A . Gel filtration analysis to assess the AIFM1 - MIA40 complex in HEK293 cells . HEK293 cells , cells lacking AIFM1 ( AIFM1 knockout , AIFM1 KO ) or AIFM1 knockout cells complemented with AIFM1 - HA were lysed under native conditions ( triton X - 100 ) , and the cleared lysates subjected to gel filtration analysis . Eluted fractions were subjected to TCA precipitation , resuspension in loading buffer containing SDS and DTT , and subsequent immunoblotting against AIFM1 and MIA40 . Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa ( as judged by comparison to protein markers : apoferritin 443 kDa ; β - amylase 200 kDa ; alcohol dehydrogenase 150 kDa ; bovine serum albumin 66 kDa ; carbonic anhydrase 29 kDa ) . Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40 . This behavior is partially rescued by complementation with AIFM1 - HA . B . Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids ( ∆ 1 - 40 MIA40 / CHCHD4 - Strep ) , which constitute the AIFM1 interaction motif . Experiment was performed as described in ( A ) . AIFM1 and endogenous MIA40 / CHCHD4 migrate in a complex with a size larger than 150 kDa , while N - terminally truncated MIA40 / CHCHD4 migrates as monomer . For number of biological replicates and quantifications , see 5C . C . Quantification of 5A , B and Appendix Figure S9 . Quantification of the areas representing the region of monomeric MIA40 / CHCHD4 and the AIFM1 - MIA40 / CHCHD4 complex , respectively was performed . Quantifications consistently show that AIFM1 knockout , siRNA - mediated depletion of AIFM1 and overexpression of MIA40 / CHCHD4 all resulted in an increase of monomeric MIA40 / CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40 / CHCHD4 are tightly regulated . The numbers below the bars indicate the numbers of biological replicates performed . Averages and standard deviations are presented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Gel filtration analysis to assess the AIFM1-MIA40 complex in HEK293 cells. HEK293 cells, cells lacking AIFM1 (AIFM1 knockout, AIFM1 KO) or AIFM1 knockout cells complemented with AIFM1-HA were lysed under native conditions (triton X-100), and the cleared lysates subjected to gel filtration analysis. Eluted fractions were subjected to TCA precipitation, resuspension in loading buffer containing SDS and DTT, and subsequent immunoblotting against AIFM1 and MIA40. Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa (as judged by comparison to protein markers: apoferritin 443 kDa; β-amylase 200 kDa; alcohol dehydrogenase 150 kDa; bovine serum albumin 66 kDa; carbonic anhydrase 29 kDa). Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40. This behavior is partially rescued by complementation with AIFM1-HA. B. Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids (∆1-40 MIA40/CHCHD4-Strep), which constitute the AIFM1 interaction motif. Experiment was performed as described in (A). AIFM1 and endogenous MIA40/CHCHD4 migrate in a complex with a size larger than 150 kDa, while N-terminally truncated MIA40/CHCHD4 migrates as monomer. For number of biological replicates and quantifications, see 5C. C. Quantification of 5A,B and Appendix Figure S9. Quantification of the areas representing the region of monomeric MIA40/CHCHD4 and the AIFM1-MIA40/CHCHD4 complex, respectively was performed. Quantifications consistently show that AIFM1 knockout, siRNA-mediated depletion of AIFM1 and overexpression of MIA40/CHCHD4 all resulted in an increase of monomeric MIA40/CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40/CHCHD4 are tightly regulated. The numbers below the bars indicate the numbers of biological replicates performed. Averages and standard deviations are presented.


D . Levels of AIFM1 and MIA40 / CHCHD4 in mitochondria isolated from the indicated tissues . Ratio of AIFM and MIA40 / CHCHD4 was formed and normalized to ratio in HEK293 cells ( = 1 ) . AIFM1 is in liver and kidney more abundant than MIA40 / CHCHD4 .

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D. Levels of AIFM1 and MIA40/CHCHD4 in mitochondria isolated from the indicated tissues. Ratio of AIFM and MIA40/CHCHD4 was formed and normalized to ratio in HEK293 cells (= 1). AIFM1 is in liver and kidney more abundant than MIA40/CHCHD4.


