Figure 2A Lytic growth of intracellular wild type Toxoplasma gondii tachyzoite stages over a period of 8 days results in plaques within mononolayers of host cells . Destabilisation of PKAr - TyDD leads to the absence of plaque formation . B A drop in PKAr - TyDD expression is detected 5 hours after Shld - 1 removal and a significant parasite growth defect is observed as early as 12 hours later . Profilin ( αPRF ) served as loading control . C IFA analysis showed that the down regulation of PKAr - TyDD leads to the abnormal segregation of the apicoplast - associated thioredoxin family protein ( ATrx1 ) 24 hours after Shld - 1withdrawal . D Replication assay of PKAr - TyDD parasites grown for 24 hours + / - Shld - 1 indicates that no more than four parasites are observed per vacuole upon PKAr destabilisation . ( 100 parasites were counted in three independent replicates ) . E , F Stabilisation of a second copy of DDmyc - PKAc1 leads to the complete block of cell growth indicated by plaque assay ( E ) E , F Stabilisation of a second copy of DDmyc - PKAc1 leads to the complete block of cell growth indicated by western blot analysis ( F ) . G , H Stabilisation of a second copy of DDmyc - PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld - 1 ( 100 parasites were counted in three independent replicates ) . Data information : In C and G , data are presented as mean ± SEM . Scale bars = 2 μm ( C and G ) , 3 mm ( A and E ) . G , H Stabilisation of a second copy of DDmyc - PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld - 1 ( 100 parasites were counted in three independent replicates ) .

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

Figure 2A Lytic growth of intracellular wild type Toxoplasma gondii tachyzoite stages over a period of 8 days results in plaques within mononolayers of host cells. Destabilisation of PKAr-TyDD leads to the absence of plaque formation.B A drop in PKAr-TyDD expression is detected 5 hours after Shld-1 removal and a significant parasite growth defect is observed as early as 12 hours later. Profilin (αPRF) served as loading control.C IFA analysis showed that the down regulation of PKAr-TyDD leads to the abnormal segregation of the apicoplast-associated thioredoxin family protein (ATrx1) 24 hours after Shld-1withdrawal.D Replication assay of PKAr-TyDD parasites grown for 24 hours +/- Shld-1 indicates that no more than four parasites are observed per vacuole upon PKAr destabilisation. (100 parasites were counted in three independent replicates).E, F Stabilisation of a second copy of DDmyc-PKAc1 leads to the complete block of cell growth indicated by plaque assay (E)E, F Stabilisation of a second copy of DDmyc-PKAc1 leads to the complete block of cell growth indicated by western blot analysis (F).G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).Data information: In C and G, data are presented as mean ± SEM. Scale bars = 2 μm (C and G), 3 mm (A and E).G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).


Figure 3A A drop in PKAc1 - Ty - iKD expression is detected as early as 24 hours upon ATc treatment with almost no protein detectable after 36 hours . B Conditional down - regulation of PKAc1 - Ty - iKD leads to the absence of plaque formation . Scale bars = 1 mm ( B ) C , D Down - regulation of PKAc1 - Ty - iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress ( data is from three independent biological replicates ) . Scale bars = 20 μm ( C ) C , D Down - regulation of PKAc1 - Ty - iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress ( data is from three independent biological replicates ) . Data information : In D , data are presented as mean ± SD . E Prematurely egressed PKAc1 - Ty - iKD parasites after 24 hours of ATc treatment invade and exit fully lysing the monolayer of HFF cells while the non - treated parasites invade and initiate a new lytic cycle . Scale bars = 2 . 5 mm ( E )

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

Figure 3A A drop in PKAc1-Ty-iKD expression is detected as early as 24 hours upon ATc treatment with almost no protein detectable after 36 hours.B Conditional down-regulation of PKAc1-Ty-iKD leads to the absence of plaque formation.Scale bars = 1 mm (B)C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Scale bars = 20 μm (C)C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Data information: In D, data are presented as mean ± SD.E Prematurely egressed PKAc1-Ty-iKD parasites after 24 hours of ATc treatment invade and exit fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle. Scale bars = 2.5 mm (E)


Figure 4B Addition of Shld - 1 leads to the rapid stabilisation of DDmyc - PKArWT - Ty and DDmyc - PKArG321E - Ty . C PKAc1 is co - immunoprecipitated with DDmyc - PKArWT - Ty in absence of cAMP but is not recovered in presence of 20 µM cAMP as revealed with [ 35S ] - labeled methionine / cysteine metabolic labelling . Conversely , PKAc1 is co - immunoprecipitated with DDmyc - PKArG321E - Ty in presence or absence of cAMP indicating the G321E substitution prevents the release of active PKAc1 in presence of cAMP . The autoradiogram shown is representative of two independent biological replicates . Data information : In C and E - H , data are presented as mean ± SEM . D Conditional overexpression of a dominant - negative DDmyc - PKArG321E - Ty that cannot bind cAMP leads a dramatic reduction in plaque formation while over - expression of DDmyc - PKArWT - Ty is well tolerated by the parasite . Scale bars = 3 mm ( D ) E Overexpression of the dominant negative DDmyc - PKArG321E - Ty triggers premature egress from infected host cells and prevents initiation of a new lytic cycle as revealed by IFA . Images are representative of ~ 80 % of the parasite population . Blue = DAPI ; red = GAP45 ; green = GRA3 . Data information : In C and E - H , data are presented as mean ± SEM . Scale bars = 10 μm ( E ) F In heavily infected cells , addition of Shld - 1 at 32 hours post - inoculation for 4 hours leads to premature egress of DDmyc - PKArG321E - Ty parasites but not of the DDmyc - PKAr - Ty control line as assessed by IFA . Data information : In C and E - H , data are presented as mean ± SEM . Scale bars = 5 µm ( F ) G Quantification of extracellular DDmyc - PKArG321E - Ty parasites released from heavily infected cells after addition of Shld - 1 at 32 hours post - inoculation for 4 hours ( 100 parasites were counted in three independent replicates ) . Data information : In C and E - H , data are presented as mean ± SEM . H A non - selective PKA inhibitor , KT5270 , significantly enhances the premature egress of DDmyc - PKArWT - Ty parasites in presence of Shld - 1 ( data is from three independent biological replicates ; statistical analysis was done by two - tailed t - test ) . Data information : In C and E - H , data are presented as mean ± SEM . I Prematurely egressed DDmyc - PKArG321E - Ty parasites after 4 hours of Shld - 1 treatment invade and exit host cells , fully lysing the monolayer of HFF cells while the non - treated parasites invade and initiate a new lytic cycle . Scale bars = 2 . 5 mm ( I ) .

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 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 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 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 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 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

Figure 4B Addition of Shld-1 leads to the rapid stabilisation of DDmyc-PKArWT-Ty and DDmyc-PKArG321E-Ty.C PKAc1 is co-immunoprecipitated with DDmyc-PKArWT-Ty in absence of cAMP but is not recovered in presence of 20 µM cAMP as revealed with [35S]-labeled methionine/cysteine metabolic labelling. Conversely, PKAc1 is co-immunoprecipitated with DDmyc-PKArG321E-Ty in presence or absence of cAMP indicating the G321E substitution prevents the release of active PKAc1 in presence of cAMP. The autoradiogram shown is representative of two independent biological replicates.Data information: In C and E-H, data are presented as mean ± SEM.D Conditional overexpression of a dominant-negative DDmyc-PKArG321E-Ty that cannot bind cAMP leads a dramatic reduction in plaque formation while over-expression of DDmyc-PKArWT-Ty is well tolerated by the parasite.Scale bars = 3 mm (D)E Overexpression of the dominant negative DDmyc-PKArG321E-Ty triggers premature egress from infected host cells and prevents initiation of a new lytic cycle as revealed by IFA. Images are representative of ~80% of the parasite population. Blue = DAPI; red = GAP45; green = GRA3.Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 10 μm (E)F In heavily infected cells, addition of Shld-1 at 32 hours post-inoculation for 4 hours leads to premature egress of DDmyc-PKArG321E-Ty parasites but not of the DDmyc-PKAr-Ty control line as assessed by IFA.Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 5 µm (F)G Quantification of extracellular DDmyc-PKArG321E-Ty parasites released from heavily infected cells after addition of Shld-1 at 32 hours post-inoculation for 4 hours (100 parasites were counted in three independent replicates).Data information: In C and E-H, data are presented as mean ± SEM.H A non-selective PKA inhibitor, KT5270, significantly enhances the premature egress of DDmyc-PKArWT-Ty parasites in presence of Shld-1 (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test).Data information: In C and E-H, data are presented as mean ± SEM.I Prematurely egressed DDmyc-PKArG321E-Ty parasites after 4 hours of Shld-1 treatment invade and exit host cells, fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle.Scale bars = 2.5 mm (I).


