Day 1 adult animals expressing mtON were exposed to 50 µM rotenone ( ETC complex I inhibitor ) for 5 hours and survival was scored . Illumination was continuous throughout toxin exposure ( see methods ) . ATR alone was protective ( - ATR , - light vs . + ATR , - light p = 0 . 016 . ) The effect of mtON activation was greater than the ATR alone effect , one - way ANOVA with Tukey ' s post hoc test * p = 0 . 01 . ( - ATR , - light vs + ATR , + light p = 0 . 0002 . - ATR , + light vs . + ATR , + light p = 0 . 0009 . n = 3 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM . mtON - expressing animals were exposed to 50 µM antimycin A ( complex III inhibitor ) and survival was scored 18 hours later . One - way ANOVA with Tukey ' s post hoc test * p = 0 . 02 ( - ATR , - light vs + ATR , + light p = 0 . 03 , - ATR , + light vs . + ATR , + light p = 0 . 01 . n = 5 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM . mtON - expressing animals were exposed to 0 . 25 M azide ( complex IV inhibitor ) for 1 hour and scored for survival 1 hour after recovering , one - way ANOVA with Tukey ' s post hoc test * p = 0 . 0002 . ( - ATR , - light vs + ATR , + light p < 0 . 0001 , - ATR , + light vs . + ATR , + light p < 0 . 0001 . n = 3 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM . mtON - expressing animals were exposed to 31 µM oligomycin A ( ATP synthase inhibitor ) and survival was scored 18 hours later . No significant differences were found by one - way ANOVA ( n = 3 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM .

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

Day 1 adult animals expressing mtON were exposed to 50 µM rotenone (ETC complex I inhibitor) for 5 hours and survival was scored. Illumination was continuous throughout toxin exposure (see methods). ATR alone was protective (-ATR, -light vs. +ATR, -light p = 0.016.) The effect of mtON activation was greater than the ATR alone effect, one-way ANOVA with Tukey's post hoc test *p = 0.01. (-ATR, -light vs +ATR, +light p = 0.0002. -ATR, +light vs. +ATR, +light p = 0.0009. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 50 µM antimycin A (complex III inhibitor) and survival was scored 18 hours later. One-way ANOVA with Tukey's post hoc test *p = 0.02 (-ATR, -light vs +ATR, +light p = 0.03, -ATR, +light vs. +ATR, +light p = 0.01. n = 5 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 0.25 M azide (complex IV inhibitor) for 1 hour and scored for survival 1 hour after recovering, one-way ANOVA with Tukey's post hoc test *p = 0.0002. (-ATR, -light vs +ATR, +light p < 0.0001, -ATR, +light vs. +ATR, +light p <0.0001. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 31 µM oligomycin A (ATP synthase inhibitor) and survival was scored 18 hours later. No significant differences were found by one-way ANOVA (n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM.


mtON - expressing animals in a complex I mutant background ( gas - 1 ) were scored for locomotion speed in the presence of food by counting body bends per minute . mtON activation rescued the decreased locomotion of the gas - 1 mutant background , one - way ANOVA with Tukey ' s post hoc test , - ATR , + light vs . + ATR , + light * p = 0 . 0145 , + ATR , + light vs . wildtype ( WT ) * p = 0 . 0008 ( - ATR , - light vs + ATR , + light p = 0 . 0272 , + ATR , - light vs . + ATR , + light p = 0 . 0332 , all conditions vs WT , p < 0 . 001 , n = 30 , 30 , 30 , 41 , 41 animals each bar from left to right ) . Data show mean ± standard deviation .

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mtON-expressing animals in a complex I mutant background (gas-1) were scored for locomotion speed in the presence of food by counting body bends per minute. mtON activation rescued the decreased locomotion of the gas-1 mutant background, one-way ANOVA with Tukey's post hoc test, -ATR, +light vs. +ATR, +light *p = 0.0145, +ATR, +light vs. wildtype (WT) *p = 0.0008 (-ATR, -light vs +ATR, +light p = 0.0272, +ATR, -light vs. +ATR, +light p = 0.0332, all conditions vs WT, p < 0.001, n = 30, 30,30,41,41 animals each bar from left to right). Data show mean ± standard deviation.


Top : Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status . Top bands ( ~ 62 kDa ) are phosphorylated AMPK signal , and bottom bands ( ~ 43 kDa ) are actin signal . Image is from the same membrane cut to separately probe for phosphorylated AMPK ( pAMPK ) and actin . Bottom : Densitometry analysis showing decreased pAMPK to actin ratio , as there is no known antibody directed against total AMPK in C . elegans . Phosphorylation increases in the absence of food , but low phosphorylation is preserved when mtON is activated , two - way ANOVA with Sidak ' s test for multiple comparisons , * p = 0 . 0203 , n = 3 , 3 , 3 , 4 , 4 biological replicates each bar from left to right . Data show mean + standard deviation .

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Top: Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status. Top bands (~62 kDa) are phosphorylated AMPK signal, and bottom bands (~43 kDa) are actin signal. Image is from the same membrane cut to separately probe for phosphorylated AMPK (pAMPK) and actin. Bottom: Densitometry analysis showing decreased pAMPK to actin ratio, as there is no known antibody directed against total AMPK in C. elegans. Phosphorylation increases in the absence of food, but low phosphorylation is preserved when mtON is activated, two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0203, n = 3,3,3,4,4 biological replicates each bar from left to right. Data show mean + standard deviation.


Locomotion was assessed by counting body bends per minute . Wild type animals were compared to aak - 2 ( ok524 ) mutant animals . 2 - sample , 2 - tailed unpaired t - test * p < 0 . 0001 , wild type n = 35 , aak - 2 n = 39 animals across at least 3 days . Data show mean ± standard deviation .

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Locomotion was assessed by counting body bends per minute. Wild type animals were compared to aak-2(ok524) mutant animals. 2-sample, 2-tailed unpaired t-test *p < 0.0001, wild type n = 35, aak-2 n = 39 animals across at least 3 days. Data show mean ± standard deviation.


Locomotion in response to mtON activation . Illumination was continuous throughout body bends measurement ( see methods ) . For AAK - 2 activation , animals were exposed to 1 mM AICAR for 4 hours before body bends measurement . One - way ANOVA with Tukey ' s post hoc test , - ATR , + light vs + ATR , + light * p < 0 . 0001 , + ATR , + light vs . AICAR * p < 0 . 0001 , + ATR , + light vs AICAR + light * p < 0 . 0001 . n = 36 , 39 , 37 , 46 , 36 , 36 animals each bar from left to right . Data show mean ± standard deviation .

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Locomotion in response to mtON activation. Illumination was continuous throughout body bends measurement (see methods). For AAK-2 activation, animals were exposed to 1 mM AICAR for 4 hours before body bends measurement. One-way ANOVA with Tukey's post hoc test, -ATR, +light vs +ATR, +light *p < 0.0001, +ATR, +light vs. AICAR *p < 0.0001, +ATR,+light vs AICAR +light *p < 0.0001. n = 36, 39, 37, 46, 36, 36 animals each bar from left to right. Data show mean ± standard deviation.


