( F ) Treatment with K + channel blockers reduces number of GGGGCC toxic foci in ALSC9orf72 MN . n = 3 independent treatments with each hiPSC ( one - way ANOVA followed by Dunnett ' s multiple comparison ) . Scale bar : 5 µm . Dashed represents soma . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent . Arrow indicates structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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(F) Treatment with the K+ channel blockers reduces the number of GGGGCC toxic RNA foci in ALSC9orf72 MN. n=3 independent treatments with each hiPSC line (one-way ANOVA followed by Dunnett's multiple comparison test). Scale bar: 5 µm. Dashed line represents the cell soma. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( E ) Apamin XE991 increase of pCREBS133 in ALSC9orf72 lines ( one - way ANOVA followed by Dunnett ' s multiple comparison ) . n = 3 independent treatments with each hiPSC . Scale bar : 10 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 . Error bars represent . Exact p - values are reported in Appendix Table S1 .

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(E) Apamin and XE991 increase the levels of pCREBS133 in all the ALSC9orf72 lines (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 10 µm. Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1.


( F ) ALSC9orf72 MN showed an increased number of Homer1b / c + synaptic contacts after treatment with K + channel blockers , which in contrast had no significant effect on Healthy MN . n = 3 independent treatments with each hiPSC ( two - way ANOVA ) . Scale bar : 5 µm Data information : * p < 0 . 05 ; * * p < 0 . 01 . Error bars represent . Exact p - values are reported in Appendix Table S1 .

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(F) ALSC9orf72 MN showed an increased number of Homer1b/c+ synaptic contacts after treatment with the K+ channel blockers, which in contrast had no significant effect on Healthy MN. n=3 independent treatments with each hiPSC line (two-way ANOVA). Scale bar: 5 µm Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1.


( K ) Synapse - neuronal processes - related significantly up - regulated by both treatments .

(K) Synapse- and neuronal processes-related proteins significantly up-regulated by both treatments.


response of mitochondrial matrix - localized roGFP2 - Tsa2ΔCR ( C ) probes to boli of exogenous H2O2 at indicated concentrations . Probes were expressed in wild - type BY4742 yeast grown to exponential phase in SGal ( - Leu ) medium .

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The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (C) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


response of mitochondrial matrix - localized Grx1 - roGFP2 ( D ) probes to boli of exogenous H2O2 at indicated concentrations . Probes were expressed in wild - type BY4742 yeast grown to exponential phase in SGal ( - Leu ) medium .

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

The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (D) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


response of mitochondrial matrix - localized roGFP2 - Tsa2ΔCR ( E ) probes to 10 µM antimycin A ( a III inhibitor ) . Probes were expressed in wild - type BY4742 yeast grown to exponential phase in SGal ( - Leu ) medium .

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The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (E) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


response of mitochondrial matrix - localized Grx1 - roGFP2 ( F ) probes to 10 µM antimycin A ( a III inhibitor ) . Probes were expressed in wild - type BY4742 yeast grown to exponential phase in SGal ( - Leu ) medium .

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

The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (F) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


( G ) response of mitochondrial matrix - localized ( left panel ) ( right panel ) roGFP2 - Tsa2ΔCR probes expressed in BY4742 wild - type Δtsa1Δtsa2 , to addition of exogenous H2O2 at indicated concentrations . were grown to exponential phase in SGal ( - Leu ) medium . lighter colored curves are controls , showing probe response upon addition of water .

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(G) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes expressed in BY4742 wild-type and Δtsa1Δtsa2 cells, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls, showing the probe response upon the addition of water.


( H ) response of mitochondrial matrix - localized ( left panel ) ( right panel ) roGFP2 - Tsa2ΔCR probes , expressed in wild - type Δtsa1Δtsa2 , to addition of 10 µM antimycin A . were grown to exponential phase in SGal ( - Leu ) medium . lighter colored curves are controls showing probe responses upon addition of 0 . 1 % ( v / v ) ethanol .

