Figure 4B . Anti - PCNA immunoprecipitation , showing that HUWE1 co - precipitates . C . Reciprocal experiment showing that PCNA co - precipitates with HUWE1 in anti - HUWE1 immunoprecipitation . D . As control , endogenous HUWE1 was depleted by siRNA treatment , and extracts were subjected to anti - HUWE1 immunoprecipitation . The HUWE1 blot shows that much less HUWE1 is precipitated by anti - HUWE1 antibodies from HUWE1 - knockdown cells than control , as expected . PCNA is co - precipitated by anti - HUWE1 antibodies from control , but not HUWE1 - depleted cells , showing that the interaction is specific and not due to unspecific binding of PCNA to anti - HUWE1 antibodies . E , F . HUWE1 co - precipitates with PCNA in anti - PCNA immunoprecipitation from extracts of MCF7 ( E ) and 8988T ( F ) cells , showing that the HUWE1 - PCNA interaction can be detected in different cell lines . G . Co - immunoprecipitation experiment from control and RAD18 - knockout 293T cells , showing that the HUWE1 - PCNA interaction is not affected by loss of PCNA ubiquitination . RAD18 - knockout cells were obtained by CRISPR / Cas9 technology . The RAD18 and PCNA blots show loss of RAD18 , and of PCNA ubiquitination respectively . H . Myc - HUWE1C - ter fragment was transfected in 293T cells . Anti - PCNA immunoprecipitation showed that the C - terminal HUWE1 fragment interacts with PCNA . I . Coomassie staining of recombinant GST - PCNA expressed and purified from bacteria . J . GST - pulldowns using GST - PCNA or GST - empty as control , and extracts of 293T cells transfected with Myc - HUWE1C - ter , showing interaction of this fragment with recombinant PCNA .

O B-PANEL_START 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START 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 4B. Anti-PCNA immunoprecipitation, showing that HUWE1 co-precipitates.C. Reciprocal experiment showing that PCNA co-precipitates with HUWE1 in anti-HUWE1 immunoprecipitation.D. As control, endogenous HUWE1 was depleted by siRNA treatment, and extracts were subjected to anti-HUWE1 immunoprecipitation. The HUWE1 blot shows that much less HUWE1 is precipitated by anti-HUWE1 antibodies from HUWE1-knockdown cells than control, as expected. PCNA is co-precipitated by anti-HUWE1 antibodies from control, but not HUWE1-depleted cells, showing that the interaction is specific and not due to unspecific binding of PCNA to anti-HUWE1 antibodies. E, F. HUWE1 co-precipitates with PCNA in anti-PCNA immunoprecipitation from extracts of MCF7 (E) and 8988T (F) cells, showing that the HUWE1-PCNA interaction can be detected in different cell lines.G. Co-immunoprecipitation experiment from control and RAD18-knockout 293T cells, showing that the HUWE1-PCNA interaction is not affected by loss of PCNA ubiquitination. RAD18-knockout cells were obtained by CRISPR/Cas9 technology. The RAD18 and PCNA blots show loss of RAD18, and of PCNA ubiquitination respectively.H. Myc-HUWE1C-ter fragment was transfected in 293T cells. Anti-PCNA immunoprecipitation showed that the C-terminal HUWE1 fragment interacts with PCNA.I. Coomassie staining of recombinant GST-PCNA expressed and purified from bacteria.J. GST-pulldowns using GST-PCNA or GST-empty as control, and extracts of 293T cells transfected with Myc-HUWE1C-ter, showing interaction of this fragment with recombinant PCNA.


Figure 5A , B . GST - pulldowns showing that HUWE1 PIP - box mutants do not interact with PCNA . A . Recombinant GST - tagged HUWE1C - ter , either wild - type or PIP - box mutant variants ( QVFF and VFF - the indicated residues , critical for the PIP - box , were mutated to A ) , were purified from bacteria ( see Coomassiestaining ) and employed for GST - pulldowns using extracts of 293T cells . PCNA co - precipitated with wild - type , but not PIP - box mutants . B . Reciprocal GST - pulldown using recombinant GST - PCNA and extracts of 293T cells overexpressing Myc - HUWE1C - ter . Wild - type , but not the PIP - box mutant ( VFF ) HUWE1 fragment bound to GST - PCNA . C , D . Reciprocal co - immunoprecipitationexperiments showing that PIP - box mutation abolishes PCNA interaction . C . 293T cells were transfected with Myc - HUWE1C - ter variants , and subjected to anti - PCNA ( C ) or anti - Myc ( D ) immunoprecipitation . Only wild - type , but not PIP - box mutant ( VFF ) HUWE1 interacted with PCNA in both experimental setups . E . Mutation of the PIP - box in full - length HUWE1 also blocks PCNA interaction . Full - length Myc - tagged HUWE1 ( wild - type or PIP - mutant ) was transfected in 293T cells . Following Myc - immunoprecipitation , endogenous PCNA was detected in wild - type , but not PIP - box mutant ( FF ) complexes .

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

Figure 5A, B. GST-pulldowns showing that HUWE1 PIP-box mutants do not interact with PCNA. A. Recombinant GST-tagged HUWE1C-ter, either wild-type or PIP-box mutant variants (QVFF and VFF -the indicated residues, critical for the PIP-box, were mutated to A), were purified from bacteria (see Coomassiestaining) and employed for GST-pulldowns using extracts of 293T cells. PCNA co-precipitated with wild-type, but not PIP-box mutants. B. Reciprocal GST-pulldown using recombinant GST-PCNA and extracts of 293T cells overexpressing Myc-HUWE1C-ter. Wild-type, but not the PIP-box mutant (VFF) HUWE1 fragment bound to GST-PCNA.C, D. Reciprocal co-immunoprecipitationexperiments showing that PIP-box mutation abolishes PCNA interaction. C. 293T cells were transfected with Myc-HUWE1C-ter variants, and subjected to anti-PCNA (C) or anti-Myc (D) immunoprecipitation. Only wild-type, but not PIP-box mutant (VFF) HUWE1 interacted with PCNA in both experimental setups.E. Mutation of the PIP-box in full-length HUWE1 also blocks PCNA interaction. Full-length Myc-tagged HUWE1 (wild-type or PIP-mutant) was transfected in 293T cells. Following Myc-immunoprecipitation, endogenous PCNA was detected in wild-type, but not PIP-box mutant (FF) complexes.


Figure 6A . Stable expression of Myc - tagged wild - type , PIP - box mutant , and catalytic inactive HUWE1 variants in HUWE1 - knockout 293T cells . The Western blot shows that expression of the Myc - tagged variants stably transfected in the knockout cell lines , is similar to the endogenous HUWE1 expression . B . Myc - tag immunoprecipitation using extracts of the corrected cell lines described above . As expected , PCNA co - precipitates with wild - type , but not PIP - box mutant HUWE1 ( FF ) . C . iPOND experiment using HUWE1 - knockout 293T cells , corrected with wild - type or PIP - mutant HUWE1 . The PIP - mutant shows reduced crosslinking to nascent DNA , indicated defective recruitment to replication forks . D . Clonogenic assay showing that stable expression of wild - type HUWE1 , but not of the PIP box mutant or the catalytic mutant , corrects the HU sensitivity of the HUWE1 - knockout 293T cells . Shown is the average of 3 independent experiments - / + SD . If not indicated otherwise , the p - values shown specify the statistical significance relative to 293T ( for the 0 . 5mM HU condition ) . P - values are : 0 . 0003 ( 293T vs HUWE1 - / - ; 0 . 157 ( 293T vs HUWE1 - / - + WT ) ; 0 . 0045 ( 293T vs HUWE1 - / - + FF ) ; 0 . 0006 ( 293T vs HUWE1 - / - + C4341A ; 0 . 047 ( HUWE1 - / - + WT vs HUWE1 - / - + FF ) . E . The S - phase arrest phenotype is also rescued by wild - type , but not PIP - mutant or catalytic mutant HUWE1 . S - phase arrested cells were quantified using the BrdU / PI bi - dimensional flow cytometry assay . Shown is the average of 3 independent experiments - / + SD . The p - values shown specify the statistical significance relative to 293T ( 0 . 018 , 0 . 957 , 0 . 015 , and 0 . 024 respectively ) . A quantification of the same data , showing the percentage of cells arrested in S - phase , is shown in Fig EV3A . F . Normal replication tract length , quantified using the DNA fiber assay , is restored by expressing wild - type , but not PIP - mutant HUWE1 , in the HUWE1 - knockout 293T cells . The " n " numbers of fibers analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . If not indicated otherwise , the p - values shown specify the statistical significance relative to 293T . P - values are : 1 . 47x10 - 11 ( 293T vs HUWE - / - samples ) , 0 . 094 ( 293T vs HUWE - / - + WT samples ) , 1 . 40x10 - 7 ( 293T vs HUWE - / - + FF samples ) , 1 . 22x10 - 7 ( HUWE - / - vs HUWE - / - + WT samples ) , 0 . 679 ( HUWE - / - vs HUWE - / - + FF samples ) . G . Alkaline Comet Assay showing that wild - type , but not the FF mutant can correct the breakage phenotype of HUWE1 - knockout 293T cells . The " n " numbers of comet tails analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . The p - values indicated are : 4 . 18x10 - 43 ( 293T vs HUWE - / - samples ) , 6 . 89x10 - 41 ( HUWE1 - / - + WT vs FF samples ) , and 5 . 97x10 - 50 ( HUWE1 - / - + WT vs C4341A samples ) .

