figf4 ( a ) Beclin - 1 alone ( lanes 1 - 4 ) or Beclin - 1 and ATG14L ( lanes 5 - 8 ) were overexpressed in HEK293 cells . Beclin - 1 was purified either by direct immunoprecipitation ( lanes 1 - 4 ) or by ATG14L immunoprecipitation ( lanes 5 - 8 ) . Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1 . Reactions were immunoblotted with the indicated antibodies . Black line denotes discontinuous lanes from the same gel . Two unique experiments were performed . ( b ) Beclin - 1 alone or bound to ATG14L was purified as described in a . Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points . Western blotting was performed with the indicated antibodies . ( c ) An ATG14L - FLAG - 6His - inducible U2OS cell line was induced for 16 h in the presence of amino acids . Endogenous Beclin - 1 was immunoprecipitated and immunoblotted as in Fig . 3a . ATG14L input levels were detected by immunoblotting . Two unique experiments were performed . ( d ) HEK293 cells transfected with either ATG14L or Beclin - 1 , or both , in conjunction with ULK1 , were immunoprecipitated as indicated and blotted with the indicated antibodies . ( e ) HEK293 cells were transfected with Beclin - 1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD , which is defective in Beclin - 1 binding , under nutrient - rich conditions . Lysates were resolved by SDS - PAGE and blotted with the indicated antibodies . ( f ) HEK293 cells were transfected with ULK1 and Beclin - 1 in conjunction with either ATG14L WT , or one of two mutants ( ΔBATS , ΔN ) that are defective in phagophore localization . Samples were handled as in e . Uncropped images of blots are shown in Supplementary Fig . S4 . Lysates were resolved by SDS - PAGE and blotted with the indicated antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in HEK293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L immunoprecipitation (lanes 5-8). Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed.(b) Beclin-1 alone or bound to ATG14L was purified as described in a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blotting was performed with the indicated antibodies.(c) An ATG14L-FLAG-6His-inducible U2OS cell line was induced for 16 h in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig. 3a. ATG14L input levels were detected by immunoblotting. Two unique experiments were performed.(d) HEK293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1, were immunoprecipitated as indicated and blotted with the indicated antibodies.(e) HEK293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient-rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.(f) HEK293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L WT, or one of two mutants (ΔBATS,ΔN) that are defective in phagophore localization. Samples were handled as in e. Uncropped images of blots are shown in Supplementary Fig. S4. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.


figf5 ( a ) HEK293 cells were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated in the presence of amino acids . Lysates were immunoblotted with the indicated antibodies . A representative experiment of three repeats is shown . ( b ) UVRAG bridges the interaction between Beclin - 1 and ULK1 . HEK293 cells were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated . Lysates were immunoprecipitated with anti - HA ( Beclin - 1 ) antibody and blotted with the indicated antibodies . A representative experiment of three repeats is shown . Uncropped images of blots are shown in Supplementary Fig . S4 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids. Lysates were immunoblotted with the indicated antibodies. A representative experiment of three repeats is shown.(b) UVRAG bridges the interaction between Beclin-1 and ULK1. HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated. Lysates were immunoprecipitated with anti-HA(Beclin-1) antibody and blotted with the indicated antibodies. A representative experiment of three repeats is shown. Uncropped images of blots are shown in Supplementary Fig. S4.