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(H) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes, expressed in wild-type and Δtsa1Δtsa2 cells, to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls showing the probe responses upon addition of 0.1% (v/v) ethanol.


( I ) response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type Δpor1 , to boli at exogenous H2O2 at indicated concentrations . were grown to exponential phase in SGal ( - Leu ) medium . Lighter colored curves are controls showing probe response upon addition of water .

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(I) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells, to boli at exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of water.


( J ) response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type Δpor1 to addition of 10 µM antimycin A . were grown to exponential phase in SGal ( - Leu ) medium . Lighter colored curves are controls showing probe response upon addition of 0 . 1 % ( v / v ) ethanol .

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(J) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of 0.1% (v/v) ethanol.


( A ) response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type Δprx1 , to addition of exogenous H2O2 at indicated concentrations . were grown in SGal ( - Leu ) medium harvested at early exponential phase . Lighter colored curves are controls showing probe response to addition of water .

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(A) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δprx1, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Lighter colored curves are controls showing the probe response to the addition of water.


( B , C ) response of a roGFP2 - Tsa2ΔCR probe , expressed in wild - type , Δtrx3 Δprx1 , to addition of 0 . 1 mM ( B ) or 1 mM ( C ) exogenous H2O2 . were grown in SGal ( - Leu ) medium harvested at early exponential phase .

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(B,C) The response of a cytosolic roGFP2-Tsa2ΔCR probe, expressed in wild-type, Δtrx3 and Δprx1 cells, to the addition of 0.1 mM (B) or 1 mM (C) exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.


( D ) response of a roGFP2 - Tsa2ΔCR probe , in Δtrx3 transformed with a Trx3 plasmid Δprx1 transformed with a Prx1 plasmid , to addition of 0 . 1 mM exogenous H2O2 . were grown in SGal medium lacking appropriate for plasmid selection harvested at early exponential phase . grey curve in both panels represents wild - type control , treated with same of exogenous H2O2 .

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(D) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δtrx3 cells transformed with a Trx3 plasmid and Δprx1 cells transformed with a Prx1 plasmid, to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for plasmid selection and harvested at early exponential phase. The light grey curve in both panels represents the wild-type control, treated with the same concentration of exogenous H2O2.


( E ) profile of mRNA expression of yeast redox or redox - related enzymes in Δprx1 + empty vector , compared to Δprx1 + Prx1 - WT , both grown to an early exponential phase in SGal ( - Ura ) medium ( n = 3 biological replicates , with obtained from 3 independent cultures ) .

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(E) The profile of mRNA expression of the yeast redox or redox-related enzymes in Δprx1+empty vector cells, compared to Δprx1+Prx1-WT cells, both grown to an early exponential phase in SGal (-Ura) medium (n = 3 biological replicates, with cells obtained from 3 independent cultures).


( F ) response of a roGFP2 - Tsa2ΔCR probe , in Δprx1 or Δctt1Δprx1 transformed with either an empty plasmid or a Prx1 - WT plasmid to addition of 0 . 1 mM exogenous H2O2 . were grown in SGal medium lacking appropriate for selection harvested in early exponential

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(F) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δprx1 cells or Δctt1Δprx1 cells transformed with either an empty plasmid or a Prx1-WT plasmid to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for selection and harvested in early exponential phase


( A ) response of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in wild - type , Δtsa1Δtsa2 , Δpor1 , Δtrr2 , Δtrx3 or Δprx1 to a bolus of exogenous H2O2 at indicated concentrations . were grown in SGal ( - Leu ) medium harvested at early exponential phase .

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(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtsa1Δtsa2, Δpor1, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.


( B ) Left panel : response of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in wild - type , Δtrr2 , Δtrx3 or Δprx1 to a bolus of 2 . 5 mM H2O2 . were grown in SGal ( - Leu ) medium harvested at early exponential phase . Right panel : Scheme depicting Prx1 its peroxidatic cysteine at position 91 .

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(B) Left panel: The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of 2.5 mM H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: Scheme depicting Prx1 and its peroxidatic cysteine at position 91.


