( B ) The response of a mitochondrial matrix - localized roGFP2 - Prx1 - P233stop probe expressed in wild - type cells to the indicated concentrations of exogenous H2O2 . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Error bars represent the standard deviation ( n = 3 biological replicates , with cells obtained from 3 independent cultures for every strain and probe combinations . For each biological replicate three technical replicates were performed ) .

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(B) The response of a mitochondrial matrix-localized roGFP2-Prx1-P233stop probe expressed in wild-type cells to the indicated concentrations of exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).


( C ) Maleimide - based gel - shift assay to assess Prx1 cysteine redox state . Wild - type cells transformed with an empty plasmid or ∆ prx1 cells with either an empty plasmid or a plasmid encoding wild - type Prx1 , the hyperoxidation - resistant Prx1 - P233stop or the inactive Prx1 - C91A variants , were grown to exponential phase in SGal ( - Ura ) medium . Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins and then directly treated with the alkylating agent mmPEG24 .

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(C) Maleimide-based gel-shift assay to assess Prx1 cysteine redox state. Wild-type cells transformed with an empty plasmid or ∆prx1 cells with either an empty plasmid or a plasmid encoding wild-type Prx1, the hyperoxidation-resistant Prx1-P233stop or the inactive Prx1-C91A variants, were grown to exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins and then directly treated with the alkylating agent mmPEG24.


( D ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 probe , expressed in Δprx1 cells containing a plasmid encoding either wild - type Prx1 , Prx1 - P233stop or Prx1C91A to the addition of 1 mM exogenous H2O2 . Cells had been pre - treated with either 10 mM H2O2 or with water as a control . For these experiments , cells were grown in SGal ( - Leu , - Ura ) medium and harvested at early exponential phase . OxD refers to the degree of sensor oxidation . Error bars represent the standard deviation ( n = 3 biological replicates , with cells obtained from 3 independent cultures for every strain and probe combinations . For each biological replicate three technical replicates were performed ) .

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(D) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe, expressed in Δprx1 cells containing a plasmid encoding either wild-type Prx1, Prx1-P233stop or Prx1C91A to the addition of 1 mM exogenous H2O2. Cells had been pre-treated with either 10 mM H2O2 or with water as a control. For these experiments, cells were grown in SGal (-Leu, -Ura) medium and harvested at early exponential phase. OxD refers to the degree of sensor oxidation. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).


( E ) Hydrogen peroxide ' acute stress ' assay . Δprx1 cells co - transformed with empty vector or with a vector encoding either wild - type Prx1 , the P233stop or the C91A variants were grown in SD ( - Ura ) and treated with the indicated concentrations of H2O2 for 30 mins . Afterwards , the cells were diluted and a fixed volume plated on YPD plates . The number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a percentage relative to the 0 mM treatment . Error bars represent standard deviation ( n = 5 - 8 biological replicates , with cells taken from independent cultures for each individual biological replicate ) .

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(E) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with empty vector or with a vector encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SD (-Ura) and treated with the indicated concentrations of H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 5-8 biological replicates, with cells taken from independent cultures for each individual biological replicate).


( A ) Hydrogen peroxide ' acute stress ' assay . Δprx1 cells co - transformed with an empty plasmid or a plasmid encoding Prx1 , Tsa1 , PRDX3 , PRDX5 , PRDX6 , PfAOP or PfAOP - L109M were grown in SD medium lacking the appropriate amino acids for plasmid selection and pre - treated with 0 , 1 , 5 , 10 , 25 mM H2O2 for 30 mins . Afterwards , the cells were diluted and a fixed volume was plated on YPD plates . The number of viable colonies was counted after 2 days of growth at 30 ° C , here represented as a percentage relative to the 0 mM pre - treatment . Error bars represent standard deviation ( n = 3 - 8 biological replicates , with cells taken from independent cultures for each individual biological replicate ) . Significance was assessed with a Student ' s 2 - tailed , unpaired , t - test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(A) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with an empty plasmid or a plasmid encoding Prx1, Tsa1, PRDX3, PRDX5, PRDX6, PfAOP or PfAOP-L109M were grown in SD medium lacking the appropriate amino acids for plasmid selection and pre-treated with 0, 1, 5, 10, 25 mM H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume was plated on YPD plates. The number of viable colonies was counted after 2 days of growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3-8 biological replicates, with cells taken from independent cultures for each individual biological replicate). Significance was assessed with a Student's 2-tailed, unpaired, t-test. *p < 0.05; **p < 0.01; ***p < 0.001.


( B ) The efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with cell death upon exposure to acute H2O2 stress . Data from viability and matrix Grx1 - roGFP2 responses during acute H2O2 stress were correlated .

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(B) The efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with cell death upon exposure to acute H2O2 stress. Data from viability and matrix Grx1-roGFP2 responses during acute H2O2 stress were correlated.


( A ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 probe to 1 mM H2O2 . in BY4742 wild - type cells with an empty vector , in Δglr1 cells transformed either with an empty vector or a vector encoding wild - type Glr1 or the cytosolic form of Glr1 , where the MTS - encoding region was removed , or in Δglr1Δprx1 cells transformed either with an empty vector or a vector encoding the cytosolic form of Glr1 . Cells were grown to exponential phase in SGal ( - Leu , - Ura ) medium . Error bars represent the standard deviation ( n = 3 biological replicates , with cells obtained from 3 independent cultures for every strain and probe combinations . For each biological replicate three technical replicates were performed ) .

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(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe to 1 mM H2O2. in BY4742 wild-type cells with an empty vector, in Δglr1 cells transformed either with an empty vector or a vector encoding wild-type Glr1 or the cytosolic form of Glr1, where the MTS-encoding region was removed, or in Δglr1Δprx1 cells transformed either with an empty vector or a vector encoding the cytosolic form of Glr1. Cells were grown to exponential phase in SGal (-Leu, -Ura) medium. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).


( B ) Growth curve of wild - type , Δprx1 , Δglr1 and Δglr1Δprx1 cells in SD medium complemented with all amino acids ( n = 3 biological replicates ) . Significance for the difference in the time the cultures reach 50 % of their maximal OD600 was assessed with the t - test . Error bars ( as ribbon ) represent the standard deviation ( n = 4 biological replicates , with cells taken from independent cultures for each individual biological replicate ) .

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(B) Growth curve of wild-type, Δprx1, Δglr1 and Δglr1Δprx1 cells in SD medium complemented with all amino acids (n = 3 biological replicates). Significance for the difference in the time the cultures reach 50% of their maximal OD600 was assessed with the t-test. Error bars (as ribbon) represent the standard deviation (n = 4 biological replicates, with cells taken from independent cultures for each individual biological replicate).


( C ) H2O2 ' acute stress ' assay . Wild - type , Δprx1 , Δglr1 and Δglr1Δprx1 cells pre - grown in SGal medium complemented with all amino acids to early exponential phase . Cells were treated with H2O2 at the indicated concentrations for 30 mins . Subsequently cells were diluted , and a fixed volume plated on YPD plates . The number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a percentage relative to the 0 mM pre - treatment . Error bars represent standard deviation ( n = 3 biological replicates , with cells taken from independent cultures for each individual biological replicate ) . An inset with a different scale on the y - axis is presented for the 1 and 5 mM concentration to allow better interpretation .

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(C) H2O2 'acute stress' assay. Wild-type, Δprx1, Δglr1 and Δglr1Δprx1 cells pre-grown in SGal medium complemented with all amino acids to early exponential phase. Cells were treated with H2O2 at the indicated concentrations for 30 mins. Subsequently cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate). An inset with a different scale on the y-axis is presented for the 1 and 5 mM concentration to allow better interpretation.


