure of α9 (extracted from the NMR structure of Bax monomer, PDB entry 1F16) is shown with the residues that are buried in the MOM or exposed to the aqueous milieu, as the IASD labeling data in (A-C) suggested, presented in yellow or cyan stick form, respectively.


Figure 6B and D . The oxidized mitochondria with radioactive single - cysteine Bax proteins were prepared and analyzed as in Figure 1B . C and E . The oxidized mitochondria with the radioactive single - cysteine Bax proteins were prepared , subjected to Na2CO3 extraction , and analyzed as in Figure 1D .

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Figure 6B and D. The oxidized mitochondria with radioactive single-cysteine Bax proteins were prepared and analyzed as in Figure 1B.C and E. The oxidized mitochondria with the radioactive single-cysteine Bax proteins were prepared, subjected to Na2CO3 extraction, and analyzed as in Figure 1D.


Figure 7B - D . The in vitro synthesized radioactive single or double - cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria , oxidized , and analyzed as in Figure 1B . Protein standards , and monomers and disulfide - linked dimers of the Bax proteins are indicated as in Figure 1B . n = 2 for all mutants .

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Figure 7B-D. The in vitro synthesized radioactive single or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria, oxidized, and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.


Figure 8A . The oxidized mitochondria with radioactive single , double , or triple - cysteine Bax proteins were prepared and analyzed by non - reducing or reducing SDS - PAGE as in Figure 1B . Protein standards , Bax monomers and disulfide - linked Bax homodimers are also indicated as in Figure 1B . Closed triangles indicate disulfide - linked higher order Bax homo - oligomers . Open triangles indicate a background band that could not be reduced , and hence is not a disulfide - linked complex . B . The BMH - treated mitochondria with the radioactive single - and double - cysteine Bax proteins were prepared and analyzed as in Figure EV3G , except that closed triangles indicate the BMH - linked Bax oligomers .

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Figure 8A. The oxidized mitochondria with radioactive single, double, or triple-cysteine Bax proteins were prepared and analyzed by non-reducing or reducing SDS-PAGE as in Figure 1B. Protein standards, Bax monomers and disulfide-linked Bax homodimers are also indicated as in Figure 1B. Closed triangles indicate disulfide-linked higher order Bax homo-oligomers. Open triangles indicate a background band that could not be reduced, and hence is not a disulfide-linked complex.B. The BMH-treated mitochondria with the radioactive single- and double-cysteine Bax proteins were prepared and analyzed as in Figure EV3G, except that closed triangles indicate the BMH-linked Bax oligomers.


Figure 9A - C . The single or double - cysteine Bax proteins with or without the indicated mutations were synthesized in vitro , activated by tBid protein and targeted to the Bax - / - / Bak - / - mitochondria . Cytochrome c release from the mitochondria was measured using ELISA . The dots are the fractions of cytochrome c release from two independent replicates after they were corrected as described in Figure EV1B , and the lines are the means . The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1 .

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Figure 9A-C. The single or double-cysteine Bax proteins with or without the indicated mutations were synthesized in vitro, activated by tBid protein and targeted to the Bax-/-/Bak-/- mitochondria. Cytochrome c release from the mitochondria was measured using ELISA. The dots are the fractions of cytochrome c release from two independent replicates after they were corrected as described in Figure EV1B, and the lines are the means. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1.


Figure 10A . The cytochrome c release by the indicated Bax mutants was measured in two independent replicates , corrected and shown as in Figure 9 . The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1 . B . Purified wild - type ( WT ) or mutant ( G179I or T182I ) Bax protein of the indicated concentration was incubated with the Bax - / - / Bak - / - mitochondria in the absence or presence of tBid protein . The proteins released from and those remaining in the mitochondria were separated and analyzed by SDS - PAGE and immunoblotting with antibody specific to either cytochrome c or Smac . n = 2 . C . Purified wild - type or mutant Bax protein of the indicated concentration alone or together with purified cBid was incubated with the Bax - / - / Bak - / - mitochondria containing Smac - mCherry protein . Release of Smac - mCherry was detected by measuring the fluorescence in the supernatant after the mitochondria were pelleted by centrifugation . The dots are the fractions of Smac - mCherry release from three independent replicates , and the lines are the means . D . The in vitro synthesized radioactive Bax proteins with indicated mutations were targeted to the mitochondria in the absence or presence of purified tBid protein , and then labeled with IASD , as in Figure 2B . The IASD - labeled and unlabeled Bax proteins were resolved using either gradient SDS - PAGE or IEF , detected by phosphor - imaging , and indicated by triangles and arrows , respectively . n = 3 for A178C , G179I ; 2 for other mutants . E . The IASD labeling data in ( D ) and the similar data from the independent replicates were quantified to derive the membrane burial indices shown in Appendix Figure S7A , from which the relative membrane burial indices were obtained as described in Figure 2C , and shown .