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

Figure 6A. Stable expression of Myc-tagged wild-type, PIP-box mutant, and catalytic inactive HUWE1 variants in HUWE1-knockout 293T cells. The Western blot shows that expression of the Myc-tagged variants stably transfected in the knockout cell lines, is similar to the endogenous HUWE1 expression.B. Myc-tag immunoprecipitation using extracts of the corrected cell lines described above. As expected, PCNA co-precipitates with wild-type, but not PIP-box mutant HUWE1 (FF).C. iPOND experiment using HUWE1-knockout 293T cells, corrected with wild-type or PIP-mutant HUWE1. The PIP-mutant shows reduced crosslinking to nascent DNA, indicated defective recruitment to replication forks.D. Clonogenic assay showing that stable expression of wild-type HUWE1, but not of the PIP box mutant or the catalytic mutant, corrects the HU sensitivity of the HUWE1-knockout 293T cells. Shown is the average of 3 independent experiments -/+SD. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T (for the 0.5mM HU condition). P-values are: 0.0003 (293T vs HUWE1-/-; 0.157 (293T vs HUWE1-/- +WT); 0.0045 (293T vs HUWE1-/- +FF); 0.0006 (293T vs HUWE1-/- +C4341A; 0.047 (HUWE1-/- +WT vs HUWE1-/- +FF).E. The S-phase arrest phenotype is also rescued by wild-type, but not PIP-mutant or catalytic mutant HUWE1. S-phase arrested cells were quantified using the BrdU/PI bi-dimensional flow cytometry assay. Shown is the average of 3 independent experiments -/+SD. The p-values shown specify the statistical significance relative to 293T (0.018, 0.957, 0.015, and 0.024 respectively). A quantification of the same data, showing the percentage of cells arrested in S-phase, is shown in Fig EV3A.F. Normal replication tract length, quantified using the DNA fiber assay, is restored by expressing wild-type, but not PIP-mutant HUWE1, in the HUWE1-knockout 293T cells. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T. P-values are: 1.47x10-11 (293T vs HUWE-/- samples), 0.094 (293T vs HUWE-/- +WT samples), 1.40x10-7 (293T vs HUWE-/- +FF samples), 1.22x10-7 (HUWE-/- vs HUWE-/- +WT samples), 0.679 (HUWE-/- vs HUWE-/- +FF samples).G. Alkaline Comet Assay showing that wild-type, but not the FF mutant can correct the breakage phenotype of HUWE1-knockout 293T cells. The "n" numbers of comet tails analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. The p-values indicated are: 4.18x10-43 (293T vs HUWE-/- samples), 6.89x10-41 (HUWE1-/- +WT vs FF samples), and 5.97x10-50 (HUWE1-/- +WT vs C4341A samples).


Figure 7A , B . The impact of HUWE1 on H2AX phosphorylation and ubiquitination , under normal conditions ( A ) or upon induction of replication stress ( 2mM HU for 18h , or 2h after exposure to 40J / m2 UV ) . Control or HUWE1 - knockout HeLa cells were analyzed . C . In vitro ubiquitination assay showing that recombinant HUWE1C - ter can mono - ubiquitinate H2AX . D . Wild - type , but not the PIP - mutant can correct the defective H2AX ubiquitination in HUWE1 - knockout 293T cells . . E . Chromatin immunoprecipitation showing that H2AX binding to the common fragile site FRA3B is reduced in HUWE1 - knockout 8988T cells . Cells were treated with 600nM aphidicolin for 24h . Binding was quantified relative to input material . Shown is the average of 4 experiments - / + SD . P - value is 4 . 4x10 - 6 . F . Western blots showing that several H2AX ubiquitin ligases participate in H2AX ubiquitination following replication stress . HUWE1 , RNF168 , and BMI1 were knocked - down in HeLa cells . The efficiency of the knockdown is shown in Fig EV4 . Cells were treated with 600nM aphidicolin for 24h , 2mM HU for 24h , or analyzed 2h after exposure to 40J / m2 UV . G . Chromatin immunoprecipitation from HeLa cells , showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B . Cells were treated with 600nM aphidicolin for 24h . Binding was quantified relative to input material . Shown is the average of 3 experiments - / + SD . The p - values shown indicate the statistical significance relative to siControl ( 0 . 0016 , 0 . 0005 , and 0 . 0009 respectively ) . H . Chromatin fractionation experiments showing that HUWE1 - knockout HeLa cells , treated with 2mM HU for 24h , fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA . In contrast , 53BP1 chromatin association is not induced by HU treatment , and not affected by HUWE1 knockout , and thus can serve as loading control . Shown are blots of whole cell extract ( WCE ) samples , representing input material , and of chromatin pellet samples .

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

Figure 7A, B. The impact of HUWE1 on H2AX phosphorylation and ubiquitination, under normal conditions (A) or upon induction of replication stress (2mM HU for 18h, or 2h after exposure to 40J/m2 UV). Control or HUWE1-knockout HeLa cells were analyzed.C. In vitro ubiquitination assay showing that recombinant HUWE1C-ter can mono-ubiquitinate H2AX.D. Wild-type, but not the PIP-mutant can correct the defective H2AX ubiquitination in HUWE1-knockout 293T cells.. E. Chromatin immunoprecipitation showing that H2AX binding to the common fragile site FRA3B is reduced in HUWE1-knockout 8988T cells. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 4 experiments -/+ SD. P-value is 4.4x10-6.F. Western blots showing that several H2AX ubiquitin ligases participate in H2AX ubiquitination following replication stress. HUWE1, RNF168, and BMI1 were knocked-down in HeLa cells. The efficiency of the knockdown is shown in Fig EV4. Cells were treated with 600nM aphidicolin for 24h, 2mM HU for 24h, or analyzed 2h after exposure to 40J/m2 UV.G. Chromatin immunoprecipitation from HeLa cells, showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 3 experiments -/+ SD. The p-values shown indicate the statistical significance relative to siControl (0.0016, 0.0005, and 0.0009 respectively).H. Chromatin fractionation experiments showing that HUWE1-knockout HeLa cells, treated with 2mM HU for 24h, fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA. In contrast, 53BP1 chromatin association is not induced by HU treatment, and not affected by HUWE1 knockout, and thus can serve as loading control. Shown are blots of whole cell extract (WCE) samples, representing input material, and of chromatin pellet samples.