( D ) H2O2 ' acute stress ' assay . Δprx1Δglr1 cells co - transformed with an empty plasmid or with a plasmid encoding either wild - type Prx1 , the P233stop or the C91A variants were grown in SGal ( - Ura ) medium to early exponential phase . Cells were treated with the indicated amounts of H2O2 for 30 mins . Subsequently , the cells were diluted , and a fixed volume plated on YPD plates . The number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a percentage relative to the 0 mM treatment . Error bars represent standard deviation ( n = 3 biological replicates , with cells taken from independent cultures for each individual biological replicate ) .

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(D) H2O2 'acute stress' assay. Δprx1Δglr1 cells co-transformed with an empty plasmid or with a plasmid encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SGal (-Ura) medium to early exponential phase. Cells were treated with the indicated amounts of H2O2 for 30 mins. Subsequently, the cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate).


( F ) Comparison of protein degradation efficiency using different degrons . Cells were incubated for indicated time periods with Asv or DOX / IAA or both ( DIA , see methods ) then collected for western blot analysis . Doxycycline was added 2 hours before addition of IAA or Asv / IAA . To determine protein degradation , either cyclin A2 or cyclin B1 were probed as indicated . Anti - myc antibody was used to check OsTIR1 - myc expression . Asterisks indicate non - specific protein crossreacting with the cyclin B1 antibody .

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(F) Comparison of protein degradation efficiency using different degrons. Cells were incubated for indicated time periods with Asv or DOX/IAA or both (DIA, see methods) then collected for western blot analysis. Doxycycline was added 2 hours before addition of IAA or Asv/IAA. To determine protein degradation, either cyclin A2 or cyclin B1 were probed as indicated. Anti-myc antibody was used to check OsTIR1-myc expression. Asterisks indicate non-specific protein crossreacting with the cyclin B1 antibody.


( G ) Timecourse of DIA induced cyclin A and B degradation , cross checking levels of each mitotic cyclin . Treatments were performed as for DIA treated Cell extracts were checked for both cyclin A2 and B1 levels following DIA

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(G) Timecourse of DIA induced cyclin A and B degradation, cross checking levels of each mitotic cyclin. Treatments were performed as for DIA treated Cell extracts were checked for both cyclin A2 and B1 levels following DIA addition


A ) Cyclin B2 knock - out and induced degradation by immunoblotting . Indicated cell lines were analysed 24 hours after mock or Dox / IAA / Asv ( DIA ) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation . (

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A) Cyclin B2 knock-out and induced degradation by immunoblotting. Indicated cell lines were analysed 24 hours after mock or Dox/IAA/Asv (DIA) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation. (


( D ) Cell proliferation of A2dd , CycB1dd and B1dd / B2ko following mock or DIA treatment . 1000 cells were plated in each well ( diameter 3 . 5cm ) and incubated for 10 days before methanol fixation and Crystal Violet staining .

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(D) Cell proliferation of A2dd, CycB1dd and B1dd/B2ko following mock or DIA treatment. 1000 cells were plated in each well (diameter 3.5cm) and incubated for 10 days before methanol fixation and Crystal Violet staining.


( E ) Kinetics of mitotic entry as measured by time lapse microscopy in A2dd and B1dd / B2ko cells following mock or four - hour DIA treatment of asynchronous cells . The cells were imaged for 16h with 5min intervals using widefield DIC , mitotic entry was manually scored by detecting cell rounding . Curves display the cumulative mitotic index ( data from three repeats , n > 500 cells per condition , s . d . indicated by shaded area ) .

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(E) Kinetics of mitotic entry as measured by time lapse microscopy in A2dd and B1dd/B2ko cells following mock or four-hour DIA treatment of asynchronous cells. The cells were imaged for 16h with 5min intervals using widefield DIC, mitotic entry was manually scored by detecting cell rounding. Curves display the cumulative mitotic index (data from three repeats, n>500 cells per condition, s. d. indicated by shaded area).


( I ) Cyclin A2 siRNA depletion causes endoreplication . Following 72 hours of siRNA transfection MCF7 and MCF10A cells were analysed by PI staining and FACS . The histograms show the changes in DNA content ( PI Int . ) towards > 4N following cyclin A2 depletion .

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(I) Cyclin A2 siRNA depletion causes endoreplication. Following 72 hours of siRNA transfection MCF7 and MCF10A cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards >4N following cyclin A2 depletion.


( J ) Cyclin A2 siRNA and degron depletion in RPE - 1 cells . RPE - 1 OsTIR1 and RPE - 1 A2dd cells were subjected to 72 hours of cyclin A2 siRNA depletion and / or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting . The longer exposure ( L . E . ) reveals incomplete depletion of cyclin 2 by siRNA .

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(J) Cyclin A2 siRNA and degron depletion in RPE-1 cells. RPE-1 OsTIR1 and RPE-1 A2dd cells were subjected to 72 hours of cyclin A2 siRNA depletion and/or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting. The longer exposure (L.E.) reveals incomplete depletion of cyclin 2 by siRNA.


( C ) Representative images from live - cell imaging of SiR - Tubulin labelled ( red ) mitotic B1dd / B2ko cells expressing FusionRed - H2B ( green ) , Mis12 - GFP ( white ) ; time in mins , scale bar = 5µm . Cells were imaged four hours after DIA treatment .

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(C) Representative images from live-cell imaging of SiR-Tubulin labelled (red) mitotic B1dd/B2ko cells expressing FusionRed-H2B (green), Mis12-GFP (white); time in mins, scale bar = 5µm. Cells were imaged four hours after DIA treatment.


( F ) Cyclin A2 levels were assessed by immunoblotting after 18 hours in 100ng / ml Nocodazole and / or 5µM STLC . 6µM proTAME was added to the indicated samples for the final six hours .

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(F) Cyclin A2 levels were assessed by immunoblotting after 18 hours in 100ng/ml Nocodazole and/or 5µM STLC. 6µM proTAME was added to the indicated samples for the final six hours.


( G ) Upper panel , confirmation of Cdc27 depletion by immunoblotting 72 hours after siRNA transfection in B1dd / B2ko cells . Lower panel , following 40 - hour siRNA transfection B1dd / B2ko cells were treated for 24 hours with Thymidine , released for 10 hours and treated with proTAME and Apcin for additional four hours . At this point , the cells were fixed and stained with tubulin and pericentrin for immunofluorescence analysis .

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(G) Upper panel, confirmation of Cdc27 depletion by immunoblotting 72 hours after siRNA transfection in B1dd/B2ko cells. Lower panel, following 40-hour siRNA transfection B1dd/B2ko cells were treated for 24 hours with Thymidine, released for 10 hours and treated with proTAME and Apcin for additional four hours. At this point, the cells were fixed and stained with tubulin and pericentrin for immunofluorescence analysis.


( H ) Representative images of mitotic spindles in control - and DIA - treated B1dd / B2ko cells following arrest in mitosis by proTAME / Apcin treatment Tubulin staining is shown in white , pericentrin in green and DAPI in red , scale bar = 5µm .

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(H) Representative images of mitotic spindles in control- and DIA-treated B1dd/B2ko cells following arrest in mitosis by proTAME/Apcin treatment Tubulin staining is shown in white, pericentrin in green and DAPI in red, scale bar = 5µm.


