A Immunofluorescence of kidney sections from diabetic nephropathy subjects non - diabetic controls stained with KDM6A , KLF10 , nephrin WT - 1 . Scale bars , 50 μm . * P < 0 . 05 by Wilcoxon two - sample ( n = 6 for each group ) .

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A Immunofluorescence analysis of kidney sections from diabetic nephropathy subjects and non-diabetic controls stained with KDM6A, KLF10, nephrin and WT-1. Scale bars, 50 μm. *P < 0.05 by Wilcoxon two-sample test (n = 6 for each group).


B Western blot of kidney tissues from non - diabetic controls diabetic nephropathy subjects . Expression of KDM6A , KLF10 , nephrin WT - 1 in kidney tissues were determined by immunoblotting using indicated antibodies . Relative in kidney tissues were normalized to actin . * P < 0 . 05 by Wilcoxon two - sample ( n = 6 for each group ) .

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B Western blot analysis of kidney tissues from non-diabetic controls and diabetic nephropathy subjects. Expression levels of KDM6A, KLF10, nephrin and WT-1 in kidney tissues were determined by immunoblotting using the indicated antibodies. Relative protein levels in kidney tissues were normalized to actin. *P < 0.05 by Wilcoxon two-sample test (n = 6 for each group).


C of KDM6A KLF10 , nephrin mRNAs in urine exosomes of diabetic nephropathy non - diabetic controls . Relative mRNA in human urinary exosomes were normalized to 18S rRNA . Horizontal lines are medians . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Wilcoxon two - sample ( n = 12 for each group ) .

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C Levels of KDM6A and KLF10, nephrin mRNAs in urine exosomes of diabetic nephropathy patients and non-diabetic controls. Relative mRNA levels in human urinary exosomes were normalized to 18S rRNA. Horizontal lines are medians. *P < 0.05, **P < 0.01, and ***P < 0.001 by Wilcoxon two-sample test (n = 12 for each group).


( A ) Western blot of NDUFS3 in homogenates from quadriceps muscle of 15 - days old ( upper panel ) 1 - month old ( lower panel ) .

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(A) Western blot analysis of NDUFS3 protein levels in homogenates from quadriceps muscle of 15-days old (upper panel) and 1-month old animals (lower panel).


( B ) comparison over time of Ndufs3 smKO mice ( filled blue squares ; n = 14 ) matched wild - type ( WTFlx ; n = 15 ) ( empty blue squares ) . Ndufs3 smKO ( filled red circles ) showed similar results ( n = 10 for smKO , n = 12 for WTFlx group ) . P values were calculated by Student ' s t ;

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(B) Body weight comparison over time of Ndufs3 smKO male mice (filled blue squares; n=14) and age matched wild-type (WTFlx; n=15) males (empty blue squares). Ndufs3 smKO females (filled red circles) showed similar results (n=10 for smKO, and n=12 for WTFlx group). P values were calculated by Student's t test;


( C ) Representative image of a smKO Ndfu3s3 - smKO mouse a WTFlx littermate of 6 - month old showing decreased .

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(C) Representative image of a smKO male Ndfu3s3-smKO mouse and a WTFlx littermate of 6-month old showing decreased body weight.


( D ) Survival curve of Ndufs3 smKO mice ( red ) ( blue ) . P values were calculated using Log - rank ( Mantel - Cox ) ; p = 0 . 0010 for ( n = 19 ) , p = 0 . 0004 for ( n = 10 ) .

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(D) Survival curve of Ndufs3 smKO female mice (red line) and males (blue line). P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0010 for males (n=19), and p=0.0004 for females (n=10).


( E ) Nocturnal ambulatory activity of Ndufs3 smKO aged matched wild - type from 1 2 months old mice . P values were calculated by Student ' s t . P values were calculated by Student ' s t to determine level of statistical difference . ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 0001 .

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(E) Nocturnal ambulatory activity of Ndufs3 smKO and aged matched wild-type littermates from 1 and 2 months old male mice. P values were calculated by Student's t test. P values were calculated by Student's t test to determine the level of statistical difference. ; *p<0.05, **p<0.01 and ***p<0.0001.


( F ) number of falls in treadmill of WTFlx Ndufs3 smKO mice , starting at 2 months ( n = 8 ) . Bars represent means ± standard error ( SE ) . P values were calculated by Student ' s t to determine level of statistical difference . n . s . - not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 0001 .

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(F) The number of falls in the treadmill of WTFlx and Ndufs3 smKO male mice, starting at 2 months (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.


( G ) Rotarod performed by Ndufs3 smKO wild - type mice . ( n = 8 ) . Bars represent means ± standard error ( SE ) . P values were calculated by Student ' s t to determine level of statistical difference . n . s . - not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 0001 .

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(G) Rotarod performed by Ndufs3 smKO and wild-type male mice. (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.


( A ) Representative image of quadriceps from WTFlx Ndufs3 smKO of 8 months of . Muscle from Ndufs3 smKO ( black bar ) wild - type mice ( bar ) from 6 , 8 months of ( n = 5 each group ) . Bars represent means ± standard error ( ) .

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(A) Representative image of quadriceps from WTFlx and Ndufs3 smKO animals of 8 months of age. Muscle weight from Ndufs3 smKO males (black bar) and wild-type mice (white bar) from 6, and 8 months of age (n=5 each group). Bars represent means ± standard error (SEM).


( B ) Representative H & E , COX , SDH of quadriceps from WTFlx Ndufs3 - Mlc1f smKO at 8 months old .

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(B) Representative H&E, COX, and SDH staining of quadriceps from WTFlx and Ndufs3-Mlc1f smKO at 8 months old.


( C ) Cross - sectional ( CSA ) of quadriceps muscle . A minimum of 100 / sample were analyzed ( n = 3 / genotype ) . of central nuclei in skeletal muscle of quadriceps muscle of WTFlx Ndufs3 smKO at 8 months ( n = 3 mice for WTFlx n = 4 mice for Ndufs3 smKO ) . P values were calculated by Student ' s t .

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(C) Cross-sectional area (CSA) of quadriceps muscle fibers. A minimum of 100 fibers/sample were analyzed (n =3/genotype). The percentage of central nuclei in skeletal muscle fibers of quadriceps muscle of WTFlx and Ndufs3 smKO at 8 months (n =3 mice for WTFlx and n=4 mice for Ndufs3 smKO). P values were calculated by Student's t test.


( D ) Serum lactate of WTFlx Ndufs3 - smKO at different ages . Ndufs3 smKO mice ( filled black squares ) showed increased compared to matched WTFlx ( empty black squares ) . Error bars represent .

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(D) Serum lactate levels of WTFlx and Ndufs3-smKO animals at different ages. Ndufs3 smKO male mice (filled black squares) showed increased levels compared to matched WTFlx males (empty black squares). Error bars represent SEM.


( E ) BN - PAGE In - Gel Activities of I IV measured in homogenates from quadriceps of wild - type Ndufs3 - Mlc1f smKO at 2 4 months .

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(E) BN-PAGE In-Gel Activities of Complex I and IV measured in homogenates from quadriceps of wild-type and Ndufs3-Mlc1f smKO animals at 2 and 4 months.


( F - G ) Western blots of muscle homogenates ( quadriceps ) of WTFlx Ndufs3 - Mlc1f smKO at different ages ( 2 4 months , respectively ) using antibodies against PGC - 1α , COX1 ( IV subunit ) , NDUFS3 , VDAC1 , SDHA ( II subunit ) , β - actin . ( H ) of western blots in panels F G . Bars represent means ± of n = 4 for each group . P values were determined by Student ' s t .

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(F-G) Western blots of muscle homogenates (quadriceps) of WTFlx and Ndufs3-Mlc1f smKO animals at different ages (2 and 4 months, respectively) using antibodies against PGC-1α, COX1 (complex IV subunit), NDUFS3, VDAC1, SDHA (complex II subunit), and β-actin. (H) Quantification of the western blots in panels F and G. Bars represent means ±SEM of n=4 for each group. P values were determined by Student's t test.


( C ) KO - GFP mouse was visibly smaller weaker than a KO - NDUFS3 mouse , at 7 months .

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(C) The KO-GFP mouse was visibly smaller and weaker than a KO-NDUFS3 mouse, at 7 months.


