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 .

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

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 .

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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 .

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

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 ) .

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

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

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.


A , 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 .

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

A, 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 .

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

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.


Seedlings 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 ) .

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

Seedlings 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).


B 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 .

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

B 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 .

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

C 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 .

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

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.


A 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 .

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

A 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 .

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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 .

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

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.


B Table summaries of the microscale thermophoresis binding assay The sequence of the respective synthetic peptides is indicated .

B 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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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.


A 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 .

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

A 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 ) .

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

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 .

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

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.


C 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 ) .

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

C 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 .

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

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 ) .

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

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).


( 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 ) .

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

(A) 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 .

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(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 .

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(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 .

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(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 .

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(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 .

(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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 ) .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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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(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.


( 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 .

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(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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(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.


A - 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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A-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.


A - 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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A-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.


A - 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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A-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.


A - 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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A-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.


A - 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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A-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.


A - 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

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A-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 ) .

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(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 .

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(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.


A - 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 .

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A-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 .

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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.


A - 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 ) . .

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A-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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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.


( 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 .

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(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 ) .

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(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 .

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(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 .

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(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 ) .

(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 .

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(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.


Representative western blot showing level of IKKε in IKBKE ( IKKε ) - silenced ( A ) T47D breast cancer cell lines .

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Representative 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 .

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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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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.


( A ) Representative western blot showing level of ATF4 in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( 16 hours ) .

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(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 )

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(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 .

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(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 ) .

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(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 .

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(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.


( A ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines . Data are expressed as fold changes , relative to levels in a non - silenced control of each cell line and normalised to β - Actin ( n ≥ 3 biological replicates ) .

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(A) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. Data are expressed as fold changes, relative to levels in a non-silenced control of each cell line and normalised to β-Actin (n≥3 biological replicates).


( B ) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE ( IKKε ) - silenced ZR - 75 - 1 , T47D , MDA - MB - 468 and MCF7 breast cancer cell lines .

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(B) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE (IKKε)-silenced ZR-75-1, T47D, MDA-MB-468 and MCF7 breast cancer cell lines.


( C - E ) Levels of the SBP enzymes in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines . ( C ) PHGDH , ( D ) PSAT1 and ( E ) PSPH levels in indicated cell lines normalised to Vinculin . Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation ( n ≥ 3 biological replicates ) .

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(C-E) Levels of the SBP enzymes in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. (C) PHGDH, (D) PSAT1 and (E) PSPH levels in indicated cell lines normalised to Vinculin. Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation (n≥3 biological replicates).


( F ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in ATF4 - silenced ZR - 75 - 1 , T47D and MDA - MB - 468 breast cancer cell lines . Data are expressed as fold changes , relative to levels in non - silenced control cells and normalised to β - Actin ( n = 3 biological replicates ) .

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(F) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced ZR-75-1, T47D and MDA-MB-468 breast cancer cell lines. Data are expressed as fold changes, relative to levels in non-silenced control cells and normalised to β-Actin (n=3 biological replicates).


( G ) Representative western blot showing levels of PHGDH , PSAT1 and PSPH in ATF4 - silenced ZR - 75 - 1 , MDA - MB - 468 , MDA - MB - 231 , T47D and HCC1143 breast cancer cell lines .

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(G) Representative western blot showing levels of PHGDH, PSAT1 and PSPH in ATF4-silenced ZR-75-1, MDA-MB-468, MDA-MB-231, T47D and HCC1143 breast cancer cell lines.


( A ) Correlation of change in OCR ( ΔOCR ) in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines ( from Fig . 3C ) and the change in cell confluency ( Δconfluency ) upon treatment of the panel of cell lines with NCT502 ( from Fig . EV5A ) .

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(A) Correlation of change in OCR (ΔOCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the change in cell confluency (Δconfluency) upon treatment of the panel of cell lines with NCT502 (from Fig. EV5A).