( L ) Immunofluorescence images from mitotic Ctr and DIA treated B1dd / B2ko cells after P / A synchronisation stained with anti - alpha - tubulin ( green ) , anti - gamma - tubulin ( red ) antibodies and DAPI ( blue ) , scale bar = 5µm . Cells were exposed to ice - cold medium and either fixed immediately after cold exposure , or incubated for 5 , 10 , or 20 minutes at 37 ° C before fixation .

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(L) Immunofluorescence images from mitotic Ctr and DIA treated B1dd/B2ko cells after P/A synchronisation stained with anti-alpha-tubulin (green), anti-gamma-tubulin (red) antibodies and DAPI (blue), scale bar = 5µm. Cells were exposed to ice-cold medium and either fixed immediately after cold exposure, or incubated for 5,10, or 20 minutes at 37° C before fixation.


( A ) Time - lapse images of PCNA - mRuby tagged A2dd cells . The imaging sequence was started at the time of Doxycycline addition , and degron activity was triggered three hours later by addition of IAA and Asv or PBS ( indicated by dashed line ) . Time is shown as hh : min , scale bar equals 10µm . For further analysis cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase .

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(A) Time-lapse images of PCNA-mRuby tagged A2dd cells. The imaging sequence was started at the time of Doxycycline addition, and degron activity was triggered three hours later by addition of IAA and Asv or PBS (indicated by dashed line). Time is shown as hh:min, scale bar equals 10µm. For further analysis cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase.


( D ) Images from time - lapse sequence of mitosis showing cell division in controls and after addition of 0 . 5µM PD - 166285 in DIA treated A2dd labelled with SiR - DNA ( time is indicated as hh : min , the scale bars represent 20µm ) .

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(D) Images from time-lapse sequence of mitosis showing cell division in controls and after addition of 0.5µM PD-166285 in DIA treated A2dd labelled with SiR-DNA (time is indicated as hh:min, the scale bars represent 20µm).


( D ) Cyclin A2dd cells with inducible CycB1 - YFP ( B1 - WT ) and CycB1 - YFP - NLS ( B1 - NLS ) were analysed for DIA induced cyclin B1 expression / cyclin A2 depletion . Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and cyclin A2

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(D) Cyclin A2dd cells with inducible CycB1-YFP (B1-WT) and CycB1-YFP-NLS (B1-NLS) were analysed for DIA induced cyclin B1 expression / cyclin A2 depletion. Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and cyclin A2 antibodies


( I ) Immunoprecipitation of YFP from extract from A2dd following induction of YFP ( Y ) , CycB1 - YFP ( BY ) and CycB1 - YFP - NLS ( BYN ) . The samples in the odd lanes ( IAA / Asv - ) were treated with 1µg / ml doxycycline alone , while samples in even lanes ( IAA / Asv + ) were treated with Auxin and Asv to induce cyclin A2 depletion . Total cell extract and immune - precipitates were probed by immunoblotting with the indicated antisera .

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(I) Immunoprecipitation of YFP from extract from A2dd following induction of YFP (Y), CycB1-YFP (BY) and CycB1-YFP-NLS (BYN). The samples in the odd lanes (IAA/Asv-) were treated with 1µg/ml doxycycline alone, while samples in even lanes (IAA/Asv+) were treated with Auxin and Asv to induce cyclin A2 depletion. Total cell extract and immune-precipitates were probed by immunoblotting with the indicated antisera.


( E ) Quantification of changes in selected cyclin B - dependent mitotic phosphorylations including Ki - 67 ( left panel ) , INCENP / Borealin ( middle panel ) and Top2B , CDCA2 and Tpx2 .

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(E) Quantification of changes in selected cyclin B-dependent mitotic phosphorylations including Ki-67 (left panel), INCENP/Borealin (middle panel) and Top2B, CDCA2 and Tpx2.


( F ) Representative images of Ki - 67 immunofluorescent staining of chromosome spreads from control ( Ctr ) and DIA treated P / A synchronised B1dd / B2ko cells and quantification of Ki - 67 staining intensity using cross - sections of

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(F) Representative images of Ki-67 immunofluorescent staining of chromosome spreads from control (Ctr) and DIA treated P/A synchronised B1dd/B2ko cells and quantification of Ki-67 staining intensity using cross-sections of chromosomes


( G ) Representative images from live - cell imaging of DIA - treated SiR - Tubulin labelled ( red ) B1dd / B2ko cells expressing FusionRed - H2B ( green ) and AurB - GFP ( white ) ; time is indicated in minutes , scale bar = 5µm . Bottom panel shows frequencies of aberrant AurB localisation in P / A synchronised B1dd / B2ko cells .

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(G) Representative images from live-cell imaging of DIA-treated SiR-Tubulin labelled (red) B1dd/B2ko cells expressing FusionRed-H2B (green) and AurB-GFP (white); time is indicated in minutes, scale bar = 5µm. Bottom panel shows frequencies of aberrant AurB localisation in P/A synchronised B1dd/B2ko cells.


( J ) Tpx2 shows reduced spread on mitotic chromosomes in DIA treated B1dd / B2ko cells . The top panel shows immunofluorescence images , for quantification we measured the mean intensity of Tpx2 staining on the centrosomes and spindle and plotted the ratio of these values per cell . We also analysed the centrosome / spindle distribution of AurA that does not change following DIA treatment .

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(J) Tpx2 shows reduced spread on mitotic chromosomes in DIA treated B1dd/B2ko cells. The top panel shows immunofluorescence images, for quantification we measured the mean intensity of Tpx2 staining on the centrosomes and spindle and plotted the ratio of these values per cell. We also analysed the centrosome/spindle distribution of AurA that does not change following DIA treatment.


( B ) Confirmation of ENSA / ARPP19 S67 phosphorylation in mitotic extracts from Ctr and DIA treated cells , before and after ENSA / ARPP19 siRNA depletion .

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(B) Confirmation of ENSA/ARPP19 S67 phosphorylation in mitotic extracts from Ctr and DIA treated cells, before and after ENSA/ARPP19 siRNA depletion.


( E ) Dynamics of Greatwall , PP1 , and Cdc27 dephosphorylation following Cdk1 inhibition in proTAME / Apcin treated mitotic cell extracts from Ctr or DIA treated cells . Cell extracts were prepared at the indicated times following treatment with 1µM Flavopiridol and analysed by

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(E) Dynamics of Greatwall, PP1, and Cdc27 dephosphorylation following Cdk1 inhibition in proTAME/Apcin treated mitotic cell extracts from Ctr or DIA treated cells. Cell extracts were prepared at the indicated times following treatment with 1µM Flavopiridol and analysed by immunoblotting


A ) Representative images from live - cell imaging of siRNA transfected B1dd / B2ko cells ( FusionRed - H2B in red , SiR - Tubulin in white , scale bar = 10µm ) . Bar - plot panels on the right show single - cell analysis of 10 cells manually scored for length mitosis pre - NEBD and post - NEBD . Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus .

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A) Representative images from live-cell imaging of siRNA transfected B1dd/B2ko cells (FusionRed-H2B in red, SiR-Tubulin in white, scale bar = 10µm). Bar-plot panels on the right show single-cell analysis of 10 cells manually scored for length mitosis pre-NEBD and post-NEBD. Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus.


( B ) Widefield Imaging . DIC ( Grey ) , SiR - DNA ( b / w ) , of ENSA / ARPP19 siRNA transfected DIA treated B1dd / B2ko cells . Time is indicated in hh : min , scale bar = 10µm .