Survival after HR in day 1 adult animals is shown with hypoxic preconditioning ( PC , represented by diagonal stripes ) 2 - sample 2 - tailed unpaired t - test , ctrl vs . PC * p = 0 . 0035 , n = 4 , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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Survival after HR in day 1 adult animals is shown with hypoxic preconditioning (PC, represented by diagonal stripes) 2-sample 2-tailed unpaired t-test, ctrl vs. PC *p = 0.0035, n = 4, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Protection ( % ) is percent survival minus percent survival of control condition . PC data calculated from panel B . FCCP final concentrations 0 . 001 , 0 . 01 , 0 . 1 , 1 nM . One - way ANOVA with Tukey ' s post hoc test , PC vs . 0 . 001 nM FCCP , * p = 0 . 0012 , 0 . 001 nM FCCP vs 0 . 01 FCCP , * p = 0 . 0142 , n = 4 , 3 , 3 , 3 , 3 independent experiments each bar from left to right , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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Protection (%) is percent survival minus percent survival of control condition. PC data calculated from panel B. FCCP final concentrations 0.001, 0.01, 0.1, 1 nM. One-way ANOVA with Tukey's post hoc test, PC vs. 0.001 nM FCCP, *p = 0.0012, 0.001 nM FCCP vs 0.01 FCCP, *p = 0.0142, n = 4,3,3,3,3 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Illumination was continuous throughout PC alone ; control illumination was for the same duration under normoxic conditions ( see methods & panel A ) . Two - way ANOVA comparing + ATR vs - ATR in each group . * p = 0 . 016 , n = 11 , 11 , 4 , 4 , 6 , 6 independent experiments each bar from left to right , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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Illumination was continuous throughout PC alone; control illumination was for the same duration under normoxic conditions (see methods & panel A). Two-way ANOVA comparing +ATR vs -ATR in each group. *p = 0.016, n = 11,11,4,4,6,6 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


light during hypoxia . 2 sample 2 - tailed unpaired t - test , * p = 0 . 0276 , n = 7 independent experiments , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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light during hypoxia. 2 sample 2-tailed unpaired t-test, *p = 0.0276, n = 7 independent experiments, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Immunoblot of expression levels of MYC , TSC1 , TSC2 , and β - actin loading control in high MYC Burkitt ' s lymphoma ( BL ) cells compared to low MYC Hodgkin lymphoma ( HL ) cells .

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Immunoblot of expression levels of MYC, TSC1, TSC2, and β-actin loading control in high MYC Burkitt's lymphoma (BL) cells compared to low MYC Hodgkin lymphoma (HL) cells.


Immunoblots showing expression levels of MYC , TSC1 , TSC2 or β - actin loading control in P493 - 6 cells treated with tetracycline for 72 hours ( + Tet ) or in untreated cells ( - Tet ) .

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Immunoblots showing expression levels of MYC, TSC1, TSC2 or β-actin loading control in P493-6 cells treated with tetracycline for 72 hours (+Tet) or in untreated cells (-Tet).


Relative TSC1 and TSC2 mRNA expression levels determined by qRT - PCR for high MYC ( - Tet ) versus low MYC ( + Tet ) P493 - 6 cells treated for 24 h with tetracycline ( mean ± st . dev . , n = 3 technical replicates ) * p < 0 . 05 ; * * p < 0 . 01 ; statistical relevance was determined by unpaired t - test ( two - tailed ) .

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Relative TSC1 and TSC2 mRNA expression levels determined by qRT-PCR for high MYC (-Tet) versus low MYC (+Tet) P493-6 cells treated for 24 h with tetracycline (mean ± st.dev., n=3 technical replicates) *p< 0.05; **p< 0.01; statistical relevance was determined by unpaired t-test (two-tailed).


qRT - PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h - 72h ( + Tet ) . Immunoblots for 24h and 48h ( + Tet ) show S6K and phosphorylation ( P - ) of S6K as downstream mTORC1 target , and β - actin loading control . For 72 h ( + Tet ) the immunoblots show expression of MYC and phosphorylation ( P - ) of downstream mTORC1 targets S6K and S6 , and α - tubulin as loading control .

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qRT-PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h-72h (+Tet). Immunoblots for 24h and 48h (+Tet) show S6K and phosphorylation (P-) of S6K as downstream mTORC1 target, and β-actin loading control. For 72 h (+Tet) the immunoblots show expression of MYC and phosphorylation (P-) of downstream mTORC1 targets S6K and S6, and α-tubulin as loading control.


Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1 - specific sh - RNAs compared to scrambled control sh - RNA in P493 - 6 cells . Other blots show the expression levels of TSC2 , S6K / P - S6K , S6 / P - S6 and α - tubulin for loading control .

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Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1-specific sh-RNAs compared to scrambled control sh-RNA in P493-6 cells. Other blots show the expression levels of TSC2, S6K/P-S6K, S6/P-S6 and α-tubulin for loading control.


Immunoblots of indicated proteins in P493 - 6 cells with high MYC ( - Tet , 72 hours ) or low MYC ( + Tet , 72 hours ) levels either treated with rapamycin or solvent .

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Immunoblots of indicated proteins in P493-6 cells with high MYC (-Tet, 72 hours) or low MYC (+Tet, 72 hours) levels either treated with rapamycin or solvent.


Elevated TSC1 and MYC expression in BL ( cohort 1 ) . Example of immune staining of TSC1 , MYC , the B - cell marker CD20 , and DAPI nuclear DNA - staining in germinal centers of control lymph nodes ( upper rows ) and samples from BL patients ( lower rows ) . Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples ( see materials and methods ) ( the horizontal line shows the median , whiskers show maximum and minimum data points and the box represents the first to the third quartiles , n = 56 fields for tumor samples and n = 21 fields for control germinal centers ; scale bar = 100 μm ) .

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Elevated TSC1 and MYC expression in BL (cohort 1). Example of immune staining of TSC1, MYC, the B-cell marker CD20, and DAPI nuclear DNA-staining in germinal centers of control lymph nodes (upper rows) and samples from BL patients (lower rows). Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples (see materials and methods) (the horizontal line shows the median, whiskers show maximum and minimum data points and the box represents the first to the third quartiles, n=56 fields for tumor samples and n=21 fields for control germinal centers; scale bar = 100 μm).


Quantification of immunoblot analysis of TSC expression in BL ( cohort 2 ) ( n = 13 ) compared to healthy tonsils ( n = 3 ) or reactive lymph nodes ( n = 3 ) ( mean ± st . dev . ) shown in Fig EV2B .

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Quantification of immunoblot analysis of TSC expression in BL (cohort 2) (n=13) compared to healthy tonsils (n=3) or reactive lymph nodes (n=3) (mean ± st. dev.) shown in Fig EV2B.


Left graph shows the multiplication rate of P493 - 6 ( - Tet ) cells expressing either a scrambled control sh - RNA or a TSC1 - specific sh - RNA determined by viable cell counting 3 days after seeding of equal number of viable cells ( determined by Trypan blue exclusion ; mean ± st . dev . , n = 3 biological replicates ) . Right graph shows percentage of apoptotic P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or a TSC1 - specific sh - RNA determined by FACS analysis of AnnexinV / 7AAD stained cells ( mean ± st . dev . , n = 3 biological replicates ) .

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Left graph shows the multiplication rate of P493-6 (-Tet) cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA determined by viable cell counting 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± st.dev., n=3 biological replicates). Right graph shows percentage of apoptotic P493-6 (-Tet) cells expressing scrambled control shRNA or a TSC1-specific sh-RNA determined by FACS analysis of AnnexinV/7AAD stained cells (mean ± st.dev., n=3 biological replicates).


Rapamycin treatment recovers survival of TSC1 knockdown in P493 - 6 cells . Relative viable cell number counts of P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or TSC1 - specific sh - RNA 14 days after seeding equal number of viable cells ( Trypan blue exclusion ) , in the presence of 30pM rapamycin where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

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Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates).


TSC1 knockdown is synthetic lethal with MYC deregulation . U2OS - MYC - ER cells expressing either scrambled control sh - RNA or TSC1 - specific sh - RNA were treated with hydroxytamoxifen to induce MYC and rapamycin ( 100 nM ) where indicated . Percentage of apoptotic cells was determined with Annexin / 7AAD staining 4 days after MYC induction ( mean ± st . dev . , n = 3 biological replicates ) .

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TSC1 knockdown is synthetic lethal with MYC deregulation. U2OS-MYC-ER cells expressing either scrambled control sh-RNA or TSC1-specific sh-RNA were treated with hydroxytamoxifen to induce MYC and rapamycin (100 nM) where indicated. Percentage of apoptotic cells was determined with Annexin/7AAD staining 4 days after MYC induction (mean ± st.dev., n=3 biological replicates).


Survival rate of different BL cell lines upon TSC1 knockdown . Graphs show numbers of viable cells expressing either a scrambled control sh - RNA or a TSC1 - specific sh - RNA 3 days after seeding of equal number of viable cells ( determined by trypan blue exclusion ; mean ± st . dev . , n = 3 biological replicates ) .