Figure 5B Upon Shld - 1 addition , a putative cGMP - specific PDE , PDE2 was more phosphorylated at Serine 1317 in DDmyc - PKArG321E - Ty parasites compared with DDmyc - PKArWT - Ty parasites suggesting a crosstalk between cAMP and cGMP signaling . C Chemical inhibition of the cGMP - dependent PKG by Compound 1 and Compound 2 blocks egress induced by 5 µM BIPPO , a PDE inhibitor , or by stabilisation of DDmyc - PKArG321E - Ty in presence of Shld - 1 . A PKGT761M substitution that renders PKG resistant to both compounds indicates that the block in egress in only mediated by PKG when 0 . 2 µM Compound 1 is used while the block in egress associated with C2 is not specific to PKG inhibition only ( data is from three independent biological replicates ) . Data information : In C and D , data are presented as mean ± SD . D Genetic inhibition of PKA triggers MIC2 secretion in intracellular buffer . Anti - GRA1 antibodies were used as a loading control and anti - catalase antibodies served as a lysis control ( data is from three independent biological replicates ; statistical analysis was done by two - tailed t - test ) Data information : In C and D , data are presented as mean ± SD .

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 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

Figure 5B Upon Shld-1 addition, a putative cGMP-specific PDE, PDE2 was more phosphorylated at Serine 1317 in DDmyc-PKArG321E-Ty parasites compared with DDmyc-PKArWT-Ty parasites suggesting a crosstalk between cAMP and cGMP signaling.C Chemical inhibition of the cGMP-dependent PKG by Compound 1 and Compound 2 blocks egress induced by 5 µM BIPPO, a PDE inhibitor, or by stabilisation of DDmyc-PKArG321E-Ty in presence of Shld-1. A PKGT761M substitution that renders PKG resistant to both compounds indicates that the block in egress in only mediated by PKG when 0.2 µM Compound 1 is used while the block in egress associated with C2 is not specific to PKG inhibition only (data is from three independent biological replicates).Data information: In C and D, data are presented as mean ± SD. D Genetic inhibition of PKA triggers MIC2 secretion in intracellular buffer. Anti-GRA1 antibodies were used as a loading control and anti-catalase antibodies served as a lysis control (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test) Data information: In C and D, data are presented as mean ± SD.


Figure 6A The premature egress induced by PKA genetic inhibition is exacerbated when host cells are heavily infected ( data is from three independent biological replicates ) . B Transferring supernatants of Shld - 1 treated cultures from cells infected with DDmyc - PKArG321E - Typarasites at a MOI of 8 induces egress of parasites from cells infected at a MOI of 2 within three hours ( data is from three independent biological replicates ; statistical analysis was done by two - tailed t - test ) . C Acidification of the culture medium induces parasite egress in less than 10 minutes . The effect is inhibited in presence of compound 1 ( data is from two independent biological replicates ) . D Neutralisation of cultures with the weak base NH4Cl blocks premature egress induced by DDmyc - PKArG321E - Ty stabilisation . This block is circumvented by addition of the Ca2 + ionophore A23187 or the PDE inhibitor BIPPO ( data is from three independent biological replicates ) . Data information : Data are presented as mean ± SD .

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 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 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

Figure 6A The premature egress induced by PKA genetic inhibition is exacerbated when host cells are heavily infected (data is from three independent biological replicates).B Transferring supernatants of Shld-1 treated cultures from cells infected with DDmyc-PKArG321E-Typarasites at a MOI of 8 induces egress of parasites from cells infected at a MOI of 2 within three hours (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test).C Acidification of the culture medium induces parasite egress in less than 10 minutes. The effect is inhibited in presence of compound 1 (data is from two independent biological replicates). D Neutralisation of cultures with the weak base NH4Cl blocks premature egress induced by DDmyc-PKArG321E-Ty stabilisation. This block is circumvented by addition of the Ca2+ ionophore A23187 or the PDE inhibitor BIPPO (data is from three independent biological replicates). Data information: Data are presented as mean ± SD.


Figure 7A - D Localization of PDE1 - 3Ty ( A ) shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , and myc - GAP70 were used as markers of the plasma membrane , IMC , residual body , and apical pole , respectively . Data information : Scale bars = 2 μm . A - D Localization of PDE2 - 3Ty ( B ) shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , and myc - GAP70 were used as markers of the plasma membrane , IMC , residual body , and apical pole , respectively . Data information : Scale bars = 2 μm . A - D Localization of GCα - 3Ty ( C ) shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , and myc - GAP70 were used as markers of the plasma membrane , IMC , residual body , and apical pole , respectively . Data information : Scale bars = 2 μm . A - D Localization of PKG - 3Ty shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , and myc - GAP70 were used as markers of the plasma membrane , IMC , residual body , and apical pole , respectively . Data information : Scale bars = 2 μm . E Western blot analysis indicates that GCα - 3Ty is present in three distinct isoforms . A PKG - 3Ty western blot also shows three immune reactive isoforms . Catalase or actin were used as loading controls and ΔKu80 as the parental strain . F PKG - 3Ty parasites were solubilised in either PBS or 1 % Triton X - 100 ( TX100 ) and split into soluble ( S ) and pellet ( P ) fractions . Total lysate is also shown . G , H The signal of Myc - ACβ - iKD followed by IFA ( G ) after 48 hours ± ATc . Data information : Scale bars = 2 μm . G , H The signal of Myc - ACβ - iKD followed by western blot ( H ) after 48 hours ± ATc . I Invasion of ACα - KO tachyzoites or Myc - Acβ - iKD after 48 hours ± ATc is not affected . However , addition of ATc for 48 hours reduces the invasion efficiency of ACα - KO / Myc - Acβ - iKD ( data is from three independent biological replicates ) . J As for destabilisation of PKAc1 - iKD or stabilisation of DDmyc - PKArG321E - Ty , ATc - KO / Myc - Acβ - iKD tachyzoites treated with lytic cycle for 33 or 40 hours , exit host cells and invade and exit the monolayer of HFF cells leading to lysis while the non - treated parasites invade and initiate a new lytic cycle . Data information : Scale bars = 2 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 7A-D Localization of PDE1-3Ty (A) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.A-D Localization of PDE2-3Ty (B) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.A-D Localization of GCα-3Ty (C) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.A-D Localization of PKG-3Ty shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.E Western blot analysis indicates that GCα-3Ty is present in three distinct isoforms. A PKG-3Ty western blot also shows three immune reactive isoforms. Catalase or actin were used as loading controls and ΔKu80 as the parental strain.F PKG-3Ty parasites were solubilised in either PBS or 1% Triton X-100 (TX100) and split into soluble (S) and pellet (P) fractions. Total lysate is also shown.G, H The signal of Myc-ACβ-iKD followed by IFA (G) after 48 hours ± ATc.Data information: Scale bars = 2 μm.G, H The signal of Myc-ACβ-iKD followed by western blot (H) after 48 hours ± ATc.I Invasion of ACα-KO tachyzoites or Myc-Acβ-iKD after 48 hours ± ATc is not affected. However, addition of ATc for 48 hours reduces the invasion efficiency of ACα-KO/ Myc-Acβ-iKD (data is from three independent biological replicates). J As for destabilisation of PKAc1-iKD or stabilisation of DDmyc-PKArG321E-Ty, ATc -KO/ Myc-Acβ-iKD tachyzoites treated with lytic cycle for 33 or 40 hours, exit host cells and invade and exit the monolayer of HFF cells leading to lysis while the non-treated parasites invade and initiate a new lytic cycle. Data information: Scale bars = 2 μm.


Figure 1A , B Hypocotyl length of ChWT , ( A ) RNAi - ChHFR1 transgenic and ( B ) chfr1 mutant seedlings grown under different R : FR . Seedlings were grown for 7 days in continuous W ( R : FR > 1 . 5 ) or for 3 days in W then transferred to W supplemented with increasing amounts of FR ( W + FR ) for 4 more days , producing various R : FR . Aspect of representative 7 - day old ChWT , RNAi - HFR1 and chfr1 - 1 seedlings grown in W or W + FR ( R : FR , 0 . 02 ) , as indicated , is shown in lower panel . Data information : Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h . Asterisks mark significant differences ( Student t - test : * * p - value < 0 . 01 ; * p - value < 0 . 05 ) relative to ChWT value at the same time point . C , D Effect of W + FR exposure on the expression of PIL1 , YUC8 and XTR7 genes in seedlings of ChWT , ( C ) RNAi - HFR1 and ( D ) chfr1 mutant lines . Expression was analyzed in 7 - day old W - grown seedlings transferred to W + FR ( R : FR , 0 . 02 ) for 0 , 1 , 4 , 8 and 12 h . Transcript abundance is normalized to EF1α levels . Data information : Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h . Asterisks mark significant differences ( Student t - test : * * p - value < 0 . 01 ; * p - value < 0 . 05 ) relative to ChWT value at the same time point .