( D ) injected with rAAV9 - Ndufs3 ( heretofore referred to as KO - NDUFS3 ) mice were similar to WTFlx mice significantly higher than rAAV9 - eGFP injected mice ( KO - GFP ) starting at 5 months . Statistical was performed by One - way ANOVA followed by Tukey post - . P values were calculated using Log - rank ( Mantel - Cox ) ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates number of used per group .

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(D) Females body weight injected with rAAV9-Ndufs3 (heretofore referred to as KO-NDUFS3) mice were similar to WTFlx mice body weight and significantly higher than the rAAV9-eGFP injected mice (KO-GFP) starting at 5 months. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( E ) of KO - NDUFS3 mice were similar to WTFlx mice , significantly different from KO - GFP from 4 months onwards . Statistical was performed by One - way ANOVA followed by a Tukey post - . P values were calculated using Log - rank ( Mantel - Cox ) ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates number of used per group .

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(E) Males body weight of KO-NDUFS3 mice were similar to WTFlx mice, and significantly different from KO-GFP from 4 months onwards. Statistical analysis was performed by One-way ANOVA followed by a Tukey post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( F ) Treadmill performance was significantly improved in KO - NDUFS3 mice when compared to KO - GFP , from 2 months onwards . P values were calculated using Log - rank ( Mantel - Cox ) ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates number of used per group .

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(F) Treadmill performance was significantly improved in the KO-NDUFS3 mice when compared to the KO-GFP, from 2 months onwards. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( G ) Four limb strength was evaluated by grid hang . KO - NDUFS3 mice performed significantly better starting at 3 months , when compared to KO - GFP mice . F ) G ) Statistical was performed by One - way ANOVA followed by Bonferroni post - . P values were calculated using Log - rank ( Mantel - Cox ) ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates number of used per group .

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(G) Four limb strength was evaluated by the grid hang test. KO-NDUFS3 mice performed significantly better starting at 3 months, when compared to the KO-GFP mice. F) and G) Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( H ) Quadriceps gastrocnemius wet of 6 months old mice was similar between WTFlx KO - NDUFS3 mice . Quadriceps was significantly increased in KO - NDUFS3 mice when compared to KO - GFP injected . Statistical was performed by One - way ANOVA followed by Bonferroni post - . ns - not significant . P values were calculated using Log - rank ( Mantel - Cox ) ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates number of used per group .

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(H) Quadriceps and gastrocnemius wet weight of 6 months old mice was similar between WTFlx and KO-NDUFS3 mice. Quadriceps weight was significantly increased in KO-NDUFS3 mice when compared to KO-GFP injected. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. ns- not significant. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( I ) Survival curve of Ndufs3 smKO mice injected with rAVV9 - Ndufs3 ( blue ) with rAVV9 - eGFP ( green ) . WTFlx mice are depicted in black . rAAV9 - Ndufs3 injections extended survival of smKO mice .

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(I) Survival curve of Ndufs3 smKO mice injected with rAVV9-Ndufs3 (blue line) and with rAVV9-eGFP (green line). WTFlx mice are depicted in black. rAAV9-Ndufs3 injections extended the survival of the smKO mice.


( A ) Western blots of 6 months old quadriceps of WTFlx , KO KO - NDUFS3 injected mice at 15 - 18 days old . GFP expression was observed only in KO - GFP mice . loading was obtained with stain - free gel technology . ( B ) of NDUFS3 , COXI SDHA expression in mice showed in A .

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(A) Western blots of 6 months old quadriceps of WTFlx, KO and KO-NDUFS3 injected mice at 15-18 days old. GFP expression was observed only in the KO-GFP mice. Protein loading was obtained with stain-free gel technology. (B) Quantification of NDUFS3, COXI and SDHA expression levels in female mice showed in A.


( C ) Western blot of BN - PAGE of quadriceps of 6 - month - old mice . We analyzed : CI ( NDUFA9 , NDUFB8 ) , CIV ( COX1 ) , CII ( SDHA ) . ( D ) of BN - PAGE western blots from mice shown in panel C .

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(C) Western blot of BN-PAGE of quadriceps of 6-month-old mice. We analyzed: CI (NDUFA9, NDUFB8), CIV (COX1), CII (SDHA). (D) Quantification of the BN-PAGE western blots from female mice shown in panel C.


( E ) BN - PAGE in - gel activity of CI CIV in quadriceps of 6 - month - old mice . ( F ) of BN - PAGE in - gel activities in .

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(E) BN-PAGE in-gel activity of CI and CIV in quadriceps of 6-month-old mice. (F) Quantification of BN-PAGE in-gel activities in females.


( G ) Total mtDNA in quadriceps from were determined by qPCR using ND1 / 18S . Inset shows appearance of quadriceps , reflecting normalization of mitochondrial after replacement of NDUFS3 .

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(G) Total mtDNA levels in quadriceps from males and females were determined by qPCR using the ratio ND1/18S. Inset shows the appearance of the quadriceps, reflecting normalization of mitochondrial levels after replacement of NDUFS3.


( H ) Spectrophotometric CI / citrate synthase activity was measured in quadriceps homogenates of 6 - month - old .

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(H) Spectrophotometric CI/citrate synthase activity ratio was measured in quadriceps homogenates of 6-month-old males and females.


( I ) Plasma lactate were determined as described in methods . Statistical was performed by One - way ANOVA followed by Tukey post - .

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(I) Plasma lactate levels were determined as described in methods. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test.


( B ) smKO mice group that received retro - orbital injections at 2 months was tested prior to rAAV9 - Ndufs3 injection showing a significant exercise intolerance attested by number of falls in treadmill . KO - NDUFS3 gradually recovered over time . KO - NDUFS3 mice are color coded . Green triangles represent injected with GFP whereas triangles represent non - injected mice . Statistical was performed by One - way ANOVA followed by Bonferroni post - .

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(B) The smKO mice group that received retro-orbital injections at 2 months was tested prior to the rAAV9-Ndufs3 injection showing a significant exercise intolerance attested by the number of falls in the treadmill. The KO-NDUFS3 gradually recovered over time. Individual KO-NDUFS3 mice are color coded. Green triangles represent the animals injected with GFP whereas white triangles represent non-injected mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.


( C ) hang was performed in symptomatic mice which had a significant decrease in four - limb muscle strength coordination , at 2 months . KO - NDUFS3 mice showed a significant recovery 3 . 5 months after injection . Statistical was performed by One - way ANOVA followed by Bonferroni post - .

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(C) The hang test was performed in symptomatic mice which had a significant decrease in four-limb muscle strength and coordination, at 2 months. The KO-NDUFS3 mice showed a significant recovery 3.5 months after injection. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.


( D ) KO - NDUFS3 muscle was similar to WTFlx mice , it was significantly increased in gastrocnemius when compared to KO - GFP mice . Statistical was performed by One - way ANOVA followed by Bonferroni post - .

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(D) KO-NDUFS3 muscle weight was similar to WTFlx mice, and it was significantly increased in gastrocnemius when compared to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.


( E ) Immunohistochemical with NDUFB8 COX1 antibodies in quadriceps muscle sections of 6 - month - old . expression of NDUFB8 ( representative of assembled CI ) was recovered in KO - NDUFS3 mice COX1 expression was similar to WTFlx .

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(E) Immunohistochemical analysis with NDUFB8 and COX1 antibodies in quadriceps muscle sections of 6-month-old animals. The expression of NDUFB8 (representative of assembled CI) was recovered in the KO-NDUFS3 mice and COX1 expression was similar to WTFlx animals.


( F ) COX COX / SDH activity showed that KO - NDUFS3 quadriceps were reverted to found in wild - type , at 6 months . n - indicates number of used per group , scale : 100 µm .

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(F) COX and COX/SDH activity staining showed that KO-NDUFS3 quadriceps were reverted to levels found in wild-type, at 6 months. n-indicates the number of animals used per group, scale: 100 µm.


( A ) Western blots showed NDUFS3 expression in KO - NDUFS3 GFP expression in KO - GFP quadriceps samples . PGC - 1α expression was normalized in KO - NDUFS3 mice which was abnormally elevated in KO - GFP . same was observed for CIV ( COX1 ) , CIII ( 2 ) CII ( SDHA ) . ( B ) of western blots showed in A . Statistical was performed by One - way ANOVA followed by Tukey post - . Data represent means ± .