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Survival rate of different BL cell lines upon TSC1 knockdown. Graphs show numbers of viable cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA 3 days after seeding of equal number of viable cells (determined by trypan blue exclusion; mean ± st.dev., n=3 biological replicates).


Immunoblots of control - or TSC1 - shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity ( 10nM ) or titrate the activity to control levels ( 30 pM ) , and survival rate of these cells over 7 days ( mean ± st . dev . , n = 3 biological replicates ) ; ( Bl2 cells were selected for stable knockdown with Puromycin , DG75 cells without selection ) .

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Immunoblots of control- or TSC1-shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity (10nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± st.dev., n=3 biological replicates); (Bl2 cells were selected for stable knockdown with Puromycin, DG75 cells without selection).


Ramos cells expressing either a TSC1 - specific or a control shRNA were inoculated into NOD / SCID mice and tumor volume was measured regularly . The immunoblot shows the level of knockdown of TSC1 ( sh - TSC1b ) compared to control ( sh - contr ) , and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation . Tumor growth curves show mean ± SEM ( n = 8 / group ) . Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation . The top panels show tumors derived from Ramos cells stably expressing control sh - RNA ( sh - contr ) ; the lower panels show tumors derived from Ramos cells stably expressing TSC1 - specific sh - RNA ( sh - TSC1b ) .

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

Ramos cells expressing either a TSC1-specific or a control shRNA were inoculated into NOD/SCID mice and tumor volume was measured regularly. The immunoblot shows the level of knockdown of TSC1 (sh-TSC1b) compared to control (sh-contr), and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation. Tumor growth curves show mean ± SEM (n=8/group). Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation. The top panels show tumors derived from Ramos cells stably expressing control sh-RNA (sh-contr); the lower panels show tumors derived from Ramos cells stably expressing TSC1-specific sh-RNA (sh-TSC1b).


TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner . Rate of basal oxygen consumption in P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or TSC - specific sh - RNA , treated with 20 nM rapamycin for 12 h , or tetracycline to repress MYC for 48 h where

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

TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in P493-6 (-Tet) cells expressing scrambled control sh-RNA or TSC-specific sh-RNA, treated with 20 nM rapamycin for 12 h, or tetracycline to repress MYC for 48 h where indicated


TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner . Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated ( B ) ( mean ± st . dev . , n = 6 biological replicates ) .

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

TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated (B) (mean ± st. dev., n=6 biological replicates).


Ratio of mitochondrial to genomic DNA determined by qRT - PCR in P493 - 6 ( - Tet ) cells expressing either scrambled control sh - RNA or a TSC1 - specific sh - RNA , and treated with 20 nM rapamycin for 12 h where indicated ( mean ± st . dev . , n = 3 technical replicates ) .

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

Ratio of mitochondrial to genomic DNA determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).


expression of cytochrome C ( CYCS ) or ATP5G1 mRNAs ( D ) determined by qRT - PCR in P493 - 6 ( - Tet ) cells expressing either scrambled control sh - RNA or a TSC1 - specific sh - RNA , and treated with 20 nM rapamycin for 12 h where indicated ( mean ± st . dev . , n = 3 technical replicates ) .

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

expression of cytochrome C (CYCS) or ATP5G1 mRNAs (D) determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).


TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner . FACS analysis of DCF - DA stained P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or a TSC1 - specific sh - RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

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TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner. FACS analysis of DCF-DA stained P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated (mean ± st.dev., n=3 biological replicates).


TSC1 knockdown results in increased phosphorylation of SAPK / JNK . Immunoblot of SAPK / JNK Thr183 / Tyr185 - phosphorylation in P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or a TSC1 - specific sh - RNA . α - tubulin expression serves as loading control .

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

TSC1 knockdown results in increased phosphorylation of SAPK/JNK. Immunoblot of SAPK/JNK Thr183/Tyr185-phosphorylation in P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA. α-tubulin expression serves as loading control.


Antioxidant treatment rescues cells from death caused by TSC1 knockdown . Relative viable cell number counts of P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or TSC1 - specific sh - RNA 3 days after seeding equal number of viable cells ( Trypan blue exclusion ) , in the presence of 10 μM of the antioxidant BHA where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

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

Antioxidant treatment rescues cells from death caused by TSC1 knockdown. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 3 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 10 μM of the antioxidant BHA where indicated (mean ± st.dev., n=3 biological replicates).


Accumulation of EU - labeled ( Click - It , Invitrogen ) TSC1 - mRNA over 3 h in high MYC ( - Tet ) versus low MYC ( + Tet ) P493 - 6 cells ( mean ± st . dev . , n = 3 technical replicates ) .

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

Accumulation of EU-labeled (Click-It, Invitrogen) TSC1-mRNA over 3 h in high MYC (-Tet) versus low MYC (+Tet) P493-6 cells (mean ± st. dev., n=3 technical replicates).


Drawing of the TSC1 promoter and luciferase - reporter constructs with the predicted E - box ( CACGTG , pos . - 317 / - 322 ) indicated . Relative luciferase units ( RLU ) of co - transfection experiments using the indicated TSC1 - promoter - reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells ( mean ± st . dev . , n = 3 ) . Immunoblot shows MYC overexpression and α - tubulin as loading control .

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

Drawing of the TSC1 promoter and luciferase-reporter constructs with the predicted E-box (CACGTG, pos. -317/-322) indicated. Relative luciferase units (RLU) of co-transfection experiments using the indicated TSC1-promoter-reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells (mean ± st. dev., n=3). Immunoblot shows MYC overexpression and α-tubulin as loading control.


TSC1 mRNA turnover determined by pulse and chase ( Click - It , Invitrogen ) over 8 h in P493 - 6 cells , either treated with tetracycline to repress MYC or left untreated ( mean ± st . dev . , n = 3 technical replicates ) .

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

TSC1 mRNA turnover determined by pulse and chase (Click-It, Invitrogen) over 8 h in P493-6 cells, either treated with tetracycline to repress MYC or left untreated (mean ± st.dev., n=3 technical replicates).


miR - 15a expression determined by qRT - PCR in P493 - 6 cells treated with tetracycline for 3 days ( + Tet ) to suppress MYC expression or in untreated ( - Tet ) cells ( mean ± st . dev . , n = 3 technical replicates ) .

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

miR-15a expression determined by qRT-PCR in P493-6 cells treated with tetracycline for 3 days (+Tet) to suppress MYC expression or in untreated (-Tet) cells (mean ± st. dev., n=3 technical replicates).


Immunoblots showing expression levels of TSC1 , S6K / P - S6K and α - tubulin in P493 - 6 and HEK293T cells overexpressing miR - 15a or a control miRNA . At the right miR - 15a overexpression was determined by qRT - PCR in P493 - 6 and HEK293T cells ( mean ± st . dev . , n = 3 technical replicates ) .

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

Immunoblots showing expression levels of TSC1, S6K/P-S6K and α-tubulin in P493-6 and HEK293T cells overexpressing miR-15a or a control miRNA. At the right miR-15a overexpression was determined by qRT-PCR in P493-6 and HEK293T cells (mean ± st. dev., n=3 technical replicates).


Immunoblot showing TSC1 expression , S6K / P - S6K and α ‑ tubulin in MCF - 7 cells transfected with LNA modified anti - sense miR - 15a oligos or control LNAs . At the right , successful miR - 15a knockdown determined by qRT - PCR in MCF - 7 cells is shown ( mean ± st . dev . , n = 3 technical replicates ) .

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

Immunoblot showing TSC1 expression, S6K/P-S6K and α‑tubulin in MCF-7 cells transfected with LNA modified anti-sense miR-15a oligos or control LNAs. At the right, successful miR-15a knockdown determined by qRT-PCR in MCF-7 cells is shown (mean ± st. dev., n=3 technical replicates).