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Figure 1A, B Hypocotyl length of ChWT, (A) RNAi-ChHFR1 transgenic and (B) chfr1 mutant seedlings grown under different R:FR. Seedlings were grown for 7 days in continuous W (R:FR>1.5) or for 3 days in W then transferred to W supplemented with increasing amounts of FR (W+FR) for 4 more days, producing various R:FR. Aspect of representative 7-day old ChWT, RNAi-HFR1 and chfr1-1 seedlings grown in W or W+FR (R:FR, 0.02), as indicated, is shown in lower panel. Data information: Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h. Asterisks mark significant differences (Student t-test: ** p-value <0.01; * p-value <0.05) relative to ChWT value at the same time point.C, D Effect of W+FR exposure on the expression of PIL1, YUC8 and XTR7 genes in seedlings of ChWT, (C) RNAi-HFR1 and (D) chfr1 mutant lines. Expression was analyzed in 7-day old W-grown seedlings transferred to W+FR (R:FR, 0.02) for 0, 1, 4, 8 and 12 h. Transcript abundance is normalized to EF1α levels. Data information: Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h. Asterisks mark significant differences (Student t-test: ** p-value <0.01; * p-value <0.05) relative to ChWT value at the same time point.


Figure 2Seedlings of ChWT and AtWT were grown for 3 days in W then either kept under the same conditions or transferred to W + FR ( R : FR , 0 . 02 ) for the indicated times . Plant material was harvested every 24 h . Transcript abundance of HFR1 and PIF7 was normalized to three reference genes ( EF1α , SPC25 , and YLS8 ) . Expression values are the means ± SE of three independent biological replicates relative to the data of AtWT grown in continuous W at day 3 . Asterisks mark significant differences ( 2 - way ANOVA : * * p - value < 0 . 01 , * * * p - value < 0 . 001 ) between ChWT and AtWT when grown under W ( black asterisks ) or W + FR ( red asterisks ) .

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Figure 2Seedlings of ChWT and AtWT were grown for 3 days in W then either kept under the same conditions or transferred to W+FR (R:FR, 0.02) for the indicated times. Plant material was harvested every 24 h. Transcript abundance of HFR1 and PIF7 was normalized to three reference genes (EF1α, SPC25, and YLS8). Expression values are the means ± SE of three independent biological replicates relative to the data of AtWT grown in continuous W at day 3. Asterisks mark significant differences (2-way ANOVA: ** p-value <0.01, *** p-value <0.001) between ChWT and AtWT when grown under W (black asterisks) or W+FR (red asterisks).


Figure 3B Relative expression of HFR1 in seedlings of AtWT , At hfr1 - 5 , hfr1 > pAt : ChHFR1 ( in blue ) and hfr1 > pAt : AtHFR1 ( in red ) lines grown under W + FR ( R : FR , 0 . 02 ) . Expression values are the means ± SE of three independent biological replicates relative to the data of 7 days old AtWT . Transcript abundance is normalized to UBQ10 levels . Data information : Different letters denote significant differences ( one - way ANOVA with Tukey test , p - value < 0 . 05 ) among means . C Elongation response of seedlings of the indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W + FR ( R : FR , 0 . 02 ) . The mean hypocotyl length in W ( HypW ) and W + FR ( HypW + FR ) of at least four biological replicates was used to calculate HypW + FR - HypW . Error bars represent SE . Data information : Different letters denote significant differences ( one - way ANOVA with Tukey test , p - value < 0 . 05 ) among means . D Relative HFR1 protein levels in seedlings of the indicated lines , normalized to actin protein levels , are the means ± SE of three independent biological replicates relative to hfr1 > pAt : ChHFR1 line # 22 that is taken as 1 . Seedlings were grown for 7 days in continuous W ( ~ 20 µmol m - 2 s - 1 ) after which they were incubated for 3 h in high W ( ~ 100 µmol m - 2 s - 1 ) and transferred to W + FR ( R : FR , 0 . 06 ) for 3 h . Data information : Different letters denote significant differences ( one - way ANOVA with Tukey test , p - value < 0 . 05 ) among means .

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Figure 3B Relative expression of HFR1 in seedlings of AtWT, At hfr1-5, hfr1>pAt:ChHFR1 (in blue) and hfr1>pAt:AtHFR1 (in red) lines grown under W+FR (R:FR, 0.02). Expression values are the means ± SE of three independent biological replicates relative to the data of 7 days old AtWT. Transcript abundance is normalized to UBQ10 levels. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.C Elongation response of seedlings of the indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W+FR (R:FR, 0.02). The mean hypocotyl length in W (HypW) and W+FR (HypW+FR) of at least four biological replicates was used to calculate HypW+FR-HypW. Error bars represent SE. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.D Relative HFR1 protein levels in seedlings of the indicated lines, normalized to actin protein levels, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 line #22 that is taken as 1. Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and transferred to W+FR (R:FR, 0.06) for 3 h. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.


Figure 4A Expression of HFR1 and protein levels of HFR1 - 3xHA in seedlings of hfr1 > pAt : ChHFR1 ( line # 22 ) and hfr1 > pAt : AtHFR1 ( line # 13 ) . Seedlings were grown for 7 days in continuous W ( ~ 20 µmol m - 2 s - 1 ) after which they were incubated for 3 h in high W ( ~ 100 µmol m - 2 s - 1 ) and then either kept at high W or transferred to W + FR ( R : FR , 0 . 06 ) for 3 or 6 h , as indicated in the cartoon at the top . Relative HFR1 transcript levels , normalized to UBQ10 , are the means ± SE of three independent biological replicates relative to hfr1 > pAt : ChHFR1 # 22 grown for 3 h under W + FR . Relative protein levels , normalized to actin , are the means ± SE of three independent biological replicates relative to hfr1 > pAt : ChHFR1 # 22 . Samples were collected at data points marked in the cartoon with asterisks . C Relative HFR1 transcript levels transiently expressed in tobacco leaves , normalized to the GFP , are the means ± SE of three independent biological replicates ( left ) . Relative HFR1 protein levels , normalized to the GFP levels , are the means ± SE of four independent biological replicates ( right ) . In A and C , asterisks mark significant differences ( Student t - test : * p - value < 0 . 05 , * * p - value < 0 . 01 ) between the indicated pairs . D Degradation of ChHFR1 ( 35S : ChHFR1 ) and AtHFR1 ( 35S : AtHFR1 ) in tobacco leaf discs treated with cycloheximide ( CHX , 100 µM ) for the indicated times . Tobacco plants were kept under high W ( ~ 200 µmol m - 2 s - 1 ) for 3 days after agroinfiltration and then leaf circles were treated with W + FR ( R : FR , 0 . 2 ) and CHX . Relative HFR1 protein levels ( ChHFR1 , blue bars ; AtHFR1 , red bars ) , normalized to the GFP levels , are the means ± SE of four biological replicates relative to data point 0 , taken as 1 for each line . Asterisks mark significant differences ( 2 - way ANOVA : * p - value < 0 . 05 ) between ChHFR1 and AtHFR1 at the same time point .

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Figure 4A Expression of HFR1 and protein levels of HFR1-3xHA in seedlings of hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 (line #13). Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and then either kept at high W or transferred to W+FR (R:FR, 0.06) for 3 or 6 h, as indicated in the cartoon at the top. Relative HFR1 transcript levels, normalized to UBQ10, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22 grown for 3 h under W+FR. Relative protein levels, normalized to actin, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22. Samples were collected at data points marked in the cartoon with asterisks.C Relative HFR1 transcript levels transiently expressed in tobacco leaves, normalized to the GFP, are the means ± SE of three independent biological replicates (left). Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates (right). In A and C, asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the indicated pairs.D Degradation of ChHFR1 (35S:ChHFR1) and AtHFR1 (35S:AtHFR1) in tobacco leaf discs treated with cycloheximide (CHX, 100 µM) for the indicated times. Tobacco plants were kept under high W (~200 µmol m-2 s-1) for 3 days after agroinfiltration and then leaf circles were treated with W+FR (R:FR, 0.2) and CHX. Relative HFR1 protein levels (ChHFR1, blue bars; AtHFR1, red bars), normalized to the GFP levels, are the means ± SE of four biological replicates relative to data point 0, taken as 1 for each line. Asterisks mark significant differences (2-way ANOVA: * p-value <0.05) between ChHFR1 and AtHFR1 at the same time point.