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(A) Western blots showed NDUFS3 expression in KO-NDUFS3 and GFP expression in the KO-GFP quadriceps samples. PGC-1α expression was normalized in the KO-NDUFS3 mice which was abnormally elevated in the KO-GFP. The same was observed for CIV (COX1), CIII (Core 2) and CII (SDHA). (B) Quantification of western blots showed in A. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( C ) BN - PAGE western blots showed recovery of I assembly into supercomplexes in KO - NDUFS3 injected mice . CIV CII were similar to wild - type in KO - NDUFS3 samples , at 6 months .

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(C) BN-PAGE western blots showed recovery of Complex I assembly into complex and supercomplexes in KO-NDUFS3 injected mice. CIV and CII were similar to wild-type in the KO-NDUFS3 samples, at 6 months.


( D ) of BN - PAGE western blots Statistical was performed by One - way ANOVA followed by Tukey post - . Data represent means ± .

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(D) Quantification of BN-PAGE western blots Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( E ) BN - PAGE In - gel CI IV activities of KO - NDUFS3 mice were also reverted to similar to WTFlx samples ( F ) of in - gel activities . Statistical was performed by One - way ANOVA followed by Tukey post - . Data represent means ± .

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(E) BN-PAGE In-gel CI and IV activities of KO-NDUFS3 mice were also reverted to levels similar to the WTFlx samples (F) Quantification of in-gel activities. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( G ) I / Citrate synthase activity of KO - NDUFS3 samples measured spectrophotometrically were similar to WTFlx samples increased in comparison to KO - GFP mice . Statistical was performed by One - way ANOVA followed by Tukey post - . Data represent means ± .

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(G) Complex I/Citrate synthase activity of KO-NDUFS3 samples measured spectrophotometrically were similar to WTFlx samples and increased in comparison to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( H ) Total mtDNA were determined by qPCR . MtDNA in KO - NDUFS3 mice were decreased when compared to KO - GFP mice not different from in WTFlx mice . Statistical was performed by One - way ANOVA followed by Bonferroni post - . Statistical was performed by One - way ANOVA followed by Tukey post - . Data represent means ± .

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(H) Total mtDNA levels were determined by qPCR. MtDNA levels in KO-NDUFS3 mice were decreased when compared to KO-GFP mice and not different from the levels in WTFlx mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( I ) serum lactate were similar to WTFlx in KO - NDUFS3 samples significantly decreased when compared to KO - GFP . KO - NDUFS3 are color coded . Green triangles represent injected with rAAV9 - eGFP whereas black triangles represent which did not receive an rAAV9 - eGFP injection . Statistical was performed by One - way ANOVA followed by Tukey post - . Data represent means ± .

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(I) The serum lactate levels were similar to WTFlx in KO-NDUFS3 samples and significantly decreased when compared to KO-GFP levels. The individual KO-NDUFS3 animals are color coded. Green triangles represent the animals injected with rAAV9-eGFP whereas black triangles represent animals which did not receive an rAAV9-eGFP injection. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( B ) knotting probability ( Pkn ) of intracellular ( observed ) does not scale proportionally to of ( expected ) . slope of Pkn is reduced in stretches larger than 20 nucleosomes . Data from ( Valdes et al . , 2018 ) .

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(B) DNA knotting probability (Pkn) of intracellular chromatin (observed) does not scale proportionally to the length of DNA (expected). The slope of Pkn is reduced in chromatin stretches larger than 20 nucleosomes. Data from (Valdes et al., 2018).


( B ) Lanes 1 2 : negatively supercoiled plasmid ( YEp24 ) its equilibrium distribution of Lk topoisomers upon its relaxation with Topo I . Lanes 3 4 : distribution of Lk topoisomers of YEp24 upon its relaxation in lysates of ∆ top1 TOP2 yeast in absence after addition of ICRF - 193 . Plots compare relative of topoisomers of Lk distributions in lanes 2 , 3 4 .

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(B) Lanes 1 and 2: negatively supercoiled plasmid (YEp24) and its equilibrium distribution of Lk topoisomers upon its relaxation with Topo I. Lanes 3 and 4: distribution of Lk topoisomers of YEp24 upon its relaxation in lysates of ∆top1 TOP2 yeast cells in absence and after the addition of ICRF-193. Plots compare the relative intensity of individual topoisomers of the Lk distributions in lanes 2, 3 and 4.


( C ) Top : 2D gel electrophoresis of Lk distributions of YEp13 minichromosome present in lysates of ∆ top1 TOP2 yeast before after addition of ICRF - 193 ( see details in Fig EV3 ) . Plots compare relative of Lk distributions ( divided into ten sections ) . Bottom : 2D gel electrophoresis of same samples upon nicking in order to reveal occurrence of knots ( see details in Fig EV4 ) . graph shows Pkn of YEp13 ( mean ± SD from three independent experiments ) . P - values ( Student ' s t ) : ns , p > 0 . 05 .

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(C) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome present in the lysates of ∆top1 TOP2 yeast cells before and after the addition of ICRF-193 (see details in Fig EV3). Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots (see details in Fig EV4). The graph shows Pkn of YEp13 (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.


( E ) Lk distributions of control plasmid ( YEp24 ) relaxed by lysates of ∆ top1 top2 - 4 yeast that expressed TOP2 or Top2 - ∆ 83 . Plots compare relative of Lk topoisomers .

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(E) Lk distributions of the control plasmid (YEp24) relaxed by lysates of ∆top1 top2-4 yeast cells that expressed TOP2 or Top2-∆83. Plots compare the relative intensity of individual Lk topoisomers.


( F ) Top : 2D gel electrophoresis of Lk distributions of YEp13 minichromosome produced in presence of TOP2 or Top2 - ∆ 83 . Plots compare relative of Lk distributions ( divided into ten sections ) . Bottom : 2D gel electrophoresis of same samples upon nicking in order to reveal occurrence of knots . Graph : Pkn of YEp13 ( mean ± SD from three independent experiments ) .

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(F) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome produced in the presence of TOP2 or Top2-∆83. Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots. Graph: Pkn of YEp13 (mean ± SD from three independent experiments).


( A ) Top , content ( n / 2n ) of exponentially growing ( OD600 = 0 . 6 - 0 . 8 ) smc2 - 8 yeast . First blot : 2D gel electrophoresis of distribution of Lk topoisomers ( Lk ) of YEp13 in quenched at 26oC after shifting culture to 35oC for 60 min . Second blot : 2D gel electrophoresis of same samples upon nicking YEp13 in order to reveal occurrence of knots ( kn ) . Graph : Pkn of YEp13 before after inactivation of condensin .

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(A) Top, DNA content (n/2n) of exponentially growing (OD600 = 0.6-0.8) smc2-8 yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). Graph: Pkn of YEp13 before and after the inactivation of condensin.


Top , content ( n / 2n ) of exponentially growing yeast . First blot : 2D gel electrophoresis of distribution of Lk topoisomers ( Lk ) of YEp13 in quenched at 26oC after shifting culture to 35oC for 60 min . Second blot : 2D gel electrophoresis of same samples upon nicking YEp13 in order to reveal occurrence of knots ( kn ) . ( B ) Experiments conducted as in ( A ) but in scc1 - 73 yeast . Graph : Pkn of YEp13 before after inactivation of cohesin .

Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (B) Experiments conducted as in (A) but in scc1-73 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of cohesin.


Top , content ( n / 2n ) of exponentially growing yeast . First blot : 2D gel electrophoresis of distribution of Lk topoisomers ( Lk ) of YEp13 in quenched at 26oC after shifting culture to 35oC for 60 min . Second blot : 2D gel electrophoresis of same samples upon nicking YEp13 in order to reveal occurrence of knots ( kn ) . ( C ) Experiments conducted as in ( A ) but in smc6 - 9 yeast . Graph : Pkn of YEp13 before after inactivation of Smc5 / 6 .

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Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (C) Experiments conducted as in (A) but in smc6-9 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of the Smc5/6 complex.