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Figure 10A. The cytochrome c release by the indicated Bax mutants was measured in two independent replicates, corrected and shown as in Figure 9. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1.B. Purified wild-type (WT) or mutant (G179I or T182I) Bax protein of the indicated concentration was incubated with the Bax-/-/Bak-/- mitochondria in the absence or presence of tBid protein. The proteins released from and those remaining in the mitochondria were separated and analyzed by SDS-PAGE and immunoblotting with antibody specific to either cytochrome c or Smac. n = 2.C. Purified wild-type or mutant Bax protein of the indicated concentration alone or together with purified cBid was incubated with the Bax-/-/Bak-/- mitochondria containing Smac-mCherry protein. Release of Smac-mCherry was detected by measuring the fluorescence in the supernatant after the mitochondria were pelleted by centrifugation. The dots are the fractions of Smac-mCherry release from three independent replicates, and the lines are the means.D. The in vitro synthesized radioactive Bax proteins with indicated mutations were targeted to the mitochondria in the absence or presence of purified tBid protein, and then labeled with IASD, as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using either gradient SDS-PAGE or IEF, detected by phosphor-imaging, and indicated by triangles and arrows, respectively. n = 3 for A178C,G179I; 2 for other mutants.E. The IASD labeling data in (D) and the similar data from the independent replicates were quantified to derive the membrane burial indices shown in Appendix Figure S7A, from which the relative membrane burial indices were obtained as described in Figure 2C, and shown.


Figure 1A . In situ hybridization for GemC1 and McIdas mRNA was performed on sagittal sections of E10 . 5 dpc mouse brain . GemC1 and McIdas mRNA is specifically detected in the developing choroid plexuses of the ventricles . Lower panels show higher magnifications . Scale bars , 100 µm . B . In situ hybridization on sagittal ( upper ) and coronal ( middle and lower ) sections of the airways of E15 . 5 dpc mouse embryos , depicting specific GemC1 and McIdas mRNA expression in the respiratory epithelium of the nasal and oral cavity and in the upper bronchial tree in the mouse lungs . Inserts show higher magnification images . Scale bars , 200 µm . C . MTECs were cultured ex - vivo and transitioned to ALI culture condition . Quantitative real time PCR was used to measure the expression levels of GemC1 , McIdas , FoxJ1 and Geminin mRNA levels at different time points during multiciliogenesis . Values indicate expression levels ( + / - standard error , s . e . ) relative to ALI DAY 1 ( marked by * ) , following normalization against the house - keeping HPRT mRNA . qPCR data were analyzed by the REST - MCS beta software from at least three independent experiments .

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Figure 1A. In situ hybridization for GemC1 and McIdas mRNA was performed on sagittal sections of E10.5 dpc mouse brain. GemC1 and McIdas mRNA is specifically detected in the developing choroid plexuses of the ventricles. Lower panels show higher magnifications. Scale bars, 100 µm.B. In situ hybridization on sagittal (upper) and coronal (middle and lower) sections of the airways of E15.5 dpc mouse embryos, depicting specific GemC1 and McIdas mRNA expression in the respiratory epithelium of the nasal and oral cavity and in the upper bronchial tree in the mouse lungs. Inserts show higher magnification images. Scale bars, 200 µm.C. MTECs were cultured ex-vivo and transitioned to ALI culture condition. Quantitative real time PCR was used to measure the expression levels of GemC1, McIdas, FoxJ1 and Geminin mRNA levels at different time points during multiciliogenesis. Values indicate expression levels (+/- standard error, s.e.) relative to ALI DAY 1 (marked by *), following normalization against the house-keeping HPRT mRNA. qPCR data were analyzed by the REST-MCS beta software from at least three independent experiments.


Figure 2A . and B . MTEC cultures were infected with a lentivirus expressing either GFP - GemC1 ( GemC1 ) or GFP as a control and two days later transitioned to ALI culture conditions . Cells harvested at different time points during differentiation were co - stained with antibodies against GFP ( green ) to mark infected cells and endogenous McIdas ( A ) or Foxj1 ( B ) ( red ) . Arrows indicate infected cells that are McIdas or Foxj1 positive . Scale bars , 10 µm . C . and D . The percentage of infected cells expressing McIdas ( C ) or Foxj1 ( D ) at each time point was quantified . Data are presented as the mean values of at least 10 independent fields obtained from at least two independent experiments for each condition . Error bars indicate ± SEM . * p < 0 . 05 , * * * p < 0 . 001 . P - values were calculated by the non - parametrical two - tailed Mann - Whitney test .