Figure 5B Table summaries of the microscale thermophoresis binding assay The sequence of the respective synthetic peptides is indicated . D Relative HFR1 protein levels , normalized to the GFP levels , are the means ± SE of four independent biological replicates . Asterisks mark significant differences ( Student t - test : * p - value < 0 . 05 , * * p - value < 0 . 01 ) between the indicated pairs .

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Figure 5B Table summaries of the microscale thermophoresis binding assay The sequence of the respective synthetic peptides is indicated.D Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates. Asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the indicated pairs.


Figure 6A Y2H growth assay showing the interaction between AtHFR1 and AtPIF7 . The BD - and the AD - derivative constructs used in the assay are shown on the left side of the panel . SD - LW or SD - HLW refer to the selective medium ( plated as drops in dilutions of 1 , 1 : 10 and 1 : 100 ) indicative of transformed cells or interaction between the hybrid proteins , respectively . Truncated forms of murine p53 ( BD - fused ) and SV40 large T - antigen ( AD - fused ) , known to interact , were used as a positive control . Empty vectors ( / ) were used as negative controls . Hypocotyl length of seedlings of AtWT , ( B ) pif7 - 1 , hfr1 - 5 , pif7 - 1 hfr1 - 5 ( top graph ) , pif7 - 2 , hfr1 - 5 and pif7 - 2 hfr1 - 5 ( bottom graph ) mutants grown under different R : FR . Seedlings were grown in W ( R : FR > 1 . 5 ) for 7 days or for 2 days in W and then transferred to two W + FR treatments ( R : FR 0 . 06 or 0 . 02 ) for 5 additional days . Values of hypocotyl length are the means ± SE of three independent biological replicates ( at least 10 seedlings per replica ) . C Hypocotyl length of seedlings of AtWT , ( C ) transgenic 35S : GFP - ΔNt - HFR1 ( 35S : ΔNt - HFR1 ) , two lines of 35S : PIF7 - CFP ( 35S : PIF7 # 1 and # 2 ) , and 35S : GFP - ΔNt - HFR1 35S : PIF7 - CFP double transgenic ( 35S : ΔNt - HFR1 x 35S : PIF7 # 1 and # 2 ) seedlings grown under different R : FR . Seedlings were grown in W ( R : FR > 1 . 5 ) for 7 days or for 2 days in W and then transferred to two W + FR treatments ( R : FR 0 . 06 or 0 . 02 ) for 5 additional days . Values of hypocotyl length are the means ± SE of three independent biological replicates ( at least 10 seedlings per replica ) . D Aspect of representative 7 - day - old W - grown seedlings shown in C . Scale bar is 1 cm .

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Figure 6A Y2H growth assay showing the interaction between AtHFR1 and AtPIF7. The BD- and the AD- derivative constructs used in the assay are shown on the left side of the panel. SD-LW or SD-HLW refer to the selective medium (plated as drops in dilutions of 1, 1:10 and 1:100) indicative of transformed cells or interaction between the hybrid proteins, respectively. Truncated forms of murine p53 (BD-fused) and SV40 large T-antigen (AD-fused), known to interact, were used as a positive control. Empty vectors (/) were used as negative controls.Hypocotyl length of seedlings of AtWT, (B) pif7-1, hfr1-5, pif7-1 hfr1-5 (top graph), pif7-2, hfr1-5 and pif7-2 hfr1-5 (bottom graph) mutants grown under different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days in W and then transferred to two W+FR treatments (R:FR 0.06 or 0.02) for 5 additional days. Values of hypocotyl length are the means ± SE of three independent biological replicates (at least 10 seedlings per replica).C Hypocotyl length of seedlings of AtWT, (C) transgenic 35S:GFP-ΔNt-HFR1 (35S:ΔNt-HFR1), two lines of 35S:PIF7-CFP (35S:PIF7 #1 and #2), and 35S:GFP-ΔNt-HFR1 35S:PIF7-CFP double transgenic (35S:ΔNt-HFR1 x 35S:PIF7 #1 and #2) seedlings grown under different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days in W and then transferred to two W+FR treatments (R:FR 0.06 or 0.02) for 5 additional days. Values of hypocotyl length are the means ± SE of three independent biological replicates (at least 10 seedlings per replica). D Aspect of representative 7-day-old W-grown seedlings shown in C. Scale bar is 1 cm.


Figure 7C Hypocotyl length of seedlings of ( left ) AtWT , pifq , pif7 - 2 , ChWT , ( middle ) 35S : GFP - ΔNt - HFR1 ( ΔNtHFR1 ) , hfr1 - 5 and ( right ) chfr1 - 1 and chfr1 - 2 lines grown at warm temperatures . Hypocotyl lengths are the means ± SE of three biological replicates . Asterisks mark significant differences ( Student t - test : * p - value < 0 . 05 , * * p - value < 0 . 01 ) relative to the same genotype grown at 22ºC ( left and right graphs , black asterisks ) , and between the indicated pairs ( middle graph , red asterisks ) . F Relative chlorophylls levels of ( left ) AtWT , pifq , ChWT , ( middle ) ΔNtHFR1 , hfr1 - 5 and ( right ) chfr1 - 1 and chfr1 - 2 lines after DIS was promoted for the indicated time . For each genotype , values are relative to pigment levels at time 0 ( 7 days in W ) . Data are the means ± SE of four independent biological replicates . G Aspect of 4 - day old dark - grown seedlings of AtWT , pifq , pif7 - 2 , hfr1 - 5 and ΔNt - HFR1 ( left panel ) , and AtWT , ChWT and chfr1 - 1 ( right panel ) .

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Figure 7C Hypocotyl length of seedlings of (left) AtWT, pifq, pif7-2, ChWT, (middle) 35S:GFP-ΔNt-HFR1 (ΔNtHFR1), hfr1-5 and (right) chfr1-1 and chfr1-2 lines grown at warm temperatures. Hypocotyl lengths are the means ± SE of three biological replicates. Asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) relative to the same genotype grown at 22ºC (left and right graphs, black asterisks), and between the indicated pairs (middle graph, red asterisks).F Relative chlorophylls levels of (left) AtWT, pifq, ChWT, (middle) ΔNtHFR1, hfr1-5 and (right) chfr1-1 and chfr1-2 lines after DIS was promoted for the indicated time. For each genotype, values are relative to pigment levels at time 0 (7 days in W). Data are the means ± SE of four independent biological replicates.G Aspect of 4-day old dark-grown seedlings of AtWT, pifq, pif7-2, hfr1-5 and ΔNt-HFR1 (left panel), and AtWT, ChWT and chfr1-1 (right panel).


Figure 1 ( A ) Upper panel : immunoblot analysis of COS7 cells treated for 12 h with MG132 ( 25 μM ) and vinblastine ( 25 μM ) . Lower panel : real ‐ time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure ( 25 μM , 12 h ) . ( B , C ) Immunofluorescence staining of indicated proteins in COS7 cells treated as in ( A ) . Scale bar , 20 μm . ( D ) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 ( 25 μM , 12 h ) . Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online ) . Diagram shows the percentage of aggresome ‐ positive cells . ( E ) Aggresome quantification as in supplementary Fig S1D online from cells analysed in ( H ) . Images are shown in supplementary Fig S1G online . Diagram shows the percentage of aggresome ‐ positive cells at 4 , 24 and 48 h after MG132 wash out . ( F ) Cells were treated as in ( H ) ; however , 4 h after MG132 wash out , BafA1 ( 5 μM ) was added for 20 h to inhibit aggresome clearance . Diagrams show the percentage of aggresome ‐ positive cells and the mean size of aggresomes ( supplementary Fig S1H online ) . Images are shown in supplementary Fig S1G online . ( G ) Dot ‐ blot ( upper panel , quantification in supplementary Fig S1B online ) and western blot ( lower panel ) analyses of 0 . 5 % NP40 ‐ soluble and ‐ insoluble proteins from cells shown in Fig 1D ; supplementary Fig S1D online . ( H ) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 ( 25 μM , 18 h ) or DMSO as a control . After MG132 wash out , cells were allowed to recover for 4 , 24 and 48 h and 0 . 5 % NP40 ‐ soluble and ‐ insoluble proteins were analysed by immunoblotting . ( I ) COS7 cells were transfected for 24 h with GST or GST fused to DIC ( GST ‐ DIC ) and treated with MG132 ( 25 μM , 12 h ) . Indicated proteins in whole ‐ cell lysates and in precipitates obtained by GST pull ‐ down with glutathione beads were analysed by immunoblotting . BafA1 , bafilomycin A1 ; ctrl , control ; DIC , dynein intermediate chain ; DMSO , dimethyl sulphoxide ; GST , glutathione ‐ S ‐ transferase ; Hsp70 , heat ‐ shock protein 70 ; nons , nonsense ; siRNA , short ‐ interfering RNA ; vinblast , vinblastine .