( C ) response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe to exogenous 2 . 5 mM H2O2 . probe was expressed in wild - type transformed with an empty vector or in ∆ prx1 transformed with either an empty vector , a vector encoding wild - type Prx1 or a vector encoding Prx1 - C91A , grown in SGal ( - Leu , - Ura ) . ( D ) response to of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in either wild - type transformed with an empty vector or in ∆ prx1 transformed with either an empty vector , a Prx1 - WT or a Prx1 - C91A plasmid to exogenous H2O2 at a of 2 . 5 mM . were grown to SGal ( - Leu , - Ura ) medium harvested at early exponential phase .

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(C) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe to exogenous 2.5 mM H2O2. The probe was expressed in wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a vector encoding wild-type Prx1 or a vector encoding Prx1-C91A, grown in SGal (-Leu, -Ura). (D) The response to of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in either wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a Prx1-WT or a Prx1-C91A plasmid to exogenous H2O2 at a concentration of 2.5 mM. Cells were grown to SGal (-Leu, -Ura) medium and harvested at early exponential phase.


( B ) ' Acute stress - washout ' . Δprx1 containing a Prx1 - WT plasmid expressing a mitochondrial matrix - localized Grx1 - roGFP2 probe were grown to exponential phase in SGal ( - Leu , - Ura ) medium harvested treated with either 0 , 1 , 5 , 10 , 25 or 50 mM H2O2 for 10 mins . After removal of H2O2 resuspension in fresh buffer response of a mitochondrial matrix - localized Grx1 - roGFP2 probe to addition of 1 mM H2O2 was measured . middle panel is an enlargement of left panel . graph in right panel shows steady state Grx1 - roGFP2 oxidation following H2O2 pre - treatment as well as maximum Grx1 - roGFP2 oxidation in response to subsequent second H2O2 treatment .

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(B) 'Acute stress-washout' assay. Δprx1 cells containing a Prx1-WT plasmid and expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. After removal of H2O2 and resuspension in fresh buffer the response of a mitochondrial matrix-localized Grx1-roGFP2 probe to the addition of 1 mM H2O2 was measured. The middle panel is an enlargement of the left panel. The graph in the right panel shows the steady state Grx1-roGFP2 oxidation following H2O2 pre-treatment as well as the maximum Grx1-roGFP2 oxidation in response to the subsequent second H2O2 treatment.


' Acute stress - washout ' . Δprx1 expressing a mitochondrial matrix - localized Grx1 - roGFP2 probe were grown to exponential phase in SGal ( - Leu , - Ura ) medium harvested treated with either 0 , 1 , 5 , 10 , 25 or 50 mM H2O2 for 10 mins . ( C ) same experiment as in ( B ) performed with Δprx1 transformed with a Prx1 - C91A plasmid .

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'Acute stress-washout' assay. Δprx1 cells expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. (C) The same experiment as in (B) performed with Δprx1 cells transformed with a Prx1-C91A plasmid.


( D ) ∆ prx1 transformed with a vector encoding wild - type Prx1 , a vector encoding matrix - targeted D - aminooxidase ( su9 - DAO ) expressing a mitochondrial matrix - localized Grx1 - roGFP2 were grown to exponential phase in SGal ( - Leu , - Ura , - His ) then pre - treated with 0 . 15 M D - alanine for 0 , 0 . 5 , 1 . 5 hours . Afterwards , were washed response of Grx1 - roGFP2 to addition of 1 mM H2O2 was measured .

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(D) ∆prx1 cells transformed with a vector encoding wild-type Prx1, a vector encoding the matrix-targeted D-aminooxidase (su9-DAO) and expressing a mitochondrial matrix-localized Grx1-roGFP2 were grown to exponential phase in SGal (-Leu, -Ura, -His) and then pre-treated with 0.15 M D-alanine for 0, 0.5, 1.5 hours. Afterwards, the cells were washed and the response of Grx1-roGFP2 to the addition of 1 mM H2O2 was measured.


