( 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 .

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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 ) The KO - GFP mouse was visibly smaller and 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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 line ) and with rAVV9 - eGFP ( green line ) . WTFlx mice are depicted in black . rAAV9 - Ndufs3 injections extended the survival of the 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 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 .

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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 ) Quantification of the BN - PAGE western blots from female 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 and CIV in quadriceps of 6 - month - old mice . ( F ) Quantification of BN - PAGE in - gel activities in females .

(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 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 .

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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 ratio was measured in quadriceps homogenates of 6 - month - old males and females .

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

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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 ) 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 .

O O O O B-TISSUE B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

(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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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) 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 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 .

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

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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 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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.


Population doublings ( PDs ) determined in BJ cells cultured with and without folate for 28 days .

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Population doublings (PDs) determined in BJ cells cultured with and without folate for 28 days.


Example of a single combed DNA molecule labeled with IdU ( green ) and CldU ( red ) , showing replication from three adjacent origins . Horizontal white arrows indicate fork orientation .

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Example of a single combed DNA molecule labeled with IdU (green) and CldU (red), showing replication from three adjacent origins. Horizontal white arrows indicate fork orientation.


Representative examples of single combed DNA molecules from control cells and cells grown for 7 days in a folate - free medium .

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Representative examples of single combed DNA molecules from control cells and cells grown for 7 days in a folate-free medium.


Fork rate ( Kb / min ) distribution . Light blue bars : BJ cells ( n = 126 ) ; black bars : BJ cells that were cultured for 7 days in a folate - free medium ( n = 131 ) ; blue bars : BJ cells cultured for 7 days in a folate - free medium and supplemented with A , G , C , and T nucleosides for the last 48 h of the experiment ( n = 138 ) . Box plot summarizing the fork rate distribution ( Kb / min ) of three independent experiments . Control ( n = 360 ) ; − folate ( n = 372 ) ; − folate + AGCT ( n = 361 ) .

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Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 126); black bars: BJ cells that were cultured for 7 days in a folate-free medium (n = 131); blue bars: BJ cells cultured for 7 days in a folate-free medium and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 138).Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 360); −folate (n = 372); −folate + AGCT (n = 361).


Fork distance ( Kb ) distribution . The color code is as in ( D ) . Control ( n = 72 ) ; − folate ( n = 71 ) ; − folate + AGCT ( n = 75 ) . Box plot summarizing the fork distance distribution ( Kb ) of three independent experiments . Control ( n = 212 ) ; − folate ( n = 215 ) ; − folate + AGCT ( n = 209 ) .

B-SUBCELLULAR 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-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O

Fork distance (Kb) distribution. The color code is as in (D). Control (n = 72); −folate (n = 71); −folate + AGCT (n = 75).Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 212); −folate (n = 215); −folate + AGCT (n = 209).


A Population doublings ( PDs ) determined in BJ cells cultured at the indicated folate concentrations for 48 days .

O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O

A Population doublings (PDs) determined in BJ cells cultured at the indicated folate concentrations for 48 days.


B , C The average replication rate ± SEM ( B ) and the average fork distance ± SEM ( C ) in the indicated folate concentrations at 14 and 21 days . At least 115 DNA fibers were analyzed at each concentration and at each time point to determine the average replication rate . At least 71 replication forks were analyzed at each concentration and at each time point to determine the average fork distance .

O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O

B, C The average replication rate ± SEM (B) and the average fork distance ± SEM (C) in the indicated folate concentrations at 14 and 21 days. At least 115 DNA fibers were analyzed at each concentration and at each time point to determine the average replication rate. At least 71 replication forks were analyzed at each concentration and at each time point to determine the average fork distance.