( D , E ) Experiments conducted as in ( A ) , but in arrested in G1 with alfa - factor ( D ) or in metaphase with nocodazole ( E ) for 2 h at 26oC for one additional hour at 26oC or 35oC .

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(D, E) Experiments conducted as in (A), but in cells arrested in G1 with alfa-factor (D) or in metaphase with nocodazole (E) for 2 h at 26oC and for one additional hour at 26oC or 35oC.


( F , G ) Experiments conducted as in ( B ) , but in arrested in G1 with alfa - factor for 2 h at 26oC ( F ) or in metaphase with nocodazole ( G ) for one additional hour at 26oC or 35oC .

(F, G) Experiments conducted as in (B), but in cells arrested in G1 with alfa-factor for 2 h at 26oC (F) or in metaphase with nocodazole (G) and for one additional hour at 26oC or 35oC.


( H , I ) Experiments conducted as in ( C ) , but in arrested in G1 with alfa - factor ( H ) or in metaphase with nocodazole ( I ) for 2 h at 26oC for one additional hour at 26oC or 35oC .

(H, I) Experiments conducted as in (C), but in cells arrested in G1 with alfa-factor (H) or in metaphase with nocodazole (I) for 2 h at 26oC and for one additional hour at 26oC or 35oC.


( A - E ) topology of indicated minichromosomes of increasing ( kb ) before ( 26oC ) after inactivation of condensin ( 35oC ) in smc2 - 8 . In each case , first 2D gel resolves Lk topoisomers ( Lk ) , second 2D gel uncovers knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare Pkn before after inactivation of condensin ( mean ± SD from three independent experiments ) . P - values ( Student ' s t ) : ns , p > 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(A-E) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of condensin (35oC) in smc2-8 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of condensin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05; **p < 0.01; ***p < 0.001.


( F ) Plot of Pkn values of minichromosomes of increasing ( including YEp13 ) before after inactivation of condensin .

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(F) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of condensin.


( A - D ) topology of indicated minichromosomes of increasing ( kb ) before ( 26oC ) after inactivation of cohesin ( 35oC ) in scc1 - 73 . In each case , first 2D gel resolves Lk topoisomers ( Lk ) , second 2D gel uncovers knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare Pkn before after inactivation of cohesin ( mean ± SD from three independent experiments ) . P - values ( Student ' s t ) : ns , p > 0 . 05 .

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(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of cohesin (35oC) in scc1-73 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of cohesin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.


( E ) Plot of Pkn values of minichromosomes of increasing ( including YEp13 ) before after inactivation of cohesin . P - values ( Student ' s t ) : * * p < 0 . 01 .

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(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of cohesin. P-values (Student's t test): **p < 0.01.


( A - D ) topology of indicated minichromosomes of increasing ( kb ) before ( 26oC ) after inactivation of Smc5 / 6 ( 35oC ) in smc6 - 9 . In each case , first 2D gel resolves Lk topoisomers ( Lk ) , second 2D gel uncovers knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare Pkn before after inactivation of Smc5 / 6 ( mean ± SD from two independent experiments ) .

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(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of Smc5/6 complex (35oC) in smc6-9 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of Smc5/6 complex (mean ± SD from two independent experiments).


( E ) Plot of Pkn values of minichromosomes of increasing ( including YEp13 ) before after inactivation of Smc5 / 6 .

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(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of Smc5/6 complex.


( a , b ) Multiple MBds associate with a PC3 ( a ) a B - lymphoblast ( b ) . Insets : MBd labelling ( a ) merged differential interference contrast microscopy image with MBd labelling to show boundaries ( b ) . MKLP1 , MBd marker ( a , b ; red ) ; CD44 , ( a ; green ) ; DAPI , ( a ; blue ) . Scale bar , 5 μm ( a ) 2 μm ( b ) .

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(a,b) Multiple MBds associate with a PC3 cell (a) and a B-lymphoblast (b). Insets: MBd labelling (a) and merged differential interference contrast microscopy image with MBd labelling to show cell boundaries (b). MKLP1, MBd marker (a,b; red); CD44, membrane (a; green); DAPI, DNA (a; blue). Scale bar, 5 μm (a) and 2 μm (b).


( c , d ) Three - dimensional reconstructions of polarized in a monolayer ( c ) a HeLa ( d ) show intracellular MBds . ( c ) ZO - 1 , tight junction ; MKLP1 , MBds . Scale bar , 2 μm . Enlargement ( bottom panel ) of box in top panel shows five MBds ( arrows ) . ( d ) Wheat - germ agglutinin , plasma ( red ) ; MKLP1 - GFP , MBds ( green ) ; DAPI , ( blue ) . Scale bar , 5 μm .

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(c,d) Three-dimensional reconstructions of polarized cells in a monolayer (c) and a HeLa cell (d) show intracellular MBds. (c) ZO-1, tight junction; MKLP1, MBds. Scale bar, 2 μm. Enlargement (bottom panel) of the box in the top panel shows five MBds (arrows). (d) Wheat-germ agglutinin, plasma membrane (red); MKLP1-GFP, MBds (green); DAPI, DNA (blue). Scale bar, 5 μm.


( e ) Electron microscopy of an MBd in a permeabilized MCF - 7 showing immungold labelling with MKLP1 antibodies . Inset , lower magnification of MBd ( boxed ) in ; nucleus , right . Scale bar , 200 nm .

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(e) Electron microscopy of an MBd in a permeabilized MCF-7 cell showing immungold labelling with MKLP1 antibodies. Inset, lower magnification of the MBd (boxed) in the cell; nucleus, right. Scale bar, 200 nm.


( f ) Time - lapse during extracellular trypsin treatment of HeLa show retention of most MBds ( MKLP1 - GFP , red ) . Two MBds ( yellow arrows ) are lost on treatment , indicating digestion / or dissociation . Time ( h : min ) post trypsin . Scale bar , 5 μm .

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(f) Time-lapse images during extracellular trypsin treatment of HeLa cells show retention of most MBds (MKLP1-GFP, red). Two MBds (yellow arrows) are lost on treatment, indicating digestion and/or dissociation. Time (h:min) post trypsin. Scale bar, 5 μm.


( g ) Two - day co - cultures of HeLa expressing either MKLP1 - GFP ( MBd marker ) or RFP . Green MBds ( arrows ) associated with red ( asterisk ) indicate post - mitotic transfer of MBds between . Scale bar , 10 μm .

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(g) Two-day co-cultures of HeLa cells expressing either MKLP1-GFP (MBd marker) or cytosolic RFP. Green MBds (arrows) associated with red cells (asterisk) indicate post-mitotic transfer of MBds between cells. Scale bar, 10 μm.


( a ) CETN1 - GFP signal is brighter in upper centrosome / spindle pole of a mitotic spindle . merged differential interference contrast microscopy image with CETN1 - GFP labelling at two centrosomes shows a metaphase chromosome . Insets : enlargements semi - quantitative integrated profiles of centrioles . Scale bar , 5 μm . ( b ) brighter CETN1 - GFP signal represents older centrosome , as it co - stains more intensely for hCenexin1 remains more intense throughout division ( Supplementary Fig . S1a ) . Scale bar , 5 μm . Lower left , merge .

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(a) The CETN1-GFP signal is brighter in the upper centrosome/spindle pole of a mitotic spindle. The merged differential interference contrast microscopy image with CETN1-GFP labelling at two centrosomes shows a metaphase chromosome. Insets: enlargements and semi-quantitative integrated intensity profiles of centrioles. Scale bar, 5 μm. (b) The brighter CETN1-GFP signal represents the older centrosome, as it co-stains more intensely for hCenexin1 and remains more intense throughout cell division (Supplementary Fig. S1a). Scale bar, 5 μm. Lower left, merge.


( c , d ) Time - lapse show that mitotic midbody is preferentially inherited by daughter with older centrosome in HeLa ( c ) hESCs ( d ) . were imaged at indicated times ( h : min ) from telophase by phase - contrast microscopy ( c ) from metaphase by differential interference contrast microscopy ( d ) . Middle panel of ( c ) upper left panel of ( d ) : CETN1 - GFP at centrosomes ; enlargements integrated profiles show that daughter with older centrosome ( c , upper ; d , lower ) inherits MBd ( lower right in c , d ) . Mitotic midbody MBds ( c , d ; arrows ) . MKLP1 , MBd marker ( red ) ; α - tubulin , mitotic midbody - boundary marker ( green ) ; DAPI , ( blue ) . Scale bars , 10 μm ( c , d ) .