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(B) Widefield Imaging. DIC (Grey), SiR-DNA (b/w), of ENSA/ARPP19 siRNA transfected DIA treated B1dd/B2ko cells. Time is indicated in hh:min, scale bar = 10µm.


( C ) Panels of representative immunofluorescence images of siRNA transfected and P / A treated cells treated as indicated , scale bar = 10µm .

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(C) Panels of representative immunofluorescence images of siRNA transfected and P/A treated cells treated as indicated, scale bar = 10µm.


( B ) High - content screening ( HCS ) of 5 , 406 compounds in two batches in duplicate and calculation of Z ' - factors between high ( MERS - CoV infection , black ) and low ( mock , green ) values .

(B) High-content screening (HCS) of 5,406 compounds in two batches in duplicate and calculation of Z'-factors between high (MERS-CoV infection, black) and low (mock, green) values.


( C ) Correlation of duplicate screening sets . Compounds with MERS - CoV inhibition and cell viability > 70 % were regarded as primary hits . Scatter plot shows MERS - CoV inhibition ratios overlaid with cell viability ratios .

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(C) Correlation of duplicate screening sets. Compounds with MERS-CoV inhibition and cell viability >70% were regarded as primary hits. Scatter plot shows MERS-CoV inhibition ratios overlaid with cell viability ratios.


Time - of - addition study with selected FDA - approved drugs . Five FDA - approved drugs were analyzed by time - course experiments to determine the stage in the MERS - CoV life cycle that is inhibited by the drugs . Vero cells were infected at a multiplicity of infection ( MOI ) of 5 with MERS - CoV , and FDA - approved drugs were administered at six - time points prior to or post - infection as indicated . Drug concentrations were higher than the 90 % inhibitory concentration ( IC90 ) values of the drugs , and chloroquine was used as an early stage infection inhibitor control .

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Time-of-addition study with selected FDA-approved drugs.Five FDA-approved drugs were analyzed by time-course experiments to determine the stage in the MERS-CoV life cycle that is inhibited by the drugs. Vero cells were infected at a multiplicity of infection (MOI) of 5 with MERS-CoV, and FDA-approved drugs were administered at six-time points prior to or post-infection as indicated. Drug concentrations were higher than the 90% inhibitory concentration (IC90) values of the drugs, and chloroquine was used as an early stage infection inhibitor control.


A Detection of the activation of IRE1α , PERK and ATF6 UPR branches by immunoblotting in control ( shCtrl ) and Fam20C stable knockdown ( shFAM20C ) HepG2 cells treated with or without 5 μM Tg for 30 min . ATF6 - FL , full - length ATF6 ; ATF6 - N , N - terminal cleavage product of ATF6 ; Asterisk , unspecific background bands . Fam20C knockdown was verified by protein immunoblotting of Concanavalin A - Sepharose ( Con A ) - enriched culture medium . Ponceau staining was shown as a loading control .

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A Detection of the activation of IRE1α, PERK and ATF6 UPR branches by immunoblotting in control (shCtrl) and Fam20C stable knockdown (shFAM20C) HepG2 cells treated with or without 5 μM Tg for 30 min. ATF6-FL, full-length ATF6; ATF6-N, N-terminal cleavage product of ATF6; Asterisk, unspecific background bands. Fam20C knockdown was verified by protein immunoblotting of Concanavalin A-Sepharose (Con A)-enriched culture medium. Ponceau staining was shown as a loading control.


B ( Left ) Detection of spliced XBP1 ( S ) and unspliced XBP1 ( U ) mRNA in shCtrl and shFAM20C HepG2 cells treated with or without 5 μM Tg for 1 h . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) .

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B (Left) Detection of spliced XBP1 (S) and unspliced XBP1 (U) mRNA in shCtrl and shFAM20C HepG2 cells treated with or without 5 μM Tg for 1 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).


C ( Left ) Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing RNAi - resistant codon - altered Fam20C wild - type ( WT ) or its inactive D478A mutant ( DA ) treated with or without 5 μM Tg for 1 h . Fam20C expression levels were shown by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) .

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C (Left) Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing RNAi-resistant codon-altered Fam20C wild-type (WT) or its inactive D478A mutant (DA) treated with or without 5 μM Tg for 1 h. Fam20C expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).


( Left ) Detection of XBP1 mRNA splicing in shCtrl and shFAM20C HepG2 cells ( D ) treated with or without 2 μg / ml Tm for 8 h . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) .

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(Left) Detection of XBP1 mRNA splicing in shCtrl and shFAM20C HepG2 cells (D) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).


Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing Fam20C WT or DA ( E ) treated with or without 2 μg / ml Tm for 8 h . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) .

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Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing Fam20C WT or DA (E) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).


F Volcano plot depicting the log2 of fold change versus - log10 ( p - value ) for Fam20C - interacting proteins in Tg - treated HepG2 cells . The red dots represent proteins showing statistically significant increase of binding with Fam20C ( log2 fold change ≥ 1 ; - log10 ( p - value ) ≥ 1 . 3 ) ; the blue dots represent proteins showing statistically significant decrease of binding with Fam20C ( log2 fold change ≤ - 1 ; - log10 ( p - value ) ≥ 1 . 3 ) ; the green dot is Fam20C .

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F Volcano plot depicting the log2 of fold change versus - log10 (p-value) for Fam20C-interacting proteins in Tg-treated HepG2 cells. The red dots represent proteins showing statistically significant increase of binding with Fam20C (log2 fold change ≥ 1; - log10 (p-value) ≥ 1.3); the blue dots represent proteins showing statistically significant decrease of binding with Fam20C (log2 fold change ≤ - 1; - log10 (p-value) ≥ 1.3); the green dot is Fam20C.


G HepG2 cells expressing FLAG - tagged Fam20C were treated with or without 5 μM Tg for 30 min . FLAG - immunoprecipitates were analyzed by PDI immunoblotting .

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G HepG2 cells expressing FLAG-tagged Fam20C were treated with or without 5 μM Tg for 30 min. FLAG-immunoprecipitates were analyzed by PDI immunoblotting.


A Representative MS / MS fragmentation spectrum of tryptic phosphorylated PDI peptide ( Ile351 - Lys370 ) depicting PDI Ser357 phosphorylation enriched from HepG2 cells treated with 5 μM Tg for 10 min .

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A Representative MS/MS fragmentation spectrum of tryptic phosphorylated PDI peptide (Ile351-Lys370) depicting PDI Ser357 phosphorylation enriched from HepG2 cells treated with 5 μM Tg for 10 min.


C Time - dependent incorporation of phosphate group into PDI WT or S357A catalyzed by recombinant Fam20C . Phosphorylated PDI was determined by anti - pS357 - PDI immunoblotting on both regular and phostag gels .

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C Time-dependent incorporation of phosphate group into PDI WT or S357A catalyzed by recombinant Fam20C. Phosphorylated PDI was determined by anti-pS357-PDI immunoblotting on both regular and phostag gels.


D Phosphorylated PDI catalyzed by Fam20C were analyzed by dual - color immunoblotting with anti - PDI ( red ) and anti - pS357 - PDI ( green ) antibodies . The yellow signal depicts the phosphorylated PDI species .

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D Phosphorylated PDI catalyzed by Fam20C were analyzed by dual-color immunoblotting with anti-PDI (red) and anti-pS357-PDI (green) antibodies. The yellow signal depicts the phosphorylated PDI species.


Detection of pS357 - PDI in HepG2 cells treated with 5 μM Tg ( E ) for indicated times .

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Detection of pS357-PDI in HepG2 cells treated with 5 μM Tg (E) for indicated times.