( B ) Correlation of ΔOCR in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines ( from Fig . 3C ) and the Δconfluency upon treatment of the panel of cell lines with 6 - Diazo - 5 - oxo - L - norleucine ( DON ) ( from Fig . EV5C ) .

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(B) Correlation of ΔOCR in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the Δconfluency upon treatment of the panel of cell lines with 6-Diazo-5-oxo-L-norleucine (DON) (from Fig. EV5C).


( C ) Association between IKBKE ( IKKε ) and PSAT1 mRNA overexpression evaluated by a chi - squared independence test . The + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number and percentage of samples , as well as the Chi - square values are shown .

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(C) Association between IKBKE (IKKε) and PSAT1 mRNA overexpression evaluated by a chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.


Expression of IKBKE ( IKKε ) and the SBP enzymes PSAT1 , in the METABRIC dataset . Samples were stratified by Pam50 intrinsic subtypes and ER status . Brown - Forsythe and Welch ANOVA test with unpaired t - test with Welch correction were applied . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Expression of IKBKE (IKKε) and the SBP enzymes PSAT1, in the METABRIC dataset. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.


Expression of SBP enzymes PHGDH and PSPH in the METABRIC dataset . The expression of a proliferation related gene set and ATF4 are also shown . Samples were stratified by Pam50 intrinsic subtypes and ER status . Brown - Forsythe and Welch ANOVA test with unpaired t - test with Welch correction were applied . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Expression of SBP enzymes PHGDH and PSPH in the METABRIC dataset. The expression of a proliferation related gene set and ATF4 are also shown. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.


( J ) Association between IKBKE ( IKKε ) and ATF4 mRNA overexpression evaluated by chi - squared independence test . The + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number and percentage of samples , as well as the Chi - square values are shown .

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(J) Association between IKBKE (IKKε) and ATF4 mRNA overexpression evaluated by chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.


( K , L ) Correlation of IKBKE ( IKKε ) and PSAT1 ( K ) or IKBKE ( IKKε ) and ATF4 ( L ) expression in the ER negative sample subset .

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(K, L) Correlation of IKBKE (IKKε) and PSAT1 (K) or IKBKE (IKKε) and ATF4 (L) expression in the ER negative sample subset.


( A ) Negative stain electron micrograph showing the tendency of S . cerevisiae prApe1 ( sc - prApe1 ) dodecamers to form higher - order assemblies or chains ( black arrowheads ) . ( B ) Negative stain electron micrograph of S . cerevisiae mApe1 ( sc - mApe1 ) showing monodisperse dodecameric particles ( circles ) as well as hexameric particles ( white arrowheads ) . ( C ) Bar plot showing the occurrence of single , double , triple or multiple aggregates of sc - prApe1 in comparison with sc - mApe1 . Insets show single , double , triple or multiple dodadecamers . Data information : n = 3 , 186 particles were counted in the case of sc - prApe1 and n = 3 , 107 particles were counted in the case of sc - mApe1 . Particles were picked from 20 different micrographs . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared test ) .

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(A) Negative stain electron micrograph showing the tendency of S. cerevisiae prApe1 (sc-prApe1) dodecamers to form higher-order assemblies or chains (black arrowheads). (B) Negative stain electron micrograph of S. cerevisiae mApe1 (sc-mApe1) showing monodisperse dodecameric particles (circles) as well as hexameric particles (white arrowheads). (C) Bar plot showing the occurrence of single, double, triple or multiple aggregates of sc-prApe1 in comparison with sc-mApe1. Insets show single, double, triple or multiple dodadecamers. Data information: n=3,186 particles were counted in the case of sc-prApe1 and n=3,107 particles were counted in the case of sc-mApe1. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).


( D ) Negative stain electron micrograph of GraFixed sc - mApe1 showing dodecameric particles ( circles ) . ( E ) Class averages of sc - mApe1 dodecamers including views of two - fold and three - fold symmetry . ( F ) Fourier shell correlation of the EM reconstruction of the sc - mApe1 dodecamer indicates a resolution of 24 Å .