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(c,d) Time-lapse images show that the mitotic midbody is preferentially inherited by the daughter cell with the older centrosome in HeLa cells (c) and hESCs (d). Cells were imaged at the indicated times (h:min) from telophase by phase-contrast microscopy (c) and from metaphase by differential interference contrast microscopy (d). Middle panel of (c) and upper left panel of (d): CETN1-GFP at centrosomes; enlargements and integrated intensity profiles show that the daughter cell with the older centrosome (c, upper; d, lower) inherits the MBd (lower right images in c,d). Mitotic midbody and MBds (c,d; arrows). MKLP1, MBd marker (red); α-tubulin, mitotic midbody and cell-boundary marker (green); DAPI, DNA (blue). Scale bars, 10 μm (c,d).


( a ) A histological section through mouse seminiferous tubules labelled for MKLP1 shows several MKLP1 + in of basal layer where reside . Scale bar , 20 μm . Inset : Enlargement of marked by arrow .

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(a) A histological section through mouse seminiferous tubules labelled for MKLP1 shows several MKLP1+ puncta in cells of the basal layer where stem cells reside. Scale bar, 20 μm. Inset: Enlargement of the cell marked by the arrow.


( b , c ) Electron of mitotic midbody ( b , arrow ) multiple midbody - like structures in interphase with similar shapes sizes in a juxtanuclear position ( c , arrows ) in basal of mouse seminiferous tubules . N , nucleus . Scale bars , 1 μm .

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(b,c) Electron micrographs of mitotic midbody (b, arrow) and multiple midbody-like structures in interphase cells with similar shapes and sizes in a juxtanuclear position (c, arrows) in basal cells of mouse seminiferous tubules. N, nucleus. Scale bars, 1 μm.


( d ) Representative planes of a neural progenitor in ventricular zone ( Sox2 + , bottom left panel ) of an E13 . 5 mouse brain show that an intracellular MBd ( asterisk ) is associated with ventricle - facing daughter in asymmetrically dividing ( top row ) . bottom row emphasizes position of paired chromosomes in a dividing anaphase . CD133 , midbody / MBd marker ( green ) ; Na / K - ATPase , - border marker ( red ) ; DRAQ5 , ( blue ) ; DAPI , . v , ventricle . Scale bar , 5 μm . Note that abscission occurs apically in these .

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(d) Representative planes of a neural progenitor cell in the ventricular zone (Sox2+, bottom left panel) of an E13.5 mouse brain show that an intracellular MBd (asterisk) is associated with the ventricle-facing daughter in the asymmetrically dividing cell (top row). The bottom row emphasizes the position of paired chromosomes in a dividing anaphase cell. CD133, midbody/MBd marker (green); Na/K-ATPase, cell-border marker (red); DRAQ5, DNA (blue); DAPI, DNA. v, ventricle. Scale bar, 5 μm. Note that abscission occurs apically in these cells.


( e ) A histological section through a hair follicle ( left , phase - contrast microscopy ) stained for - marker keratin 15 ( K15 ) to identify bulge region ( dotted box ) , - niche . stain ( DAPI ) phase - contrast microscopy image show full follicle architecture . ( f ) Upper panels show MBd - accumulating in bulge region ( boxed ) co - labelled with K15 MKLP1 . Enlargements ( lower panels ) of boxed region highlight a with four MBds ( asterisks ) . N , nucleus . Scale bar , 5 μm .

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(e) A histological section through a hair follicle (left, phase-contrast microscopy) stained for the stem-cell marker keratin 15 (K15) to identify the bulge region (dotted box), the stem-cell niche. DNA stain (DAPI) and the phase-contrast microscopy image show full follicle architecture. (f) Upper panels show MBd-accumulating cells in the bulge region (boxed) co-labelled with K15 and MKLP1. Enlargements (lower panels) of the boxed region highlight a cell with four MBds (asterisks). N, nucleus. Scale bar, 5 μm.


( g - i ) Quantitative representative show a decrease in MBd - accumulating on differentiation of pluripotent ( g ) to fibroblast - like ( h ) , an increase in MBd - accumulating after reprogramming differentiated ( h ) to induced pluripotent ( i ) . Numbers refer to mean ± s . d . , n = 3 . MKLP1 , MBds ; ZO - 1 , tight junctions ; α - tubulin , microtubules ; Aurora B , midbodies . Scale bar , 10 μm .

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(g-i) Quantitative analysis and representative images show a decrease in MBd-accumulating cells on the differentiation of pluripotent stem cells (g) to fibroblast-like cells (h), and an increase in MBd-accumulating cells after reprogramming differentiated cells (h) to induced pluripotent stem cells (i). Numbers refer to mean±s.d., n=3. MKLP1, MBds; ZO-1, tight junctions; α-tubulin, microtubules; Aurora B, midbodies. Scale bar, 10 μm.


( b ) - chased with EdU show a decrease in EdU ( x axis ) over time ( y axis ) , reflecting dilution of dye after divisions . ( c , d ) After a 96 h chase period , EdU were compared between with MBd numbers of > 1 , 1 0 ( y axis ) in HeLa ( c ) SAOS - 2 ( d ) . In both cases , no significant differences were noted ( c , P = 0 . 2101 ; d , P = 0 . 5609 , one - way of variance , with at least 800 analysed for each experiment , n = 3 ) , indicating similar cycling rates among different subpopulations of . ( b - d ) Each graph is a representative experiment . analysed are shown by green points , medians are depicted by vertical red lines , horizontal red lines with ticks illustrate interquartile range .

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(b) Cells pulse-chased with EdU show a decrease in EdU intensity (x axis) over time (y axis), reflecting dilution of dye after cell divisions. (c,d) After a 96 h chase period, EdU levels were compared between cells with MBd numbers of >1, 1 and 0 (y axis) in HeLa (c) and SAOS-2 cells (d). In both cases, no significant differences were noted (c, P=0.2101; d, P=0.5609, one-way analysis of variance, with at least 800 cells analysed for each experiment, n=3), indicating similar cycling rates among different subpopulations of cells. (b-d) Each graph is a representative experiment. Cells analysed are shown by green points, the medians are depicted by vertical red lines, and horizontal red lines with ticks illustrate the interquartile range.


( a ) Depiction of FPP . Digitonin selectively permeabilizes plasma but not internal membranes . Proteinase K degrades components but membranous compartments remain intact . Under these conditions , MKLP1 - GFP - labelled MBds ( blue ellipse ) in cytoplasm will be degraded whereas those inside - bound compartments will not . ( b ) MBds in MBd - poor hRPE - 1 are largely protected ( ∼ 90 % in membranous compartments , 10 analysed ) , whereas most MBds in HeLa are not ( ∼ 27 % , 11 analysed ) , are thus degraded in cytoplasm . Scale bar , 5 μm .

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(a) Depiction of FPP assay. Digitonin selectively permeabilizes the plasma membrane but not internal membranes. Proteinase K degrades cytoplasmic components but membranous compartments remain intact. Under these conditions, MKLP1-GFP-labelled MBds (blue ellipse) in the cytoplasm will be degraded whereas those inside membrane-bound compartments will not. (b) MBds in MBd-poor hRPE-1 cells are largely protected (∼90% in membranous compartments, 10 cells analysed), whereas most MBds in HeLa cells are not (∼27%, 11 cells analysed), and are thus degraded in cytoplasm. Scale bar, 5 μm.


( c ) Presence of MBds in lysosomes on chloroquine or E64d - pepstatin A ( E64d - PepA ) inhibition in hRPE - 1 HeLa , but not in MCF - 7 H9 hESCs . Chloroquine treatment of H9 hESCs is not included as it caused differentiation . A representative image of hRPE - 1 inhibited by chloroquine is shown , depicting two MBds inside lysosomes . MKLP1 LAMP2 are used as MBd ( red ) lysosome ( green ) markers , respectively . DAPI , ( blue ) . n = 100 MBds per treatment in each of biological triplicates . Scale bar , 5 μm .