Relative luciferase activities ( RLU ) derived from reporter constructs containing TSC1 - 3 ´ UTR wild type sequences or with mutated miR - 15a seed sequence binding site on co - transfection with miR - 15a in MCF - 7 cells ( mean ± st . dev . , n = 3 biological replicates ) . The drawing at the left shows the TSC1 - 3 ' UTR with the position of the miR - 15a / 16 - 1 seed sequence and used mutation indicated .

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

Relative luciferase activities (RLU) derived from reporter constructs containing TSC1-3´UTR wild type sequences or with mutated miR-15a seed sequence binding site on co-transfection with miR-15a in MCF-7 cells (mean ± st. dev., n=3 biological replicates). The drawing at the left shows the TSC1-3'UTR with the position of the miR-15a/16-1 seed sequence and used mutation indicated.


Rate of oxygen consumption in P493 - 6 ( - Tet ) cells ectopically expressing miR - 15a or control - miRNA vector , under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated ( mean ± st . dev . , n = 8 biological replicates ) .

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

Rate of oxygen consumption in P493-6 (-Tet) cells ectopically expressing miR-15a or control-miRNA vector, under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated (mean ± st. dev., n=8 biological replicates).


a , Polychromasia ( Wright - Giemsa stain , indicated by arrows ) and increased reticulocytes ( brilliant cresyl blue stain ) revealed by staining blood smears from 6 - week - old wild - type ( WT ) or Nix - / - mice ( scale bar , 20 µm ) .

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a, Polychromasia (Wright-Giemsa stain, indicated by arrows) and increased reticulocytes (brilliant cresyl blue stain) revealed by staining blood smears from 6-week-old wild-type (WT) or Nix-/- mice (scale bar, 20 µm).


b , c , Ter119 ( b ) or Mitotracker deep red ( c ) versus CD71 staining of RBCs from 3 - or 6 - week - old mice . Cell counts were also calculated ( mean ± s . e . m . ; n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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b, c, Ter119 (b) or Mitotracker deep red (c) versus CD71 staining of RBCs from 3- or 6-week-old mice. Cell counts were also calculated (mean ± s.e.m.; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.


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


d , Analyses of RBCs from 3 - or 6 - week - oldmice by

d, Analyses of RBCs from 3- or 6-week-oldmice by immunocytochemistry


Analyses of RBCs from 3 - or 6 - week - oldmice by transmission electron

Analyses of RBCs from 3- or 6-week-oldmice by transmission electron microscopy


a , Quantification of NHS - biotin - labelled RBCs or transferred CMFDA - labelled RBCs ( n = 3 ) .

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a, Quantification of NHS-biotin-labelled RBCs or transferred CMFDA-labelled RBCs (n = 3).


b , Western blot analyses of caspases in RBCs after in vitro culture . Asterisks denote processed caspases . FL , full length .

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b, Western blot analyses of caspases in RBCs after in vitro culture. Asterisks denote processed caspases. FL, full length.


c , The relative luminescence units ( RLU ) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix - / - RBCs after 12 - h culture in the presence of qVD - oph or solvent control ( DMSO ) ( n = 3 ) .

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c, The relative luminescence units (RLU) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix-/- RBCs after 12-h culture in the presence of qVD-oph or solvent control (DMSO) (n = 3).


d , Mitotracker versus annexin V staining of RBCs after in vitro culture .

d, Mitotracker versus annexin V staining of RBCs after in vitro culture.


e , Mean fluorescent intensity ( MFI ) of ROS staining in RBCs after in vitro culture , and caspase activities in Nix - / - RBCs after 24 h culture with solvent control , ROS scavengers or qVD - oph ( n = 3 ) . BHT , butylated hydroxytoluene ; CAT , catalase ; TEM , tempol .

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

e, Mean fluorescent intensity (MFI) of ROS staining in RBCs after in vitro culture, and caspase activities in Nix-/- RBCs after 24 h culture with solvent control, ROS scavengers or qVD-oph (n = 3). BHT, butylated hydroxytoluene; CAT, catalase; TEM, tempol.


f , Haematoxylin and eosin ( HE ) staining of spleen sections of 9 - week - old wild - type and Nix - / - mice . Arrows denote iron deposits within macrophagecytoplasm . Iron deposits were also stained with Prussian blue , followed by counterstain with nuclear - fast red . Scale bar , 20 µm .

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f, Haematoxylin and eosin (HE) staining of spleen sections of 9-week-old wild-type and Nix-/- mice. Arrows denote iron deposits within macrophagecytoplasm. Iron deposits were also stained with Prussian blue, followed by counterstain with nuclear-fast red. Scale bar, 20 µm.


g , Real - time RT - PCR of erythropoietin ( Epo ) ( n = 12 ) . For all relevant panels , statistical significance of the data ( mean ± s . e . m . ) is : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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g, Real-time RT-PCR of erythropoietin (Epo) (n = 12). For all relevant panels, statistical significance of the data (mean ± s.e.m.) is: *P < 0.05; **P < 0.01; ***P < 0.001.


a , Ter119 + CD71 + reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 ( top row ) , 2 ( middle row ) or 4 ( bottom row ) days , followed by Mitotracker deep red and Ter119 staining .

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a, Ter119+CD71+ reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 (top row), 2 (middle row) or 4 (bottom row) days, followed by Mitotracker deep red and Ter119 staining.


b , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue . Scale bar , 20 µm .

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b, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue. Scale bar, 20 µm.


c , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 and COX IV and analysed by deconvolution microscopy . Scale bar , 5 µm . The Pearson coefficiency for LC3 and COX IV co - localization at day 0 ( mean ± s . e . m . ) is : wild type , 0 . 81 ± 0 . 001 ; Nix - / - , 0 . 44 ± 0 . 036 ( n = 35 , P = 0 . 0006 ) .

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

c, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 and COX IV and analysed by deconvolution microscopy. Scale bar, 5 µm. The Pearson coefficiency for LC3 and COX IV co-localization at day 0 (mean ± s.e.m.) is: wild type, 0.81 ± 0.001; Nix-/-, 0.44 ± 0.036 (n = 35, P = 0.0006).


d , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy . Scale bar , 0 . 5 µm .

d, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy. Scale bar, 0.5 µm.


a , Ter119 + CD71 + reticulocytes were sorted from the peripheral blood of wild - type and Nix - / - mice at day 6 after PHZ treatment . The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT - 737 , followed by staining with Mitotracker deep red and phycoerythrin - conjugated anti - Ter119 .

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

a, Ter119+CD71+ reticulocytes were sorted from the peripheral blood of wild-type and Nix-/- mice at day 6 after PHZ treatment. The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT-737, followed by staining with Mitotracker deep red and phycoerythrin-conjugated anti-Ter119.


b , c , Reticulocytes were cultured with FCCP ( b ) or ABT - 737 ( c ) for 24 h and analysed by transmission electron microscopy . Scale bar , 0 . 5 µm .

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b, c, Reticulocytes were cultured with FCCP (b) or ABT-737 (c) for 24 h and analysed by transmission electron microscopy. Scale bar, 0.5 µm.


( B ) BW25113 ( ycbB , relA ' ) and its derivatives obtained by individual deletion of endopeptidase genes . BW25113 ( ycbB , relA ' ) is an abbreviated name for BW25113 ΔrelA pKT2 ( ycbB ) pKT8 ( relA ' ) .

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(B) BW25113(ycbB, relA') and its derivatives obtained by individual deletion of endopeptidase genes. BW25113(ycbB, relA') is an abbreviated name for BW25113 ΔrelA pKT2(ycbB) pKT8(relA').


Growth of BW25113 ( ycbB , relA ' ) , BW25113 ( ycbB , relA ' ) ΔmepM , and its derivatives harboring plasmids encoding MepM , MepM H393A , and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg / ml ( + ceftriaxone ) or in the absence of the drug ( - ceftriaxone ) in BHI agar plates supplemented with 40 µM IPTG and 1 % L - arabinose for induction of ycbB and relA ' , respectively . The genes encoding MepM , MepM H393A , and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter , which is inducible by 2 , 4 - diacetylphloroglucinol ( DAPG ) . Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer .