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(D) Negative stain electron micrograph of GraFixed sc-mApe1 showing dodecameric particles (circles). (E) Class averages of sc-mApe1 dodecamers including views of two-fold and three-fold symmetry. (F) Fourier shell correlation of the EM reconstruction of the sc-mApe1 dodecamer indicates a resolution of 24 Å.


( G ) C . thermophilum mApe1 crystal structure fitted into electron microscopy ( EM ) map of sc - mApe1 dodecamer . Each of the 12 chains is depicted by a different color . A black sphere represents the N - terminus where the propeptide would emanate from the dodecamer . From left to right : two - fold axis view from the edge , three - fold axis view from the facet and three - fold axis view from the vertex .

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(G) C. thermophilum mApe1 crystal structure fitted into electron microscopy (EM) map of sc-mApe1 dodecamer. Each of the 12 chains is depicted by a different color. A black sphere represents the N-terminus where the propeptide would emanate from the dodecamer. From left to right: two-fold axis view from the edge, three-fold axis view from the facet and three-fold axis view from the vertex.


( A ) Electron micrograph of negatively stained Ams1 with particles and higher - order assemblies ( arrows ) . Class averages exhibit two distinct two - fold views consistent with D2 symmetry . Scale bar 10 nm . ( B ) Electron cryomicrograph of Ams1 with particles highlighted ( circles ) . ( C ) Selected particles ( top row ) are shown with corresponding class averages ( bottom row ) . Scale bar 10 nm . ( D ) Fourier shell correlation indicates a resolution of 6 . 3 Å according to the 0 . 143 criterion .

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(A) Electron micrograph of negatively stained Ams1 with particles and higher-order assemblies (arrows). Class averages exhibit two distinct two-fold views consistent with D2 symmetry. Scale bar 10 nm. (B) Electron cryomicrograph of Ams1 with particles highlighted (circles). (C) Selected particles (top row) are shown with corresponding class averages (bottom row). Scale bar 10 nm. (D) Fourier shell correlation indicates a resolution of 6.3 Å according to the 0.143 criterion.


( E ) Top view of the cryo - EM structure , four Ams1 molecules were segmented and colored ( dark blue , beige , red , light blue ) .

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(E) Top view of the cryo-EM structure, four Ams1 molecules were segmented and colored (dark blue, beige, red, light blue).


( F ) Molecular architecture of Ams1 monomer . Primary structure , two map segments with fitted pseudo - atomic models are shown . Left . The N - terminal portion was built from distant homology models : a four - helix bundle ( 17 - 27 , 209 - 271 ) in yellow and a jelly - roll fold ( 45 - 203 ) in red . Right . The α - mannosidase core ( 287 - 1083 ) with an α / β barrel ( 287 - 573 ) in blue , a three - helix bundle ( 574 - 671 ) in green , and the β - sandwich domain ( 672 - 1083 ) in purple .

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(F) Molecular architecture of Ams1 monomer. Primary structure, two map segments with fitted pseudo-atomic models are shown. Left. The N-terminal portion was built from distant homology models: a four-helix bundle (17 - 27, 209 - 271) in yellow and a jelly-roll fold (45 - 203) in red. Right. The α-mannosidase core (287 - 1083) with an α/β barrel (287 - 573) in blue, a three-helix bundle (574 - 671) in green, and the β-sandwich domain (672 - 1083) in purple.


( G ) Density section through the Ams1 monomer with three - helix bundle in the center .

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(G) Density section through the Ams1 monomer with three-helix bundle in the center.


( H ) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo - EM map . Corresponding 3D structure in Movie EV1 .

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(H) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo-EM map. Corresponding 3D structure in Movie EV1.


( J ) The β - sandwich domain ( purple ) makes up the transverse interface between chain A and chain B .