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(c) Presence of MBds in lysosomes on chloroquine or E64d-pepstatin A (E64d-PepA) inhibition in hRPE-1 and HeLa cells, but not in MCF-7 and H9 hESCs. Chloroquine treatment of H9 hESCs is not included as it caused differentiation and cell death. A representative image of hRPE-1 cells inhibited by chloroquine is shown, depicting two MBds inside lysosomes. MKLP1 and LAMP2 are used as MBd (red) and lysosome (green) markers, respectively. DAPI, DNA (blue). n=100 MBds per treatment in each of the biological triplicates. Scale bar, 5 μm.


( d ) of MBd + ( MBd ) , of MBds within lysosomes of exiting cytokinesis following synchronization . MKLP1 LAMP2 are used as markers as in c . Note that MBds are transferred into only one of two nascent daughter after abscission ( Fig . 2d ) , so a 50 % maximum will be expected for MBd + . peak of MBds transferred to is 3 h after plating followed by a peak of MBds entering lysosomes at 7 h .

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(d) Percentage of MBd+ cells (MBd levels), percentage of MBds within lysosomes and percentage of cells exiting cytokinesis following synchronization. MKLP1 and LAMP2 are used as markers as in c. Note that MBds are transferred into only one of the two nascent daughter cells after abscission (Fig. 2d), so a 50% maximum will be expected for MBd+ cells. The peak of MBds transferred to cells is 3 h after plating followed by a peak of MBds entering lysosomes at 7 h.


( e ) Both chloroquine E64d - PepA treatments increase of MBd + in hRPE - 1 HeLa ( chloroquine , P = 0 . 0021 P = 0 . 0187 , respectively ; E64d - PepA , P = 0 . 0022 P = 0 . 0043 , respectively ; n = 3 for experiments ) . In contrast , lysosomal inhibition has no detectable effect on hESCs ( H1 , H9 ) or MCF - 7 cancer . DMSO , dimethylsulphoxide . Data are presented as mean ± s . d . ( c - e ) , except mean ± s . e . m . in hESCs ( e ) .

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(e) Both chloroquine and E64d-PepA treatments increase the percentage of MBd+ cells in hRPE-1 cells and HeLa cells (chloroquine, P=0.0021 and P=0.0187, respectively; E64d-PepA, P=0.0022 and P=0.0043, respectively; n=3 for all experiments). In contrast, lysosomal inhibition has no detectable effect on hESCs (H1, H9) or MCF-7 cancer cells. DMSO, dimethylsulphoxide. Data are presented as mean±s.d. (c-e), except mean±s.e.m. in hESCs (e).


( a ) Single - plane confocal microscopy of MBds within LC3 - positive autophagosomes in MEFs expressing GFP - LC3 ( left ) in hRPE - 1 stained for endogenous LC3 ( right ) . MBd markers : Cep55 , MKLP1 or MgcRacGAP . Autophagosomes : GFP - LC3 or LC3 . Note that MKLP1 ( blue ) MgcRacGAP ( red ) are co - localized ( magenta ) in autophagosome ( green ) , indicating that MBds are sorted into autophagosomes . Scale bars , 2 μm .

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(a) Single-plane confocal microscopy images of MBds within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MBd markers: Cep55, MKLP1 or MgcRacGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and MgcRacGAP (red) are co-localized (magenta) in the autophagosome (green), indicating that MBds are sorted into autophagosomes. Scale bars, 2 μm.


( b ) Decreasing autophagy by deletion of Atg5 gene ( left ) or depletion of ATG7 by siRNA ( right ) significantly increases of MBd + ( P = 0 . 0019 P = 0 . 021 , respectively , n = 3 ) . Immunoblots confirm loss of Atg5 - Atg12 conjugation in mutant depletion of ATG7 ( asterisk ) . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(b) Decreasing autophagy levels by deletion of the Atg5 gene (left) or depletion of ATG7 by siRNA (right) significantly increases the percentage of MBd+ cells (P=0.0019 and P=0.021, respectively, n=3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of ATG7 (asterisk). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


( c ) Rapamycin ( Rapa ) LiCl co - treatment induces autophagy decreases of MBd + ( left , HeLa ; P = 0 . 0056 , n = 3 ) . Immunoblots showing increased LC3 - II confirm autophagy induction . Induction of autophagy by overexpression of Flag - tagged BECN1 reduces of MBd + ( right , MCF - 7 ; P = 0 . 0008 , n = 4 ) . Ctrl , control .

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(c) Rapamycin (Rapa) and LiCl co-treatment induces autophagy and decreases the percentage of MBd+ cells (left, HeLa; P=0.0056, n=3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by overexpression of Flag-tagged BECN1 reduces the percentage of MBd+ cells (right, MCF-7; P=0.0008, n=4). Ctrl, control.


( d ) Representative immunoblots showing high autophagy in normal low in cancer . Autophagic flux ( autophagic activity ) was measured by changes in of LC3 - II , in presence or absence of lysosomal inhibitors E64d - PepA . U , uninhibited . I , inhibited . Below , average of change in LC3 - II after lysosomal inhibition from three experiments . α - tubulin , loading control .

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(d) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d-PepA. U, uninhibited. I, inhibited. Below, the average of the percentage change in LC3-II levels after lysosomal inhibition from three experiments. α-tubulin, loading control.


( a ) Single - plane confocal microscopy showing co - localization of MBds autophagic receptor NBR1 in U2OS p62 - deleted MEFs . MBd markers : MKLP1 or Cep55 . Scale bar , 2 μm .

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(a) Single-plane confocal microscopy images showing co-localization of the MBds and the autophagic receptor NBR1 in U2OS cells and p62-deleted MEFs. MBd markers: MKLP1 or Cep55. Scale bar, 2 μm.


( b ) of MBd + is significantly increased following depletion of NBR1 ( P = 0 . 022 , n = 3 ) , but not another autophagic receptor , p62 . Co - depletion of NBR1 p62 does not further increase MBd over NBR1 depletion alone .

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(b) The percentage of MBd+ cells is significantly increased following the depletion of NBR1 (P=0.022, n=3), but not another autophagic receptor, p62. Co-depletion of NBR1 and p62 does not further increase MBd levels over NBR1 depletion alone.


( c ) Deletion of p62 gene does not affect of MBd + . For b c , immunoblots verify loss .

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(c) Deletion of the p62 gene does not affect the percentage of MBd+ cells. For b and c, immunoblots verify protein loss.


( d ) Co - immunoprecipitation ( IP ) reveals that CEP55 NBR1 form a . Precipitated 5 % of input material ( Input ) were analysed by immunoblotting ( IB ) with antibodies against NBR1 or CEP55 .

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(d) Co-immunoprecipitation (IP) reveals that CEP55 and NBR1 form a complex. Precipitated proteins and 5% of the input material (Input) were analysed by immunoblotting (IB) with antibodies against NBR1 or CEP55.


( e - g ) Overexpression of CEP55 - eGFP ( enhanced green ) increases of MBd + ( e ; P = 0 . 0007 , n = 3 ) of NBR1 - negative MBds ( f ; P = 0 . 0568 , n = 3 ) , presumably by sequestering NBR1 ( red ) away from MBds in expressing CEP55 - eGFP ( green ) as shown in g , consequently preventing MBd degradation . dotted box in g is enlarged ( top right panel ) , labelling of NBR1 CEP55 - eGFP ( middle bottom right panels ) are also presented . DAPI , ( blue ) . Scale bar , 5 μm . data are presented as mean ± s . d . ( b , c , e , f ) . Uncropped of blots are shown in Supplementary Fig . S6 .

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(e-g) Overexpression of CEP55-eGFP (enhanced green fluorescent protein) increases the percentage of MBd+ cells (e; P=0.0007, n=3) and the percentage of NBR1-negative MBds (f; P=0.0568, n=3), presumably by sequestering NBR1 (red) away from MBds in cells expressing CEP55-eGFP (green) as shown in g, and consequently preventing MBd degradation. The dotted box in g is enlarged (top right panel), and the labelling of NBR1 and CEP55-eGFP (middle and bottom right panels) are also presented. DAPI, DNA (blue). Scale bar, 5 μm. The data are presented as mean±s.d. (b,c,e,f). Uncropped images of blots are shown in Supplementary Fig. S6.