( E ) Left panel : response of mitochondrial matrix - localized roGFP2 - Prx1 roGFP2 probes expressed in wild - type to exogenous H2O2 at indicated concentrations . were grown in SGal ( - Leu ) medium harvested at early exponential phase . Right panel : model illustrating that hyperoxidation of Prx1 moiety in roGFP2 - Prx1 sensor leads to a roGFP2 - like behavior .

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(E) Left panel: the response of mitochondrial matrix-localized roGFP2-Prx1 and roGFP2 probes expressed in wild-type cells to exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: model illustrating that hyperoxidation of the Prx1 moiety in the roGFP2-Prx1 sensor leads to a roGFP2-like behavior.


( H ) Redox shift assays of Prx1 . Δprx1 transformed with a plasmid encoding wild - type Prx1 were grown to early exponential phase in SGal ( - Ura ) medium . were subsequently treated with indicated concentrations of H2O2 for 10 mins . Exposure to H2O2 oxidizes single cysteine of Prx1 to a state that cannot be reduced with TCEP .

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(H) Redox shift assays of Prx1. Δprx1 cells transformed with a plasmid encoding wild-type Prx1 were grown to early exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins. Exposure to H2O2 oxidizes the single cysteine of Prx1 to a state that cannot be reduced with TCEP.


( J ) response of a mitochondrial matrix - localized Grx1 - roGFP2 , expressed in wild - type grown to addition of exogenous 1 mM H2O2 . were grown in SGal ( - Leu ) harvested at early exponential phase . Left panel , pre - treatment efficiently inhibits ability of Prx1 to transfer oxidizing equivalents to EGSH . Right panel , newly synthesized Prx1 is required to recover transfer of oxidizing equivalents to EGSH in mitochondrial matrix .

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(J) The response of a mitochondrial matrix-localized Grx1-roGFP2, expressed in wild-type cells grown to the addition of exogenous 1 mM H2O2. Cells were grown in SGal (-Leu) and harvested at early exponential phase. Left panel, the pre-treatment efficiently inhibits the ability of Prx1 to transfer oxidizing equivalents to EGSH. Right panel, newly synthesized Prx1 is required to recover the transfer of oxidizing equivalents to EGSH in the mitochondrial matrix.