Detection of pS357 - PDI in HepG2 cells treated with 2 . 5 μg / ml Tm ( F ) for indicated times .

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Detection of pS357-PDI in HepG2 cells treated with 2.5 μg/ml Tm (F) for indicated times.


Detection of pS357 - PDI in HepG2 cells treated with 1 mM DTT ( G ) for indicated times .

Detection of pS357-PDI in HepG2 cells treated with 1 mM DTT (G) for indicated times.


( Left ) Detection of p - PDI in WT and PDI KO HepG2 cells ( H ) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels . ( Right ) Quantification of p - PDI percentage based on band intensities on phostag gels . Data information : Data were shown as mean ± SEM of three ( H ) independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) .

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(Left) Detection of p-PDI in WT and PDI KO HepG2 cells (H) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of three (H) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).


( Left ) Detection of p - PDI in WT and Fam20C KO HeLa cells ( I ) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels . ( Right ) Quantification of p - PDI percentage based on band intensities on phostag gels . Data information : Data were shown as mean ± SEM of four ( I ) independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) .

(Left) Detection of p-PDI in WT and Fam20C KO HeLa cells (I) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of four (I) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).


A Fluorescent photomicrographs of HepG2 cells co - transfected with PDI - mEmerald - KDEL ( green ) and Fam20C - mApple ( red ) . The cells were treated with DMSO ( Upper ) or 5 μM Tg ( Middle ) for 30 min . After Tg treatment , the cells were washed with PBS and then returned to culture medium for 60 min ( Lower ) . GM130 was immunostained ( blue ) as a cis - Golgi marker . Scale bar = 10 μm .

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A Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red). The cells were treated with DMSO (Upper) or 5 μM Tg (Middle) for 30 min. After Tg treatment, the cells were washed with PBS and then returned to culture medium for 60 min (Lower). GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.


B Pearson correlation coefficient calculated Data were shown as mean ± SEM of N > 80 cells from four independent experiments . * * * p < 0 . 001 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) .

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B Pearson correlation coefficient calculated Data were shown as mean ± SEM of N > 80 cells from four independent experiments. ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).


C Detection of pS357 - PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times .

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C Detection of pS357-PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times.


D HepG2 cells were treated with 25 ng / μl CHX for indicated times and analyzed by immunoblotting for pS357 - PDI and UPR signaling . 5 μM Tg or DMSO was added 30 min before cell harvest .

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D HepG2 cells were treated with 25 ng/μl CHX for indicated times and analyzed by immunoblotting for pS357-PDI and UPR signaling. 5 μM Tg or DMSO was added 30 min before cell harvest.


E Fluorescent photomicrographs of HepG2 cells co - transfected with PDI - mEmerald - KDEL ( green ) and Fam20C - mApple ( red ) pretreated with 25 ng / μl CHX for 7 . 5 h , followed by introduction of DMSO ( Upper ) or 5 μM Tg ( Lower ) for 30 min , respectively . GM130 was immunostained ( blue ) as a cis - Golgi marker . Scale bar = 10 μm .

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E Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red) pretreated with 25 ng/μl CHX for 7.5 h, followed by introduction of DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.


F Fluorescent photomicrographs of HepG2 cells co - transfected with PDI - mEmerald - KDEL ( green ) and Fam20C - mApple - KDEL ( red ) treated with DMSO ( Upper ) or 5 μM Tg ( Lower ) for 30 min , respectively . Scale bar = 10 μm .

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F Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple-KDEL (red) treated with DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. Scale bar = 10 μm.


G HepG2 cells were transfected with empty vector ( - ) , FLAG - tagged Fam20C or Fam20C - KDEL followed by introduction without or with 5 μM Tg for 30 min , respectively . PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels . p - PDI percentage was quantified based on band intensities on phostag gels .

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G HepG2 cells were transfected with empty vector (-), FLAG-tagged Fam20C or Fam20C-KDEL followed by introduction without or with 5 μM Tg for 30 min, respectively. PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels. p-PDI percentage was quantified based on band intensities on phostag gels.


B The time course of the distance between the geometrical centers of domains a and a ′ and the angle among domains b , b ′ and a ′ from the simulation of pS357 - PDI ( red ) and PDI ( black ) ( Yang et al . , 2014 ) .

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B The time course of the distance between the geometrical centers of domains a and a′ and the angle among domains b, b′ and a′ from the simulation of pS357-PDI (red) and PDI (black) (Yang et al., 2014).


C ( Left ) ANS fluorescence spectra of PDI WT and S357E . Controls with ANS alone was shown . A . U . , arbitrary units . ( Right ) Enhancement factor were quantified . Data information : All data were shown as mean ± SEM of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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C (Left) ANS fluorescence spectra of PDI WT and S357E. Controls with ANS alone was shown. A.U., arbitrary units. (Right) Enhancement factor were quantified. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).


D Limited proteolysis of PDI WT ( Left ) and S357E ( Right ) proteins by proteinase K ( Pro K ) for indicated times . Main digested fragments were indicated on the right margin . Band abb ' x * is a digested product with cleavage after Met356 in the x - linker region ( Wang et al . , 2010 ) .

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D Limited proteolysis of PDI WT (Left) and S357E (Right) proteins by proteinase K (Pro K) for indicated times. Main digested fragments were indicated on the right margin. Band abb'x* is a digested product with cleavage after Met356 in the x-linker region (Wang et al., 2010).


E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min , and in some aliquots of cells Tg was removed for 30 min . Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis .

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E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min, and in some aliquots of cells Tg was removed for 30 min. Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis.


F HepG2 cells expressing HA - tagged PDI and FLAG - tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis .

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F HepG2 cells expressing HA-tagged PDI and FLAG-tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis.


Chaperone activities of PDI WT and S357E ( G , H ) or phosphorylated PDI WT and S357A by Fam20C for indicated times ( I Denatured and reduced GAPDH ( G , I ) and rhodanese ( H , were used as substrates , respectively . The chaperone activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . In ( I , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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Chaperone activities of PDI WT and S357E (G, H) or phosphorylated PDI WT and S357A by Fam20C for indicated times (I Denatured and reduced GAPDH (G, I) and rhodanese (H, were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , **p < 0.01, ***p < 0.001 (two-tailed Student's t-test). In (I , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Chaperone activities of phosphorylated PDI WT and S357A by Fam20C for indicated times J ) . Denatured and reduced rhodanese J ) were used as substrates , respectively . The chaperone activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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Chaperone activities of phosphorylated PDI WT and S357A by Fam20C for indicated times J). Denatured and reduced rhodanese J) were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Reductase activities of PDI WT and S357E ( K ) measured by insulin reduction assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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Reductase activities of PDI WT and S357E (K) measured by insulin reduction assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).


Isomerase activities of PDI WT and S357E ( L ) measured by scrambled RNase A reactivation assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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Isomerase activities of PDI WT and S357E (L) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).


Reductase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times ( M ) measured by insulin reduction assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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Reductase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times (M) measured by insulin reduction assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Isomerase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times ( N ) measured by scrambled RNase A reactivation assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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Isomerase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times (N) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER ( TMR - labeled , red ) . Endogenous PDI was immunostained ( green ) as an ER marker . Scale bar = 10 μm .

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B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER (TMR-labeled, red). Endogenous PDI was immunostained (green) as an ER marker. Scale bar = 10 μm.


C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 ( green ) and TMR ( red ) , followed by introduction of DMSO or 5 μM Tg for 30 min . Scale bar = 10 μm .