E . Gel filtration analysis of lysates prepared from mitochondria isolated from the indicated tissues and from HepG2 and HEK293 cells . Experiment was performed as described in ( A ) . AIFM1 and MIA40 / CHCHD4 co - migrate in all samples . Excess AIFM1 migrates at lower molecular mass . Number of biological replicates in quantification plot . Where appropriate , standard deviations are presented .

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E. Gel filtration analysis of lysates prepared from mitochondria isolated from the indicated tissues and from HepG2 and HEK293 cells. Experiment was performed as described in (A). AIFM1 and MIA40/CHCHD4 co-migrate in all samples. Excess AIFM1 migrates at lower molecular mass. Number of biological replicates in quantification plot. Where appropriate, standard deviations are presented.


F . Assessing the solubility of MIA40 / CHCHD4 in the IMS . Mitochondria were isolated form the indicated cell lines and the outer membrane destroyed by hypoxic swelling . MIA40 / CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype cells or AIFM1 knockout cells that had been complemented with AIFM1 - HA . It was released from cells lacking AIFM1 . This indicates that MIA40 / CHCHD4 in wildtype cells is membrane - associated via AIFM1 .

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

F. Assessing the solubility of MIA40/CHCHD4 in the IMS. Mitochondria were isolated form the indicated cell lines and the outer membrane destroyed by hypoxic swelling. MIA40/CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype cells or AIFM1 knockout cells that had been complemented with AIFM1-HA. It was released from cells lacking AIFM1. This indicates that MIA40/CHCHD4 in wildtype cells is membrane-associated via AIFM1.


A . Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132 . HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1 - HA were analysed by pulse - chase experiments coupled to redox state determination . Levels and occurrence of oxidized NDUFB7 was not changed in the presence of MG132 . Conversely , NDUFS5 levels were strongly increased . In AIFM1 knockout cells , large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation - dependent protein import . The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132 . N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5 .

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

A. Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analysed by pulse-chase experiments coupled to redox state determination. Levels and occurrence of oxidized NDUFB7 was not changed in the presence of MG132. Conversely, NDUFS5 levels were strongly increased. In AIFM1 knockout cells, large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation-dependent protein import. The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132. N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5.


The heat map shows the position ( left to right ) , identity ( top row ) and frequency ( color code ) of variant nucleotide positions detected by SARS - CoV - 2 full genome amplicon sequencing in ( A ) 20 samples of MPP - R workers tested positive on May 20 or 25 , ( B ) 15 samples of MPP - R workers tested positive between June 5 and 17 , and ( C ) two workers from MPP - D who may have transmitted the virus to cases B1 and / or B2 . Individual collection dates are shown to the right of each sample . Variant sequences are given relative to the Wuhan reference strain NC _ 045512 . The two silent mutations which define the prototype of the investigated outbreak are marked with an asterisk . Frequencies below 100 % mean that only a fraction of viral genomes shows nucleotide variations , indicating the presence of viral intra - host sub - populations . White rectangles denote nucleotide positions which were not covered by amplicon - seq reads within the respective sample . For nucleotide positions in coding regions , the corresponding viral ORF ( s ) are shown above the variant position . Variants without such information are located in non - coding regions .

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

The heat map shows the position (left to right), identity (top row) and frequency (color code) of variant nucleotide positions detected by SARS-CoV-2 full genome amplicon sequencing in (A) 20 samples of MPP-R workers tested positive on May 20 or 25, (B) 15 samples of MPP-R workers tested positive between June 5 and 17, and (C) two workers from MPP-D who may have transmitted the virus to cases B1 and/or B2. Individual collection dates are shown to the right of each sample. Variant sequences are given relative to the Wuhan reference strain NC_045512. The two silent mutations which define the prototype of the investigated outbreak are marked with an asterisk. Frequencies below 100% mean that only a fraction of viral genomes shows nucleotide variations, indicating the presence of viral intra-host sub-populations. White rectangles denote nucleotide positions which were not covered by amplicon-seq reads within the respective sample. For nucleotide positions in coding regions, the corresponding viral ORF(s) are shown above the variant position. Variants without such information are located in non-coding regions.


( A ) Distance ( in meters ) of PCR - tested workers from the suspected index case B1 at the workplace . For workers without fixed position in the beef processing plant ( marked with an asterisk ) coordinates indicate estimated average location during the early shift . Squares and diamonds denote prototype or variant SARS - CoV - 2 genotypes , respectively . Filled blue circles denote cases for which viral genomes were not sequenced ( i . e . , workers tested positive after 5 / 25 / 2020 ) .