Figure 1 ( A ) Growth curves of S . pombe cultures in minimal medium without ( EMM ) or with antimycin A ( EMM + antA ) . Cultures were supplemented with either the complete amino - acid mix ( all AA mix , black ) , without any amino acids ( no AA , red ) , with arginine only ( R , blue ) , or with individual amino acids other than arginine ( individual AAs , grey ) . ( B ) Growth curves as in ( A ) for cultures supplemented with complete amino - acid mix ( black ) , without any amino acids ( red ) , or amino - acid mix without arginine ( green ) . ( C ) Scatter plot of normalised colony sizes of cells grown on EMM against normalised colony sizes of cells grown on EMM supplemented with arginine ( EMM + R ) ( Dataset file 1 ) . Mutants that passed our filtering ( Methods ) and whose colony - size ratios were 30 % bigger when supplemented with arginine are shown in red ; mutants encoding known genes of the arginine biosynthesis pathway are shown with blue circles . ( E ) Box plot of mean colony size ratios of mutants grown with vs without arginine for all mutants ( grey ) and respiratory deficient mutants ( orange , based on previous screens ( Malecki & Bähler , 2016 ) ( Welch ' s t - test pval * * < 0 . 002 ) . The same analysis was performed for mutants grown on EMM ( left ; 4 biological repeats , Dataset file 1 ) or on EMM supplemented with antimycin A ( right ; 2 biological repeats , Dataset file 2 ) . Boxplots were created using the R boxplot command with default settings . The central band indicates the median , the upper and lower limits of the box indicate first and third quartiles , and the whiskers show the most extreme data , limited to 1 . 5 - time interquartile range .

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 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 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

Figure 1(A) Growth curves of S. pombe cultures in minimal medium without (EMM) or with antimycin A (EMM + antA). Cultures were supplemented with either the complete amino-acid mix (all AA mix, black), without any amino acids (no AA, red), with arginine only (R, blue), or with individual amino acids other than arginine (individual AAs, grey).(B) Growth curves as in (A) for cultures supplemented with complete amino-acid mix (black), without any amino acids (red), or amino-acid mix without arginine (green).(C) Scatter plot of normalised colony sizes of cells grown on EMM against normalised colony sizes of cells grown on EMM supplemented with arginine (EMM+R) (Dataset file 1). Mutants that passed our filtering (Methods) and whose colony-size ratios were 30% bigger when supplemented with arginine are shown in red; mutants encoding known genes of the arginine biosynthesis pathway are shown with blue circles.(E) Box plot of mean colony size ratios of mutants grown with vs without arginine for all mutants (grey) and respiratory deficient mutants (orange, based on previous screens (Malecki & Bähler, 2016) (Welch's t-test pval** < 0.002). The same analysis was performed for mutants grown on EMM (left; 4 biological repeats, Dataset file 1) or on EMM supplemented with antimycin A (right; 2 biological repeats, Dataset file 2). Boxplots were created using the R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


Figure 2 ( A ) Normalised concentration values of 16 amino acids in cells grown in EMM without ( grey ) or with ( blue ) antimycin A . For each condition , four independent samples were analysed . Concentrations are presented relative to the mean value for the given amino acid in EMM medium . * Ornithine ( Orn ) is abbreviated as O . Boxplots were created using R boxplot command with default settings . The central band indicates the median , the upper and lower limits of the box indicate first and third quartiles , and the whiskers show the most extreme data , limited to 1 . 5 - time interquartile range . ( B ) Amino - acid concentrations before and after addition of antimycin A to cells exponentially growing in EMM . Left : heat map showing concentration changes ( log2 fold change ) of each measured amino acid during the time course relative to time point 0 ( before antimycin A addition ) . Right : simplified scheme of amino - acid biosynthetic pathways . Amino acids for which any biosynthetic enzyme has an annotated mitochondrial location are highlighted in yellow . * Ornithine ( Orn ) is abbreviated as O . ( C ) Left panels : cell growth in EMM without ( red ) or with ( blue ) arginine supplementation for five selected respiratory mutants as indicated ( graphs selected from Fig EV3 ) . Right panels : normalised intracellular amino - acid concentrations as in ( A ) for the same 5 respiratory mutants ( blue ) and wild - type control cells ( grey ) . For each mutant and the wild type strain , four independent samples were analysed . * Ornithine ( Orn ) is abbreviated as O . Boxplot features as in ( A ) .

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 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 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

Figure 2(A) Normalised concentration values of 16 amino acids in cells grown in EMM without (grey) or with (blue) antimycin A. For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. Boxplots were created using R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.(B) Amino-acid concentrations before and after addition of antimycin A to cells exponentially growing in EMM. Left: heat map showing concentration changes (log2 fold change) of each measured amino acid during the time course relative to time point 0 (before antimycin A addition). Right: simplified scheme of amino-acid biosynthetic pathways. Amino acids for which any biosynthetic enzyme has an annotated mitochondrial location are highlighted in yellow. *Ornithine (Orn) is abbreviated as O.(C) Left panels: cell growth in EMM without (red) or with (blue) arginine supplementation for five selected respiratory mutants as indicated (graphs selected from Fig EV3). Right panels: normalised intracellular amino-acid concentrations as in (A) for the same 5 respiratory mutants (blue) and wild-type control cells (grey). For each mutant and the wild type strain, four independent samples were analysed. *Ornithine (Orn) is abbreviated as O. Boxplot features as in (A).


Figure 3 ( A ) Normalised amino - acid concentrations in cells grown in EMM without ( grey ) or with arginine ( red ) . For each condition , four independent samples were analysed . Concentrations are presented relative to the mean value for the given amino acid in EMM medium . * Ornithine ( Orn ) is abbreviated as O . The central band indicates the median , the upper and lower limits of the box indicate first and third quartiles , and the whiskers show the most extreme data , limited to 1 . 5 - time interquartile range . ( C ) Growth curves of strains deleted for car1 , car2 or car3 , and a wild - type control . Strains were grown in EMM ( black ) , EMM with antimycin A ( red ) , EMM with arginine ( light blue ) or EMM with both arginine and antimycin A ( dark blue ) as indicated . ( D ) Growth curves of strains deleted for car2 ( top ) or car3 ( bottom ) . Strains were grown in EMM ( black ) , EMM with antimycin A ( red ) , EMM with arginine and antimycin A ( dark blue ) , EMM with mix of all amino acids and antimycin A ( bright blue ) , and EMM with antimycin A and each individual amino acid from the mix ( grey ) . ( E ) Growth curves of wild - type control ( WT ) and car2 deletion strain ( Δcar2 ) . Strains were grown in EMM media with antimycin A and with either a mix of all amino acids ( + AA mix ) or different combinations of lysine ( K ) , glutamine ( Q ) , glutamic acid ( E ) , or arginine ( R ) as indicated . Dashed line : time point where wild - type strain saturates in EMM with amino - acid mix .

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 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 O O O O O O O O O O O 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

Figure 3(A) Normalised amino-acid concentrations in cells grown in EMM without (grey) or with arginine (red). For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.(C) Growth curves of strains deleted for car1, car2 or car3, and a wild-type control. Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine (light blue) or EMM with both arginine and antimycin A (dark blue) as indicated.(D) Growth curves of strains deleted for car2 (top) or car3 (bottom). Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine and antimycin A (dark blue), EMM with mix of all amino acids and antimycin A (bright blue), and EMM with antimycin A and each individual amino acid from the mix (grey).(E) Growth curves of wild-type control (WT) and car2 deletion strain (Δcar2). Strains were grown in EMM media with antimycin A and with either a mix of all amino acids (+AA mix) or different combinations of lysine (K), glutamine (Q), glutamic acid (E), or arginine (R) as indicated. Dashed line: time point where wild-type strain saturates in EMM with amino-acid mix.