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Figure 2A. and B. MTEC cultures were infected with a lentivirus expressing either GFP-GemC1 (GemC1) or GFP as a control and two days later transitioned to ALI culture conditions. Cells harvested at different time points during differentiation were co-stained with antibodies against GFP (green) to mark infected cells and endogenous McIdas(A) or Foxj1(B) (red). Arrows indicate infected cells that are McIdas or Foxj1 positive. Scale bars, 10 µm.C. and D. The percentage of infected cells expressing McIdas(C) or Foxj1(D) at each time point was quantified. Data are presented as the mean values of at least 10 independent fields obtained from at least two independent experiments for each condition. Error bars indicate ± SEM. *p<0.05, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test.


Figure 3MCIDAS ( A ) regulatory elements ( see materials and methods ) cloned upstream of the luciferase gene were co - transfected into HEK293T cells with vectors expressing GemC1 , McIdas and Geminin , as indicated or an empty vector ( - ) as a control . All luciferase experiments ( A , B , D ) were normalized for transfection efficiency with an expression vector for Renilla luciferase . Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector . Data are the mean values of at least three independent experiments and error bars indicate ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by the non - parametrical two - tailed Mann - Whitney test . Abbreviations : RLF : Relative Luciferase Fold induction . FOXJ1 ( B , D ) regulatory elements ( see materials and methods ) cloned upstream of the luciferase gene were co - transfected into HEK293T cells with vectors expressing GemC1 , McIdas and Geminin , as indicated or an empty vector ( - ) as a control . All luciferase experiments ( A , B , D ) were normalized for transfection efficiency with an expression vector for Renilla luciferase . Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector . Data are the mean values of at least three independent experiments and error bars indicate ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by the non - parametrical two - tailed Mann - Whitney test . Abbreviations : RLF : Relative Luciferase Fold induction . McIdasmRNA levels after McIdas RNAi were assessed by qPCR ( C ) . FOXJ1 ( B , D ) regulatory elements ( see materials and methods ) cloned upstream of the luciferase gene were co - transfected into HEK293T cells with vectors expressing GemC1 , McIdas and Geminin , as indicated or an empty vector ( - ) as a control . In D , McIdas siRNAs or a control siRNA were also co - transfected . All luciferase experiments ( A , B , D ) were normalized for transfection efficiency with an expression vector for Renilla luciferase . Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector . Data are the mean values of at least three independent experiments and error bars indicate ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by the non - parametrical two - tailed Mann - Whitney test . Abbreviations : RLF : Relative Luciferase Fold induction .

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 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 B-PANEL_START 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

Figure 3MCIDAS (A) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.McIdasmRNA levels after McIdas RNAi were assessed by qPCR (C).FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. In D, McIdas siRNAs or a control siRNA were also co-transfected. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.


Figure 4A . and B . MCIDAS ( A ) and FOXJ1 ( B ) regulatory elements cloned upstream of the luciferase gene were co - transfected into HEK293T cells with vectors expressing GemC1 , McIdas , Geminin and E2F5 , as indicated , or an empty vector ( - ) as a control . Luciferase values were normalized against co - transfected Renilla luciferase and depicted as fold induction over the control empty vector . Data shown are mean values of at least three independent experiments and error bars indicate ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by the non - parametrical two - tailed Mann - Whitney test . Abbreviations : RLF : Relative Luciferase Fold induction . C . and D . Chromatin immunoprecipitation of MCIDAS and FOXJ1 promoter fragments by GemC1 . HEK293T cells were co - transfected with vectors expressing GemC1 - GFP ( GemC1 ) or GFP as a control , E2F5 and DP1 , together with the regulatory elements of either MCIDAS ( C ) or FOXJ1 ( D ) . Following chromatin immunoprecipitation , DNA fragments of the MCIDAS and FOXJ1 promoters were detected in immunoprecipitates by qPCR . Data from a representative experiment are shown as fold enrichment of MCIDAS and FOXJ1 promoter fragments in anti - GFP immunoprecipitates over control IgG immunoprecipitates . At least two independent experiments were performed .