figf6 ( a ) HEK293 cells were transfected with the indicated plasmids . Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated . Lysates were immunoblotted with the indicated antibodies . ( b ) HEK293 cells transfected with Beclin - 1 ATG14L were grown in the presence or absence of amino acids . Lysates were immunoblotted with the indicated antibodies ( left panel ) and quantified by densitometry ( right panel ) . Data represent mean + s . d . of three unique experiments . ( c ) Beclin - 1 - shRNA reconstituted lines ( WT or mutant ) and controls ( scramble shRNA or Beclin - 1 shRNA ) were grown with or without amino acids and assessed for autophagy ( left panel ) and Beclin - 1 levels ( right panel ) . ( d ) Autophagosome ( denoted by arrowheads ) generation on amino - acid withdrawal was analysed by electron microscopy . Cell lines and conditions from c were used and representative images from the indicated amino - acid - starved lines are shown ; scale bars , 0 . 4 μm . ( d ) Autophagosome ( denoted by arrowheads ) generation on amino - acid withdrawal was analysed by electron microscopy . Cell lines and conditions from c were used and representative images from the indicated amino - acid - starved lines are shown ; scale bars , 0 . 4 μm . ( e ) Quantification of d . Fold induction was determined by arbitrarily making the nutrient - rich condition 1 ( solid bars ) for each line . Error bars represent the s . d . of the mean value over an average of 20 fields of view within a representative experiment . NS , not significant . ( f ) HA - Beclin - 1 WT or S14D was transiently expressed in FIP200 − / − MEFs grown under nutrient - rich conditions . Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA - Beclin - 1 . Scale bars , 20 μm . ( g ) Quantification of LC3B puncta from confocal in f . In the HA - Beclin - 1 - or HA - Beclin - 1 - S14D - transfected samples , only the HA - positive cells were counted for LC3B puncta . Error bars were processed as in e . Mean value shown ; P values determined by Student ' s t - test using 10 unique fields of view from f . Uncropped images of blots are shown in Supplementary Fig . S4 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) HEK293 cells were transfected with the indicated plasmids. Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated. Lysates were immunoblotted with the indicated antibodies.(b) HEK293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Data represent mean+s.d. of three unique experiments.(c) Beclin-1-shRNA reconstituted lines (WT or mutant) and controls (scramble shRNA or Beclin-1 shRNA) were grown with or without amino acids and assessed for autophagy (left panel) and Beclin-1 levels (right panel).(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm.(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (e) Quantification of d. Fold induction was determined by arbitrarily making the nutrient-rich condition 1 (solid bars) for each line. Error bars represent the s.d. of the mean value over an average of 20 fields of view within a representative experiment. NS, not significant.(f) HA-Beclin-1 WT or S14D was transiently expressed in FIP200−/− MEFs grown under nutrient-rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA-Beclin-1. Scale bars, 20 μm. (g) Quantification of LC3B puncta from confocal in f. In the HA-Beclin-1- or HA-Beclin-1-S14D-transfected samples, only the HA-positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value shown; P values determined by Student's t-test using 10 unique fields of view from f. Uncropped images of blots are shown in Supplementary Fig. S4.


figf7 ( a ) Bec - 1C . elegans were reconstituted with either WT or mutant GFP - BEC - 1 . Stable worm lines with Bec - 1 rescue were obtained and embryos were stained with anti - PGL - 1 antibody . Arrowheads indicate normal PGL - 1 staining in germline cells . ( b ) Quantification of PGL - 1 puncta outside germline cells ( left panel ) . Error bars represents s . d . between 3 unique embryos in a representative experiment . Reconstituted Bec - 1 ( WT and mutant ) levels in Bec − / − stable worms were compared by western blotting . Mean value presented . ( c ) Spectrum of defects in PGL granule degradation in bec - 1 mutant rescue embryos . Mutant embryos exhibited either high levels of diffuse PGL - 1 staining ( middle - left panel ; one - third of the embryos ) , or large punctuate PGL - 1 structures in somatic cells ( bottom - left panel ; two - thirds of the embryos ) . Both diffuse or punctuate PGL - 1 staining in somatic cells have been described in autophagy - deficient embryos . ( d ) Embryos from the lines described in a were labelled with anti - LGG - 1 , along with WT and unc - 51 worms . Representative embryos at ∼ 100 cell stage are shown . ( e ) Quantification of LGG - 1 per embryo from labelling in d . Error bars generated as in b . Mean value presented . Uncropped images of blots are shown in Supplementary Fig . S4 . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) Bec-1C. elegans were reconstituted with either WT or mutant GFP-BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti- PGL-1 antibody. Arrowheads indicate normal PGL-1 staining in germline cells. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents s.d. between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mutant) levels in Bec−/− stable worms were compared by western blotting. Mean value presented.(c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos exhibited either high levels of diffuse PGL-1 staining (middle-left panel; one-third of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel; two-thirds of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy-deficient embryos.(d) Embryos from the lines described in a were labelled with anti-LGG-1, along with WT and unc-51 worms. Representative embryos at ∼ 100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labelling in d. Error bars generated as in b. Mean value presented. Uncropped images of blots are shown in Supplementary Fig. S4. Scale bar, 10 μm.