Figure 4 ( A ) Changes in transcript abundance 30 and 120 min after addition of antimycin A to exponentially growing cells without ( EMM ) or with arginine ( EMM + R ) . Transcripts annotated as up - or down - regulated Core Environmental Stress Response ( CESR ) are indicated in red and blue , respectively . All data is normalised to EMM time point 0 . ( B ) Graphs as in ( A ) showing expression profiles of transcripts encoding enzymes for arginine catabolism ( blue ) or arginine biosynthesis ( red ) . ( C ) Heat map of expression changes for transcripts repressed over 2 - fold at 30 min in cells supplemented with arginine ( EMM + R ) relative to cells without supplementation ( EMM ) . Values for timepoints 0 and 30 min after antimycin A addition are shown for cells grown in EMM , EMM with arginine and ssp1 deletion mutant grown in EMM . The data are normalised to EMM timepoint 0 . ( D ) Heat map of expression changes of the same genes as in ( B ) after one hour of inhibiting TOR kinase with torin1 or caffeine / rapamycin treatment relative to cell before treatment ; cells were grown in rich media - YES ( data from ( Rodríguez - López et al . , 2019 ) )

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 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

Figure 4(A) Changes in transcript abundance 30 and 120 min after addition of antimycin A to exponentially growing cells without (EMM) or with arginine (EMM+R). Transcripts annotated as up- or down-regulated Core Environmental Stress Response (CESR) are indicated in red and blue, respectively. All data is normalised to EMM time point 0.(B) Graphs as in (A) showing expression profiles of transcripts encoding enzymes for arginine catabolism (blue) or arginine biosynthesis (red).(C) Heat map of expression changes for transcripts repressed over 2-fold at 30 min in cells supplemented with arginine (EMM +R) relative to cells without supplementation (EMM). Values for timepoints 0 and 30 min after antimycin A addition are shown for cells grown in EMM, EMM with arginine and ssp1 deletion mutant grown in EMM. The data are normalised to EMM timepoint 0.(D) Heat map of expression changes of the same genes as in (B) after one hour of inhibiting TOR kinase with torin1 or caffeine/rapamycin treatment relative to cell before treatment; cells were grown in rich media - YES (data from (Rodríguez-López et al., 2019))


Figure 1B ) Volcano plot of quantified proteins . Log2 fold change was determined as the difference between the mean LFQ intensity of the four replicates of telomeric to control sequence and p - values were calculated with a Welch t - test . Proteins above the threshold p = 0 . 05 , s0 = 1 , c = 0 . 5 ( dashed line ) are considered enriched . Abbreviations : LFQ : Label free quantificationD ) Heatmap of LFQ intensity of proteins enriched at DNA baits . Telomere interactors are indicated on the right side of the heatmap . Color code indicates measured log2 LFQ intensities in the telomere pull - down . RNA binding proteins are marked with a black box on the left side of the figure . Abbreviations : LFQ : Label free quantification RBP : RNA binding proteinE ) Critically short telomeres arise after 90 population doublings in the absence of telomerase ( tlc1 ) . Genomic DNA was extracted at indicated population doublings and digested with XhoI . A radioactive probe was used to recognize telomeric DNA in a southern blot . Abbreviations : PD : population doubling . F ) tlc1 cells reach senescence at PD 80 - 100 . % viability of indicated strains after propagation in YPD is indicated at different population doublings n = 3 . Asterisk indicates population doubling used in G ) to determine binding of Npl3 to short telomeres by TAP - ChIP . 3 independent spores from the indicated genotypes were used . Population doublings are counted from germination of meiotic products and hence begin at approx . PD40 . Abbreviations : TAP : tandem affinity purification tagG ) Npl3 - TAP associates strongly to short telomeres . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 ( paired t - test one tailed * p < 0 . 05 , * * p < 0 . 01 ) . PD90 refers to 90 population doublings in the absence of TLC1 ( telomerase RNA subunit ) . The no tag control serves as an indicator of non - specific background signal . Abbreviations : TAP : tandem affinity purification tag , PD : population doubling .

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

Figure 1B) Volcano plot of quantified proteins. Log2 fold change was determined as the difference between the mean LFQ intensity of the four replicates of telomeric to control sequence and p-values were calculated with a Welch t-test. Proteins above the threshold p=0.05, s0=1, c=0.5 (dashed line) are considered enriched. Abbreviations: LFQ: Label free quantificationD) Heatmap of LFQ intensity of proteins enriched at DNA baits. Telomere interactors are indicated on the right side of the heatmap. Color code indicates measured log2 LFQ intensities in the telomere pull-down. RNA binding proteins are marked with a black box on the left side of the figure. Abbreviations: LFQ: Label free quantification RBP: RNA binding proteinE) Critically short telomeres arise after 90 population doublings in the absence of telomerase (tlc1). Genomic DNA was extracted at indicated population doublings and digested with XhoI. A radioactive probe was used to recognize telomeric DNA in a southern blot. Abbreviations: PD: population doubling.F) tlc1 cells reach senescence at PD 80-100. %viability of indicated strains after propagation in YPD is indicated at different population doublings n=3. Asterisk indicates population doubling used in G) to determine binding of Npl3 to short telomeres by TAP-ChIP. 3 independent spores from the indicated genotypes were used. Population doublings are counted from germination of meiotic products and hence begin at approx. PD40. Abbreviations: TAP: tandem affinity purification tagG) Npl3-TAP associates strongly to short telomeres. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). PD90 refers to 90 population doublings in the absence of TLC1 (telomerase RNA subunit). The no tag control serves as an indicator of non-specific background signal. Abbreviations: TAP: tandem affinity purification tag, PD: population doubling.


Figure 2A ) Telomere interactors were identified and plotted as a volcano plot as in Figure 1 A , B and using tlc1 protein extracts . Interactors were identified using untreated protein extracts from tlc1 cells ( left panel ) or digested with RNase A and RNase H ( right panel ) . B ) Npl3 associates to TERRA . Cross - linked samples from the indicated strains were subjected to Npl3 - TAP RIP . Enrichment of the indicated RNAs was determined by quantitative PCR on pulled - down reverse - transcribed RNA . Data represents mean % input + / - SEM n = 3 ( unpaired t - test two tailed * p < 0 . 05 , * * p < 0 . 01 ) . Npl3 associates strongly to telomeres in the absence of Rat1 . C ) The Rat1 - AID tagged protein is strongly degraded following 1h of 1mM IAA treatment . Protein levels are determined using anti - FLAG ( Rat1 - AID ) and anti - actin antibodies . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron . Npl3 associates strongly to telomeres in the absence of Rat1 . D ) TERRA levels increase in cells expressing the Rat1 - AID variant after 1h treatment with 1mM IAA . TERRA levels were determined by quantitative PCR on reverse - transcribed RNAs after phenol - chloroform extraction . RNA levels are normalized to 7S ncRNA levels . Data represents fold increase to corresponding - IAA samples n = 2 . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron . Npl3 associates strongly to telomeres in the absence of Rat1 . E ) Npl3 - TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1 - AID . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 ( paired t - test one tailed * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron . F - G ) Npl3 associates to telomeres in an R - loop dependent manner . E ) RNH1 was overexpressed from a galactose - inducible promoter . Protein levels are determined using anti - HA and anti - actin antibodies . F ) Npl3 - TAP associates to telomeres in an R - loop - dependent manner . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 ( paired t - test one tailed * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations : eV : empty vector control , oE :

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

Figure 2A) Telomere interactors were identified and plotted as a volcano plot as in Figure 1 A, B and using tlc1 protein extracts. Interactors were identified using untreated protein extracts from tlc1 cells (left panel) or digested with RNase A and RNase H (right panel).B) Npl3 associates to TERRA. Cross-linked samples from the indicated strains were subjected to Npl3-TAP RIP. Enrichment of the indicated RNAs was determined by quantitative PCR on pulled-down reverse-transcribed RNA. Data represents mean % input +/- SEM n=3 (unpaired t-test two tailed *p<0.05, **p<0.01).Npl3 associates strongly to telomeres in the absence of Rat1. C) The Rat1-AID tagged protein is strongly degraded following 1h of 1mM IAA treatment. Protein levels are determined using anti-FLAG (Rat1-AID) and anti-actin antibodies. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.Npl3 associates strongly to telomeres in the absence of Rat1. D) TERRA levels increase in cells expressing the Rat1-AID variant after 1h treatment with 1mM IAA. TERRA levels were determined by quantitative PCR on reverse-transcribed RNAs after phenol-chloroform extraction. RNA levels are normalized to 7S ncRNA levels. Data represents fold increase to corresponding -IAA samples n=2. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.Npl3 associates strongly to telomeres in the absence of Rat1. E) Npl3-TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1-AID. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.F-G) Npl3 associates to telomeres in an R-loop dependent manner. E) RNH1 was overexpressed from a galactose-inducible promoter. Protein levels are determined using anti-HA and anti-actin antibodies. F) Npl3-TAP associates to telomeres in an R-loop-dependent manner. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: eV: empty vector control, oE: overexpression