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

Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM H393A, and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) in BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. The genes encoding MepM, MepM H393A, and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter, which is inducible by 2,4-diacetylphloroglucinol (DAPG). Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer.


Growth of BW25113 ( ycbB , relA ' ) , BW25113 ( ycbB , relA ' ) ΔmepM , and its derivatives harboring plasmids encoding MepM ,

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

Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM


The mepM gene was expressed under the control of TIS1 or TIS2 ( translation initiation ) and of PrhaBAD ( the L - rhamnose - inducible promoter of vector pHV30 ) . Growth of BW25113 ( ycbB , relA ' ) ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg / ml , IPTG 40 µM ( induction of ycbB ) and L - arabinose 1 % ( induction of relA ' ) . ( A ) For TIS2 , growth around paper disks containing 1 or 2 mg of L - rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance . In contrast , a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer . ( B ) The experiment was repeated with bacteria pre - exposed to L - rhamnose that were harvested at the vicinity of the disk containing 2 mg of L - rhamnose . Expression of ceftriaxone resistance remained dependent on the presence of L - rhamnose indicating that the requirement for MepM is not transient . The diameter of the growth zones is larger in panel ( B ) than in panel ( A ) as expected from the fact that bacteria in the inoculum used in ( B ) had been grown in the presence of the inducer and already contained MepM . In ( A ) growth is only possible after diffusion of L - rhamnose in the medium prior to the action of ceftriaxone . At a distance from the disk , diffusion was not sufficiently rapid to observe resistance .

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

The mepM gene was expressed under the control of TIS1 or TIS2 (translation initiation) and of PrhaBAD (the L-rhamnose-inducible promoter of vector pHV30). Growth of BW25113(ycbB, relA') ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg/ml, IPTG 40 µM (induction of ycbB) and L-arabinose 1% (induction of relA'). (A) For TIS2, growth around paper disks containing 1 or 2 mg of L-rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance. In contrast, a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer. (B) The experiment was repeated with bacteria pre-exposed to L-rhamnose that were harvested at the vicinity of the disk containing 2 mg of L-rhamnose. Expression of ceftriaxone resistance remained dependent on the presence of L-rhamnose indicating that the requirement for MepM is not transient. The diameter of the growth zones is larger in panel (B) than in panel (A) as expected from the fact that bacteria in the inoculum used in (B) had been grown in the presence of the inducer and already contained MepM. In (A) growth is only possible after diffusion of L-rhamnose in the medium prior to the action of ceftriaxone. At a distance from the disk, diffusion was not sufficiently rapid to observe resistance.


( A ) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme ( upper panel ) or by lysozyme and 5 µM MepM ( lower panel ) . Absorbance was monitored at 205 nm ( mAU , milli - absorbance unit ) .

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

(A) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme (upper panel) or by lysozyme and 5 µM MepM (lower panel). Absorbance was monitored at 205 nm (mAU, milli-absorbance unit).


Functional complementation of the mepM deletion in BW25113 ( ycbB , relA ' ) ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal . Induction of endopeptidase ( ED ) genes was performed with 0 . 2 % L - rhamnose in the presence or absence of 8 µg / ml ceftriaxone . BHI agar plates contained 40 µM IPTG and 1 % L - arabinose for induction of ycbB and relA ' , respectively .

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

Functional complementation of the mepM deletion in BW25113(ycbB, relA') ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal. Induction of endopeptidase (ED) genes was performed with 0.2% L-rhamnose in the presence or absence of 8 µg/ml ceftriaxone. BHI agar plates contained 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively.


( B ) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53 ( mepM ) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter . In this assay , functional replacement of MepM is detected based on growth in media containing 0 . 2 % or 1 % L - arabinose for expression of the endopeptidase gene carried by vector pHV7 , while by - passing the requirement for induction of the mepM copy of pHV53 by L - rhamnose . Complementation was observed with both concentrations of inducer for mepH , mepS , and pbpG . Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L - arabinose ( 1 % ) was lethal . The right panel presents the growth control performed in the presence of 0 . 2 % L - rhamnose for induction of the mepM copy carried by pHV53 .

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

(B) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53(mepM) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter. In this assay, functional replacement of MepM is detected based on growth in media containing 0.2% or 1% L-arabinose for expression of the endopeptidase gene carried by vector pHV7, while by-passing the requirement for induction of the mepM copy of pHV53 by L-rhamnose. Complementation was observed with both concentrations of inducer for mepH, mepS, and pbpG. Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L-arabinose (1%) was lethal. The right panel presents the growth control performed in the presence of 0.2% L-rhamnose for induction of the mepM copy carried by pHV53.


( B ) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter . Induction of ycbB and of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose and 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

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

(B) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


( A ) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with the pHV30 vector or recombinant plasmids encoding mepA , mepK , mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter . ( B ) The complementation assay was repeated for BW25113 Δ7EDs pHV63 ( ycbB ) , which was obtained by deletion of all chromosomal endopeptidase genes except mepM . Induction of ycbB and of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose and 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

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

(A) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding mepA, mepK, mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter. (B) The complementation assay was repeated for BW25113 Δ7EDs pHV63(ycbB), which was obtained by deletion of all chromosomal endopeptidase genes except mepM. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


( A ) Growth curves of ( i ) BW25113 ( ycbB , relA ' ) and its ΔsltY derivative ; and ( ii ) strain M1 and its derivatives ( M1 . 1 to 1 . 4 ) harboring mutations in sltY . The growth medium contained ( i ) 40 µM IPTG , 1 % L - arabinose , and 8 µg / ml ceftriaxone for BW25113 ( ycbB , relA ' ) and its ΔsltY derivative or ( ii ) 50 µM IPTG and 16 µg / ml ampicillin for M1 and its derivatives .

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

(A) Growth curves of (i) BW25113(ycbB, relA') and its ΔsltY derivative; and (ii) strain M1 and its derivatives (M1.1 to 1.4) harboring mutations in sltY. The growth medium contained (i) 40 µM IPTG, 1% L-arabinose, and 8 µg/ml ceftriaxone for BW25113(ycbB, relA') and its ΔsltY derivative or (ii) 50 µM IPTG and 16 µg/ml ampicillin for M1 and its derivatives.


a , Pancreatic sections stained with Atg7 , LC3 and p62 showing Atg7 − / − next to Atg7 + / + regions after Pdx - Cre - mediated recombination . Right - side images are cropped and magnified regions ( boxed ) from left - side images . The magnification is × 2 . 5 .

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

a, Pancreatic sections stained with Atg7, LC3 and p62 showing Atg7−/− next to Atg7+/+ regions after Pdx-Cre-mediated recombination. Right-side images are cropped and magnified regions (boxed) from left-side images. The magnification is × 2.5.


b , Haematoxylin and eosin ( HE ) , p53 and caspase - 3 staining of exocrine tissue in Atg7 + / + and Atg7 − / − pancreases .

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

b, Haematoxylin and eosin (HE), p53 and caspase-3 staining of exocrine tissue in Atg7+/+ and Atg7−/− pancreases.


c , Quantification ( median , s . e . m . ) of p53 and caspase - 3 activation in Atg7 - deficient acinar tissue .

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

c, Quantification (median, s.e.m.) of p53 and caspase-3 activation in Atg7-deficient acinar tissue.


d , Kaplan - Meier analysis comparing overall survival of mice either wild type ( blue ) or Atg7 − / − ( red ) in the pancreas . Mantel - Cox test was used for statistics . ' n / a ' , 14 / 16 mice were killed > 500 days when completely healthy .

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

d, Kaplan-Meier analysis comparing overall survival of mice either wild type (blue) or Atg7−/− (red) in the pancreas. Mantel-Cox test was used for statistics. 'n/a', 14/16 mice were killed >500 days when completely healthy.


e , Biochemical analysis of endocrine function in moribund Atg7 − / − or age - matched healthy control mice . Detailed information about the mice is tabulated . A Mann - Whitney U - test was used for statistics . Scale bars : 50 µm . * * P 0 . 01 and * * * P 0 . 001 .