D - G BJ cells were grown for 14 days in 100 nM folate with and without nucleoside supplementation . ( D ) Fork rate ( Kb / min ) distribution . Light blue bars : BJ cells ( n = 115 ) ; gray bars : BJ cells that were cultured for 14 days in 100 nM folate ( n = 117 ) ; blue bars : BJ cells cultured for 14 days in 100 nM folate and supplemented with A , G , C , and T nucleosides for the last 48 h of the experiment ( n = 117 ) . ( E ) Box plot summarizing the fork rate distribution ( Kb / min ) of three independent experiments . Control ( n = 352 ) ; 100 nM folate ( n = 364 ) ; 100 nM folate + AGCT ( n = 355 ) . Main box represents the values from the lower to upper quartile ( 25th to 75th percentile ) . The middle line represents the median . ( F ) Fork distance ( Kb ) distribution . The color code is as in ( D ) . Control ( n = 69 ) ; 100 nM folate ( n = 74 ) ; 100 nM folate + AGCT ( n = 72 ) . ( G ) Box plot summarizing the fork distance distribution ( Kb ) of three independent experiments . Control ( n = 201 ) ; 100 nM folate ( n = 220 ) ; 100 nM folate + AGCT ( n = 228 ) . Main box represents the values from the lower to upper quartile ( 25th to 75th percentile ) . The middle line represents the median . * * P < 0 . 001 .

O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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-SUBCELLULAR O 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-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-G BJ cells were grown for 14 days in 100 nM folate with and without nucleoside supplementation. (D) Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 115); gray bars: BJ cells that were cultured for 14 days in 100 nM folate (n = 117); blue bars: BJ cells cultured for 14 days in 100 nM folate and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 117). (E) Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 352); 100 nM folate (n = 364); 100 nM folate + AGCT (n = 355). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. (F) Fork distance (Kb) distribution. The color code is as in (D). Control (n = 69); 100 nM folate (n = 74); 100 nM folate + AGCT (n = 72). (G) Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 201); 100 nM folate (n = 220); 100 nM folate + AGCT (n = 228). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. **P < 0.001.


Cyclin E - expressing BJ cells were grown for 7 days with and without folate . Fork rate ( Kb / min ) distribution . White bars : BJ cells expressing an empty vector ( n = 145 ) ; dark gray bars : BJ cells expressing the cyclin E oncogene ( n = 147 ) ; light gray bars : BJ cells cultured for 7 days in a folate - free medium ( n = 135 ) ; black bars : BJ cells expressing the cyclin E oncogene cultured for 7 days in a folate - free medium ( n = 138 ) .

B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Fork rate (Kb/min) distribution. White bars: BJ cells expressing an empty vector (n = 145); dark gray bars: BJ cells expressing the cyclin E oncogene (n = 147); light gray bars: BJ cells cultured for 7 days in a folate-free medium (n = 135); black bars: BJ cells expressing the cyclin E oncogene cultured for 7 days in a folate-free medium (n = 138).


Cyclin E - expressing BJ cells were grown for 7 days with and without folate . Fork distance distribution ( Kb ) . The color code is as in ( A ) . Empty vector ( n = 78 ) ; CycE ( n = 79 ) ; empty vector − folate ( n = 71 ) ; CycE − folate ( n = 80 ) .

B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR 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-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O

Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Fork distance distribution (Kb). The color code is as in (A). Empty vector (n = 78); CycE (n = 79); empty vector −folate (n = 71); CycE −folate (n = 80).


Cyclin E - expressing BJ cells were grown for 7 days with and without folate . Percent of origins with the indicated progression ratio between sister forks . Empty vector ( n = 158 ) ; CycE ( n = 155 ) ; empty vector − folate ( n = 160 ) ; CycE − folate ( n = 154 ) . * P < 0 . 05 .

B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O

Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Percent of origins with the indicated progression ratio between sister forks. Empty vector (n = 158); CycE (n = 155); empty vector −folate (n = 160); CycE −folate (n = 154). *P < 0.05.


Cyclin E - expressing BJ cells were grown for 7 days with and without folate . Examples of nuclei with γH2AX and 53BP1 foci following cyclin E expression ( CycE ) ( n = 65 ) , empty vector ( n = 65 ) , folate - free medium for 7 days ( empty vector − folate ) ( n = 67 ) or oncogene expression under folate - free conditions ( CycE − folate ) ( n = 70 ) . Red : γH2AX ; green : 53BP1 ; blue : DAPI staining . Percent of nuclei with the indicated number of γH2AX - 53BP1 co - localized foci . * * P < 0 . 01 .