Figure 3A - B ) Npl3 associates to telomeres in early S . A ) Cell cycle profiles of indicated strains . Cells were synchronized in G1 with alpha - factor for 2 . 5h and released into the cell cycle at 30 ° C in pre - warmed medium supplemented with the indicated HU concentrations . B ) Npl3 - TAP associates strongly to telomeres in early S ( 250 mM HU ) . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM relative to cells arrested in alpha factor n = 3 ( paired t - test two tailed * p < 0 . 05 , * * p < 0 . 01 Abbreviations : ns : not significant HU : hydroxyurea . * p < 0 . 05 , * * p < 0 . 01C ) NPL3 over expression stabilizes telomeric R - loops . The indicated strains were grown on 1 % raffinose 2 % galactose to induce NPL3 over expression . Cross - linked samples were subjected to DRIP . DNA - RNA hybrids were immunoprecipitated using the S9 . 6 antibody . R - loop levels were determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM relative to 250 mM HU eV n = 3 ( paired t - test two tailed * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations eV : empty vector control , oE : over expression HU : hydroxyurea . * p < 0 . 05 , * * p < 0 . 01D ) Deletion of NPL3 decreases telomeric R - loops . The indicated strains were crosslinked and subjected to DRIP . DNA - RNA hybrids were immunoprecipitated using the S9 . 6 antibody . R - loop levels were determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 . Specificity of S9 . 6 antibody was determined treating a fraction of the samples with RNase H . E - G ) NPL3 stabilizes R - loops . E ) R - loop dot blot . The indicated strains were grown on 1 % raffinose 2 % galactose for 72h and subsequently released into 1 % raffinose 2 % galactose or 2 % glucose medium for 6 PDs . R - loop levels and dsDNA levels were determined using the S9 . 6 antibody and anti - dsDNA antibodies . Specificity of the S9 . 6 antibody was confirmed by treatment with RNase H . Loaded genomic DNA content is indicated at the bottom of the figure . F ) R - loop dot blot quantification . Data represents R - loop signal as the S9 . 6 antibody signal relative to corresponding dsDNA signal n = 3 ( unpaired t - test two tailed * p < 0 . 05 ) . G ) Serial dilutions of indicated strains were assayed on 1 % raffinose 2 % galactose media . Plates were imaged after 72h incubation at indicated temperatures . Abbreviations : V : empty vector control , oE : over expression , ds : double - strand ,

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

Figure 3A-B) Npl3 associates to telomeres in early S. A) Cell cycle profiles of indicated strains. Cells were synchronized in G1 with alpha-factor for 2.5h and released into the cell cycle at 30°C in pre-warmed medium supplemented with the indicated HU concentrations. B) Npl3-TAP associates strongly to telomeres in early S (250 mM HU). Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to cells arrested in alpha factor n=3 (paired t-test two tailed *p<0.05, **p<0.01 Abbreviations: ns: not significant HU: hydroxyurea. *p<0.05, **p<0.01C) NPL3 over expression stabilizes telomeric R-loops. The indicated strains were grown on 1% raffinose 2% galactose to induce NPL3 over expression. Cross-linked samples were subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to 250 mM HU eV n=3 (paired t-test two tailed *p<0.05, **p<0.01). Abbreviations eV: empty vector control, oE: over expression HU: hydroxyurea. *p<0.05, **p<0.01D) Deletion of NPL3 decreases telomeric R-loops. The indicated strains were crosslinked and subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3. Specificity of S9.6 antibody was determined treating a fraction of the samples with RNase H.E-G) NPL3 stabilizes R-loops. E) R-loop dot blot. The indicated strains were grown on 1% raffinose 2% galactose for 72h and subsequently released into 1% raffinose 2% galactose or 2% glucose medium for 6 PDs. R-loop levels and dsDNA levels were determined using the S9.6 antibody and anti-dsDNA antibodies. Specificity of the S9.6 antibody was confirmed by treatment with RNase H. Loaded genomic DNA content is indicated at the bottom of the figure. F) R-loop dot blot quantification. Data represents R-loop signal as the S9.6 antibody signal relative to corresponding dsDNA signal n=3 (unpaired t-test two tailed *p<0.05). G) Serial dilutions of indicated strains were assayed on 1% raffinose 2% galactose media. Plates were imaged after 72h incubation at indicated temperatures. Abbreviations: V: empty vector control, oE: over expression, ds: double-strand,


Figure 4A ) Serial dilutions of indicated strains were assayed on YPD media . Cells were plated after 30 - 50 generations propagated on YPD agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 72h growth at 30 ° C . B ) Serial dilutions of indicated strains were assayed on YPD media . Cells were plated after 30 - 50 generations propagated on YPD agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 48h growth at 30 ° C . C ) Serial dilutions of indicated strains were assayed on SC - His media . Cells were plated after 30 - 50 generations propagated on SC - His agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 72h growth at 30 ° C .

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

Figure 4A) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C.B) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 48h growth at 30°C.C) Serial dilutions of indicated strains were assayed on SC-His media. Cells were plated after 30-50 generations propagated on SC-His agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C.


Figure 1 ( B ) Western blot analysis of Atxn1 levels in cerebellar granule neurons ( CGNs ) after knockdown of Zbtb7a and / or Zbtb7b ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA . ( C ) Western blot analysis of Atxn1 levels in the cerebella of wild - type ( n = 3 ) , Zbtb7a + / - ( n = 4 ) , Zbtb7b + / - ( n = 3 ) , Zbtb7a + / - ; Zbtb7b + / - ( n = 4 ) mice . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA . ( D ) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and / or Zbtb7b ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA . ( E ) Genetic interaction of wild - type human ATXN1 ( 30Q ) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes . Co - expression of ATXN1 ( 30Q ) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels . Scale bar : 100

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

Figure 1(B) Western blot analysis of Atxn1 levels in cerebellar granule neurons (CGNs) after knockdown of Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.(C) Western blot analysis of Atxn1 levels in the cerebella of wild-type (n = 3), Zbtb7a+/- (n = 4), Zbtb7b+/- (n = 3), Zbtb7a+/-; Zbtb7b+/- (n = 4) mice. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.(D) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.(E) Genetic interaction of wild-type human ATXN1(30Q) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes. Co-expression of ATXN1(30Q) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels. Scale bar: 100 μm


Figure 2 ( C ) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild - type ( WT ) , zinc - finger domain mutant ( ZFmut ) or BTB domain mutants ( BTBmut1 and BTBmut2 ) ( n = 3 ) . Data information : Mean ± SD , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( D ) qRT - PCR results of Atxn1 mRNA levels in the cerebella of wild - type ( n = 3 ) , Zbtb7b + / - ( n = 4 ) , and Zbtb7b - / - mice ( n = 4 ) . Data information : Mean ± SD , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( E ) MA plot ( M : log ratio ; A : mean average ) of the differentially expressed genes ( DEG ) analysis from RNA - seq samples of the cells overexpressing either WT or ZFmut ZBTB7B . Four filters and the number of genes kept after applying each filter are described . Two genes that passed the 4th filter are labeled . ( F ) Expression heatmap of the 16 genes that passed the 3rd filter . Color scale : row z - score . ( G ) qRT - PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines ( n = 2 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( H ) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation . ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line ( sgRSK3 ) and control cell lines ( WT and sgControl ) ( n = 3 ) . ZBTB7B + RSK3 : co - overexpression of both ZBTB7B and RSK3 . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( I ) qRT - PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells ( n = 2 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( J ) qRT - PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice ( n = 6 ) . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test . ( K ) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter . Start and End position numbers are described based on TSS . The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test . ( L ) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test .