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

e, Biochemical analysis of endocrine function in moribund Atg7−/− or age-matched healthy control mice. Detailed information about the mice is tabulated. A Mann-Whitney U-test was used for statistics. Scale bars: 50 µm. **P 0.01 and ***P 0.001.


a , Alcian blue / PAS ( AB / PAS ) staining in pancreases from 150 - day - old Atg7 - proficient or Atg7 - deficient KrasG12D / + mice ( wild - type mice with expression of KrasG12D in their pancreases ) .

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

a, Alcian blue/PAS (AB/PAS) staining in pancreases from 150-day-old Atg7-proficient or Atg7-deficient KrasG12D/+ mice (wild-type mice with expression of KrasG12D in their pancreases).


b , Quantification of PanIN from mice of the indicated genotype and age . Fold increase and male / female ratios are shown . Box plots express the first ( Q1 ) and third ( Q3 ) quartiles set by the upper and lower limits of the box , whereas the median is shown by the horizontal line inside the box . The whiskers extend upward and downward to the highest or lowest observation within the upper ( Q3 + 1 . 5 × the interquartile range ) and lower ( Q1 − 1 . 5 × interquartile range ) limits . Values outside the upper and lower limits are ' outliers ' and are shown by individual symbols .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Quantification of PanIN from mice of the indicated genotype and age. Fold increase and male/female ratios are shown. Box plots express the first (Q1) and third (Q3) quartiles set by the upper and lower limits of the box, whereas the median is shown by the horizontal line inside the box. The whiskers extend upward and downward to the highest or lowest observation within the upper (Q3 + 1.5 × the interquartile range) and lower (Q1 − 1.5 × interquartile range) limits. Values outside the upper and lower limits are 'outliers' and are shown by individual symbols.


c , d , Kaplan - Meier analysis comparing overall survival ( c ) or PDAC - free survival ( d ) of Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / + mice .

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

c, d, Kaplan-Meier analysis comparing overall survival (c) or PDAC-free survival (d) of Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ mice.


c , d , Kaplan - Meier analysis comparing overall survival ( c ) or PDAC - free survival ( d ) of Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / +

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

c, d, Kaplan-Meier analysis comparing overall survival (c) or PDAC-free survival (d) of Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ mice


e , PanIN grading of pancreases from 150 - day - old Atg7 + / + or Atg7 − / − Pdx - Cre KrasG12D / + mice . Values are mean and error bars are s . d . b , e , A Mann - Whitney U - test was used for statistics .

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e, PanIN grading of pancreases from 150-day-old Atg7+/+ or Atg7−/− Pdx-Cre KrasG12D/+ mice. Values are mean and error bars are s.d. b, e, A Mann-Whitney U-test was used for statistics.


f , Representative p53 staining from 150 - day - old mice of both genotypes .

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

f, Representative p53 staining from 150-day-old mice of both genotypes.


g , PDAC - free survival of autophagy - proficient ( blue ) and Atg5 - deficient ( red ) KrasG12D / + mice . d , g , Median survival ± s . d . and male / female ( m / f ) ratio are provided . ' n / a ' , no animals succumbed to PDAC . A log - rank test ( Mantel - Cox ) was used for statistics . a , f , Scale bars : 500 µm ( a ) , 50 µm ( f ) . * P 0 . 05 , * * P 0 . 01 ; NS , not significant .

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g, PDAC-free survival of autophagy-proficient (blue) and Atg5-deficient (red) KrasG12D/+ mice. d, g, Median survival ± s.d. and male/female (m/f) ratio are provided. 'n/a', no animals succumbed to PDAC. A log-rank test (Mantel-Cox) was used for statistics. a, f, Scale bars: 500 µm (a), 50 µm (f). *P  0.05, **P  0.01; NS, not significant.


a , Kaplan - Meier analysis comparing PDAC - free survival in Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / + p53 − / − mice .

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

a, Kaplan-Meier analysis comparing PDAC-free survival in Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ p53−/− mice.


b , Pancreatic tumours of indicated genotypes stained for Atg7 , LC3 and p62 . Insets are magnified crops from the same images to show specifics of LC3 and p62 staining . The magnification is × 4 .

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b, Pancreatic tumours of indicated genotypes stained for Atg7, LC3 and p62. Insets are magnified crops from the same images to show specifics of LC3 and p62 staining. The magnification is × 4.


c , Tumour incidence and tumour weight ( median , s . e . m . ) in mice from indicated genotypes , killed at 21 , 29 and 38 days of age . Numbers in the incidence diagram represent tumour - bearing mice versus all mice . Significance was assessed with Fisher ' s exact test ( incidence ) and Mann - Whitney U - test ( weight ) .

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

c, Tumour incidence and tumour weight (median, s.e.m.) in mice from indicated genotypes, killed at 21, 29 and 38 days of age. Numbers in the incidence diagram represent tumour-bearing mice versus all mice. Significance was assessed with Fisher's exact test (incidence) and Mann-Whitney U-test (weight).


d , Representative haematoxylin and eosin stainings of pancreases from indicated time points and genotypes .

d, Representative haematoxylin and eosin stainings of pancreases from indicated time points and genotypes.


e , Kaplan - Meier analysis comparing PDAC - free survival of Atg5 + / + ( blue ) or Atg5 − / − ( green ) KrasG12D / + p53 − / − mice .

e, Kaplan-Meier analysis comparing PDAC-free survival of Atg5+/+ (blue) or Atg5−/− (green) KrasG12D/+ p53−/− mice.


f , Kaplan - Meier analysis comparing PDAC - free survival of KrasG12D / + p53 − / − mice that were chloroquine - treated ( CQ , red ) , vehicle - treated ( PBS , black ) or untreated ( blue ) . a , e , f , The blue curve in all Kaplan - Meier plots represents the same colony of mice ( KrasG12D / + , p53 − / − , Atg7 + / + , Atg5 + / + ) . A Mantel - Cox test was used for statistics . b , d , Scale bars : 100 µm ( b ) , 200 µm ( d ) . * P 0 . 05 , * * P 0 . 01 .

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

f, Kaplan-Meier analysis comparing PDAC-free survival of KrasG12D/+ p53−/− mice that were chloroquine-treated (CQ, red), vehicle-treated (PBS, black) or untreated (blue). a, e, f, The blue curve in all Kaplan-Meier plots represents the same colony of mice (KrasG12D/+, p53−/−, Atg7+/+, Atg5+/+). A Mantel-Cox test was used for statistics. b, d, Scale bars: 100 µm (b), 200 µm (d). *P 0.05, **P 0.01.


a , LC3 staining of representative pancreatic tumours from mice of indicated genotypes . Scale bars : 50 µm .

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a, LC3 staining of representative pancreatic tumours from mice of indicated genotypes. Scale bars: 50 µm.


b , Western blotting for LC3 , p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53 .

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b, Western blotting for LC3, p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53.


c , d , Average oxygen consumption rate ( OCR , c ) and extracellular acidification rate ( ECAR ,

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c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR,


c , d , Average oxygen consumption rate ( OCR , c ) and extracellular acidification rate ( ECAR , d ) of eight Atg7 + / + and seven Atg7 − / − cell lines revealed increased ECAR in autophagy - deficient cells . Error bars are s . e . m .

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c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR, d) of eight Atg7+/+ and seven Atg7−/− cell lines revealed increased ECAR in autophagy-deficient cells. Error bars are s.e.m.


e , Treatment with 2 - deocyglucose ( 2 - DG ) reduces ECAR and abrogates the difference between cell lines from Atg7 + / + and Atg7 − / − tumours . Values are the average of three biological replicates . Error bars are s . e . m .

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e, Treatment with 2-deocyglucose (2-DG) reduces ECAR and abrogates the difference between cell lines from Atg7+/+ and Atg7−/− tumours. Values are the average of three biological replicates. Error bars are s.e.m.


, g , LC - MS analysis revealed increased glucose consumption from medium ( f ) and extracellular lactate accumulation ( g ) in Atg7 - null cell lines . Values are the average of eight ( Atg7 + / + ) and seven ( Atg7 − / − ) biological replicates .