( B ) response of a mitochondrial matrix - localized roGFP2 - Prx1 - P233stop probe expressed in wild - type to indicated concentrations of exogenous H2O2 . were grown in SGal ( - Leu ) medium harvested at early exponential phase . Error bars represent standard deviation ( n = 3 biological replicates , with obtained from 3 independent cultures for every 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 - gel - shift to assess Prx1 cysteine redox state . Wild - type transformed with an empty plasmid or ∆ prx1 with either an empty plasmid or a plasmid encoding wild - type Prx1 , hyperoxidation - resistant Prx1 - P233stop or inactive Prx1 - C91A variants , were grown to exponential phase in SGal ( - Ura ) medium . were subsequently treated with indicated concentrations of H2O2 for 10 mins then directly treated with 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 ) response of a mitochondrial matrix - localized Grx1 - roGFP2 probe , expressed in Δprx1 containing a plasmid encoding either wild - type Prx1 , Prx1 - P233stop or Prx1C91A to addition of 1 mM exogenous H2O2 . had been pre - treated with either 10 mM H2O2 or with water as a control . For these experiments , were grown in SGal ( - Leu , - Ura ) medium harvested at early exponential phase . OxD refers to degree of sensor oxidation . Error bars represent standard deviation ( n = 3 biological replicates , with obtained from 3 independent cultures for every 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 ' . Δprx1 co - transformed with empty vector or with a vector encoding either wild - type Prx1 , P233stop or C91A variants were grown in SD ( - Ura ) treated with indicated concentrations of H2O2 for 30 mins . Afterwards , were diluted a fixed volume plated on YPD plates . number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a relative to 0 mM treatment . Error bars represent standard deviation ( n = 5 - 8 biological replicates , with taken from independent cultures for each 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 ' . Δprx1 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 appropriate for plasmid selection pre - treated with 0 , 1 , 5 , 10 , 25 mM H2O2 for 30 mins . Afterwards , were diluted a fixed volume was plated on YPD plates . number of viable colonies was counted after 2 days of growth at 30 ° C , here represented as a relative to 0 mM pre - treatment . Error bars represent standard deviation ( n = 3 - 8 biological replicates , with taken from independent cultures for each biological replicate ) . Significance was assessed with a Student ' s 2 - , unpaired , t - . * 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 ) efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with upon exposure to acute H2O2 stress . Data from viability 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 ) response of a mitochondrial matrix - localized Grx1 - roGFP2 probe to 1 mM H2O2 . in BY4742 wild - type with an empty vector , in Δglr1 transformed either with an empty vector or a vector encoding wild - type Glr1 or form of Glr1 , where MTS - encoding region was removed , or in Δglr1Δprx1 transformed either with an empty vector or a vector encoding form of Glr1 . were grown to exponential phase in SGal ( - Leu , - Ura ) medium . Error bars represent standard deviation ( n = 3 biological replicates , with obtained from 3 independent cultures for every 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 Δglr1Δprx1 in SD medium complemented with ( n = 3 biological replicates ) . Significance for difference in time cultures reach 50 % of their maximal OD600 was assessed with t - . Error bars ( as ribbon ) represent standard deviation ( n = 4 biological replicates , with taken from independent cultures for each 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 ' . Wild - type , Δprx1 , Δglr1 Δglr1Δprx1 pre - grown in SGal medium complemented with to early exponential phase . were treated with H2O2 at indicated concentrations for 30 mins . Subsequently were diluted , a fixed volume plated on YPD plates . number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a relative to 0 mM pre - treatment . Error bars represent standard deviation ( n = 3 biological replicates , with taken from independent cultures for each biological replicate ) . An inset with a different scale on y - axis is presented for 1 5 mM 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 ' . Δprx1Δglr1 co - transformed with an empty plasmid or with a plasmid encoding either wild - type Prx1 , P233stop or C91A variants were grown in SGal ( - Ura ) medium to early exponential phase . were treated with indicated amounts of H2O2 for 30 mins . Subsequently , were diluted , a fixed volume plated on YPD plates . number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a relative to 0 mM treatment . Error bars represent standard deviation ( n = 3 biological replicates , with taken from independent cultures for each 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 degradation efficiency using different degrons . were incubated for indicated time periods with Asv or DOX / IAA or both ( DIA , see methods ) then collected for western blot . Doxycycline was added 2 hours before addition of IAA or Asv / IAA . To determine 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 crossreacting with 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 B degradation , cross checking of each mitotic cyclin . Treatments were performed as for DIA treated extracts were checked for both cyclin A2 B1 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 induced degradation by immunoblotting . Indicated lines were analysed 24 hours after mock or Dox / IAA / Asv ( DIA ) treatment using indicated antibodies to confirm homozygous gene tagging efficiency of 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 ) proliferation of A2dd , CycB1dd B1dd / B2ko following mock or DIA treatment . 1000 were plated in each well ( diameter 3 . 5cm ) incubated for 10 days before methanol fixation Crystal Violet .

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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 B1dd / B2ko following mock or four - hour DIA treatment of asynchronous . were imaged for 16h with 5min intervals using widefield DIC , mitotic entry was manually scored by detecting rounding . Curves display cumulative mitotic index ( data from three repeats , n > 500 per condition , s . d . indicated by shaded ) .