B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O

Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Examples of nuclei with γH2AX and 53BP1 foci following cyclin E expression (CycE) (n = 65), empty vector (n = 65), folate-free medium for 7 days (empty vector −folate) (n = 67) or oncogene expression under folate-free conditions (CycE −folate) (n = 70). Red: γH2AX; green: 53BP1; blue: DAPI staining.Percent of nuclei with the indicated number of γH2AX-53BP1 co-localized foci. **P < 0.01.


Cyclin E - expressing BJ cells grown for 7 days with and without folate . Immunoblotting with anti - phosphorylated ATM and anti - phosphorylated CHK1 antibodies . Anti - β - catenin and anti - actin antibodies were used as loading controls . Protein level quantification .

B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O

Cyclin E-expressing BJ cells grown for 7 days with and without folate.Immunoblotting with anti-phosphorylated ATM and anti-phosphorylated CHK1 antibodies. Anti-β-catenin and anti-actin antibodies were used as loading controls.Protein level quantification.


Cyclin E - expressing BJ cells grown for 7 days with and without folate . Examples of nuclei with RAD51 foci following cyclin E expression ( CycE ) ( n = 67 ) , empty vector ( n = 65 ) , folate - free medium for 7 days ( − folate ) ( n = 71 ) or oncogene expression under folate - free conditions ( CycE − folate ) ( n = 75 ) . Green : RAD51 , blue : DAPI staining . Percent of nuclei with the indicated number of RAD51 foci ( n = 66 ) . * * P < 0 . 01 .

B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O

Cyclin E-expressing BJ cells grown for 7 days with and without folate.Examples of nuclei with RAD51 foci following cyclin E expression (CycE) (n = 67), empty vector (n = 65), folate-free medium for 7 days (−folate) (n = 71) or oncogene expression under folate-free conditions (CycE −folate) (n = 75). Green: RAD51, blue: DAPI staining.Percent of nuclei with the indicated number of RAD51 foci (n = 66). **P < 0.01.


A , B Cyclin E - and Ras ( H - RasV12 ) - expressing 3T3 cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium . Control cells were grown in a normal medium for the whole period . ( A ) Examples of anchorage - independent growth in soft agar of 3T3 cells . ( B ) Average number of colonies per soft agar plate of 3T3 cells . The number of colonies per plate is expressed as the average ± SEM from three independent experiments .

O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O

A, B Cyclin E- and Ras (H-RasV12)-expressing 3T3 cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (A) Examples of anchorage-independent growth in soft agar of 3T3 cells. (B) Average number of colonies per soft agar plate of 3T3 cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments.


C - E Ras ( H - RasV12 ) - expressing MCF10A cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium . Control cells were grown in a normal medium for the whole period . ( C ) Examples of anchorage - independent growth in soft agar of MCF10A cells . ( D ) Average number of colonies per soft agar plate of MCF10A cells . The number of colonies per plate is expressed as the average ± SEM from three independent experiments . ( E ) Percentage of tumor - free flanks at the indicated time points after cell injection . Ten mice were injected in both sides in each group .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-CELL_LINE O O O O O O O O O O O O O O B-CELL_LINE O 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-TISSUE O O B-TISSUE O O O O O O O O O O B-ORGANISM O O O O O O O O O

C-E Ras (H-RasV12)-expressing MCF10A cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (C) Examples of anchorage-independent growth in soft agar of MCF10A cells. (D) Average number of colonies per soft agar plate of MCF10A cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments. (E) Percentage of tumor-free flanks at the indicated time points after cell injection. Ten mice were injected in both sides in each group.