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 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

Figure 4A. and B. MCIDAS (A) and FOXJ1 (B) regulatory elements cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas, Geminin and E2F5, as indicated, or an empty vector (-) as a control. Luciferase values were normalized against co-transfected Renilla luciferase and depicted as fold induction over the control empty vector. Data shown are mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.C. and D. Chromatin immunoprecipitation of MCIDAS and FOXJ1 promoter fragments by GemC1. HEK293T cells were co-transfected with vectors expressing GemC1-GFP (GemC1) or GFP as a control, E2F5 and DP1, together with the regulatory elements of either MCIDAS (C) or FOXJ1 (D). Following chromatin immunoprecipitation, DNA fragments of the MCIDAS and FOXJ1 promoters were detected in immunoprecipitates by qPCR. Data from a representative experiment are shown as fold enrichment of MCIDAS and FOXJ1 promoter fragments in anti-GFP immunoprecipitates over control IgG immunoprecipitates. At least two independent experiments were performed.


Figure 5B . GemC1KO / KO mice compared to their wild type littermates ( GemC1KO / WT ) at P5 . C . Survival plot of mice homozygous for the GemC1 deletion ( GemC1KO / KO , n = 8 ) and control wild type or heterozygous littermates GemC1 ( GemC1WT , n = 14 ) . GemC1 knock - out mice exhibit markedly reduced growth after birth and die within the first postnatal week .

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Figure 5B. GemC1KO/KO mice compared to their wild type littermates (GemC1KO/WT) at P5.C. Survival plot of mice homozygous for the GemC1 deletion (GemC1KO/KO, n=8) and control wild type or heterozygous littermates GemC1 (GemC1WT, n=14). GemC1 knock-out mice exhibit markedly reduced growth after birth and die within the first postnatal week.


Figure 6A . , B . Transverse sections from trachea isolated from GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) P0 mice , immunostained with antibodies against acetylated y - tubulin ( ACT , cilia marker , red ) , pericentrin ( PCNT , marker for nascent centrioles , green ) , γ tubulin ( centriole marker , green ) and E - cadherin ( marker for cell boundaries , blue ) . Serial images have been merged to show the complete trachea section ( left ) . Inserts show higher magnification images for acetylated y - tubulin and pericentrin staining in GemC1WT / WT and GemC1KO / KO trachea , respectively . Scale bars , 10 µm . C . McIdas and Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) P0 mice . Arrows show representative positive cells for McIdas ( green ) and FoxJ1 ( red ) in the GemC1WT / WT mice . DNA was stained with Draq - 5 ( blue ) . Scale bars , 10 µm . D . Transverse sections from P0 GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) trachea , immunostained with antibodies against Keratin 5 ( Krt5 , basal cell marker , green ) , and acetylated y - tubulin ( ACT , cilia marker , red ) . DNA was stained with Draq - 5 ( blue ) . Serial images have been merged to show the complete trachea section on the left . Higher magnification images are shown on the right . Scale bars , 10 µm . E . Transverse sections from P0 GemC1WT / WT ( left two images ) or GemC1KO / KO ( right two images ) trachea , immunostained with antibodies against Muc5ac ( marker for mucus - expressing cells , red ) and E - cadherin ( marker for cell boundaries , green ) . DNA was stained with Draq - 5 ( blue ) . Serial images have been merged to show the complete trachea section ( left ) . Higher magnifications from the same section are shown on the right . Scale bars , 10 µm .

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 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 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 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

Figure 6A., B. Transverse sections from trachea isolated from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice, immunostained with antibodies against acetylated y-tubulin (ACT, cilia marker, red), pericentrin (PCNT, marker for nascent centrioles, green), γ tubulin (centriole marker, green) and E-cadherin (marker for cell boundaries, blue). Serial images have been merged to show the complete trachea section (left). Inserts show higher magnification images for acetylated y-tubulin and pericentrin staining in GemC1WT/WT and GemC1KO/KO trachea, respectively. Scale bars, 10 µm.C. McIdas and Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice. Arrows show representative positive cells for McIdas (green) and FoxJ1 (red) in the GemC1WT/WT mice. DNA was stained with Draq-5 (blue). Scale bars, 10 µm.D. Transverse sections from P0 GemC1WT/WT (upper) or GemC1KO/KO (lower) trachea, immunostained with antibodies against Keratin 5 (Krt5, basal cell marker, green), and acetylated y-tubulin (ACT, cilia marker, red). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section on the left. Higher magnification images are shown on the right. Scale bars, 10 µm.E. Transverse sections from P0 GemC1WT/WT (left two images) or GemC1KO/KO (right two images) trachea, immunostained with antibodies against Muc5ac (marker for mucus-expressing cells, red) and E-cadherin (marker for cell boundaries, green). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section (left). Higher magnifications from the same section are shown on the right. Scale bars, 10 µm.