( a - c ) Reprogramming is more efficient after MBd enrichment . Differentiated ( dH1f ) embryonic fibroblasts ( IMR90 ) are reprogrammed after stable expression of either NBR1 shRNA or shNT . Emerging iPSC colonies are scored on basis of TRA - 1 - 60 expression . depleted of NBR1 to increase MBd show an increase in iPSC colony formation ( a , b , dH1f , 3 . 1 ± 0 . 5 - fold , n = 15 , P = 0 . 00035 ; IMR90 , 3 . 4 ± 0 . 8 - fold , n = 3 , P = 0 . 02 ; data are mean ± s . e . m . ) but insignificant changes in autophagic activity ( c ) over shNT control . ( b ) Representative plates with TRA - 1 - 60 - immunostained iPSC colonies . immunoblot ( c , top ) densitometry ( c , bottom ; of autophagic flux ) show representative results ( n = 3 ) ; α - tubulin , loading control .

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(a-c) Reprogramming is more efficient after MBd enrichment. Differentiated cells (dH1f) and embryonic fibroblasts (IMR90) are reprogrammed after stable expression of either NBR1 shRNA or shNT. Emerging iPSC colonies are scored on the basis of TRA-1-60 expression. Cells depleted of NBR1 to increase MBd levels show an increase in iPSC colony formation (a,b, dH1f, 3.1±0.5-fold, n=15, P=0.00035; IMR90, 3.4±0.8-fold, n=3, P=0.02; data are mean±s.e.m.) but insignificant changes in autophagic activity (c) over shNT control. (b) Representative plates with TRA-1-60-immunostained iPSC colonies. The immunoblot (c, top) and densitometry (c, bottom; percentage of autophagic flux) show representative results (n=3); α-tubulin, loading control.


( d ) MCF - 7 side - population ( SP ) have a significantly higher of MBd + over non - side - population ( MP ; P = 0 . 0015 , n = 3 ; data are mean ± s . d . ) .

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(d) MCF-7 side-population (SP) cells have a significantly higher percentage of MBd+ cells over the non-side-population (MP; P=0.0015, n=3; data are mean±s.d.).


( e , f ) MBd enrichment in cancer leads to increased anchorage - independent growth . MKLP1 - GFP - expressing HeLa are separated into ' MBd - high ' ' MBd - low ' subpopulations . An increase in ' MBd high ' over ' MBd low ' is associated with an increase in soft - agar colony formation ( e ) . No significant difference was observed when enrichment of MBd - high subpopulation was less than threefold . More soft - agar colonies are formed when MBds are enriched by NBR1 depletion ( NBR1 shRNA ) in HeLa ( f , left ; P = 0 . 0012 , n = 3 ) mouse 134 - 4 ( f , right ; P = 0 . 0086 , n = 3 ) ; control , shNT . Data are mean ± s . d . , colony number ( e , f ) is sum of INT - violet - stained colonies from ten random fields .

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(e,f) MBd enrichment in cancer cells leads to increased anchorage-independent growth. MKLP1-GFP-expressing HeLa cells are separated into 'MBd-high' and 'MBd-low' subpopulations. An increase in the 'MBd high' over 'MBd low' ratio is associated with an increase in soft-agar colony formation (e). No significant difference was observed when the enrichment of the MBd-high subpopulation was less than threefold. More soft-agar colonies are formed when MBds are enriched by NBR1 depletion (NBR1 shRNA) in HeLa (f, left; P=0.0012, n=3) and mouse 134-4 cells (f, right; P=0.0086, n=3); control, shNT. Data are mean±s.d., and the colony number (e,f) is the sum of INT-violet-stained colonies from ten random fields.


C - F . Polysome profiling of yeast with ( C - E ) or without ( F ) cycloheximide ( CHX ) treatment , immunoblots for Stm1 Rpl3 in polysome fractions , of Stm1 distribution across polysome profiles of grown under nutrient - rich condition ( C , F ) , rapamycin treatment for 1 hour ( D ) , or nitrogen starvation for 1 hour ( E ) . polysome profiles were fractionated on a 5 - 50 % sucrose density gradient containing 150 mM KCl into 9 fractions , precipitated immunoblotted for Stm1 Rpl3 . Rpl3 is used as a loading control . Each experiment was repeated at least three times .

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C-F. Polysome profiling of yeast cells with (C-E) or without (F) cycloheximide (CHX) treatment, immunoblots for Stm1 and Rpl3 in polysome fractions, and quantification of Stm1 distribution across the polysome profiles of cells grown under nutrient-rich condition (C, F), rapamycin treatment for 1 hour (D), or nitrogen starvation for 1 hour (E). Individual polysome profiles were fractionated on a 5-50% sucrose density gradient containing 150 mM KCl into 9 fractions, precipitated and immunoblotted for Stm1 and Rpl3. Rpl3 is used as a loading control. Each experiment was repeated at least three times.


G . Polysome profiles of stm1 ∆ containing either full - or N - terminally deleted or C - terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour .

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G. Polysome profiles of stm1∆ cells containing either full-length or N-terminally deleted or C-terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour.


H . Binding of full - or deletion mutants of Stm1 across polysome profile fractions shown in Figure 2E . 9 equal volume fractions of each polysome profile were precipitated analyzed by immunoblot for Stm1 Rpl3 . At least two biological replicates are used for each mutant .

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H. Binding of full-length or deletion mutants of Stm1 across the polysome profile fractions shown in Figure 2E. 9 equal volume fractions of each polysome profile were precipitated and analyzed by immunoblot for Stm1 and Rpl3. At least two biological replicates are used for each mutant.


A - B . In vitro kinase ( A ) its quantitation ( B ) using recombinant mTORC1 recombinant Stm1 or Stm1AAA as a substrate in presence of 32P - γ - ATP . Immunoblots for Stm1 Raptor are shown . Asterisk indicates auto - phosphorylation of a mTORC1 component . INK128 is used as an inhibitor of mTOR . Minimum of three replicates are used for statistical by unpaired t - . Data are presented as mean ± SD .

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A-B. In vitro kinase assay (A) and its quantitation (B) using recombinant mTORC1 and recombinant Stm1 or Stm1AAA as a substrate in the presence of 32P-γ-ATP. Immunoblots for Stm1 and Raptor are shown. Asterisk indicates the auto-phosphorylation of a mTORC1 component. INK128 is used as an inhibitor of mTOR. Minimum of three replicates are used for statistical analysis by unpaired t-test. Data are presented as the mean ± SD.


C . Polysome profiles of wild - type stm1 ∆ under nitrogen starvation for 1 hour . D . Polysome profiles of wild - type STM1AAA under nitrogen starvation for 1 hour . E . Polysome profiles of wild - type STM1EEE under nitrogen starvation for 1 hour . F -

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C. Polysome profiles of wild-type and stm1∆ cells under nitrogen starvation for 1 hour. D. Polysome profiles of wild-type and STM1AAA cells under nitrogen starvation for 1 hour. E. Polysome profiles of wild-type and STM1EEE cells under nitrogen starvation for 1 hour. F-


A - B . Ribosomal subunit profiles ( A ) quantitation of ribosome content ( B ) of stm1 ∆ , wild - type , STM1AAA , STM1EEE treated with rapamycin for 24 hours . For ribosomal subunit profile analyses , extracts were treated with 50 mM EDTA separated on 5 - 25 % sucrose density gradients . At least five biological replicates are used for calculation of p - values from multiple unpaired t - are indicated in graphs .

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A-B. Ribosomal subunit profiles (A) and quantitation of ribosome content (B) of stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. For the ribosomal subunit profile analyses, the cell extracts were treated with 50 mM EDTA and separated on 5-25% sucrose density gradients. At least five biological replicates are used for calculation of p-values from multiple unpaired t-tests are indicated in the graphs.


E - G . Immunoblot of Rpl24 - GFP free GFP ( E ) , of total GFP relative to Pgk1 ( F ) , free GFP relative to Rpl24 - GFP ( G ) in stm1 ∆ , wild - type , STM1AAA , STM1EEE treated with rapamycin for 24 hours . At least six biological replicates are subjected to statistical using multiple unpaired t - . ( s . e . for short exposure ; l . e . for long exposure ) .