Figure 1 ( F ) Single tube qPCR experiments showing the down - regulation of the synaptic genes NLGN3 , SLC6A , TRIM9 , SYT1 , SYNGR1 , and SNAP91 in ALSC9orf72 MN ( Welch ' s t - test ) . n = 3 independent cultures for each hiPSC line . 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 . ( G - H ) Time course analysis of nuclear pCREBS133 levels and MN survival in ALSC9orf72 and Healthy cultures ( two - way ANOVA ) . n = 3 independent cultures for each hiPSC line ( for each time point ) . Scale bar : 25 µm . 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(F) Single tube qPCR experiments showing the down-regulation of the synaptic genes NLGN3, SLC6A, TRIM9, SYT1, SYNGR1, and SNAP91 in ALSC9orf72 MN (Welch's t-test). n=3 independent cultures for each hiPSC line. 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.(G-H) Time course analysis of nuclear pCREBS133 levels and MN survival in ALSC9orf72 and Healthy cultures (two-way ANOVA). n=3 independent cultures for each hiPSC line (for each time point). Scale bar: 25 µm. 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.


Figure 2 ( A ) Both K + channel blockers exert a neuroprotective effect in ALSC9orf72 cultures by increasing the neurite length of mutant MN ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments with each hiPSC line . Scale bar : 50 µm 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 . ( C ) Apamin and XE991 decrease the size of aberrant aggresomes , confirming enhanced autophagy degradation ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments with the ALSC9orf72 I line . Scale bar : 5 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 ) The load of aggregated SQSTM1 / p62 is also reduced by the treatments in ALSC9orf72 cultures ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments for each cell line . Scale bar : 20 µm . 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Both K+ channel blockers exert a neuroprotective effect in ALSC9orf72 cultures by increasing the neurite length of mutant MN (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 50 µm 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.(C) Apamin and XE991 decrease the size of aberrant aggresomes, confirming enhanced autophagy degradation (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with the ALSC9orf72 I line. Scale bar: 5 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) The load of aggregated SQSTM1/p62 is also reduced by the treatments in ALSC9orf72 cultures (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments for each cell line. Scale bar: 20 µm. 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.(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.


Figure 3 ( 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 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 - and neuronal processes - related proteins significantly up - regulated by both treatments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 3(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 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- and neuronal processes-related proteins significantly up-regulated by both treatments.


Figure 1The 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 . 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 . 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 . 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 ) 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 ) 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 ) 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 ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1The 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.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.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.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) 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) 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) 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) 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.


Figure 2 ( 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 ) 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 ) 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 ) 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 ) 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

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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) 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) 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) 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) 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


Figure 3 ( 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 : 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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: 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) 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.


Figure 4 ( 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 ' 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 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 : 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 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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' 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 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: 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 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) 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.


Figure 5 ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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).


Figure 6 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 7 ( 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 ) . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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).(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).


Figure 1 ( 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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Figure 1(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 addition


Figure 2A ) 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 . ( 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 . ( 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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Figure 2A) 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.(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.(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.


Figure 3 ( 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 . ( 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 . ( 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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Figure 3(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.(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.(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.


Figure 4 ( 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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Figure 4(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).


Figure 5 ( 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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Figure 5(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.


Figure 6 ( 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 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 . ( 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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Figure 6(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 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.(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.


Figure 7 ( 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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Figure 7(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 immunoblotting


Figure 8A ) 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 . ( C ) Panels of representative immunofluorescence images of siRNA transfected and P / A treated cells treated as indicated , scale bar = 10µm .

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Figure 8A) 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.(C) Panels of representative immunofluorescence images of siRNA transfected and P/A treated cells treated as indicated, scale bar = 10µm.


Fig . 1 . ( 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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Fig. 1.(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.


Fig . 3 . 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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Fig. 3.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.


Figure 1A 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 ) . 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 ) . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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).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).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.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.


Figure 2A 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 . 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 . 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 . ( 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 ) .

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Figure 2A 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.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.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.(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).


Figure 3A 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 ) . 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 . 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 . 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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Figure 3A 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).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.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.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.