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 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 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 O O O O O O O O O O O O O O O O O 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 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 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 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 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 2(C) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild-type (WT), zinc-finger domain mutant (ZFmut) or BTB domain mutants (BTBmut1 and BTBmut2) (n = 3). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.(D) qRT-PCR results of Atxn1 mRNA levels in the cerebella of wild-type (n = 3), Zbtb7b+/- (n = 4), and Zbtb7b-/- mice (n = 4). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.(E) MA plot (M: log ratio; A: mean average) of the differentially expressed genes (DEG) analysis from RNA-seq samples of the cells overexpressing either WT or ZFmut ZBTB7B. Four filters and the number of genes kept after applying each filter are described. Two genes that passed the 4th filter are labeled.(F) Expression heatmap of the 16 genes that passed the 3rd filter. Color scale: row z-score.(G) qRT-PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(H) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation. ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line (sgRSK3) and control cell lines (WT and sgControl) (n = 3). ZBTB7B+RSK3: co-overexpression of both ZBTB7B and RSK3. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(I) qRT-PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(J) qRT-PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice (n = 6). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.(K) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter. Start and End position numbers are described based on TSS. The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays. Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.(L) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector (n = 3). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


Figure 3 ( A ) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells . Bottom left panel : qRT - PCR results of RSK3 mRNA . Bottom right panel : densitometry of the western blot image in the top panel ( n = 3 except for shATXN1 where n = 1 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( B ) Western blot analysis of ATXN1 after overexpression of RSK3 , ZBTB7B , or eGFP in Daoy cells ( n = 3 ) . Data information : RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3 . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( C ) Western blot analysis of ATXN1 ( 82Q ) after overexpression of either wild - type or inactive RSK3 in Daoy cells expressing ATXN1 ( 82Q ) ( n = 3 ) . Data information : RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3 . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( D ) In vitro kinase assay of RSK3 with ATXN1 . MSK1 was used as a positive control . Asterisk : auto - phosphorylated RSK3 . ( E ) In vitro kinase assay of four different RSK members with ATXN1 . ( F ) In vitro kinase assay of RSK3 with N - terminal ( F1 ) , middle ( F2 ) , and C - terminal fragment ( F3 ) of ATXN1 . ( G ) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3 . ( H ) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor ( BI - D1870 ) on SCA1 patient iPSC - derived neurons ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( I ) Western blot analysis of total and phospho - S776 ATXN1 after a co - treatment with MSK1 ( SB - 747651A ) and RSK3 inhibitor ( BI - D1870 ) on SCA1 patient iPSC - derived neurons ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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 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 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 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 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 O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells. Bottom left panel: qRT-PCR results of RSK3 mRNA. Bottom right panel: densitometry of the western blot image in the top panel (n = 3 except for shATXN1 where n = 1). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(B) Western blot analysis of ATXN1 after overexpression of RSK3, ZBTB7B, or eGFP in Daoy cells (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(C) Western blot analysis of ATXN1(82Q) after overexpression of either wild-type or inactive RSK3 in Daoy cells expressing ATXN1(82Q) (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(D) In vitro kinase assay of RSK3 with ATXN1. MSK1 was used as a positive control. Asterisk: auto-phosphorylated RSK3.(E) In vitro kinase assay of four different RSK members with ATXN1.(F) In vitro kinase assay of RSK3 with N-terminal (F1), middle (F2), and C-terminal fragment (F3) of ATXN1.(G) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3.(H) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(I) Western blot analysis of total and phospho-S776 ATXN1 after a co-treatment with MSK1 (SB-747651A) and RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


Figure 4 ( A ) Images of Drosophila eyes expressing human ATXN1 ( 82Q ) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II . Scale bar : 100 μm ( top ) and 20 μm ( bottom ) . ( B ) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1 ( 82Q ) in the central nervous system . Mean ± SEM , * P < 0 . 05 , Linear mixed - effect model ANOVA , n = 10 / genotype , four times of measurements . ( C ) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice ( Atxn1154Q / 2Q ) cerebellum . Scale bar : 2 mm . ( D ) Western blot analysis of wild - type ( 2Q ) and mutant Atxn1 ( 154Q ) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl ( n = 3 ) , shRsk3 ( n = 3 ) , or shAtxn1 ( n = 2 ) . Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . ( E ) Western blot analysis of wild - type ( 2Q ) and mutant Atxn1 ( 154Q ) in the brainstem ( BS ) and cerebella ( CBM ) of 8 weeks old Atxn1154Q / 2Q ( n = 6 ) and Atxn1154Q / 2Q ; Rsk3 + / - mice ( n = 7 ) . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test . ( F ) Rotarod motor performance test result of Atxn1154Q / 2Q ; Rsk3 + / - mice at 11 - 12 weeks old . Mean ± SEM , * P < 0 . 05 , two - way ANOVA , n = 35 , 31 , 34 , 38 for WT , Rsk3 + / - , Atxn1154Q / 2Q , Atxn1154Q / 2Q ; Rsk3 + / - genotype , respectively .

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 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 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 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 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

Figure 4(A) Images of Drosophila eyes expressing human ATXN1(82Q) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II. Scale bar: 100 μm (top) and 20 μm (bottom).(B) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1(82Q) in the central nervous system. Mean ± SEM, *P < 0.05, Linear mixed-effect model ANOVA, n = 10/genotype, four times of measurements.(C) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice (Atxn1154Q/2Q) cerebellum. Scale bar: 2 mm.(D) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl (n = 3), shRsk3 (n = 3), or shAtxn1 (n = 2). Mean ± SD, *P<0.05, ***P<0.001 ****P<0.0001, one-way ANOVA.(E) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the brainstem (BS) and cerebella (CBM) of 8 weeks old Atxn1154Q/2Q (n = 6) and Atxn1154Q/2Q; Rsk3+/- mice (n = 7). Mean ± SD, *P<0.05, **P<0.01, two-tailed t-test.(F) Rotarod motor performance test result of Atxn1154Q/2Q; Rsk3+/- mice at 11-12 weeks old. Mean ± SEM, *P<0.05, two-way ANOVA, n = 35, 31, 34, 38 for WT, Rsk3+/-, Atxn1154Q/2Q, Atxn1154Q/2Q; Rsk3+/- genotype, respectively.


Figure 5 ( A ) Fluorescent western blot analysis of Atxn1 , Msk1 , and Rsk3 in seven different brain regions of wild - type mice at 9 weeks old ( n = 3 ) . Each protein levels were represented relative to the protein levels in brainstem . Mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . Data information : BS : brainstem , CBM : cerebellum , MB : midbrain , ST : striatum , HC : hippocampus , CTX : cortex , OB : olfactory bulbs . ( B ) Representative BaseScope assay images of Rsk3 in the whole brain sagittal sections of wild - type ( top ) and Rsk3 - / - mice ( bottom ) at 5 - 6 weeks old . The areas within the black squares are magnified in the right panels in each genotype . Scale bar = 2 mm in whole brain images ; 50 μm in the magnified images . Data information : BS : brainstem , CBM : cerebellum ( C ) Representative IF images of Msk1 in the whole brain sagittal sections of wild - type ( top ) and Msk1 - / - mice ( bottom ) at 6 months old . The areas within the white squares are magnified in the right panels in each genotype . Scale bar = 2 mm in whole brain images ; 50 μm in the magnified images . Data information : BS : brainstem , CBM : cerebellum ( D ) Western blot analysis of total and active form of Rsk3 and Msk1 in the cerebella and brainstem of 12 weeks old WT and SCA1 mice ( n = 4 ) . Relative Rsk3 and Msk1 levels were represented into log2 scale . The protein levels of the two kinases are significantly different between the two brain regions . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - way ANOVA . Data information : BS : brainstem , CBM : cerebellum ( E ) Western blot analysis of ATXN1 , RSK3 , pRSK3 , MSK1 , and pMSK1 in the postmortem cerebella , pons , and medulla oblongata of human controls and SCA1 patients ( n = 3 per genotype ) . Total protein was used for normalization , and protein levels are represented relative to cerebellar protein levels in control group . C : Cerebellum , P : Pons , M : medulla oblongata . Values from P and M are combined and presented in BS . Mean ± SEM , * P < 0 . 05 , * * P < 0 . 01 , two - way ANOVA , n = 3 ( CBM ) and 6 ( BS ) . Data information : BS : brainstem , CBM :