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(J) The β-sandwich domain (purple) makes up the transverse interface between chain A and chain B.


( K ) The N - terminal four - helix bundle ( orange ) mediates the longitudinal interface . Asterisk denotes position of W234E mutation between chain A and C / D .

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(K) The N-terminal four-helix bundle (orange) mediates the longitudinal interface. Asterisk denotes position of W234E mutation between chain A and C/D.


( L ) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild - type tetramers . Representative class averages show a two - lobed structure consistent with reprojections of chain A and B ( dark blue and beige ) dimers depicted in ribbon and surface representation below . Scale bar 10 nm .

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(L) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild-type tetramers. Representative class averages show a two-lobed structure consistent with reprojections of chain A and B (dark blue and beige) dimers depicted in ribbon and surface representation below. Scale bar 10 nm.


( A ) SEC - MALS profile of Atg19 yields a molecular weight estimate of 142 kDa ( measured in triplicate ) .

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(A) SEC-MALS profile of Atg19 yields a molecular weight estimate of 142 kDa (measured in triplicate).


( B ) Right . In vitro pull - down binding assay and subsequent SDS - PAGE . Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP - Atg19 ( lane 1 ) whereas purified mApe1 dodecamers are not capable of interacting with MBP - Atg19 ( lane 3 ) . As controls , MBP does not bind prApe1 and mApe1 ( lanes 2 and 4 ) . Left . SDS - PAGE showing input of the corresponding pull - down assay .

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(B) Right. In vitro pull-down binding assay and subsequent SDS-PAGE. Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP-Atg19 (lane 1) whereas purified mApe1 dodecamers are not capable of interacting with MBP-Atg19 (lane 3). As controls, MBP does not bind prApe1 and mApe1 (lanes 2 and 4). Left. SDS-PAGE showing input of the corresponding pull-down assay.


( C ) Co - expression of MBP - Atg19 and prApe1 in Sf21 cells ; SDS - PAGE of eluate from amylose beads .

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(C) Co-expression of MBP-Atg19 and prApe1 in Sf21 cells; SDS-PAGE of eluate from amylose beads.


( D ) Size - exclusion chromatography profile ( Superose 6 column ) of the prApe1 / Atg19 complex after anti MBP - Atg19 purification and cleavage of the MBP tag ( top ) . SDS - PAGE of peak fractions of the gel filtration profile ( bottom ) . Samples were run on two separate gels .

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(D) Size-exclusion chromatography profile (Superose 6 column) of the prApe1/Atg19 complex after anti MBP-Atg19 purification and cleavage of the MBP tag (top). SDS-PAGE of peak fractions of the gel filtration profile (bottom). Samples were run on two separate gels.


( E ) Negative stain electron micrograph corresponding to the size - exclusion chromatography peak at 10 ml retention volume of ( D ) . Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets .

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(E) Negative stain electron micrograph corresponding to the size-exclusion chromatography peak at 10 ml retention volume of (D). Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets.


( F ) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co - purified with Atg19 .

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(F) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co-purified with Atg19.


( G ) Bar plot of occurrence of single , double , triple and multiple dodecamers from the prApe1 and the prApe1 / Atg19 sample . Data information : n = 3 , 186 particles were counted in the case of prApe1 and n = 3 , 340 particles were counted in the case of prApe1 / Atg19 . Particles were picked from 20 different micrographs . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared test ) .

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(G) Bar plot of occurrence of single, double, triple and multiple dodecamers from the prApe1 and the prApe1/Atg19 sample. Data information: n=3,186 particles were counted in the case of prApe1 and n=3,340 particles were counted in the case of prApe1/Atg19. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).


H ) Epifluorescence microscopy images of the prApe1 - GFP / Atg19 - mCherry / ypt7Δ , prApe1 - GFP / Atg19Δ / ypt7Δ S . cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1 - GFP spots . BF represents bright - field images .

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H) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1-GFP spots. BF represents bright-field images.