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, g, LC-MS analysis revealed increased glucose consumption from medium (f) and extracellular lactate accumulation (g) in Atg7-null cell lines. Values are the average of eight (Atg7+/+) and seven (Atg7−/−) biological replicates.


h , LC - MS analysis shows increased accumulation of 2 - DG and its metabolite 2 - DG - 6 - phosphate in Atg7 - deficient cell lines . Values are averages of five biological replicates for each genotype .

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h, LC-MS analysis shows increased accumulation of 2-DG and its metabolite 2-DG-6-phosphate in Atg7-deficient cell lines. Values are averages of five biological replicates for each genotype.


i , Background levels of 18F - FDG uptake in brain and heart can be seen in sagittal and coronal views ( middle and right panels , respectively ) of wild - type Atg7 mice ( top row ) . In contrast , intense 18F - FDG uptake is seen in pancreatic tumours of Atg7 deficient mice ( bottom row ) .

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i, Background levels of 18F-FDG uptake in brain and heart can be seen in sagittal and coronal views (middle and right panels, respectively) of wild-type Atg7 mice (top row). In contrast, intense 18F-FDG uptake is seen in pancreatic tumours of Atg7 deficient mice (bottom row).


j , LC - MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7 − / − tumour cell lines . Values are average of eight ( Atg7 + / + ) and six ( Atg7 − / − ) biological replicates . f - h , j , Error bars are s . e . m . a , f - h , j , A Mann - Whitney U - test was used to ascertain significance . * P 0 . 05 , * * P 0 . 01 .

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j, LC-MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7−/− tumour cell lines. Values are average of eight (Atg7+/+) and six (Atg7−/−) biological replicates. f-h, j, Error bars are s.e.m. a, f-h, j, A Mann-Whitney U-test was used to ascertain significance. *P  0.05, **P  0.01.


C Subcellular fractionation of the WT and NLS - deficient FolSrpk1 - GFP proteins . N , nucleus ; C , cytosol . The amount of nuclear FolSrpk1 - GFP was set as 1 . Data information : For C , each gel shown is a representative experiment carried out at least three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and Coomassie brilliant blue ( CBB ) staining .

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C Subcellular fractionation of the WT and NLS-deficient FolSrpk1-GFP proteins. N, nucleus; C, cytosol. The amount of nuclear FolSrpk1-GFP was set as 1. Data information: For C, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and Coomassie brilliant blue (CBB) staining.


D Quantitative measurement of H2O2 in tomato roots after infection by the WT and FolSrpk1p : : FolSrpk1 - GFP strains at 0 - 5 days post incubation ( dpi ) . Data are presented as mean ± SD . The presence of different letters above the mean values of three replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) .

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D Quantitative measurement of H2O2 in tomato roots after infection by the WT and FolSrpk1p::FolSrpk1-GFP strains at 0-5 days post incubation (dpi). Data are presented as mean ± SD. The presence of different letters above the mean values of three replicates indicates a significant difference between different samples (P < 0.05, ANOVA).


E Subcellular fractionation of FolSrpk1 - GFP during the early infection stage . Total proteins were extracted from tomato roots at 0 ( conidia ) , 1 , 3 and 5 dpi . Equal amount of total , nuclear and cytoplasm proteins was separated by SDS - PAGE , and FolSrpk1 was detected using anti - GFP . The amount of total and nuclear FolSrpk1 - GFP at 0 dpi was each set as 1 . Data information : For E , each gel shown is a representative experiment carried out at least three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and Coomassie brilliant blue ( CBB ) staining .

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E Subcellular fractionation of FolSrpk1-GFP during the early infection stage. Total proteins were extracted from tomato roots at 0 (conidia), 1, 3 and 5 dpi. Equal amount of total, nuclear and cytoplasm proteins was separated by SDS-PAGE, and FolSrpk1 was detected using anti-GFP. The amount of total and nuclear FolSrpk1-GFP at 0 dpi was each set as 1. Data information: For E, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and Coomassie brilliant blue (CBB) staining.


F Association of FolSrpk1 with FolAha1 under ROS stress . After treatment with 10 mM H2O2 for 0 , 1 and 2 h , proteins pulled down with GFP - Trap beads were probed with anti - GFP and anti - Flag ( top ) . Input proteins were shown by Western blotting with anti - GFP and anti - FLAG antibodies ( bottom ) . The amount of immunoprecipitated FolSrpk1 - Flag and FolAha1 - GFP at 0 h was each set as 1 . Data information : For , F each gel shown is a representative experiment carried out at least three times .

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F Association of FolSrpk1 with FolAha1 under ROS stress. After treatment with 10 mM H2O2 for 0, 1 and 2 h, proteins pulled down with GFP-Trap beads were probed with anti-GFP and anti-Flag (top). Input proteins were shown by Western blotting with anti-GFP and anti-FLAG antibodies (bottom). The amount of immunoprecipitated FolSrpk1-Flag and FolAha1-GFP at 0 h was each set as 1. Data information: For , F each gel shown is a representative experiment carried out at least three times.


G Fluorescence microscopy analysis of FolSrpk1 - GFP in mycelia of the FolSrpk1p : : FolSrpk1 - GFP strain treated with 10 mM H2O2 for 0 , 1 and 2 h . Arrows indicate the nucleus . Scale bars = 5 µm .

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G Fluorescence microscopy analysis of FolSrpk1-GFP in mycelia of the FolSrpk1p::FolSrpk1-GFP strain treated with 10 mM H2O2 for 0, 1 and 2 h. Arrows indicate the nucleus. Scale bars = 5 µm.


I Subcellular fractionation analysis of FolSrpk1 - GFP in mycelia of the FolSrpk1p : : FolSrpk1 - GFP strain treated with 10 mM H2O2 for 0 , 1 and 2 h . The amount of nuclear FolSrpk1 - GFP at 0 h was set as 1 . Data information : For I , each gel shown is a representative experiment carried out at least three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and Coomassie brilliant blue ( CBB ) staining .

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I Subcellular fractionation analysis of FolSrpk1-GFP in mycelia of the FolSrpk1p::FolSrpk1-GFP strain treated with 10 mM H2O2 for 0, 1 and 2 h. The amount of nuclear FolSrpk1-GFP at 0 h was set as 1. Data information: For I, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and Coomassie brilliant blue (CBB) staining.


Co - IP ( C ) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1 . The amount of immunoprecipitated FolSrpk1 - Flag and FolAha1 - GFP in the first line was each set as 1 . Data information : For C , each gel shown is a representative experiment carried out at least three times .

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Co-IP (C) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1. The amount of immunoprecipitated FolSrpk1-Flag and FolAha1-GFP in the first line was each set as 1. Data information: For C, each gel shown is a representative experiment carried out at least three times.


Y2H ( D ) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1 .

Y2H (D) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1.


F Fluorescence microscopy of FolSrpk1 - GFP and its mutant forms . Arrows indicate the nucleus . Bars = 5 µm . G Quantification of FolSrpk1 - GFP distribution in F . The calculation was from three independent replicates with three hyphae per replicate and three cells per hyphae . Different letters indicate statistically significant differences according to one - way ANOVA ( P < 0 . 05 ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

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F Fluorescence microscopy of FolSrpk1-GFP and its mutant forms. Arrows indicate the nucleus. Bars = 5 µm. G Quantification of FolSrpk1-GFP distribution in F. The calculation was from three independent replicates with three hyphae per replicate and three cells per hyphae. Different letters indicate statistically significant differences according to one-way ANOVA (P < 0.05). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


H Subcellular fractionation of FolSrpk1 - GFP and its mutant forms . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and CBB staining . The amount of nuclear FolSrpk1 - GFP was set as 1 . Data information : For H , , each gel shown is a representative experiment carried out at least three times .

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H Subcellular fractionation of FolSrpk1-GFP and its mutant forms. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and CBB staining. The amount of nuclear FolSrpk1-GFP was set as 1. Data information: For H, , each gel shown is a representative experiment carried out at least three times.