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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 MCF10A were analysed by PI FACS . histograms show changes in 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 degron depletion in RPE - 1 . RPE - 1 OsTIR1 RPE - 1 A2dd were subjected to 72 hours of cyclin A2 siRNA depletion / or of DIA treatment as indicated probed for cyclin A2 by immunoblotting . 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 from live - imaging of SiR - Tubulin labelled ( red ) mitotic B1dd / B2ko expressing FusionRed - H2B ( green ) , Mis12 - GFP ( ) ; time in mins , scale bar = 5µm . 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 were assessed by immunoblotting after 18 hours in 100ng / ml Nocodazole / or 5µM STLC . 6µM proTAME was added to indicated samples for 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 . Lower panel , following 40 - hour siRNA transfection B1dd / B2ko were treated for 24 hours with Thymidine , released for 10 hours treated with proTAME Apcin for additional four hours . At this point , were fixed stained with tubulin pericentrin for immunofluorescence .

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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 of mitotic spindles in control - DIA - treated B1dd / B2ko following arrest in mitosis by proTAME / Apcin treatment Tubulin is shown in , pericentrin in green 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 from mitotic Ctr DIA treated B1dd / B2ko after P / A synchronisation stained with anti - alpha - tubulin ( green ) , anti - gamma - tubulin ( red ) antibodies DAPI ( blue ) , scale bar = 5µm . were exposed to ice - cold medium 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 of PCNA - mRuby tagged A2dd . imaging was started at time of Doxycycline addition , degron activity was triggered three hours later by addition of IAA Asv or PBS ( indicated by dashed ) . Time is shown as hh : min , scale bar equals 10µm . For further were chosen that had dissipated their PCNA foci before 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 ) from time - lapse of mitosis showing division in controls after addition of 0 . 5µM PD - 166285 in DIA treated A2dd labelled with SiR - ( time is indicated as hh : min , 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 with inducible CycB1 - YFP ( B1 - WT ) CycB1 - YFP - NLS ( B1 - NLS ) were analysed for DIA induced cyclin B1 expression / cyclin A2 depletion . Samples were collected at indicated time points probed by immunoblotting with cyclin B1 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 ) CycB1 - YFP - NLS ( BYN ) . samples in odd lanes ( IAA / Asv - ) were treated with 1µg / ml doxycycline alone , while samples in even lanes ( IAA / Asv + ) were treated with Auxin Asv to induce cyclin A2 depletion . Total extract immune - precipitates were probed by immunoblotting with 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 ) of changes in selected cyclin B - dependent mitotic phosphorylations including Ki - 67 ( left panel ) , INCENP / Borealin ( middle panel ) Top2B , CDCA2 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 of Ki - 67 immunofluorescent of chromosome spreads from control ( Ctr ) DIA treated P / A synchronised B1dd / B2ko of Ki - 67 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 from live - imaging of DIA - treated SiR - Tubulin labelled ( red ) B1dd / B2ko expressing FusionRed - H2B ( green ) AurB - GFP ( ) ; time is indicated in minutes , scale bar = 5µm . Bottom panel shows frequencies of aberrant AurB localisation in P / A synchronised B1dd / B2ko .

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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 . top panel shows immunofluorescence , for we measured mean of Tpx2 on centrosomes spindle plotted of these values per . We also analysed 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 DIA treated , before 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 , Cdc27 dephosphorylation following Cdk1 inhibition in proTAME / Apcin treated mitotic extracts from Ctr or DIA treated . extracts were prepared at indicated times following treatment with 1µM Flavopiridol 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 from live - imaging of siRNA transfected B1dd / B2ko ( FusionRed - H2B in red , SiR - Tubulin in , scale bar = 10µm ) . Bar - plot panels on right show single - of 10 manually scored for mitosis pre - NEBD post - NEBD . Entry into prophase was scored by rounding NEBD was identified by influx of Tubulin in 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 - ( b / w ) , of ENSA / ARPP19 siRNA transfected DIA treated B1dd / B2ko . 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 of siRNA transfected P / A treated 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 calculation of Z ' - factors between high ( MERS - CoV infection , black ) 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 viability > 70 % were regarded as hits . Scatter plot shows MERS - CoV inhibition ratios overlaid with 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 stage in MERS - CoV life cycle that is inhibited by drugs . Vero were infected at a multiplicity of infection ( MOI ) of 5 with MERS - CoV , FDA - approved drugs were administered at six - time points prior to or post - infection as indicated . Drug concentrations were higher than 90 % inhibitory ( IC90 ) values of drugs , 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 activation of IRE1α , PERK ATF6 UPR branches by immunoblotting in control ( shCtrl ) Fam20C stable knockdown ( shFAM20C ) HepG2 treated with or without 5 μM Tg for 30 min . ATF6 - FL , full - ATF6 ; ATF6 - N , N - terminal cleavage product of ATF6 ; Asterisk , unspecific background bands . Fam20C knockdown was verified by immunoblotting of Concanavalin A - Sepharose ( Con A ) - enriched culture medium . Ponceau 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 ) unspliced XBP1 ( U ) mRNA in shCtrl shFAM20C HepG2 treated with or without 5 μM Tg for 1 h . ( Right ) of XBP1 mRNA splicing . Data information : data were shown as mean ± of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - Student ' s t - ) .