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(A) Distance (in meters) of PCR-tested workers from the suspected index case B1 at the workplace. For workers without fixed position in the beef processing plant (marked with an asterisk) coordinates indicate estimated average location during the early shift. Squares and diamonds denote prototype or variant SARS-CoV-2 genotypes, respectively. Filled blue circles denote cases for which viral genomes were not sequenced (i.e., workers tested positive after 5/25/2020).


( B ) Top panel : Observed accumulated percentage of positive cases ( red line ) within the indicated distance from the suspected index case . The gray dashed line shows the average infection rate that would be expected for a random spatial distribution of positive cases . Bottom panel : - log10 p - value for the frequency of accumulated positive cases within the given distance being significantly higher than expected based on a random spatial distribution of positive cases Only employees with fixed work positions were included in the calculation .

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(B) Top panel: Observed accumulated percentage of positive cases (red line) within the indicated distance from the suspected index case. The gray dashed line shows the average infection rate that would be expected for a random spatial distribution of positive cases. Bottom panel: -log10 p-value for the frequency of accumulated positive cases within the given distance being significantly higher than expected based on a random spatial distribution of positive cases Only employees with fixed work positions were included in the calculation.


Immunoblot analysis of proteins biotinylated and isolated as in ( A ) . Parental HeLa - T - REx cells or cells expressing H3 . 1 - BirA * - HA and treated with biotin for indicated times .

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Immunoblot analysis of proteins biotinylated and isolated as in (A). Parental HeLa-T-REx cells or cells expressing H3.1-BirA*-HA and treated with biotin for indicated times.


Immunoblot analysis of TAP - H3 . 1 or - CENP - A pulldown of UBR7 - LAP ( schematic , upper ) . 293T cells were co - transfected with UBR7 - LAP and TAP - tagged constructs , which were purified on IgG - conjugated sepharose beads .

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Immunoblot analysis of TAP-H3.1 or -CENP-A pulldown of UBR7-LAP (schematic, upper). 293T cells were co-transfected with UBR7-LAP and TAP-tagged constructs, which were purified on IgG-conjugated sepharose beads.


Immunoblot of anti - FLAG IP of 3x - FLAG tagged GFP , or FLAG - HA tagged H3 . 1 or H2B , from the non - nucleosomal fraction of 293T cells .

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Immunoblot of anti-FLAG IP of 3x-FLAG tagged GFP, or FLAG-HA tagged H3.1 or H2B, from the non-nucleosomal fraction of 293T cells.


Representative images of HeLa cells transfected with UBR7 - FL . DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green . The area within yellow dotted box in ( E ) is magnified 4x in ( E ' ) . Scale bar = 5 μm . Quantification of the nuclear anti - FLAG immunofluorescence intensity in ( E ) . Error bars represent mean + / - standard deviation . One way ANOVA was used to calculate p - values ( italics ) associated with differences between treatments . Experiment in ( E - F ) was performed twice .

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Representative images of HeLa cells transfected with UBR7-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. The area within yellow dotted box in (E) is magnified 4x in (E'). Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (E). Error bars represent mean +/- standard deviation. One way ANOVA was used to calculate p-values (italics) associated with differences between treatments. Experiment in (E-F) was performed twice.


Immunoblot analysis of anti - FLAG pulldown of full length UBR7 - FLAG ( residues 1 - 425 ) mutant UBR7 bearing deletion of UBR7 C - terminus ( ∆ C - term , residues 1 - 215 ) , UBR box ( ∆ UBR , residues 117 - 425 ) , UBR box and PHD ( ∆ UBR ∆ PHD , residues 216 - 425 ) , or PHD and C - terminus ( ∆ PHD ∆ C - term , residues 1 - 116 ) . 293T cells were co - transfected with H3 . 1 - HA and UBR7 - FLAG constructs ( schematic , upper ) . Asterisk represents non - specific band . Numbers below blot indicate FLAG - normalized anti - HA immunoblot signal relative to full length UBR7 - FLAG .