I K304 acetylation of WT and mutant FolSrpk1 proteins in the indicated strains after treatment with 10 mM H2O2 for 0 , 1 and 2 h . The amount of acetylated and total FolSrpk1 - GFP at 0 h was each set as 1 . Data information : For each gel shown is a representative experiment carried out at least three times .

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I K304 acetylation of WT and mutant FolSrpk1 proteins in the indicated strains after treatment with 10 mM H2O2 for 0, 1 and 2 h. The amount of acetylated and total FolSrpk1-GFP at 0 h was each set as 1. Data information: For each gel shown is a representative experiment carried out at least three times.


A K304 acetylation ( top ) and amount ( middle ) of FolSrpk1 - GFP and FolSrpk1K304Q - GFP in WT and FolArd1 - OE strains treated with 10 mM H2O2 for 2 h . The over - expressed FolArd1 - Flag was detected by anti - Flag ( bottom ) . The amount of acetylated and total FolSrpk1 in the WT was each set as 1 . Data information : each gel shown is a representative experiment carried out three times .

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A K304 acetylation (top) and amount (middle) of FolSrpk1-GFP and FolSrpk1K304Q-GFP in WT and FolArd1-OE strains treated with 10 mM H2O2 for 2 h. The over-expressed FolArd1-Flag was detected by anti-Flag (bottom). The amount of acetylated and total FolSrpk1 in the WT was each set as 1. Data information: each gel shown is a representative experiment carried out three times.


C FolArd1 directly acetylates FolSrpk1 in vitro . Purified GST - FolSrpk1 or its K304Q isoform ( 50 ng ) was incubated with or without 50 ng of purified FolArd1 and then analyzed by immunoblotting using anti - K304ac or anti - GST antibody . Anti - GST immunoblotting was used to show equal loading . The amount of acetylated and total GST - FolSrpk1 in the first lane was each set as 1 . Data information : each gel shown is a representative experiment carried out three times .

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C FolArd1 directly acetylates FolSrpk1 in vitro. Purified GST-FolSrpk1 or its K304Q isoform (50 ng) was incubated with or without 50 ng of purified FolArd1 and then analyzed by immunoblotting using anti-K304ac or anti-GST antibody. Anti-GST immunoblotting was used to show equal loading. The amount of acetylated and total GST-FolSrpk1 in the first lane was each set as 1. Data information: each gel shown is a representative experiment carried out three times.


E Subcellular fractionation analysis of FolSrpk1 - GFP in WT , ΔFolSir2 , and FolSir2 - OE strains . The amount of nuclear FolSrpk1 - GFP in the WT was set as 1 . Data information : each gel shown is a representative experiment carried out three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and CBB staining .

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E Subcellular fractionation analysis of FolSrpk1-GFP in WT, ΔFolSir2, and FolSir2-OE strains. The amount of nuclear FolSrpk1-GFP in the WT was set as 1. Data information: each gel shown is a representative experiment carried out three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and CBB staining.


G , H Expression profile of FolArd1 ( G ) and FolSir2 ( H ) during the infection stage . Total RNA extracted from host roots at 0 ( conidia ) and 1 - 5 dpi was reverse transcribed . The expression levels were normalized to that of the Fol actin gene . Data are presented as mean ± SD . The presence of different letters above the mean values of three replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) .

G, H Expression profile of FolArd1 (G) and FolSir2 (H) during the infection stage. Total RNA extracted from host roots at 0 (conidia) and 1-5 dpi was reverse transcribed. The expression levels were normalized to that of the Fol actin gene. Data are presented as mean ± SD. The presence of different letters above the mean values of three replicates indicates a significant difference between different samples (P < 0.05, ANOVA).


A Relative expression level of six antioxidant enzymes during the infection stage . The experiments were carried out as in Fig 3 . Data are presented as mean ± SD . The presence of different letters above the mean values of three biological replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) .

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A Relative expression level of six antioxidant enzymes during the infection stage. The experiments were carried out as in Fig 3. Data are presented as mean ± SD. The presence of different letters above the mean values of three biological replicates indicates a significant difference between different samples (P < 0.05, ANOVA).


B Secretion of the FOXG _ 17180 - GFP , FOXG _ 15294 - GFP and FOXG _ 13788 - GFP proteins . The proteins extracted from mycelia ( M ) and culture supernatants ( S ) were analyzed by Western blotting with anti - GFP and anti - Actin . Each gel shown is a representative experiment carried out three times . The loading controls were Fol Actin and CBB staining .

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B Secretion of the FOXG_17180-GFP, FOXG_15294-GFP and FOXG_13788-GFP proteins. The proteins extracted from mycelia (M) and culture supernatants (S) were analyzed by Western blotting with anti-GFP and anti-Actin. Each gel shown is a representative experiment carried out three times. The loading controls were Fol Actin and CBB staining.


F qRT - PCR analysis of Fol EF - 1α transcript levels in tomato roots 14 days after infection by the indicated strains . The expression of tomato RCE1 was used as a control to ensure the use of equal amounts of RNA for RT - PCR . Data information : For F , the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

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F qRT-PCR analysis of Fol EF-1α transcript levels in tomato roots 14 days after infection by the indicated strains. The expression of tomato RCE1 was used as a control to ensure the use of equal amounts of RNA for RT-PCR. Data information: For F, the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


B Relative growth reduction of the indicated strains representing the reduction in colony diameters between normal and stress conditions normalized to colony diameters under normal conditions ( four biological replicates ) . Data information : For B , the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

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B Relative growth reduction of the indicated strains representing the reduction in colony diameters between normal and stress conditions normalized to colony diameters under normal conditions (four biological replicates). Data information: For B, the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


C Determination of H2O2 content in tomato roots infected by the indicted strains at 0 - 5 dpi . The experiments were carried out as in Fig 1 . Data are presented as mean ± SD of three biological replicates .

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C Determination of H2O2 content in tomato roots infected by the indicted strains at 0-5 dpi. The experiments were carried out as in Fig 1. Data are presented as mean ± SD of three biological replicates.


F qRT - PCR analysis of Fol EF - 1α transcript levels in tomato roots 14 days after infection by the indicated strains . The experiments ( four biological replicates ) Data information : For F , the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

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F qRT-PCR analysis of Fol EF-1α transcript levels in tomato roots 14 days after infection by the indicated strains. The experiments (four biological replicates) Data information: For F, the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


A Subcellular fractionation of BcSrpk1 - GFP in WT and BcSrpk1mutant strains . The amount of nuclear BcSrpk1 - GFP was set as 1 . Data information : , each gel shown is a representative experiment carried out three times . The fractionation controls were histone H1 ( nucleus ) , Actin ( cytosol ) and CBB staining .

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A Subcellular fractionation of BcSrpk1-GFP in WT and BcSrpk1mutant strains. The amount of nuclear BcSrpk1-GFP was set as 1. Data information: , each gel shown is a representative experiment carried out three times. The fractionation controls were histone H1 (nucleus), Actin (cytosol) and CBB staining.


B Co - IP analysis revealing the interaction of BcAha1 with BcSrpk1 and its mutant forms . Data information : each gel shown is a representative experiment carried out three times .

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B Co-IP analysis revealing the interaction of BcAha1 with BcSrpk1 and its mutant forms. Data information: each gel shown is a representative experiment carried out three times.


C K367 acetylation of BcSrpk1 during the early infection stage . Total proteins were extracted from tomato leaves at 0 ( spores ) , 12 , 24 and 36 hpi . Eluted proteins bound to the anti - GFP beads were probed by the anti - K304ac and anti - GFP antibodies . The amount of acetylated and total BcSrpk1 at 0 hpi was each set as 1 . Data information : each gel shown is a representative experiment carried out three times .

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C K367 acetylation of BcSrpk1 during the early infection stage. Total proteins were extracted from tomato leaves at 0 (spores), 12, 24 and 36 hpi. Eluted proteins bound to the anti-GFP beads were probed by the anti-K304ac and anti-GFP antibodies. The amount of acetylated and total BcSrpk1 at 0 hpi was each set as 1. Data information: each gel shown is a representative experiment carried out three times.