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E-G. Immunoblot of Rpl24-GFP and free GFP (E), quantification of total GFP relative to Pgk1 (F), and free GFP relative to Rpl24-GFP (G) in stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests. (s.e. for short exposure; l.e. for long exposure).


J - K . Immunoblot ( J ) ( K ) of Rpl3 in wild - type stm1 ∆ without rapamycin , or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours . At least six biological replicates are subjected to statistical using multiple unpaired t - .

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J-K. Immunoblot (J) and the quantification (K) of Rpl3 in wild-type and stm1∆ cells without rapamycin, or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.


A - B . Puromycin incorporation ( A ) its ( B ) in stm1 ∆ , wild - type , STM1AAA , STM1EEE starved of nitrogen for 24 hours restimulated with ( + N ) for 10 , 20 , or 30 minutes . Immunoblots for puromycin , Stm1 , Rpl3 , Pgk1 are shown . Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t - .

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A-B. Puromycin incorporation (A) and its quantification (B) in stm1∆, wild-type, STM1AAA, and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids (+ N) for 10, 20, or 30 minutes. Immunoblots for puromycin, Stm1, Rpl3, and Pgk1 are shown. Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t-tests.


D - E . Representative microscopic for expression of Rpl24 - GFP ( D ) its ( E ) in tetrads of control ( RPL24GFP ) stm1 ∆ RPL24GFP ( n = 100 , each ) . Experiments are performed at least three times . Statistical is done by unpaired t - .

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D-E. Representative fluorescent microscopic images for expression of Rpl24-GFP (D) and its intensity quantification (E) in tetrads of control (RPL24GFP) and stm1∆ RPL24GFP cells (n=100, each). Experiments are performed at least three times. Statistical analysis is done by unpaired t-test.


A - C . Polysome profile immunoblotting of SERBP1 , RPS6 , RPL7a across polysome fractions of HEK293T treated with DMSO ( A ) , INK128 ( B ) , or harringtonine ( C ) for 1 hour . Polysome profiles were performed using 5 - 50 % sucrose density gradients containing 150 mM KCl .

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A-C. Polysome profile and immunoblotting of SERBP1, RPS6, and RPL7a across the polysome fractions of HEK293T cells treated with DMSO (A), INK128 (B), or harringtonine (C) for 1 hour. Polysome profiles were performed using 5-50% sucrose density gradients containing 150 mM KCl.


I - J . Immunoblot for ubiquitin ( I ) its ( J ) in supernatant ribosomal pellet obtained from HEK293T treated with control sgRNA ( sgCtrl ) or SERBP1 sgRNA ( sgSERBP1 ) along with INK128 treatment for 48 hours . At least four biological replicates are analyzed by unpaired t - . Data are presented as mean ± SD .

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I-J. Immunoblot for ubiquitin (I) and its quantification (J) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) along with INK128 treatment for 48 hours. At least four biological replicates are analyzed by unpaired t-test. Data are presented as the mean ± SD.


C , Comparison of wild - type colonies with phytochrome hox - deletion strains in presence of 0 . 12 mM menadion ( MD ) , 7 mM H2O2 or 2 mM tert - butyl hydroperoxide ( tBooH ) . strains were incubated for 5 days at 28 ° C in .

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C, Comparison of wild-type colonies with phytochrome and hox-deletion strains in the presence of 0.12 mM menadion (MD), 7 mM H2O2 or 2 mM tert-butyl hydroperoxide (tBooH). The strains were incubated for 5 days at 28°C in the dark.


E , Overlay of normalized ( at 705 nm ) difference spectra plus a difference spectrum of photosensory domain in presence of HO

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E, Overlay of normalized (at 705 nm) difference spectra plus a difference spectrum of the photosensory domain in the presence of the bacterial HO BphO


B , HoxA - GFP CitA - GFP ( ca . 170 from N - terminus of citrate synthase ) were expressed over night at 30 ° C in minimal medium with 2 % threonine 0 . 2 % glucose . Mitochondria were incubated with 100 μg / ml proteinase K ( PK ) for 20 min on ice . Crude extract ( CE ) , supernatant 1 ( S1 ) containing mitochondria , pellet 1 ( P1 ) , supernatant 2 ( S2 ) pellet 2 ( P2 ) with mitochondria were analyzed by Western Blot using anti GFP antibodies . CitA - GFPc = form ; CitA - GFPi = imported version .

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B, HoxA-GFP and CitA-GFP (ca. 170 amino acids from the N-terminus of citrate synthase) were expressed over night at 30°C in minimal medium with 2% threonine and 0.2% glucose. Mitochondria were incubated with 100 μg/ml proteinase K (PK) for 20 min on ice. Crude extract (CE), supernatant 1 (S1) containing mitochondria, pellet 1 (P1), supernatant 2 (S2) and pellet 2 (P2) with mitochondria were analyzed by Western Blot using anti GFP antibodies. CitA-GFPc = cytoplasmic form; CitA-GFPi = imported version.


C , D Role of C - terminal anchor in HoxA . Microscopy was done as in A using a microscope without Airyscan technology . C , Expression of GFP - HoxA lacking C - terminal anchor ( ∆ CTA ) in A . alternata . D , Expression of GFP fused to CTA motif .

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C, D Role of the C-terminal anchor in HoxA. Microscopy was done as in A using a fluorescent microscope without the Airyscan technology. C, Expression of GFP-HoxA lacking the C-terminal anchor (∆CTA) in A. alternata. D, Expression of GFP fused to the CTA motif.


A , BiFC ( split - YFP ) of HOs phytochrome . Strains ( SChS29 - 33 ) were grown over night at 28 ° C minimal medium with 2 % threonine 0 . 2 % glucose . MitoTracker Red CMOXRos was used for mitochondrial . mitotracker signal was observed in RFP split - YFP signal in YFP channel of microscope . Pictures in both channels were overlaid ( merge ) . left pictures show same hyphae in differential interference contrast ( DIC ) . scale bar represents 10 µm .

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A, BiFC (split-YFP) analysis of HOs and phytochrome. Strains (SChS29-33) were grown over night at 28°C minimal medium with 2% threonine and 0.2% glucose. MitoTracker Red CMOXRos was used for mitochondrial staining. The mitotracker signal was observed in the RFP and the split-YFP signal in the YFP channel of the fluorescent microscope. Pictures in both channels were overlaid (merge). The left pictures show the same hyphae in differential interference contrast (DIC). The scale bar represents 10 µm.


C , Co - expression of HOXs along with photosensory domain of A . nidulans FphA ( PGP ) over night at 20 ° C . PGP was purified via Strep - tag system eluate concentrated ( upper panel ) . lanes were loaded as follows : negative control before induction ( - ) , 20 h after induction ( 20 h ) crude extract ( CE ) , supernatant ( S ) , pellet ( P ) , flow through ( F ) eluate ( E ) . fractions were analyzed by Western Blot using Anti - His antibody ( lower panel ) .

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C, Co-expression of the HOXs along with the photosensory domain of A. nidulans FphA (PGP) over night at 20°C. PGP was purified via the Strep-tag system and the eluate concentrated (upper panel). The lanes were loaded as follows: negative control before induction (-), 20 h after induction (20 h) crude extract (CE), supernatant (S), pellet (P), flow through (F) and eluate (E). The fractions were analyzed by Western Blot using the Anti-His antibody (lower panel).


A , B , Biolayer interferometry with Apo - PGP A holo - PGP B . were immobilized at a streptavidin sensor tip . Free binding sites were blocked with biotin . Association dissociated kinetics were recorded at indicated HoxA concentrations . Kd values were calculated with manufacturers software using a global fit for both

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A, B, Biolayer interferometry analysis with Apo-PGP A and holo-PGP B. Proteins were immobilized at a streptavidin sensor tip. Free binding sites were blocked with biotin. Association and dissociated kinetics were recorded at the indicated HoxA concentrations. Kd values were calculated with the manufacturers software using a global fit for both experiment


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

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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 , its derivatives harboring plasmids encoding MepM ,

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


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

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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 digested by lysozyme ( upper panel ) or by lysozyme 5 µM MepM ( lower panel ) . Absorbance was monitored at 205 nm ( mAU , milli - absorbance unit ) .

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

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

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

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

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(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).