Figure 1 ( A ) ( B ) CPT sensitivity assayed by 10 - fold serial dilutions of different mutant combinations on YPAD plates . Four biological replicates have been performed . ( C ) Colony - forming unit assays to increasing CPT concentrations of the same mutant combinations as in ( B ) . Mean values ± SD are plotted . Four biological replicates have been performed . ( D ) UV sensitivity assayed by 10 - fold serial dilutions of different mutant combinations with rad3 - 102 allele on YPAD plates after exposure to the indicated UV - C doses . Five biological replicates have been performed . ( F ) BIR frequencies ( Lys + survivors among total cells ) for the WT and indicated mutant strains are plotted on a logarithmic scale . Data represent the mean ± SD from 3 to 6 independent experiments . ns , non - significant difference , * P - value = 0 . 0134 , Mann - Whitney unpaired t test .

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Figure 1(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.(C) Colony-forming unit assays to increasing CPT concentrations of the same mutant combinations as in (B). Mean values ± SD are plotted. Four biological replicates have been performed.(D) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Five biological replicates have been performed.(F) BIR frequencies (Lys+ survivors among total cells) for the WT and indicated mutant strains are plotted on a logarithmic scale. Data represent the mean ± SD from 3 to 6 independent experiments. ns, non-significant difference, * P-value = 0.0134, Mann-Whitney unpaired t test.


Figure 2 ( A ) ( B ) CPT sensitivity assayed by 10 - fold serial dilutions of different mutant combinations on YPAD plates . Four biological replicates have been performed . ( C ) UV sensitivity assayed by 10 - fold serial dilutions of different mutant combinations with rad3 - 102 allele on YPAD plates after exposure to the indicated UV - C doses . Four biological replicates have been performed . ( D ) Synthetic combinations of rad3 - 102 mus81 ∆ with rad51 ∆ and pol32 ∆ . Tetrads dissected on YPAD medium are shown . Triangles indicate either a severe growth defect or lethality .

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Figure 2(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.(C) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Four biological replicates have been performed.(D) Synthetic combinations of rad3-102 mus81∆ with rad51∆ and pol32∆. Tetrads dissected on YPAD medium are shown. Triangles indicate either a severe growth defect or lethality.


Figure 3 ( A ) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type , rad52 ∆ , rad51 ∆ , mus81 ∆ and pol32 ∆ cells and further released into S phase . Cells were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 1 h , and released into S phase . Three biological replicates have been performed . ( B ) ( C ) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type , rad3 - 102 , rad3 - 102 rad51 ∆ , rad3 - 102 mus81 and rad3 - 102 mus81 - dd cells and further released into S phase . Cells were synchronized in G1 with α - factor , untreated or irradiated with 20 J / m2 UV - C , let in G1 for 2h , and released into S phase . Three biological replicates have been performed .

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Figure 3(A) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad52∆, rad51∆, mus81∆ and pol32∆ cells and further released into S phase. Cells were synchronized in G1 with α -factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Three biological replicates have been performed.(B) (C) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad3-102, rad3-102 rad51∆, rad3-102 mus81 and rad3-102 mus81-dd cells and further released into S phase. Cells were synchronized in G1 with α-factor, untreated or irradiated with 20 J/m2 UV-C, let in G1 for 2h, and released into S phase. Three biological replicates have been performed.


Figure 4 ( A ) Analysis of replicated DNA tracks length by single - molecule DNA combing in WT , rad52 ∆ , rad51 ∆ , mus81 ∆ and pol32 ∆ cells exposed to CPT . Exponentially growing cells were treated with DMSO or 50 µM CPT for 2h and then pulse - labeled with 50 µM EdU for 20min . DNA fibers were combed on silanized coverslips and EdU - labeled DNA was detected by Click chemistry . Graph depicts the distribution of EdU tracks length in kb . Box and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . Median EdU tracks length is indicated in kb ( values from two biological replicates were pooled ) . Asterisks indicate the P - value of the Mann - Whitney unpaired t test , * * : p = 0 . 0015 . The percentage of EdU track length decrease between the DMSO and CPT conditions is indicated between parentheses for each strain . Representative images of DNA fibers are shown . Red and white : EdU , green : DNA . ( C ) Analysis of replication intermediates by 2D gel electrophoresis . Replication intermediates were monitored at early origin ARS305 and region C in WT , rad3 - 102 , mus81 ∆ and rad3 - 102 mus81 ∆ cells . Cells were synchronized in G1 with α - factor and collected at the indicated time points after release into S phase . A scheme of the studied chromosomal region is shown ( drawn to scale ) . Relevant probes are indicated by gray bars , and coordinates of ARS and restriction sites are indicated in bp . Two biological replicates have been performed .