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

Figure 5(A) Fluorescent western blot analysis of Atxn1, Msk1, and Rsk3 in seven different brain regions of wild-type mice at 9 weeks old (n = 3). Each protein levels were represented relative to the protein levels in brainstem. Mean ± SD, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. Data information: BS: brainstem, CBM: cerebellum, MB: midbrain, ST: striatum, HC: hippocampus, CTX: cortex, OB: olfactory bulbs.(B) Representative BaseScope assay images of Rsk3 in the whole brain sagittal sections of wild-type (top) and Rsk3-/- mice (bottom) at 5-6 weeks old. The areas within the black squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum(C) Representative IF images of Msk1 in the whole brain sagittal sections of wild-type (top) and Msk1-/- mice (bottom) at 6 months old. The areas within the white squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum(D) Western blot analysis of total and active form of Rsk3 and Msk1 in the cerebella and brainstem of 12 weeks old WT and SCA1 mice (n = 4). Relative Rsk3 and Msk1 levels were represented into log2 scale. The protein levels of the two kinases are significantly different between the two brain regions. Mean ± SD, *P<0.05, **P<0.01, ****P <0.0001, two-way ANOVA. Data information: BS: brainstem, CBM: cerebellum(E) Western blot analysis of ATXN1, RSK3, pRSK3, MSK1, and pMSK1 in the postmortem cerebella, pons, and medulla oblongata of human controls and SCA1 patients (n = 3 per genotype). Total protein was used for normalization, and protein levels are represented relative to cerebellar protein levels in control group. C: Cerebellum, P: Pons, M: medulla oblongata. Values from P and M are combined and presented in BS. Mean ± SEM, *P<0.05, **P<0.01, two-way ANOVA, n = 3 (CBM) and 6 (BS). Data information: BS: brainstem, CBM: cerebellum


Figure 6 ( A ) Western blot analysis of total and pS776 wild - type and mutant Atxn1 in the brainstem of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA , n = 7 , 6 , 4 , 7 following the order of genotypes in the western blot , respectively . ( B ) Plethysmography data measuring breathing ability ( total ventilated gas volume per minute ) of 34 weeks old mice with different genotypes . Mean ± SD , * P < 0 . 05 , two - tailed t - test , n = 12 , 13 , 15 , 13 , 15 , 9 , 15 , 13 following the order of genotypes in the graph , respectively . ( C ) Representative immunohistochemistry images of Iba1 + reactive microglia in the coronally sectioned hypoglossal nucleus of 36 weeks old mice with different genotypes . Yellow arrows indicates Iba1 + microglia , and larger pyramidal shaped cells are motoneurons . scale bar = 50 μm . ( D ) Quantification of the number of Iba1 + cells shown in ( C ) . Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from six sections per mouse . ( E ) Representative Hematoxylin and Eosin stained motoneurons in the coronally sectioned hypoglossal nucleus ( white dashed line ) of 36 weeks old mice with different genotypes . The motoneurons are dark purple with pyramidal shape within the nucleus . C : central canal . Scale bar = 100 μm . ( F ) Quantification of the number of motoneurons shown in ( E ) . Mean ± SD , * P < 0 . 05 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from six sections per mouse . ( G ) Kaplan - Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases . The data of Msk1 + / - , Rsk3 + / - , and Msk1 + / - ; Rsk3 + / - mice overlap those of wild - type mice . * P < 0 . 05 , Gehan - Breslow - Wilcoxon test , n = 15 , 15 , 12 , 13 , 22 , 11 , 20 , 19 following the order of genotypes in the legend on top of the figure , respectively .

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

Figure 6(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the brainstem of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, *P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.(B) Plethysmography data measuring breathing ability (total ventilated gas volume per minute) of 34 weeks old mice with different genotypes. Mean ± SD, *P<0.05, two-tailed t-test, n = 12, 13, 15, 13, 15, 9, 15, 13 following the order of genotypes in the graph, respectively.(C) Representative immunohistochemistry images of Iba1+ reactive microglia in the coronally sectioned hypoglossal nucleus of 36 weeks old mice with different genotypes. Yellow arrows indicates Iba1+ microglia, and larger pyramidal shaped cells are motoneurons. scale bar = 50 μm.(D) Quantification of the number of Iba1+ cells shown in (C). Mean ± SD, *P<0.05, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.(E) Representative Hematoxylin and Eosin stained motoneurons in the coronally sectioned hypoglossal nucleus (white dashed line) of 36 weeks old mice with different genotypes. The motoneurons are dark purple with pyramidal shape within the nucleus. C: central canal. Scale bar = 100 μm.(F) Quantification of the number of motoneurons shown in (E). Mean ± SD, *P<0.05, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.(G) Kaplan-Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases. The data of Msk1+/-, Rsk3+/-, and Msk1+/-; Rsk3+/- mice overlap those of wild-type mice. *P<0.05, Gehan-Breslow-Wilcoxon test, n = 15, 15, 12, 13, 22, 11, 20, 19 following the order of genotypes in the legend on top of the figure, respectively.


Figure 7 ( A ) Western blot analysis of total and pS776 wild - type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA , n = 7 , 6 , 4 , 7 following the order of genotypes in the western blot , respectively . ( B ) Rotarod motor performance test result of 11 - 12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SEM , * P < 0 . 05 , two - way ANOVA , n = 18 , 12 , 13 , 11 , 19 , 13 , 15 , 13 following the order of genotypes in the legend on top of the figure , respectively . ( C ) Representative immunofluorescent ( IF ) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old , stained with anti - Calbindin 1 antibody . Scale bar = 100 μm . ( D ) Quantification of the thickness of top and bottom molecular layers shown in ( C ) . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from three sections per mouse .

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

Figure 7(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, **P<0.01, ***P<0.001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.(B) Rotarod motor performance test result of 11-12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SEM, *P<0.05, two-way ANOVA, n = 18, 12, 13, 11, 19, 13, 15, 13 following the order of genotypes in the legend on top of the figure, respectively.(C) Representative immunofluorescent (IF) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old, stained with anti-Calbindin 1 antibody. Scale bar = 100 μm.(D) Quantification of the thickness of top and bottom molecular layers shown in (C). Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from three sections per mouse.


Figure 1Exponential growth of E . coli K - 12 , measured using optical density ( OD600 ) , in lysogenic broth ( LB , white circles ) or minimal medium supplemented with different carbon sources , listed in the legend on the bottom right in a greyscale . All the cultures are grown in batch mode , see Methods . Bacterial viability in colony forming units ( CFU ) per ml of cultures from panel A , grown until OD600 0 . 5 , washed and re - suspended in carbon - free minimal medium . Death rates ( slopes of the exponential fits ) are listed in Table EV1 . Initial density for fast growth ( lighter shades ) is lower than for slow growth ( darker shades ) , due larger cell sizes that lead to lower cell densities per OD600 . Death rates of panel B plotted against growth rates of panel A shown as circles in a grey - scale . Squares show data obtained using eight other carbon substrates , also listed in Table EV1 . Color circles show data obtained by varying growth rate in a chemostat . The black line shows a linear weighted least square fit to log - transformed data , that takes uncertainty in data points into account ( see Methods ) . Data shown as mean ± ( standard deviation ) SD . Two or three replicates per condition .

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Figure 1Exponential growth of E. coli K-12, measured using optical density (OD600), in lysogenic broth (LB, white circles) or minimal medium supplemented with different carbon sources, listed in the legend on the bottom right in a greyscale. All the cultures are grown in batch mode, see Methods.Bacterial viability in colony forming units (CFU) per ml of cultures from panel A, grown until OD600 0.5, washed and re-suspended in carbon-free minimal medium. Death rates (slopes of the exponential fits) are listed in Table EV1. Initial density for fast growth (lighter shades) is lower than for slow growth (darker shades), due larger cell sizes that lead to lower cell densities per OD600.Death rates of panel B plotted against growth rates of panel A shown as circles in a grey-scale. Squares show data obtained using eight other carbon substrates, also listed in Table EV1. Color circles show data obtained by varying growth rate in a chemostat. The black line shows a linear weighted least square fit to log-transformed data, that takes uncertainty in data points into account (see Methods). Data shown as mean ± (standard deviation) SD. Two or three replicates per condition.