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Immunoblot analysis of anti-FLAG pulldown of full length UBR7-FLAG (residues 1-425) mutant UBR7 bearing deletion of UBR7 C-terminus (∆C-term, residues 1-215), UBR box (∆UBR, residues 117-425), UBR box and PHD (∆UBR∆PHD, residues 216-425), or PHD and C-terminus (∆PHD∆C-term, residues 1-116). 293T cells were co-transfected with H3.1-HA and UBR7-FLAG constructs (schematic, upper). Asterisk represents non-specific band. Numbers below blot indicate FLAG-normalized anti-HA immunoblot signal relative to full length UBR7-FLAG.


Immunoblot analysis of anti - H3 . 1 - HA pulldown of full - length UBR7 - LAP and UBR7PHD fragment ( residues 117 - 215 ) ( schematic , upper ) . 293T cells were transfected as in ( A ) . Values below pulldown represent H3 . 1 - HA - normalized GFP - UBR7 immunoblot signal relative to UBR7 - FL . Data were acquired from a continuous membrane , and the image is cropped to omit irrelevant samples . Numbers below blot indicate HA - normalized anti - GFP immunoblot signal relative to full length UBR7 - LAP .

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Immunoblot analysis of anti-H3.1-HA pulldown of full-length UBR7-LAP and UBR7PHD fragment (residues 117-215) (schematic, upper). 293T cells were transfected as in (A). Values below pulldown represent H3.1-HA-normalized GFP-UBR7 immunoblot signal relative to UBR7-FL. Data were acquired from a continuous membrane, and the image is cropped to omit irrelevant samples. Numbers below blot indicate HA-normalized anti-GFP immunoblot signal relative to full length UBR7-LAP.


Anti - GFP immunoblot analysis of streptavidin pulldown of the indicated biotin - conjugated H3 peptides following incubation with 293T - GFP - UBR7 cell lysate after mutation of residues highlighted in ( C ) .

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Anti-GFP immunoblot analysis of streptavidin pulldown of the indicated biotin-conjugated H3 peptides following incubation with 293T-GFP-UBR7 cell lysate after mutation of residues highlighted in (C).


Immunoblot analysis for total and modified histones in anti - FLAG pulldown of constructs from soluble cellular fractions demonstrates that UBR7 UBR box and PHD are necessary for binding to post - nucleosomal H3 / H4 .

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

Immunoblot analysis for total and modified histones in anti-FLAG pulldown of constructs from soluble cellular fractions demonstrates that UBR7 UBR box and PHD are necessary for binding to post-nucleosomal H3/H4.


Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in ( G ) . 4 . 5 h represent newly synthesized histones and 48 h represent pre - existing histones . Wedge represents loading titration : 1x > 0 . 1x > 0 . 01x > 0 . 001x . All other lanes are loaded at 1x concentration . Immunoblot signals were normalized to anti - FLAG signal . Quantification of anti - FLAG ( H3 . 1 - APEX2 - 3xFLAG ) and anti - UBR7 immunoblot represented in ( G - H ) . Lanes 8 and 11 were quantified . Error bar represents mean + / - standard deviation . A two - tailed one - sample t - test was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed line ) .

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Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in (G). 4.5 h represent newly synthesized histones and 48 h represent pre-existing histones. Wedge represents loading titration: 1x > 0.1x > 0.01x > 0.001x. All other lanes are loaded at 1x concentration. Immunoblot signals were normalized to anti-FLAG signal.   Quantification of anti-FLAG (H3.1-APEX2-3xFLAG) and anti-UBR7 immunoblot represented in (G-H). Lanes 8 and 11 were quantified. Error bar represents mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line).


Immunoblot analysis for total and modified H3 and H4 in anti - FLAG IP . Experiment was performed as in ( D ) . Dot plot error bars represent mean + / - standard deviation . Immunoblot signals were normalized to anti - H4 signal . A two - tailed one sample t - test was used to calculate p - value associated with group mean difference from a hypothetical value of 1 . 0 . Experiment was performed twice .

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Immunoblot analysis for total and modified H3 and H4 in anti-FLAG IP. Experiment was performed as in (D). Dot plot error bars represent mean +/- standard deviation. Immunoblot signals were normalized to anti-H4 signal. A two-tailed one sample t-test was used to calculate p-value associated with group mean difference from a hypothetical value of 1.0. Experiment was performed twice.