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

Figure 1(A) Upper panel: immunoblot analysis of COS7 cells treated for 12 h with MG132 (25 μM) and vinblastine (25 μM). Lower panel: real‐time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure (25 μM, 12 h).(B,C) Immunofluorescence staining of indicated proteins in COS7 cells treated as in (A). Scale bar, 20 μm.(D) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 (25 μM, 12 h). Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online). Diagram shows the percentage of aggresome‐positive cells.(E) Aggresome quantification as in supplementary Fig S1D online from cells analysed in (H). Images are shown in supplementary Fig S1G online. Diagram shows the percentage of aggresome‐positive cells at 4, 24 and 48 h after MG132 wash out.(F) Cells were treated as in (H); however, 4 h after MG132 wash out, BafA1 (5 μM) was added for 20 h to inhibit aggresome clearance. Diagrams show the percentage of aggresome‐positive cells and the mean size of aggresomes ( supplementary Fig S1H online). Images are shown in supplementary Fig S1G online.(G) Dot‐blot (upper panel, quantification in supplementary Fig S1B online) and western blot (lower panel) analyses of 0.5% NP40‐soluble and ‐insoluble proteins from cells shown in Fig 1D; supplementary Fig S1D online.(H) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 (25 μM, 18 h) or DMSO as a control. After MG132 wash out, cells were allowed to recover for 4, 24 and 48 h and 0.5% NP40‐soluble and ‐insoluble proteins were analysed by immunoblotting.(I) COS7 cells were transfected for 24 h with GST or GST fused to DIC (GST‐DIC) and treated with MG132 (25 μM, 12 h). Indicated proteins in whole‐cell lysates and in precipitates obtained by GST pull‐down with glutathione beads were analysed by immunoblotting. BafA1, bafilomycin A1; ctrl, control; DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; Hsp70, heat‐shock protein 70; nons, nonsense; siRNA, short‐interfering RNA; vinblast, vinblastine.


Figure 2 ( A ) GST pull ‐ down analysis as in Fig 1I , but performed in cells transfected for 36 h with full ‐ length FLAG ‐ BAG3 ( I ) or the BAG3 deletion mutants FLAG - ( Δ421 - 498 ) - BAG3 ( II ) , FLAG - BAG ‐ domain ( III ) , and FLAG - PxxP - BAG ‐ domain ( IV ) . Asterisk , nonspecific band . ( B ) HEK cells were transfected for 18 h with SODG85R ‐ GFP , along with the indicated FLAG ‐ tagged BAG3 constructs ( shown in A ) and HA ‐ tagged Hsp70 ( HA ‐ Hsp70 ) . SODG85R ‐ GFP showed three distinct localization patterns : cytoplasmic , pre ‐ aggresomal and aggresomal ( see representative pictures on the right and supplementary Fig S2F online ) . The percentages of cells bearing pre ‐ aggresomal ( upper panel ) and aggresomal ( lower panel ) SODG85R ‐ GFP are shown . Scale bar , 10 μm . ( C ) HEK cells transfected with SODG85R ‐ GFP , BAG3 and Hsp70 were additionally transfected with FLAG ‐ p50 , as indicated . Diagrams show the percentages of cells with pre ‐ aggresomal and aggresomal SODG85R ‐ GFP . Right panel : representative image showing pre ‐ aggresomal localization of FLAG ‐ p50 and SODG85R ‐ GFP . Scale bar , 5 μm . ( D ) GST pull ‐ down analysis as in ( A ) , but in HEK cells transfected for 18 h with SODWT ‐ GFP or mutant SODG85R ‐ GFP , along with FLAG ‐ BAG3 . ( E ) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA . At 6 h after transfection , lactacystin ( 10 μM ) or GST as vehicle control was added for GST and GST pull ‐ down analysis was performed as in ( D ) . ( F ) Upper panel : sucrose ‐ density gradient analysis of extracts from HEK cells transfected with SODG85R ‐ GFP and GST ‐ DIC , along with FLAG ‐ BAG3 or vector control . Note the immunoreactive bands in high ‐ density fraction 14 on BAG3 overexpression ( arrow ) . Migration of a 20S marker protein is indicated . Lower panel : GST ‐ DIC pull ‐ down analyses of dynein complexes present in sucrose fraction 6 and 14 . ( G ) GST pull ‐ down analysis of SODG85R ‐ GFP as in ( D ) but in cells transfected with FLAG ‐ tagged BAG3 constructs ( I - IV ; indicated as in A ) . ( H ) GST pull ‐ down analysis of SODG85R ‐ GFP as in ( D ) but in cells treated with PYR41 ( 10 μM ; 12 h ) , as indicated . DIC , dynein intermediate chain ; DMSO , dimethyl sulphoxide ; GST , glutathione ‐ S ‐ transferase ; HA , haemagglutinin ; Hsp70 , heat ‐ shock protein 70 ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short ‐ interfering RNA ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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

Figure 2(A) GST pull‐down analysis as in Fig 1I, but performed in cells transfected for 36 h with full‐length FLAG‐BAG3 (I) or the BAG3 deletion mutants FLAG-(Δ421-498)-BAG3 (II), FLAG-BAG‐domain (III), and FLAG-PxxP-BAG‐domain (IV). Asterisk, nonspecific band.(B) HEK cells were transfected for 18 h with SODG85R‐GFP, along with the indicated FLAG‐tagged BAG3 constructs (shown in A) and HA‐tagged Hsp70 (HA‐Hsp70). SODG85R‐GFP showed three distinct localization patterns: cytoplasmic, pre‐aggresomal and aggresomal (see representative pictures on the right and supplementary Fig S2F online). The percentages of cells bearing pre‐aggresomal (upper panel) and aggresomal (lower panel) SODG85R‐GFP are shown. Scale bar, 10 μm.(C) HEK cells transfected with SODG85R‐GFP, BAG3 and Hsp70 were additionally transfected with FLAG‐p50, as indicated. Diagrams show the percentages of cells with pre‐aggresomal and aggresomal SODG85R‐GFP. Right panel: representative image showing pre‐aggresomal localization of FLAG‐p50 and SODG85R‐GFP. Scale bar, 5 μm.(D) GST pull‐down analysis as in (A), but in HEK cells transfected for 18 h with SODWT‐GFP or mutant SODG85R‐GFP, along with FLAG‐BAG3.(E) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA. At 6 h after transfection, lactacystin (10 μM) or GST as vehicle control was added for GST and GST pull‐down analysis was performed as in (D).(F) Upper panel: sucrose‐density gradient analysis of extracts from HEK cells transfected with SODG85R‐GFP and GST‐DIC, along with FLAG‐BAG3 or vector control. Note the immunoreactive bands in high‐density fraction 14 on BAG3 overexpression (arrow). Migration of a 20S marker protein is indicated. Lower panel: GST‐DIC pull‐down analyses of dynein complexes present in sucrose fraction 6 and 14.(G) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells transfected with FLAG‐tagged BAG3 constructs (I-IV; indicated as in A).(H) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells treated with PYR41 (10 μM; 12 h), as indicated. DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; HA, haemagglutinin; Hsp70, heat‐shock protein 70; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short‐interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