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 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

Figure 4(A) Analysis of replicated DNA tracks length by single-molecule DNA combing in WT, rad52∆, rad51∆, mus81∆ and pol32∆ cells exposed to CPT. Exponentially growing cells were treated with DMSO or 50 µM CPT for 2h and then pulse-labeled with 50 µM EdU for 20min. DNA fibers were combed on silanized coverslips and EdU-labeled DNA was detected by Click chemistry. Graph depicts the distribution of EdU tracks length in kb. Box and whiskers indicate 25-75 and 10-90 percentiles, respectively. Median EdU tracks length is indicated in kb (values from two biological replicates were pooled). Asterisks indicate the P-value of the Mann-Whitney unpaired t test, **: p=0.0015. The percentage of EdU track length decrease between the DMSO and CPT conditions is indicated between parentheses for each strain. Representative images of DNA fibers are shown. Red and white: EdU, green: DNA.(C) Analysis of replication intermediates by 2D gel electrophoresis. Replication intermediates were monitored at early origin ARS305 and region C in WT, rad3-102, mus81∆ and rad3-102 mus81∆ cells. Cells were synchronized in G1 with α-factor and collected at the indicated time points after release into S phase. A scheme of the studied chromosomal region is shown (drawn to scale). Relevant probes are indicated by gray bars, and coordinates of ARS and restriction sites are indicated in bp. Two biological replicates have been performed.


Figure 5 ( A ) Analysis of nuclear Rad52 - GFP foci formation . An illustrative image of the experimental setup is shown . DIC , differential interference contrast ; yellow , Rad52 - GFP ; red , mCHERRY - Pus1 ( nuclear compartment marker ) . Scale bar = 5 µm . ( B ) ( C ) Kinetic analysis of Rad52 foci formation . Wild - type and mus81 ∆ cells containing Rad52 - YFP and mCHERRY - Pus1 were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 30 min , and released into S phase . Cells were collected at the indicated time points and visualized by fluorescence microscopy . Mean ± SEM of cells with Rad52 foci are shown for each time point . Flow cytometry profiles corresponding the experimental setup are shown . Four biological replicates have been performed . ( D ) Mms4 - Flag10 phosphorylation analyzed by immunoblot in wild type cells exposed to CPT . Wild - type cells were synchronized in G1 with α - factor , treated with DMSO or 50 µM CPT , let in G1 for 1 h , and released into S phase . Cells were collected at the indicated time points and Mms4 was immunodetected with anti - Flag antibodies . Clb2 immunodetection serves as a marker for G2 phase entry . FACS profiles corresponding the experimental setup are also shown . Two biological replicates have been performed . ( E ) CPT sensitivity assayed by 10 - fold serial dilutions of S - MUS81 and G2 - MUS81 alleles compared to the wild type , mus81 , mms4 ∆ and cdc5 - 2 mutants . Three biological replicates have been performed . ( F ) UV sensitivity assayed by 10 - fold serial dilutions of S - MUS81 and G2 - MUS81 alleles in combination with rad3 - 102 on YPAD plates after exposure to the indicated UV - C doses . Three biological replicates have been performed .

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 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 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 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 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

Figure 5(A) Analysis of nuclear Rad52-GFP foci formation. An illustrative image of the experimental setup is shown. DIC, differential interference contrast; yellow, Rad52-GFP; red, mCHERRY-Pus1 (nuclear compartment marker). Scale bar = 5 µm.(B) (C) Kinetic analysis of Rad52 foci formation. Wild-type and mus81∆ cells containing Rad52-YFP and mCHERRY-Pus1 were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 30 min, and released into S phase. Cells were collected at the indicated time points and visualized by fluorescence microscopy. Mean ± SEM of cells with Rad52 foci are shown for each time point. Flow cytometry profiles corresponding the experimental setup are shown. Four biological replicates have been performed.(D) Mms4-Flag10 phosphorylation analyzed by immunoblot in wild type cells exposed to CPT. Wild-type cells were synchronized in G1 with α-factor, treated with DMSO or 50 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points and Mms4 was immunodetected with anti-Flag antibodies. Clb2 immunodetection serves as a marker for G2 phase entry. FACS profiles corresponding the experimental setup are also shown. Two biological replicates have been performed.(E) CPT sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles compared to the wild type, mus81, mms4∆ and cdc5-2 mutants. Three biological replicates have been performed.(F) UV sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles in combination with rad3-102 on YPAD plates after exposure to the indicated UV-C doses. Three biological replicates have been performed.