Representative images of HeLa cells transfected with sNASP - FL . DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green . Scale bar = 5 μm . Quantification of the nuclear anti - FLAG immunofluorescence intensity in ( G ) demonstrating that sNASP - FL is highly susceptible to pre - extraction with Triton X - 100 . One way ANOVA was used to determine p - values ( italics ) associated with differences between treatments . Error bars represent mean + / - standard deviation . Experiment in ( G - H ) was performed twice .

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

Representative images of HeLa cells transfected with sNASP-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (G) demonstrating that sNASP-FL is highly susceptible to pre-extraction with Triton X-100. One way ANOVA was used to determine p-values (italics) associated with differences between treatments. Error bars represent mean +/- standard deviation. Experiment in (G-H) was performed twice.


A , C . Immunoblot analysis for modified histone in anti - FLAG pulldown of UBR7 - FL ( A ) or sNASP - FL ( C ) from non - nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors ( hydroxyurea , aphidicolin ) or transcription inhibitors ( α - amanitin , triptolide ) . B , D . Plots represent either normalized immunoblot signals or cell counts prior to fractionation . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation , with grey dotted line marking mean of No Treat condition . A two - tailed one sample t - test was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 . Experiments were performed three times . Cells numbers were counted in two of the three experiments .

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

A,C. Immunoblot analysis for modified histone in anti-FLAG pulldown of UBR7-FL (A) or sNASP-FL (C) from non-nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors (hydroxyurea, aphidicolin) or transcription inhibitors (α-amanitin, triptolide). B,D. Plots represent either normalized immunoblot signals or cell counts prior to fractionation. Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation, with grey dotted line marking mean of No Treat condition. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiments were performed three times. Cells numbers were counted in two of the three experiments.


Immunoblot analysis of H3 , UBR7 , and GAPDH in the non - nucleosomal fraction of parental and 293T - UBR7KO cells . Quantification of anti - H3 immunoblot represented in ( A ) . Immunoblot signals were normalized to anti - GAPDH signal . Error bars represent mean + / - standard deviation . A two - tailed one - sample t - test was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed line ) . Experiment in ( A - B ) was performed in three times .

Immunoblot analysis of H3, UBR7, and GAPDH in the non-nucleosomal fraction of parental and 293T-UBR7KO cells.   Quantification of anti-H3 immunoblot represented in (A). Immunoblot signals were normalized to anti-GAPDH signal. Error bars represent mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (A-B) was performed in three times.


Immunoblot analysis of anti - FLAG pulldown of sNASP - FL in parental or 293T - UBR7KO cells . 3xFLAG tagged sNASP - FL was immunoprecipitated from non - nucleosomal fraction and eluted with FLAG peptide . Experiment was performed three times . Quantification of anti - FLAG ( sNASP - FL ) , and anti - histone immunoblots represented in ( C ) . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation . A two - tailed one sample t - test was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed line ) . Experiments were performed two ( sNASP , H4 ) to three ( all other targets ) times .

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

Immunoblot analysis of anti-FLAG pulldown of sNASP-FL in parental or 293T-UBR7KO cells. 3xFLAG tagged sNASP-FL was immunoprecipitated from non-nucleosomal fraction and eluted with FLAG peptide. Experiment was performed three times.   Quantification of anti-FLAG (sNASP-FL), and anti-histone immunoblots represented in (C). Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments were performed two (sNASP, H4) to three (all other targets) times.


Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7 - FL ( left ) or sNASP - FL ( right ) following treatment with siCTRL , siLAMP2A , or MG - 132 .

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

Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7-FL (left) or sNASP-FL (right) following treatment with siCTRL, siLAMP2A, or MG-132.


Quantification of data represented in ( C , D ) . Error bars represent mean + / - standard deviation . A two - tailed one sample t - test was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed line ) . Experiments in ( C - E ) were performed three times .

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

Quantification of data represented in (C, D). Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments in (C-E) were performed three times.


Heatmaps of ATAC - seq , anti - GFP ( GFP - UBR7 ) ChIP - seq , and anti - H3K4me3 CUT & RUN in parental and UBR7KO cells . Plots are centered on TSS that show changes in ATAC - seq signal in UBR7KO versus parental cells . Sites are clustered by unsupervised k - means analysis . Cluster color key is defined by cluster labels .