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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 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 were shown by immunoblotting . ( Right ) of XBP1 mRNA splicing . Data information : data were shown as mean ± of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - Student ' s t - ) .

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

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

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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 log2 of fold change versus - log10 ( p - value ) for Fam20C - interacting in Tg - treated HepG2 . red dots represent showing statistically significant increase of binding with Fam20C ( log2 fold change ≥ 1 ; - log10 ( p - value ) ≥ 1 . 3 ) ; blue dots represent showing statistically significant decrease of binding with Fam20C ( log2 fold change ≤ - 1 ; - log10 ( p - value ) ≥ 1 . 3 ) ; 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 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 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 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 ) anti - pS357 - PDI ( green ) antibodies . yellow signal depicts 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 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 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 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 PDI KO HepG2 ( H ) treated with 5 μM Tg for indicated times by immunoblotting on both regular phostag gels . ( Right ) of p - PDI on band intensities on phostag gels . Data information : Data were shown as mean ± of three ( H ) independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - way ANOVA , post hoc Tukey ' s HSD ) .

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

(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 photomicrographs of HepG2 co - transfected with PDI - mEmerald - KDEL ( green ) Fam20C - mApple ( red ) . were treated with DMSO ( Upper ) or 5 μM Tg ( Middle ) for 30 min . After Tg treatment , were washed with PBS 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 ± of N > 80 from four independent experiments . * * * p < 0 . 001 ( one - way ANOVA , post hoc Tukey ' s HSD ) .

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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 UPR signaling in HepG2 after Tg treatment 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 were treated with 25 ng / μl CHX for indicated times analyzed by immunoblotting for pS357 - PDI UPR signaling . 5 μM Tg or DMSO was added 30 min before 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 photomicrographs of HepG2 co - transfected with PDI - mEmerald - KDEL ( green ) 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 photomicrographs of HepG2 co - transfected with PDI - mEmerald - KDEL ( green ) 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 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 immunoblotting on both regular phostag gels . p - PDI was quantified 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 time course of between geometrical centers of domains a a ′ angle among domains b , b ′ a ′ from simulation of pS357 - PDI ( red ) 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 spectra of PDI WT S357E . Controls with ANS alone was shown . A . U . , arbitrary units . ( Right ) Enhancement factor were quantified . Data information : data were shown as mean ± of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( two - Student ' s t - ) .

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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 ) S357E ( Right ) by proteinase K ( Pro K ) for indicated times . Main digested fragments were indicated on right margin . Band abb ' x * is a digested product with cleavage after Met356 in 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 shFAM20C HepG2 were treated without or with 5 μM Tg for 30 min , in some aliquots of Tg was removed for 30 min . lysates were subjected to proteinase K digestion for 3 min immunoblotting .