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C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red), followed by introduction of DMSO or 5 μM Tg for 30 min. Scale bar = 10 μm.


D Confocal live cell imaging of WT and PDI KO HepG2 cells co - transfected with AgHaloER labeled by P1 ( green ) and TMR ( red ) and empty vector ( - ) , PDI WT , S357E or S357A . Scale bar = 10 μm .

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D Confocal live cell imaging of WT and PDI KO HepG2 cells co-transfected with AgHaloER labeled by P1 (green) and TMR (red) and empty vector (-), PDI WT, S357E or S357A. Scale bar = 10 μm.


E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 ( green ) and TMR ( red ) . Scale bar = 10 μm .

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E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red). Scale bar = 10 μm.


F ( Upper ) Cell viabilities of WT and PDI KO HepG2 cells co - transfected with AgHaloER and empty vector ( - ) , PDI WT , S357E or S357A . Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining . ( Lower ) Quantification of surviving fraction of cells . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus WT HepG2 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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F (Upper) Cell viabilities of WT and PDI KO HepG2 cells co-transfected with AgHaloER and empty vector (-), PDI WT, S357E or S357A. Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining. (Lower) Quantification of surviving fraction of cells. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT HepG2 (two-way ANOVA, the post hoc Tukey's HSD test).


A ( Left ) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h . PDI KO was verified by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 ( two - tailed Student ' s t - test ) .

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A (Left) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h. PDI KO was verified by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05 (two-tailed Student's t-test).


B ( Left ) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h . PDI expression levels were shown by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) .

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B (Left) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h. PDI expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (one-way ANOVA, the post hoc Tukey's HSD test).


C Detection of IRE1α , PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min . Asterisk indicates unspecific background band .

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C Detection of IRE1α, PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min. Asterisk indicates unspecific background band.


D HepG2 cells were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI and BiP immunoblotting .

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D HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.


E HepG2 cells expressing FLAG - tagged IRE1α were treated without or with 5 μM Tg for 1 h . An aliquot of Tg - treated cells was then washed with culture medium and chased for 4 h . FLAG - immunoprecipitates were analyzed by PDI and BiP immunoblotting .

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E HepG2 cells expressing FLAG-tagged IRE1α were treated without or with 5 μM Tg for 1 h. An aliquot of Tg-treated cells was then washed with culture medium and chased for 4 h. FLAG-immunoprecipitates were analyzed by PDI and BiP immunoblotting.


F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI and BiP immunoblotting .

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F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.


G Co - immunoprecipitation of FLAG - tagged IRE1α and HA - tagged PDI in HepG2 cells expressing V5 - tagged Fam20C WT or DA .

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G Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI in HepG2 cells expressing V5-tagged Fam20C WT or DA.


H Co - immunoprecipitation of FLAG - tagged IRE1α , HA - tagged PDI WT or S357A in HepG2 cells expressing V5 - tagged Fam20C .

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H Co-immunoprecipitation of FLAG-tagged IRE1α, HA-tagged PDI WT or S357A in HepG2 cells expressing V5-tagged Fam20C.


I Co - immunoprecipitation of HA - tagged PDI and FLAG - tagged IRE1αNLD in HepG2 cells .

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I Co-immunoprecipitation of HA-tagged PDI and FLAG-tagged IRE1αNLD in HepG2 cells.


J Recombinant GST - PDI was phosphorylated by Fam20C protein for 2 h , and then used to pulldown His - IRE1αNLD protein , as visualized by Coomassie blue staining . Successful phosphorylation of GST - PDI was verified by pS357 - PDI immunoblotting .

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J Recombinant GST-PDI was phosphorylated by Fam20C protein for 2 h, and then used to pulldown His-IRE1αNLD protein, as visualized by Coomassie blue staining. Successful phosphorylation of GST-PDI was verified by pS357-PDI immunoblotting.


K Co - immunoprecipitation of FLAG - tagged IRE1αNLD WT or its mutant C109 / 148 / 332S with HA - tagged PDI ( Left ) or MYC - tagged P5 ( Right ) in HepG2 cells .

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K Co-immunoprecipitation of FLAG-tagged IRE1αNLD WT or its mutant C109/148/332S with HA-tagged PDI (Left) or MYC-tagged P5 (Right) in HepG2 cells.


L Co - immunoprecipitation of FLAG - tagged IRE1α and HA - tagged PDI WT or its binding mutant W128I / L403W in HepG2 cells expressing V5 - tagged Fam20C or not .

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L Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI WT or its binding mutant W128I/L403W in HepG2 cells expressing V5-tagged Fam20C or not.


B Sanger sequencing results of Pdi + / + ( WT ) and PdiS359A / S359A ( KI ) mice .

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B Sanger sequencing results of Pdi+/+ (WT) and PdiS359A/S359A (KI) mice.


C ( Left ) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm ( 50 ng / g , i . p . ) for indicated times . Each lane represents independent animals . ( Right ) Quantification of Xbp1 mRNA splicing levels at indicated times . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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C (Left) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm (50 ng/g, i.p.) for indicated times. Each lane represents independent animals. (Right) Quantification of Xbp1 mRNA splicing levels at indicated times. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).


Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0 . 2 μM Tg for 2 h ( D )

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Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.2 μM Tg for 2 h (D)


Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0 . 5 μg / ml Tm for 2 h ( E ) .

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Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.5 μg/ml Tm for 2 h (E).


Detection of Bip ( F ) , Pdi ( G ) , Erdj4 ( H ) mRNA levels by real - time PCR in the liver of the same animals after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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Detection of Bip (F), Pdi (G), Erdj4 (H) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Detection of Edem1 ( I ) , Il6 ( J ) , Chop ( K ) mRNA levels by real - time PCR in the liver of the same animals after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

Detection of Edem1 (I), Il6 (J), Chop (K) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


L Detection of serum alanine transaminase ( ALT ) levels of the same animals as in ( C ) after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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L Detection of serum alanine transaminase (ALT) levels of the same animals as in (C) after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


M Hematoxylin and eosin staining of liver tissue the same animals as in ( C ) after 24 h of Tm injection ( magnification × 400 ) . Immune cell infiltration was indicated with a black arrow . Scale bar = 50 μm . Representative images were shown from five animals per group analyzed .

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M Hematoxylin and eosin staining of liver tissue the same animals as in (C) after 24 h of Tm injection (magnification × 400). Immune cell infiltration was indicated with a black arrow. Scale bar = 50 μm. Representative images were shown from five animals per group analyzed.


N ( Left ) TUNEL assay of the same liver as in ( M ) . Scale bar = 40 μm ; Zoom box , scale bar = 5 μm . ( Right ) Quantification of apoptotic cells . 10 random fields were counted for each TUNEL - stained tissue sample . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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N (Left) TUNEL assay of the same liver as in (M). Scale bar = 40 μm; Zoom box, scale bar = 5 μm. (Right) Quantification of apoptotic cells. 10 random fields were counted for each TUNEL-stained tissue sample. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).


Cryo - TEM images demonstrate the lipid bilayer EV purified from Molm - 14 and U - 937 cells . Scale bars are 100nm .

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Cryo-TEM images demonstrate the lipid bilayer EV purified from Molm-14 and U-937 cells. Scale bars are 100nm.


Peripheral blood ( PB ) plasma of control animals contain no mGFP + foci ( top ) however Molm - 14 - mGFP ( middle ) and U - 937 - mGFP ( bottom ) xenografts contain numerous mGFP - labeled EV ( green ) detectible without vesicle concentration . Scale bars are 5μm .