Figure 3 ( A ) Analysis of SODG85R ‐ GFP ( green ) localization with aggresome marker vimentin , the autophagic receptors p62 and NBR1 , the autophagosome marker LC3 and the lysosomal marker LAMP2 ( all in red ) in BAG3 / Hsp70 ‐ transfected HEK cells . Representative merged images of cells with cytoplasmic , pre ‐ aggresomal and aggresomal SOD distribution are shown . Arrows indicate LC3 ‐ positive autophagosomes . Scale bar , 5 μm . ( B ) Transmission electron microscope analysis of SODG85R ‐ GFP aggresome in cells transfected as in ( A ) . Marked area in ( a ) is shown magnified in ( b ) . Arrows , autophagosomes . Arrowheads , autolysosomes . Scale bar , 1 μm . ( C ) HEK cells transfected for 8 h with SODG85R ‐ GFP along with FLAG ‐ BAG3 and HA ‐ Hsp70 , as indicated , were treated for 24 h with BafA1 ( 0 . 1 μM ) . Indicated proteins were analysed by immunoblotting . Diagram shows the ratio of SODG85R ‐ GFP levels in BafA1 ‐ treated cells to those in untreated cells . ( D ) Same analysis as in ( C ) but with SODWT ‐ GFP . ( E ) Similar analysis as in ( C ) but in cells transfected 24 h before with nonsense or atg7 siRNA . ( F ) HEK cells were transfected with SODG85R ‐ GFP and indicated FLAG ‐ tagged BAG3 constructs together with HA ‐ Hsp70 , and lysosomal degradation of SODG85R ‐ GFP was examined as in ( C ) . Corresponding immunoblots are shown in supplementary Fig S3F online . Ag , aggresome ; BafA1 , bafilomycin A1 ; GFP , green fluorescent protein ; HA , haemagglutinin ; Hsp70 , heat ‐ shock protein 70 ; N , nucleus ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short interfering RNA ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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Figure 3(A) Analysis of SODG85R‐GFP (green) localization with aggresome marker vimentin, the autophagic receptors p62 and NBR1, the autophagosome marker LC3 and the lysosomal marker LAMP2 (all in red) in BAG3/Hsp70‐transfected HEK cells. Representative merged images of cells with cytoplasmic, pre‐aggresomal and aggresomal SOD distribution are shown. Arrows indicate LC3‐positive autophagosomes. Scale bar, 5 μm.(B) Transmission electron microscope analysis of SODG85R‐GFP aggresome in cells transfected as in (A). Marked area in (a) is shown magnified in (b). Arrows, autophagosomes. Arrowheads, autolysosomes. Scale bar, 1 μm.(C) HEK cells transfected for 8 h with SODG85R‐GFP along with FLAG‐BAG3 and HA‐Hsp70, as indicated, were treated for 24 h with BafA1 (0.1 μM). Indicated proteins were analysed by immunoblotting. Diagram shows the ratio of SODG85R‐GFP levels in BafA1‐treated cells to those in untreated cells.(D) Same analysis as in (C) but with SODWT‐GFP.(E) Similar analysis as in (C) but in cells transfected 24 h before with nonsense or atg7 siRNA.(F) HEK cells were transfected with SODG85R‐GFP and indicated FLAG‐tagged BAG3 constructs together with HA‐Hsp70, and lysosomal degradation of SODG85R‐GFP was examined as in (C). Corresponding immunoblots are shown in supplementary Fig S3F online. Ag, aggresome; BafA1, bafilomycin A1; GFP, green fluorescent protein; HA, haemagglutinin; Hsp70, heat‐shock protein 70; N, nucleus; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


Figure 4 ( A ) Spinal cord sections of mice overexpressing SODWT and SODG85R ( disease end ‐ stage ) were immunostained for BAG3 and SOD1 . Arrows indicate SOD1 ‐ and BAG3 ‐ positive aggregates in the neuropil . Scale bar , 20 μm . ( B ) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons fromSODG85Rmice at the time of disease symptom onset . Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R . Scale bar , 10 μm . Magnific . , magnification ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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Figure 4(A) Spinal cord sections of mice overexpressing SODWT and SODG85R (disease end‐stage) were immunostained for BAG3 and SOD1. Arrows indicate SOD1‐ and BAG3‐positive aggregates in the neuropil. Scale bar, 20 μm.(B) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons fromSODG85Rmice at the time of disease symptom onset. Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R. Scale bar, 10 μm. Magnific., magnification; SOD, superoxide dismutase; SODWT, wild‐type SOD.


Figure 1A - G Double immunolabelling of PCNT - PCNT - ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes on ( A ) zygotene , ( B ) pachytene , ( C ) diplotene , ( D ) diakinesis , ( E ) prometaphase I , ( F ) metaphase I and ( G ) telophase I . Chromatin has been stained with DAPI ( blue ) . Scale bar in G represents 10 μm .

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Figure 1A-G Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes on (A) zygotene, (B) pachytene, (C) diplotene, (D) diakinesis, (E) prometaphase I, (F) metaphase I and (G) telophase I. Chromatin has been stained with DAPI (blue). Scale bar in G represents 10 μm.


Figure 2A - F Double immunolabelling of PCNT - PCNT - ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) interkinesis , ( B ) early prophase II , ( C ) late prophase II , ( D ) prometaphase II , ( E ) metaphase II and ( F ) telophase II . Chromatin has been stained with DAPI ( blue ) . Scale bar in F represents 10 μm .

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Figure 2A-F Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) interkinesis, (B) early prophase II, (C) late prophase II, (D) prometaphase II, (E) metaphase II and (F) telophase II. Chromatin has been stained with DAPI (blue). Scale bar in F represents 10 μm.


Figure 3A - L Double immunolabelling of CETN3 ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) leptotene , ( B ) zygotene , ( C ) pachytene , ( D ) metaphase I , ( E ) telophase I , ( F ) early interkinesis ( F ) , late interkinesis ( G ) , prophase II ( H ) , metaphase II ( I ) , telophase II ( J ) , round spermatid ( K ) , and elongated spermatid ( L ) . Amplifications at 300 % magnification for the centrosomic CETN3 signals are presented for each image . Chromatin has been stained with DAPI ( blue ) . Scale bar in L represents 10 μm .

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Figure 3A-L Double immunolabelling of CETN3 (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) leptotene, (B) zygotene, (C) pachytene, (D) metaphase I, (E) telophase I, (F) early interkinesis (F), late interkinesis (G), prophase II (H), metaphase II (I), telophase II (J), round spermatid (K), and elongated spermatid (L). Amplifications at 300% magnification for the centrosomic CETN3 signals are presented for each image. Chromatin has been stained with DAPI (blue). Scale bar in L represents 10 μm.


Figure 4A - J Double immunolabelling of PLK1S137P ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) pachytene , ( C ) diplotene , ( E ) diakinesis , ( G ) metaphase I and ( I ) metaphase II . Chromatin has been stained with DAPI ( blue ) . Fourth column shows the double Immunolabelling of PLK1 - S137P ( green ) and γ - Tubulin ( magenta ) on squashed WT mouse spermatocytes at ( B ) pachytene , ( D ) diplotene , ( F ) diakinesis , ( H ) metaphase I and ( J ) metaphase II . Insets correspond to centrosomes at 200 % magnification . White arrows indicate the location of centrosomes . Scale bar in J represents 10µm .

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Figure 4A-J Double immunolabelling of PLK1S137P (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) pachytene, (C) diplotene, (E) diakinesis, (G) metaphase I and (I) metaphase II. Chromatin has been stained with DAPI (blue). Fourth column shows the double Immunolabelling of PLK1-S137P (green) and γ-Tubulin (magenta) on squashed WT mouse spermatocytes at (B) pachytene, (D) diplotene, (F) diakinesis, (H) metaphase I and (J) metaphase II. Insets correspond to centrosomes at 200% magnification. White arrows indicate the location of centrosomes. Scale bar in J represents 10µm.


Figure 5A - L First column shows the triple immunolabelling of CETN3 ( green ) , PCNT ( red ) and SYCP3 ( grey ) on squashed WT mouse spermatocytes . Second column shows the correspondent labellings for Plk1 ( ∆ / ∆ ) . Third column shows signals for BI2536 - treated spermatocytes . Images are shown for ( A , E , I ) zygotene , ( B , F , J ) pachytene , ( C , G , K ) diplotene and ( D , H , L ) diakinesis . Amplifications at 300 % magnification for all labellings are displayed for each image . M - O Quantification of the percentage of cells without PCNT at centrosomes ( PCNT - ) in ( M ) zygotene , ( N ) pachytene and ( O ) diplotene in control , Plk1 ( ∆ / ∆ ) and BI2536 - treated spermatocytes . Data for BI2536 represent results for 100µM at 8h treatment on organotypic cultures of seminiferous tubules . Data are presented as mean ± SD for three biological replicates for each condition .

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Figure 5A-L First column shows the triple immunolabelling of CETN3 (green), PCNT (red) and SYCP3 (grey) on squashed WT mouse spermatocytes. Second column shows the correspondent labellings for Plk1(∆/∆). Third column shows signals for BI2536-treated spermatocytes. Images are shown for (A, E, I) zygotene, (B, F, J) pachytene, (C, G, K) diplotene and (D, H, L) diakinesis. Amplifications at 300% magnification for all labellings are displayed for each image. M-O Quantification of the percentage of cells without PCNT at centrosomes (PCNT-) in (M) zygotene, (N) pachytene and (O) diplotene in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data for BI2536 represent results for 100µM at 8h treatment on organotypic cultures of seminiferous tubules. Data are presented as mean ± SD for three biological replicates for each condition.