Figure 6 ( A ) ( B ) Pulsed - field gel electrophoresis ( PFGE ) analysis of wild type , mus81 ∆ , cdc5 - 2 and G2 - MUS81 cells in response to CPT . Cells were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 1 h , and released into S phase . Cells were collected at the indicated time points . DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE . Upper panel : agarose gel stained with ethidium bromide ; lower panel : Southern blot using a chromosome IV specific probe . JMs , joint molecules accumulated in the gel wells . The mean value of JMs relative to the total amount of DNA is indicated for each time point . Two to three biological replicates have been performed . ( C ) ( D ) PFGE analysis of rad52 ∆ and rad52 ∆ mus81 ∆ cells in response to CPT performed as in ( A ) ( B ) . The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the + CPT 140 min time points in rad52 ∆ and rad52 ∆ mus81 ∆ cells are shown . Two biological replicates have been performed .

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

Figure 6(A) (B) Pulsed-field gel electrophoresis (PFGE) analysis of wild type, mus81∆, cdc5-2 and G2-MUS81 cells in response to CPT. Cells were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points. DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE. Upper panel: agarose gel stained with ethidium bromide; lower panel: Southern blot using a chromosome IV specific probe. JMs, joint molecules accumulated in the gel wells. The mean value of JMs relative to the total amount of DNA is indicated for each time point. Two to three biological replicates have been performed.(C) (D) PFGE analysis of rad52∆ and rad52∆ mus81∆ cells in response to CPT performed as in (A) (B). The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the +CPT 140 min time points in rad52∆ and rad52∆ mus81∆ cells are shown. Two biological replicates have been performed.


Figure 1 ( A ) Akt is deubiquitinated during fasting . Left : Soluble fractions of TA muscles from fed and fasted mice were analyzed by SDS - PAGE and immunoblot using Akt antibody . Right : densitometric measurement of presented blots ( n = 3 ) . * p < 0 . 05 vs . fed by one - tailed t - test . Data are represented as mean ± SEM . ( B ) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted ( 2d ) mice . Percipitated were analyzed by immunoblotting using an anti - ubiquitin conjugates . Mouse IgG was used as a control for non - specific binding . ) D ( Ubiquitinated Akt is a substrate for USP1 in vitro . Top : Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice , and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi - well plate . The dividing line indicates the removal of an intervening lane for presentation purposes . Bottom : Densitometric measurements of presented blots . Data is presented as the ratio between ubiquitinated Akt to total Akt in each well . ( E ) shRNA - mediated knockdown of USP1 in Hela cells . Soluble extracts were analyzed by immunoblotting . ( F ) USP1 deubiquitinates Akt during fasting in vivo . Left : Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS - PAGE and immunoblotting using anti - Akt . The actin blot serves as a loading control . Right : densitometric measurements of presented blots ( n = 3 ) . Data is presented as ratio of ubiquitinated Akt to total Akt normalized to actin . * p < 0 . 05 vs . fed by one - tailed t - test . Data are represented as mean ± SEM . ( G ) USP1 removes K63 - linked polyubiquitin chains on Akt in vivo . Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1 ( C90S ) or control plasmids . Mouse IgG was used as a control for non - specific binding . Protein precipitates were subjected to immunoblotting with an antibody against K63 - linked polyubiquitin conjugates . ( H ) USP1 removes polyubiquitin chains linked to K8 on Akt . Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1 ( C90S ) , HA - Akt ( K8R ) or control from fasted mice , and protein precipitates were analyzed by immunoblotting with anti - Akt antibody . Mouse IgG was used as a control for non - specific binding . Right : Densitometric measurements of presented blots . Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane ( n = 2 ) .

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 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 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 B-PANEL_START 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 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 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

Figure 1(A) Akt is deubiquitinated during fasting. Left: Soluble fractions of TA muscles from fed and fasted mice were analyzed by SDS-PAGE and immunoblot using Akt antibody. Right: densitometric measurement of presented blots (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.(B) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted (2d) mice. Percipitated were analyzed by immunoblotting using an anti-ubiquitin conjugates. Mouse IgG was used as a control for non-specific binding.)D( Ubiquitinated Akt is a substrate for USP1 in vitro. Top: Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice, and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi-well plate. The dividing line indicates the removal of an intervening lane for presentation purposes. Bottom: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each well.(E) shRNA-mediated knockdown of USP1 in Hela cells. Soluble extracts were analyzed by immunoblotting.(F) USP1 deubiquitinates Akt during fasting in vivo. Left: Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using anti-Akt. The actin blot serves as a loading control. Right: densitometric measurements of presented blots (n=3). Data is presented as ratio of ubiquitinated Akt to total Akt normalized to actin. * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.(G) USP1 removes K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1(C90S) or control plasmids. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.(H) USP1 removes polyubiquitin chains linked to K8 on Akt. Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1(C90S) , HA-Akt(K8R) or control from fasted mice, and protein precipitates were analyzed by immunoblotting with anti-Akt antibody. Mouse IgG was used as a control for non-specific binding. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).