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Peripheral blood (PB) plasma of control animals contain no mGFP+ foci (top) however Molm-14-mGFP (middle) and U-937-mGFP (bottom) xenografts contain numerous mGFP-labeled EV (green) detectible without vesicle concentration. Scale bars are 5μm.


Live - cell microscopy of KSL cells ( left panel ) LT - HSC cells ( right panel ) . Cells were sorted from control mice ( top ) , Molm - 14 - mGFP ( middle ) and U - 937 - mGFP ( bottom ) , stained with Cell Mask ( red ) and nuclear stain Hoechst ( blue ) ; imaged using the GE / API Deltavision ( DV ) widefield microscope ( 60X objective ) to show the uptake of the GFP + EV ( green ) . Scale bars are 5μm .

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Live-cell microscopy of KSL cells (left panel) LT-HSC cells (right panel). Cells were sorted from control mice (top), Molm-14-mGFP (middle) and U-937-mGFP (bottom), stained with Cell Mask (red) and nuclear stain Hoechst (blue); imaged using the GE/API Deltavision (DV) widefield microscope (60X objective) to show the uptake of the GFP+ EV (green). Scale bars are 5μm.


Flow cytometric analysis showing the frequency of LK and KSL cells in lineage - negative cells ( left panel ) and LT - HSC in KSL ( right panel ) in : Molm - 14 - xenograft ( red , n = 10 ) vs . control ( black , n = 10 ) . Data were obtained from at least two independent experiments .

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Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: Molm-14-xenograft (red, n=10) vs. control (black, n=10). Data were obtained from at least two independent experiments.


Flow cytometric analysis showing the frequency of LK and KSL cells in lineage - negative cells ( left panel ) and LT - HSC in KSL ( right panel ) in : IF injection of EV from Molm - 14 cells ( red , n = 8 ) , AML plasma EV ( Orange , n = 6 ) and human CD34 EV ( Blue , n = 4 ) relative to the vehicle - injected contralateral femurs . Data were obtained from at least two independent experiments .

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Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: IF injection of EV from Molm-14 cells (red, n=8), AML plasma EV (Orange, n=6) and human CD34 EV (Blue, n=4) relative to the vehicle-injected contralateral femurs. Data were obtained from at least two independent experiments.


Cell - cycle histograms of KSL ( upper panel ) and LT - HSC ( lower panel ) from Molm - 14 - xenografts or control using Hoechst - 33342 staining .

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Cell-cycle histograms of KSL (upper panel) and LT-HSC (lower panel) from Molm-14-xenografts or control using Hoechst-33342 staining.


Flow cytometric analysis of the Ki67 - ve percentage of LT - HSC representing the G0 phase of cell - cycle in : ( left panel ) Molm - 14 - xenografts ( red , n = 7 ) vs . control ( black , n = 9 ) ; ( right panel ) IF injection of Molm - 14 - EV , U - 937 EV , HL - 60 EV ( red , n = 5 , 4 , 4 ) vs . human CD34 EV ( blue , n = 3 ) vs . controls ( black ) .

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Flow cytometric analysis of the Ki67-ve percentage of LT-HSC representing the G0 phase of cell-cycle in: (left panel) Molm-14-xenografts (red, n=7) vs. control (black, n=9); (right panel) IF injection of Molm-14-EV, U-937 EV, HL-60 EV (red, n=5,4,4) vs. human CD34 EV (blue, n=3) vs. controls (black).


Flow cytometric analysis of intracellular P53 levels shown in histograms ( F ) of LT - HSC in : ( left panel ) Molm - 14 - xenograft , n = 6 red vs . non - engrafted control , n = 4 black , or ( right panel ) IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 5 , 3 ) or human CD34 cells ( blue , n = 6 ) normalized to vehicle - injected contralateral femurs .

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Flow cytometric analysis of intracellular P53 levels shown in histograms (F) of LT-HSC in: (left panel) Molm-14-xenograft, n=6 red vs. non-engrafted control, n=4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,5,3) or human CD34 cells (blue, n=6) normalized to vehicle-injected contralateral femurs.


Flow cytometric analysis of intracellular P53 levels shown in histograms of LT - HSC in : ( left panel ) Molm - 14 - xenograft , n = 6 red vs . non - engrafted control , n = 4 black , or ( right panel ) IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 5 , 3 ) or human CD34 cells ( blue , n = 6 ) normalized to vehicle - injected contralateral femurs .

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Flow cytometric analysis of intracellular P53 levels shown in histograms of LT-HSC in: (left panel) Molm-14-xenograft, n=6 red vs. non-engrafted control, n=4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,5,3) or human CD34 cells (blue, n=6) normalized to vehicle-injected contralateral femurs.


A heatmap showing the highly - deregulated ribosomal proteins in AML - EV exposed cells .

A heatmap showing the highly-deregulated ribosomal proteins in AML-EV exposed cells.


Flow cytometric analysis showing the histograms and MFI of O - propagyl - puromysin ( OPP ) incorporation in LT - HSC in : Molm - 14 - xenografts , red n = 6 vs . non - engrafted controls , black n = 6 ,

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Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: Molm-14-xenografts, red n=6 vs. non-engrafted controls, black n=6,


Flow cytometric analysis showing the histograms and MFI of O - propagyl - puromysin ( OPP ) incorporation in LT - HSC in : IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 43 ) , AML patients plasma ( Orange , n = 6 ) , or human CD34 + cells ( blue , n = 3 ) normalized to vehicle - injected contralateral femurs after subtracting the background fluorescence .

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Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,43), AML patients plasma (Orange, n=6), or human CD34+ cells (blue, n=3) normalized to vehicle-injected contralateral femurs after subtracting the background fluorescence.


qRT - PCR gene expression analysis showing the fold change of Pi3K / mTOR associated gene panel in KSL cells sorted from : ( A ) Molm - 14 - xenografted

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qRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: (A) Molm-14-xenografted mice


qRT - PCR gene expression analysis showing the fold change of Pi3K / mTOR associated gene panel in KSL cells sorted from : IF injected mice with Molm - 14 - EV relative to their controls and normalized to Gapdh endogenous control .

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qRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: IF injected mice with Molm-14-EV relative to their controls and normalized to Gapdh endogenous control.


Flow cytometric analysis showing the histograms ( C )

Flow cytometric analysis showing the histograms (C)


Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT - HSC in : ( D ) Molm - 14 - xenograft ( red , n = 9 ) vs . non - engrafted control ( black , n = 7 ) ,

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Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: (D) Molm-14-xenograft (red, n=9) vs. non-engrafted control (black, n=7),


Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT - HSC in : IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 7 , 3 , 3 ) or human CD34 cells ( blue , n = 4 ) normalized to the control contralateral femur with subtraction of background fluorescence .

Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=7,3,3) or human CD34 cells (blue, n=4) normalized to the control contralateral femur with subtraction of background fluorescence.


Flow cytometric assessment of protein synthesis showing the MFI of : ( F ) OPP , or ( G ) pS6RP in NIH - 3T3 cells 72 hours after transfection with the indicated miRNA mimics . The results were calculated relative to control ( miR - scramble ) with the background fluorescence subtracted and performed with at least three independent experiments , in technical replicates .

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Flow cytometric assessment of protein synthesis showing the MFI of: (F) OPP, or (G) pS6RP in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The results were calculated relative to control (miR-scramble) with the background fluorescence subtracted and performed with at least three independent experiments, in technical replicates.