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

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

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

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

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

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

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

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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 photomicrographs of HepG2 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 imaging of HepG2 expressing AgHaloER labeled by P1 ( green ) 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 imaging of WT PDI KO HepG2 co - transfected with AgHaloER labeled by P1 ( green ) TMR ( red ) 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 imaging of shCtrl shFAM20C HepG2 expressing AgHaloER labeled by P1 ( green ) 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 ) viabilities of WT PDI KO HepG2 co - transfected with AgHaloER empty vector ( - ) , PDI WT , S357E or S357A . were treated by 5 μM Tg for indicated times visualized by crystal violet . ( Lower ) of surviving fraction of . Data were shown as mean ± of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus WT HepG2 ( two - way ANOVA , post hoc Tukey ' s HSD ) .

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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 PDI KO HepG2 treated with or without 5 μM Tg for 1 h . PDI KO was verified by immunoblotting . ( Right ) of XBP1 mRNA splicing . Data were shown as mean ± of three independent experiments . * p < 0 . 05 ( two - Student ' s t - ) .

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

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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 ATF6 activation by immunoblotting in PDI KO HepG2 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 were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI 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 expressing FLAG - tagged IRE1α were treated without or with 5 μM Tg for 1 h . An aliquot of Tg - treated was then washed with culture medium chased for 4 h . FLAG - immunoprecipitates were analyzed by PDI 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 shFAM20C HepG2 were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI 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 HA - tagged PDI in HepG2 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 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 FLAG - tagged IRE1αNLD in HepG2 .

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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 for 2 h , then used to pulldown His - IRE1αNLD , as visualized by Coomassie blue . 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 .

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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 HA - tagged PDI WT or its binding mutant W128I / L403W in HepG2 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 ) 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 . ( Right ) of Xbp1 mRNA splicing at indicated times . Data information : data were shown as mean ± from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - Student ' s t - ) .

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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 hepatocytes isolated from WT 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 hepatocytes isolated from WT 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 by real - time PCR in liver of same after 24 h of Tm injection . Data information : data were shown as mean ± from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , post hoc Tukey ' s HSD ) .

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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 by real - time PCR in liver of same after 24 h of Tm injection . Data information : data were shown as mean ± from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , post hoc Tukey ' s HSD ) .

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 ) of same as in ( C ) after 24 h of Tm injection . Data information : data were shown as mean ± from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , post hoc Tukey ' s HSD ) .

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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 eosin of liver tissue same as in ( C ) after 24 h of Tm injection ( magnification × 400 ) . Immune infiltration was indicated with a black arrow . Scale bar = 50 μm . Representative were shown from five 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 of same liver as in ( M ) . Scale bar = 40 μm ; Zoom box , scale bar = 5 μm . ( Right ) of apoptotic . 10 random fields were counted for each TUNEL - stained tissue sample . Data information : data were shown as mean ± from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - Student ' s t - ) .

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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 - demonstrate lipid bilayer EV purified from Molm - 14 U - 937 . Scale bars are 100nm .

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 contain no mGFP + foci ( top ) however Molm - 14 - mGFP ( middle ) U - 937 - mGFP ( bottom ) xenografts contain numerous mGFP - labeled EV ( green ) detectible without vesicle . 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 - microscopy of KSL ( left panel ) LT - HSC ( right panel ) . were sorted from control mice ( top ) , Molm - 14 - mGFP ( middle ) U - 937 - mGFP ( bottom ) , stained with Mask ( red ) stain Hoechst ( blue ) ; imaged using GE / API Deltavision ( DV ) widefield microscope ( 60X objective ) to show uptake of 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 showing frequency of LK KSL in lineage - negative ( left panel ) 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 showing frequency of LK KSL in lineage - negative ( left panel ) LT - HSC in KSL ( right panel ) in : IF injection of EV from Molm - 14 ( red , n = 8 ) , AML plasma EV ( Orange , n = 6 ) human CD34 EV ( Blue , n = 4 ) relative to vehicle - injected 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.


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

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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 of Ki67 - ve of LT - HSC representing G0 phase of - 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 of intracellular P53 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 ( blue , n = 6 ) normalized to vehicle - injected 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 of intracellular P53 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 ( blue , n = 6 ) normalized to vehicle - injected 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 showing highly - deregulated ribosomal in AML - EV exposed .

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


Flow cytometric showing histograms 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,