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

Heatmaps of ATAC-seq, anti-GFP (GFP-UBR7) ChIP-seq, and anti-H3K4me3 CUT&RUN in parental and UBR7KO cells. Plots are centered on TSS that show changes in ATAC-seq signal in UBR7KO versus parental cells. Sites are clustered by unsupervised k-means analysis. Cluster color key is defined by cluster labels.


F , Chow fed hIGFREO had similar body mass to WT , however , hIGFREO did not gain as much weight as WT after 8 weeks of HFD ( n = 6 - 10 mice per group ) .

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

F, Chow fed hIGFREO had similar body mass to WT, however, hIGFREO did not gain as much weight as WT after 8 weeks of HFD (n=6-10 mice per group).


G , Representative images of difference in fat and water distribution shown by magnetic resonance ( MR ) imaging in hIGFREO and WT . Scale bar = 1cm .

O O O O O 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, Representative images of difference in fat and water distribution shown by magnetic resonance (MR) imaging in hIGFREO and WT. Scale bar = 1cm.


hIGFREO glucose intolerance measured by glucose tolerance test ( n = 5 - 7 mice per group ) .

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

hIGFREO glucose intolerance measured by glucose tolerance test (n=5-7 mice per group).


G , H , Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve ( n = 10 - 12 mice per group ) .

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

G, H, Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve (n=10-12 mice per group).


hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates ( WT ) after HFD . ( n = 4 per genotype ) .

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

hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates (WT) after HFD. (n=4 per genotype).


D , Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT ( n = 6 per genotype ) .

O O O O O O O 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, Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT (n= 6 per genotype).


Faith ' s phylogenetic diversity ( PD ) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF - 1R endothelial overexpressing mice ( hIGFREO ) mice and wildtype littermates ( WT ) mice after high fat diet feeding ( n = 4 - 5 mice per group ) . Chao - 1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT ( n = 4 - 5 mice per group ) .

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

Faith's phylogenetic diversity (PD) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF-1R endothelial overexpressing mice (hIGFREO) mice and wildtype littermates (WT) mice after high fat diet feeding (n=4-5 mice per group). Chao-1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT (n=4-5 mice per group).


B , Bi , human IGF - 1R endothelial overexpressing mice ( hIGFREO ) had comparable weight gain as wildtype littermates ( WT ) after 8 weeks of HFD + antibiotics ( ABs ) when compared to WT . Bii , both hIGFREO and WT gained significant weight compared to chow fed mice ( n = 7 = 9 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Bi, human IGF-1R endothelial overexpressing mice (hIGFREO) had comparable weight gain as wildtype littermates (WT) after 8 weeks of HFD + antibiotics (ABs) when compared to WT. Bii, both hIGFREO and WT gained significant weight compared to chow fed mice (n=7=9 mice per group).


D , E , There was no difference in hIGFREO and WT glucose tolerance ( as measured by glucose tolerance test and area under the curve ( AUC ) ) ( n = 7 - 9 mice per group ) .

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

D, E, There was no difference in hIGFREO and WT glucose tolerance (as measured by glucose tolerance test and area under the curve (AUC)) (n=7-9 mice per group).


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . ) .

O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) .

O O O O O O O O O O O O O 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. 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).


B . 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 .

O O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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.


A . 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 .

A. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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.


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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.


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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).


( 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 .

O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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).


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


( 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 .

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(E) Western blots detecting pVP30, VP30 and NP levels in EBOV infected HeLa cells upon hnRNP L, hnRNPUL1 or PEG10 knockdown.


B . 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 ) .

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

B. 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 ) .

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


B . 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 .

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


A - 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 .

O O O O O O O O O O O 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-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 .

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

C. Oxidized mitochondria with the radioactive double-cysteine Bax protein were prepared, subjected to Na2CO3 extraction and analyzed as in Figure 1D.


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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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


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


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


B and D . The oxidized mitochondria with radioactive single - cysteine Bax proteins were prepared and analyzed as in Figure 1B .

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

B 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 .

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

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.


B - 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 .

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

B-D. 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.


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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.


A - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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.


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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.


A . 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 .

A. 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 .

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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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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.


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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.


MCIDAS ( 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 .

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MCIDAS (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 .

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

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 ) .

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

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.


A . 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 .

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A. 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 .

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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.


B . GemC1KO / KO mice compared to their wild type littermates ( GemC1KO / WT ) at P5 .

B. 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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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.