Flow cytometric analysis showing the MFI of Raptor in NIH - 3T3 cells 72 hours after transfection with the indicated miRNA mimics . The data were calculated relative to miR - scramble and presented as means bars ± SEM and the background fluorescence was subtracted . Performed with at least three independent experiments , in technical replicates .

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Flow cytometric analysis showing the MFI of Raptor in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The data were calculated relative to miR-scramble and presented as means bars ± SEM and the background fluorescence was subtracted. Performed with at least three independent experiments, in technical replicates.


Dual Luciferase reporter assay . NIH - 3T3 was transfected with the miRNA mimics . Three hours later the cells were transfected with the Raptor 3 ′ UTR cloned into the psiCheck - 2 vector for a total of 48 hours . Data are presented as % RLU ( relative luciferase units ) of the miR - scramble control as means ± SEM from at least three independent experiments , performed in technical replicates .

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Dual Luciferase reporter assay. NIH-3T3 was transfected with the miRNA mimics. Three hours later the cells were transfected with the Raptor 3′UTR cloned into the psiCheck-2 vector for a total of 48 hours. Data are presented as %RLU (relative luciferase units) of the miR-scramble control as means ± SEM from at least three independent experiments, performed in technical replicates.


Cell - cycle flow cytometric analysis using Ki67 / Hoechst - 33342 staining of the percentage of LT - HSC in the G0 phase after nucleofection of cKit + cells using the AmaxaTM P3 Primary Cell 4D - Nucleoeftcor kit , ( J ) Cells were nucleofected with miR - Scramble ( CTRL ) or miR - 1246 mimic ( n = 3 ) for 72 hours ,

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Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, (J) Cells were nucleofected with miR-Scramble (CTRL) or miR-1246 mimic (n=3) for 72 hours,


Cell - cycle flow cytometric analysis using Ki67 / Hoechst - 33342 staining of the percentage of LT - HSC in the G0 phase after nucleofection of cKit + cells using the AmaxaTM P3 Primary Cell 4D - Nucleoeftcor kit , Cells were nucleofected with Anti - miR - Scramble ( CTRL ) or Anti - miR - 1246 ( n = 5 ) and 1 - hour later co - treatment with Molm - 14 EVs for 72 hours .

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Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, Cells were nucleofected with Anti-miR-Scramble (CTRL) or Anti-miR-1246 (n=5) and 1-hour later co-treatment with Molm-14 EVs for 72 hours.


CFU - C assay showing the relative number of colonies ( left panel ) and the colony subtypes ( right panel ) from KSL cells derived from Molm - 14 - xenografted mice ( red , n = 6 ) vs . non - engrafted control mice ( black , n = 5 ) and in vitro cultured for 7 days in methylcellulose media . Experiments were performed in technical triplicates . " G " Granulocyte , " M " Monocyte , " E " Erythrocytes .

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CFU-C assay showing the relative number of colonies (left panel) and the colony subtypes (right panel) from KSL cells derived from Molm-14-xenografted mice (red, n=6) vs. non-engrafted control mice (black, n=5) and in vitro cultured for 7 days in methylcellulose media. Experiments were performed in technical triplicates. "G" Granulocyte, "M" Monocyte, "E" Erythrocytes.


LT - HSC assessment after repopulation , recipient female mice ( n = 4 per group ) were sacrificed after 21 days and their LT - HSC were compared using P53 , pS6RP and OPP flow cytometric assays . ( D - E ) Evaluation of the in vivo long - term repopulation capacity .

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LT-HSC assessment after repopulation, recipient female mice (n=4 per group) were sacrificed after 21 days and their LT-HSC were compared using P53, pS6RP and OPP flow cytometric assays. (D-E) Evaluation of the in vivo long-term repopulation capacity.


Evaluation of the in vivo long - term repopulation capacity . For secondary transplantation ( n = 6 per group ) , PB samples were collected twice weekly for 12 weeks ( WK ) and the donor chimerism was measured by qPCR of Sex - determining Region Y ( SRY ) normalized to Gapdh gatekeeper as shown in the left panel . Sixteen weeks later , mice were sacrificed and BM male chimerism percentage was assessed as shown in the right panel . Tertiary transplantation , unfractionated BM cells from the secondary recipients ( n = 6 per group , 106 cells per mouse ) were injected into tertiary 150cGy sub - lethally irradiated female recipients and blood SRY levels were assessed for 20 weeks .

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Evaluation of the in vivo long-term repopulation capacity. For secondary transplantation (n=6 per group), PB samples were collected twice weekly for 12 weeks (WK) and the donor chimerism was measured by qPCR of Sex-determining Region Y (SRY) normalized to Gapdh gatekeeper as shown in the left panel. Sixteen weeks later, mice were sacrificed and BM male chimerism percentage was assessed as shown in the right panel. Tertiary transplantation, unfractionated BM cells from the secondary recipients (n=6 per group, 106 cells per mouse) were injected into tertiary 150cGy sub-lethally irradiated female recipients and blood SRY levels were assessed for 20 weeks.


Serial replating CFU - C analysis presented as mean ± SEM showing : ( B ) the number of colonies per 1000 plated cells , and ( C ) the number of cells per colony , for each week time - point .

Serial replating CFU-C analysis presented as mean ± SEM showing: (B) the number of colonies per 1000 plated cells, and (C) the number of cells per colony, for each week time-point.


DNA - damage assessment in KSL from primary recipients and after two turns of replating . ( D , F , H ) Number of γH2AX foci in control - derived and Molm - 14 - xenograft - derived : ( D ) Primary recipient KSL , n = 211 and 321 , All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst ( blue ) , imaged using the GE / API DV widefield microscope ( 60X objective ) to show the γH2AX foci ( green ) and counted using Imaris software .

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DNA-damage assessment in KSL from primary recipients and after two turns of replating. (D, F, H) Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: (D) Primary recipient KSL, n=211 and 321, All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.


qRT - PCR gene expression analysis showing the fold - change of select DNA - damage response - genes panel in Molm - 14 - xenograft - derived : ( E ) Primary recipient KSL , n = 6 Data indicated by bars of fold - change calculated relative to matching controls , normalized to the endogenous control Gapdh and presented as means ± SEM .

qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: (E) Primary recipient KSL, n=6 Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.


Number of γH2AX foci in control - derived and Molm - 14 - xenograft - derived : cells from W1 plating , n = 218 and 369 All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst ( blue ) , imaged using the GE / API DV widefield microscope ( 60X objective ) to show the γH2AX foci ( green ) and counted using Imaris software . Scale bars are 2μm .

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Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W1 plating, n=218 and 369 All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software. Scale bars are 2μm.


qRT - PCR gene expression analysis showing the fold - change of select DNA - damage response - genes panel in Molm - 14 - xenograft - derived : Cells from W1 plating , n = 4 , Data indicated by bars of fold - change calculated relative to matching controls , normalized to the endogenous control Gapdh and presented as means ± SEM .

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qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: Cells from W1 plating, n=4, Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.


Number of γH2AX foci in control - derived and Molm - 14 - xenograft - derived : cells from W2 plating , n = 770 and 853 . All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst ( blue ) , imaged using the GE / API DV widefield microscope ( 60X objective ) to show the γH2AX foci ( green ) and counted using Imaris software .

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Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W2 plating, n=770 and 853. All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.


qRT - PCR gene expression analysis showing the fold - change of select DNA - damage response - genes panel in Molm - 14 - xenograft - derived : Cells from W2 plating n = 4 . Data indicated by bars of fold - change calculated relative to matching controls , normalized to the endogenous control Gapdh and presented as means ± SEM .

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qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: Cells from W2 plating n=4. Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.