Figure 2Death rates of wild - type and GlpK22 mutants plotted versus growth rates . Wild type is grown in glycerol minimal medium in batch cultures ( black circle ) and in ' chemostat ' continuous cultures with growth rates coded in colors , see legend on the center right . GlpK22 mutants ( white circle ) are grown in glycerol minimal medium in batch culture . Data shown as mean ± ( standard deviation ) SD . Two replicates per condition . Example experiment of the assay sketched in panel C . Cells previously grown at 0 . 3 h − 1 in continuous culture are starved ( green circles ) . After 2 days of starvation , a sample is ( 1 ) extracted and ( 2 ) UV - sterilized , see dashed line , followed by ( 3 ) inoculation of 1 % of starved and ( 4 ) growth of the culture . The difference between the maximum viability after growth , and the initial viability at point ( 3 ) is the absolute yield . The absolute yield measured at three different time points for each of the growth conditions shown in panel A is plotted against the viability of the starvation culture at the extraction time . The recycling yield is extracted as the slope of the linear fits . Recycling yield from panel E plotted versus growth rate . Data shown as mean ± SD . Two replicates per condition . Example experiment from a starved culture previously grown at a rate of 0 . 3 h − 1 with a viability after 5 days of starvation . After ( 1 ) extraction and ( 2 ) addition of 40 µM of glycerol the decay of viability is delayed ( black circles ) compared to a control without glycerol ( green symbols ) . Per viable cell , the glycerol addition in this experiment is 0 . 33 fmol CFU − 1 . After an initial period of survival , the culture with added glycerol ( black ) dies at the same rate as the control ( green ) . The ' lag time ' fitted to this data is 1 day . Lag time of the experiments of panel B for different glycerol concentrations . Lag increases linearly with glycerol concentration and maintenance rate is extracted as the inverse of the slope of the linear fits . Maintenance rate values extracted from panel I plotted versus growth rate . Data shown as mean ± SD . Two replicates per condition . ( K ) Consistency check of the equation in panel A . Fold changes of maintenance rate , FC ( β ) , divided by the fold changes of the recycling yield , FC ( α ) , plotted versus the fold changes of death rate , FC ( γ ) . All fold changes are relative to wild - type in batch , growing at 0 . 7 h − 1 . The dashed line shows the unity line FC ( γ ) = FC ( β ) / FC ( α ) .

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

Figure 2Death rates of wild-type and GlpK22 mutants plotted versus growth rates. Wild type is grown in glycerol minimal medium in batch cultures (black circle) and in 'chemostat' continuous cultures with growth rates coded in colors, see legend on the center right. GlpK22 mutants (white circle) are grown in glycerol minimal medium in batch culture. Data shown as mean ± (standard deviation) SD. Two replicates per condition.Example experiment of the assay sketched in panel C. Cells previously grown at 0.3 h − 1 in continuous culture are starved (green circles). After 2 days of starvation, a sample is (1) extracted and (2) UV-sterilized, see dashed line, followed by (3) inoculation of 1% of starved and (4) growth of the culture. The difference between the maximum viability after growth, and the initial viability at point (3) is the absolute yield.The absolute yield measured at three different time points for each of the growth conditions shown in panel A is plotted against the viability of the starvation culture at the extraction time. The recycling yield is extracted as the slope of the linear fits. Recycling yield from panel E plotted versus growth rate. Data shown as mean ± SD. Two replicates per condition.Example experiment from a starved culture previously grown at a rate of 0.3 h − 1 with a viability after 5 days of starvation. After (1) extraction and (2) addition of 40 µM of glycerol the decay of viability is delayed (black circles) compared to a control without glycerol (green symbols). Per viable cell, the glycerol addition in this experiment is 0.33 fmol CFU − 1. After an initial period of survival, the culture with added glycerol (black) dies at the same rate as the control (green). The 'lag time' fitted to this data is 1 day.Lag time of the experiments of panel B for different glycerol concentrations. Lag increases linearly with glycerol concentration and maintenance rate is extracted as the inverse of the slope of the linear fits.Maintenance rate values extracted from panel I plotted versus growth rate. Data shown as mean ± SD. Two replicates per condition.(K) Consistency check of the equation in panel A. Fold changes of maintenance rate, FC(β), divided by the fold changes of the recycling yield, FC(α), plotted versus the fold changes of death rate, FC(γ). All fold changes are relative to wild-type in batch, growing at 0.7 h − 1. The dashed line shows the unity line FC(γ)= FC(β)/FC(α).


Figure 3Cell size during starvation . Length and width of the cells are measured with phase - contrast microscopy and the volume is computed considering cell shape as a cylinder with two semi - spheres , as described in the graphical synopsis at the top ( see also Table EV3 and Methods ) . Each measurement is an average of 200 cells . ( A ) Measured length and width of wild type cells starved in batch cultures and previously grown in the chemostat at different steady - state growth rates and of GlpK22 cells starved and previously grown in batch culture at μ = 0 . 9 h − 1 ( see upper color legend ) . Note that , as shown in Schink et al . ( 2019 ) , cell widths do not change from steady state growth to starvation , while lengths decrease . Both length and width increase exponentially with growth rate . ( B ) Starvation volume of the cells described in panel A , computed as explained in the graphical synopsis ( see also Methods ) . In agreement with literature ( Schaechter et al . , 1958 ) , it increases exponentially with growth rate ( black line ) . Recycling yield and maintenance rate per cell volume . ( C ) Recycling yields and ( D ) maintenance rate measured as described in Fig . 2 are normalized per cell volume and plotted versus the previous growth rate of the cultures they refer to ( see color legend at the top and Table EV3 ) . The normalized yield is constant within the uncertainty . Maintenance rate increases significantly with a slope of ( 0 . 99 ± 0 . 55 ) h , matching the increase of the death rate , ( 1 . 1 ± 0 . 4 ) h , shown in Fig 2B . Solid black lines show linear fits on log transformed data , using weighted least square fits .

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Figure 3Cell size during starvation. Length and width of the cells are measured with phase-contrast microscopy and the volume is computed considering cell shape as a cylinder with two semi-spheres, as described in the graphical synopsis at the top (see also Table EV3 and Methods). Each measurement is an average of 200 cells. (A) Measured length and width of wild type cells starved in batch cultures and previously grown in the chemostat at different steady-state growth rates and of GlpK22 cells starved and previously grown in batch culture at μ = 0.9 h − 1 (see upper color legend). Note that, as shown in Schink et al. (2019), cell widths do not change from steady state growth to starvation, while lengths decrease. Both length and width increase exponentially with growth rate. (B) Starvation volume of the cells described in panel A, computed as explained in the graphical synopsis (see also Methods). In agreement with literature (Schaechter et al., 1958), it increases exponentially with growth rate (black line).Recycling yield and maintenance rate per cell volume. (C) Recycling yields and (D) maintenance rate measured as described in Fig. 2 are normalized per cell volume and plotted versus the previous growth rate of the cultures they refer to (see color legend at the top and Table EV3). The normalized yield is constant within the uncertainty. Maintenance rate increases significantly with a slope of (0.99±0.55) h, matching the increase of the death rate, (1.1±0.4) h, shown in Fig 2B. Solid black lines show linear fits on log transformed data, using weighted least square fits.


Figure 4Death rates of ∆ rpoS mutants versus the growth rate ( grey symbols ) , compared to wild - type E . coli K - 12 ( white symbols , data from Fig . 1 ) . Death rate increases with growth rate with a smaller slope ( ∆ rpoS : 0 . 57 ± 0 . 13 h ) compared to wild - type ( WT : 0 . 88 ± 0 . 13 h ) . Black and grey lines show linear weighted least square fits to log - transformed data , which take uncertainty in data points into account ( see Methods ) . Data shown as mean ± SD . Two or three 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

Figure 4Death rates of ∆rpoS mutants versus the growth rate (grey symbols), compared to wild-type E. coli K-12 (white symbols, data from Fig. 1). Death rate increases with growth rate with a smaller slope (∆rpoS: 0.57±0.13 h) compared to wild-type (WT: 0.88±0.13 h). Black and grey lines show linear weighted least square fits to log-transformed data, which take uncertainty in data points into account (see Methods). Data shown as mean ± SD. Two or three replicates per condition.