Figure 2 ( A ) Inhibition of USP1 increases PI3K - Akt - FoxO signaling during fasting . Soluble fractions of normal and atrophying muscles expressing shLacz or USP1 ( C90S ) were analyzed by SDS - PAGE and immunoblot . The black line indicate the removal of intervening lanes for presentation purposes . Right : densitometric measurements of presented pAkt ( T308 ) , pAkt ( S473 ) , and Akt blots . Data is presented as ratio of phosphorylated Akt to total Akt ( n = 3 ) . * p < 0 . 05 vs . fed by one - tailed t - test . Data are represented as mean ± SEM . ( B ) Downregulation of USP1 reduces MuRF1 and Atrogin1 expression during fasting . Quantitative RT - PCR of mRNA preparations from atrophying and control muscles expressing shLacz or shUSP1 ( 80 % transfection efficiency ) using primers for MuRF1 and Atrogin1 . Data are plotted as the mean fold change relative to fed control . n = 4 . * , P < 0 . 05 vs . shLacz in fed . # , P < 0 . 05 vs . shLacz in fasting by one - tailed t - test . Data are represented as mean ± SEM . ( C ( Downregulation of USP1 markedly reduces muscle fiber atrophy . Cross - sectional areas of 500 fibers transfected with shUSP1 ( that express GFP , green bars ) vs . 500 nontransfected fibers ( black bars ) in the same muscle . n = 5 mice . A representative image is shown ; Laminin staining is in red ; Bar , 50μm . ( D ) Downregulation of USP1attenuates the loss of muscle mass during fasting . shUSP1 was delivered to more than 60 % of muscle fibers . Mean weights of electroporated muscles are plotted as the percent weight loss . n = 6 . # , P < 0 . 05 vs . shLacz in fed ; * , P < 0 . 05 vs . shLacz in fasting by one - tailed t - test . Data are represented as mean ± SEM . ( E ) USP1 inhibition significantly increases glucose tolerance in mice during fasting . Left : mice injected i . p . with specific USP1 Inhibitor ( 12ug / gr body weight ) or saline during fasting ( 2d ) were subjected to glucose tolerance test . Blood glucose levels were measured at the indicated time points following glucose injection ( 1 mg / gr body weight ) . Data is depicted in a graph as mg / dL glucose ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 vs . mice injected with saline by one - tailed t - test . Data are represented as mean ± SEM . Right : soluble fractions of muscles from injected mice were analyzed by SDS - PAGE and immunoblot .

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 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 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 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

Figure 2(A) Inhibition of USP1 increases PI3K-Akt-FoxO signaling during fasting. Soluble fractions of normal and atrophying muscles expressing shLacz or USP1(C90S) were analyzed by SDS-PAGE and immunoblot. The black line indicate the removal of intervening lanes for presentation purposes. Right: densitometric measurements of presented pAkt (T308), pAkt (S473), and Akt blots. Data is presented as ratio of phosphorylated Akt to total Akt (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.(B) Downregulation of USP1 reduces MuRF1 and Atrogin1 expression during fasting. Quantitative RT-PCR of mRNA preparations from atrophying and control muscles expressing shLacz or shUSP1 (80% transfection efficiency) using primers for MuRF1 and Atrogin1. Data are plotted as the mean fold change relative to fed control. n = 4. *, P < 0.05 vs. shLacz in fed. #, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.(C( Downregulation of USP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shUSP1 (that express GFP, green bars) vs. 500 nontransfected fibers (black bars) in the same muscle. n = 5 mice. A representative image is shown; Laminin staining is in red; Bar, 50μm.(D) Downregulation of USP1attenuates the loss of muscle mass during fasting. shUSP1 was delivered to more than 60% of muscle fibers. Mean weights of electroporated muscles are plotted as the percent weight loss. n = 6. #, P < 0.05 vs. shLacz in fed; *, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.(E) USP1 inhibition significantly increases glucose tolerance in mice during fasting. Left: mice injected i.p. with specific USP1 Inhibitor (12ug/gr body weight) or saline during fasting (2d) were subjected to glucose tolerance test. Blood glucose levels were measured at the indicated time points following glucose injection (1 mg/gr body weight). Data is depicted in a graph as mg/dL glucose (n=3). * p<0.05, ** p<0.005, *** p<0.0005 vs. mice injected with saline by one-tailed t-test. Data are represented as mean ±SEM. Right: soluble fractions of muscles from injected mice were analyzed by SDS-PAGE and immunoblot.