Figure 6A - C Immunolabelling of γ - Tubulin and SYCP3 during prophase I in control , Plk1 ( ∆ / ∆ ) and BI2536 - treated spermatocytes . Images are show for pachytene , diplotene and diakinesis in Control ( A ) , Plk1 ( ∆ / ∆ ) ( B ) and BI2536 - treated spermatocytes ( C ) . First columns show the double immunolabelling of PCM - PCNT - ( green ) and SYCP3 ( red ) , and second columns shows γ - Tubulin ( γ - Tub , magenta ) and SYCP3 ( red ) . Chromatin has been stained with DAPI ( blue ) . Amplifications at 300 % magnification for all labellings are displayed for each image . Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous tubules ( D ) Immunolabelling of CETN3 and γ - Tubulin in control and Plk1 ( ∆ / ∆ ) . Double immunolabelling of CETN3 ( red ) and γ - Tubulin ( green ) in pachytene ( a - b ) and diakinesis ( c - d ) . Chromatin has been stained with DAPI ( blue ) . ( E ) Quantification of the number of cells without γ - Tubulin at centrosomes ( γ - Tub - ) in pachytene and diplotene spermatocytes in control , Plk1 ( ∆ / ∆ ) and BI2536 - treated spermatocytes . Data represent average of two biological replicates per condition .

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 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

Figure 6A-C Immunolabelling of γ-Tubulin and SYCP3 during prophase I in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Images are show for pachytene, diplotene and diakinesis in Control (A), Plk1(∆/∆) (B) and BI2536-treated spermatocytes (C). First columns show the double immunolabelling of PCM -PCNT- (green) and SYCP3 (red), and second columns shows γ-Tubulin (γ-Tub, magenta) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Amplifications at 300% magnification for all labellings are displayed for each image. Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous tubules(D) Immunolabelling of CETN3 and γ-Tubulin in control and Plk1(∆/∆). Double immunolabelling of CETN3 (red) and γ-Tubulin (green) in pachytene (a-b) and diakinesis (c-d). Chromatin has been stained with DAPI (blue).(E) Quantification of the number of cells without γ-Tubulin at centrosomes (γ-Tub -) in pachytene and diplotene spermatocytes in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data represent average of two biological replicates per condition.


Figure 7A - D Control : Double immunolabelling of SYCP3 ( cyan ) and α - Tubulin ( red ) on control squashed spermatocytes for the first and the second meiotic divisions at metaphase I ( A ) and metaphase II ( B ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) on control squashed spermatocytes at metaphase I ( C ) and metaphase II ( D ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . E - H Plk1 ( ∆ / ∆ ) mouse model : Double immunolabelling of SYCP3 ( cyan ) and α - Tubulin ( red ) in monopolar spindle I ( E ) and monopolar spindle II ( F ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) labelling in monopolar spindle I ( G ) and monopolar spindle II ( H ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . I - L BI2536 - treated spermatocytes : Double immunolabelling of SYCP3 ( cyan ) and α - Tubulin ( red ) at monopolar spindle I ( I ) and monopolar spindle II ( J ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) labelling in monopolar spindle I ( K ) and monopolar spindle II ( L ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . Cells shown were treated for 100µM BI2536 for 8h treatment on organotypic cultures of seminiferous tubules . M - P MLN8237 - treated spermatocytes : Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) in monopolar spindle I ( M ) and monopolar spindle II ( N ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) in monopolar spindle I ( O ) and monopolar spindle II ( P ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . Cells shown were treated for 10µM MLN8237 for 8h treatment on organotypic cultures of seminiferous tubules . Q - R Quantitative analysis of the incidence of monopolar spindles during the first ( Q ) and second ( R ) meiotic divisions for each experimental condition . Percentage of monopolar spindles functional analysis are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Experiments were conducted in three different biological replicates .

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

Figure 7A-D Control: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) on control squashed spermatocytes for the first and the second meiotic divisions at metaphase I (A) and metaphase II (B). Double immunolabelling of PCNT (green) and α-Tubulin (red) on control squashed spermatocytes at metaphase I (C) and metaphase II (D). Chromatin has been stained with DAPI (pseudo coloured in grey). E-H Plk1 (∆/∆) mouse model: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) in monopolar spindle I (E) and monopolar spindle II (F). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (G) and monopolar spindle II (H). Chromatin has been stained with DAPI (pseudo coloured in grey). I-L BI2536-treated spermatocytes: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) at monopolar spindle I (I) and monopolar spindle II (J). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (K) and monopolar spindle II (L). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 100µM BI2536 for 8h treatment on organotypic cultures of seminiferous tubules. M-P MLN8237-treated spermatocytes: Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (M) and monopolar spindle II (N). Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (O) and monopolar spindle II (P). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 10µM MLN8237 for 8h treatment on organotypic cultures of seminiferous tubules. Q-R Quantitative analysis of the incidence of monopolar spindles during the first (Q) and second (R) meiotic divisions for each experimental condition. Percentage of monopolar spindles functional analysis are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Experiments were conducted in three different biological replicates.


Figure 8A - B AURKP distribution in control , Plk1 ( Δ / Δ ) , 100µM 8h BI2536 - treated and 10µM 8h MLN8237 - treated spermatocytes . ( A ) Double immunolabelling of AURKP ( grey / green ) and α - Tubulin ( red ) in control , Plk1 ( Δ / Δ ) , BI2536 - treated and MLN8237 - treated spermatocytes . Images are shown for bipolar spindles I and II for each condition . ( B ) Monopolar spindles I and II are shown for Plk1 ( Δ / Δ ) , BI2536 - treated and MLN8237 - treated spermatocytes . Chromatin has been stained with DAPI ( blue ) . . ( C ) Quantification of the percentage of bipolar spindles during the first and second meiotic divisions presenting ( blue bars ) or lacking ( green bars ) centrosomic AURKP signals for each experimental condition . Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Data represent the average of two biological replicates per condition . Data are mean ± SD . ( D ) Quantification of the percentage of monopolar spindles during the first and second meiotic division presenting ( blue bars ) or lacking ( green bars ) centrosomic AURKP signal for each experimental condition . Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Data represent the average of two biological replicates per condition . Data are mean ± SD . ( E ) Quantitative analysis of AURKP signal at cell poles . Experiments were conducted for two biological replicates . Number of cells analysed : control ( n = 32 ) , Plk1 ( Δ / Δ ) ( n = 27 ) , BI2536 - treated spermatocytes ( n = 27 ) . Data are mean ± SD ; ( * * * * ) p < 0 . 0001 , Student ' s t - test .

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 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 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

Figure 8A-B AURKP distribution in control, Plk1(Δ/Δ), 100µM 8h BI2536-treated and 10µM 8h MLN8237-treated spermatocytes. (A) Double immunolabelling of AURKP (grey/green) and α-Tubulin (red) in control, Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Images are shown for bipolar spindles I and II for each condition. (B) Monopolar spindles I and II are shown for Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Chromatin has been stained with DAPI (blue)..(C) Quantification of the percentage of bipolar spindles during the first and second meiotic divisions presenting (blue bars) or lacking (green bars) centrosomic AURKP signals for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD. (D) Quantification of the percentage of monopolar spindles during the first and second meiotic division presenting (blue bars) or lacking (green bars) centrosomic AURKP signal for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD. (E) Quantitative analysis of AURKP signal at cell poles. Experiments were conducted for two biological replicates. Number of cells analysed: control (n=32), Plk1(Δ/Δ) (n=27), BI2536-treated spermatocytes (n= 27). Data are mean ± SD; (****) p<0.0001, Student's t-test.


Figure 9 ( A ) Triple immunolabelling of CEP250 ( green ) , CETN3 ( magenta ) and SYCP3 ( grey ) in ( a - b ) late diplotene and ( c - d ) metaphase I in control and Plk1 ( Δ / Δ ) spermatocytes . ( B ) Double immunolabelling of KIF11 ( grey / green ) , and SYCP3 ( red ) in ( a ) control spindle , ( b ) Plk1 ( Δ / Δ ) monopolar spindle , ( c ) BI2536 monopolar spindle and ( d ) MLN8237 monopolar spindle . Chromatin has been stained with DAPI ( bue ) . Data is presented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules .

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 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

Figure 9(A) Triple immunolabelling of CEP250 (green), CETN3 (magenta) and SYCP3 (grey) in (a-b) late diplotene and (c-d) metaphase I in control and Plk1(Δ/Δ) spermatocytes.(B) Double immunolabelling of KIF11 (grey/green), and SYCP3 (red) in (a) control spindle, (b) Plk1(Δ/Δ) monopolar spindle, (c) BI2536 monopolar spindle and (d) MLN8237 monopolar spindle. Chromatin has been stained with DAPI (bue). Data is presented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules.


Figure 1 ( A ) Top panel : Scheme illustrating the tetracycline inducible Flp - In 293 system that controls the expression of HA - IKKε or HA - GFP . Bottom panel : Representative western blot showing induced expression of HA - IKKε in Flp - In 293 cells treated with doxycycline ( Dox , 50 ng / ml ) for 16 hours compared to endogenous IKKε in T47D , MDA - MB - 231 and MDA - MB - 468 breast cancer cell lines . ( B ) Heatmap and hierarchical clustering of metabolite concentrations in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox , 50 ng / ml , 16 hours ) ( n = 5 technical replicates ) . ( C ) Serine production from glucose ( serine m + 3 , 13C6 - glucose labelling , left panel ) and glutamine ( serine m + 1 , 15N2 - glutamine labelling , right panel ) in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) metabolite levels were normalised to the internal standard HEPES . ( E ) Contribution of pyruvate and glucose - derived carbon to TCA cyle metabolites in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES . ( F ) Fractional enrichment of serine , malate and citrate 13C - isotopologues in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) ( n = 5 technical replicates ) . ( G ) Fractional enrichment of the serine 15 - N - isotopologue in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . m + 1 shows the naturally occurring 13C isotopologue ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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 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 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

Figure 1(A) Top panel: Scheme illustrating the tetracycline inducible Flp-In 293 system that controls the expression of HA-IKKε or HA-GFP. Bottom panel: Representative western blot showing induced expression of HA-IKKε in Flp-In 293 cells treated with doxycycline (Dox, 50 ng/ml) for 16 hours compared to endogenous IKKε in T47D, MDA-MB-231 and MDA-MB-468 breast cancer cell lines.(B) Heatmap and hierarchical clustering of metabolite concentrations in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (Dox, 50 ng/ml, 16 hours) (n=5 technical replicates).(C) Serine production from glucose (serine m+3, 13C6-glucose labelling, left panel) and glutamine (serine m+1, 15N2-glutamine labelling, right panel) in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) metabolite levels were normalised to the internal standard HEPES.(E) Contribution of pyruvate and glucose-derived carbon to TCA cyle metabolites in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.(F) Fractional enrichment of serine, malate and citrate 13C-isotopologues in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) (n=5 technical replicates).(G) Fractional enrichment of the serine 15-N-isotopologue in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). m+1 shows the naturally occurring 13C isotopologue (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


Figure 2Representative western blot showing level of IKKε in IKBKE ( IKKε ) - silenced ( A ) T47D breast cancer cell lines . Representative western blot showing level of IKKε in IKBKE ( IKKε ) - silenced ( B ) MDA - MB - 468 breast cancer cell lines . ( C - D ) Heatmap and hierarchical clustering of metabolite concentrations in ( C ) IKBKE ( IKKε ) - silenced T47D cells and ( D ) IKBKE ( IKKε ) - silenced MDA - MB - 468 cells ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES . ( E ) Glycine production , representative of serine production , from glutamine ( glycine m + 1 , 15N - glutamine labelling ) and glucose ( glycine m + 2 , 13C6 - glucose labelling ) , and contribution of pyruvate and glucose - derived carbon to TCA cycle metabolites ( citrate m + 2 , malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced T47D cells . ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES . ( F ) Serine production from glutamine ( serine m + 1 , 15N - glutamine labelling ) , and serine and glycine production from glucose ( glycine m + 2 , serine m + 3 , 13C6 - glucose labelling ) as well as contribution of pyruvate and glucose - derived carbon to TCA cycle metabolites ( malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced MDA - MB - 468 cells .

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 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 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 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

Figure 2Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (A) T47D breast cancer cell lines.Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (B) MDA-MB-468 breast cancer cell lines.(C-D) Heatmap and hierarchical clustering of metabolite concentrations in (C) IKBKE (IKKε)-silenced T47D cells and (D) IKBKE (IKKε)-silenced MDA-MB-468 cells (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.(E) Glycine production, representative of serine production, from glutamine (glycine m+1, 15N-glutamine labelling) and glucose (glycine m+2, 13C6-glucose labelling), and contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (citrate m+2, malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced T47D cells. (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.(F) Serine production from glutamine (serine m+1, 15N-glutamine labelling), and serine and glycine production from glucose (glycine m+2, serine m+3, 13C6-glucose labelling) as well as contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced MDA-MB-468 cells.


Figure 3 ( A ) Basal oxygen consumption in Flp - In 293 HA - IKKε wt , Flp - In 293 HA - IKKε KD - m and Flp - In 293 HA - IKKε UbLD - m cells following treatment with doxycycline ( Dox , 16 hours ) , measured using Oroboros high resolution respirometry . Data are normalised to non - treated control cells . ( B ) Average TMRM staining intensity in Flp - In 293 HA - IKKε wt , Flp - In 293 HA - IKKε KD - m and Flp - In 293 HA - IKKε UbLD - m expressing cells induced by doxycycline ( Dox , 16 hours ) . Data are normalised to non - treated control cells . ( C ) Basal mitochondrial oxygen consumption rate ( OCR ) in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines , measured using Seahorse XF96e or XF24 analysis . ( D ) OCR in mitochondria isolated from Flp - In 293 HA - GFP or HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) , measured using Oroboros high resolution respirometry . ( F ) Representative western blot showing l evel of IKKε in three independent single cell clones of Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells following treatment with doxycycline ( 100 ng / ml , 16 hours ) . ( G ) Relative pyruvate dehydrogenase ( PDH ) activity in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . ( H ) Average TMRM staining intensity in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) and DCA ( both for 16 hours ) . Data are normalised to non - treated Flp - In 293 HA - IKKε cells . ( I ) Basal OCR in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml ) in combination with dichloroacetate ( DCA ) for 16 hours , measured using Oroboros high resolution respirometry . ( J ) Basal OCR in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml ) in combination with pyruvate deprivation for 16 hours , measured using Oroboros high resolution respirometry .

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

Figure 3(A) Basal oxygen consumption in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m cells following treatment with doxycycline (Dox, 16 hours), measured using Oroboros high resolution respirometry. Data are normalised to non-treated control cells.(B) Average TMRM staining intensity in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m expressing cells induced by doxycycline (Dox, 16 hours). Data are normalised to non-treated control cells.(C) Basal mitochondrial oxygen consumption rate (OCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines, measured using Seahorse XF96e or XF24 analysis.(D) OCR in mitochondria isolated from Flp-In 293 HA-GFP or HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours), measured using Oroboros high resolution respirometry.(F) Representative western blot showing level of IKKε in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells following treatment with doxycycline (100 ng/ml, 16 hours).(G) Relative pyruvate dehydrogenase (PDH) activity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours).(H) Average TMRM staining intensity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) and DCA (both for 16 hours). Data are normalised to non-treated Flp-In 293 HA-IKKε cells.(I) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with dichloroacetate (DCA) for 16 hours, measured using Oroboros high resolution respirometry.(J) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with pyruvate deprivation for 16 hours, measured using Oroboros high resolution respirometry.


Figure 4 ( A ) Representative western blot showing level of ATF4 in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( 16 hours ) . ( B ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . Data are expressed as fold changes , relative to levels in non - treated Flp - In 293 HA - GFP cells and normalised to β - Actin ( n = 4 biological replicates ) ( C ) Representative western blot showing levels of IKKε , IRF3 , phosphorylated IRF3 ( S396 ) and SBP enzymes in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . ( D ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . Data are expressed as fold changes , relative to levels in a non - silenced , non - treated control , and normalised to β - Actin ( n = 5 biological replicates ) . ( E ) Representative western blot showing levels of IKKε , PHGDH , PSAT1 and PSPH in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours .

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 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 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 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

Figure 4(A) Representative western blot showing level of ATF4 in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (16 hours).(B) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in non-treated Flp-In 293 HA-GFP cells and normalised to β-Actin (n=4 biological replicates)(C) Representative western blot showing levels of IKKε, IRF3, phosphorylated IRF3 (S396) and SBP enzymes in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.(D) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in a non-silenced, non-treated control, and normalised to β-Actin (n=5 biological replicates).(E) Representative western blot showing levels of IKKε, PHGDH, PSAT1 and PSPH in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.