C ) Time course analysis of triubiquitin ( 2 µM ) hydrolysis using 250 nM SARS2 PLpro , resolved on a Coomassie stained SDS - PAGE gel . Linkage specific cleavage of Lys48 - linked triubiquitin to di - and monoubiquitin resembles SARS PLpro activity ( Békés et al , 2015 ; 2016 ) . See Source Data for uncropped gels , and Appendix Fig S1B - D for gel - based cleavage quantification .

O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-ORGANISM O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O

C) Time course analysis of triubiquitin (2 µM) hydrolysis using 250 nM SARS2 PLpro, resolved on a Coomassie stained SDS-PAGE gel. Linkage specific cleavage of Lys48-linked triubiquitin to di- and monoubiquitin resembles SARS PLpro activity (Békés et al, 2015; 2016). See Source Data for uncropped gels, and Appendix Fig S1B-D for gel-based cleavage quantification.


B ) Fluorescence polarisation assays against ubiquitin - TAMRA and ISG15 - TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n = 2 for each mutant , and compared to wild - type PLpro as shown in Appendix Fig S1A . Catalytic efficiencies were calculated as described in Methods .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O B-ORGANISM O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O

B) Fluorescence polarisation assays against ubiquitin-TAMRA and ISG15-TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n=2 for each mutant, and compared to wild-type PLpro as shown in Appendix Fig S1A. Catalytic efficiencies were calculated as described in Methods.


C ) Gel based analysis of PLpro variant activity against Lys48 - triubiquitin and proISG15 . Experiments were performed in duplicate , see Fig EV3 and Source Data for uncropped gels .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O

C) Gel based analysis of PLpro variant activity against Lys48-triubiquitin and proISG15. Experiments were performed in duplicate, see Fig EV3 and Source Data for uncropped gels.


C ) Calculated enzymatic efficiencies for K48 - diUb - TAMRA cleavage , followed by fluorescence polarisation with PLpro wild - type ( reproduced from Appendix Fig S1A ) , and PLpro F69S . ( Left ) Using the same concentration range of SARS2 PLpro , the F69S mutant activity could not be fitted ; ( middle ) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin - TAMRA ( 45 vs . 86 M - 1s - 1 , compare Fig 1D , Appendix Fig S1A ) . ( Right ) A ~ 3 - fold lower efficiency for PLpro F69S cleaving ISG15 - TAMRA yields values similar to cleavage of ISG15CTD - TAMRA ( Appendix Fig S1A ) , suggesting that the S2 site contributes the difference in binding for the N - terminal Ubl - fold . Experiments for F69S were performed in technical triplicate and biological duplicate ; wild - type data is reproduced from experiments shown in Fig 1D , Appendix Fig S1A .

O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O 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-ORGANISM O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Calculated enzymatic efficiencies for K48-diUb-TAMRA cleavage, followed by fluorescence polarisation with PLpro wild-type (reproduced from Appendix Fig S1A), and PLpro F69S. (Left) Using the same concentration range of SARS2 PLpro, the F69S mutant activity could not be fitted; (middle) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin-TAMRA (45 vs. 86 M-1s-1, compare Fig 1D, Appendix Fig S1A). (Right) A ~3-fold lower efficiency for PLpro F69S cleaving ISG15-TAMRA yields values similar to cleavage of ISG15CTD-TAMRA (Appendix Fig S1A), suggesting that the S2 site contributes the difference in binding for the N-terminal Ubl-fold. Experiments for F69S were performed in technical triplicate and biological duplicate; wild-type data is reproduced from experiments shown in Fig 1D, Appendix Fig S1A.


D - F ) Gel based analysis showing hydrolysis time course of triubiquitin ( D ) , proISG15CTD ( E ) and proISG15 ( F ) using wild - type PLpro ( left , wild - type gels reproduced from Appendix Fig S1B - D to enable direct comparison ) or PLpro F69S ( right ) . PLpro F69S has a strong effect on triubiquitin hydrolysis ( D ) , no marked effect on hydrolysis of proISG15CTD ( E ) and reduces proISG15 cleavage to the same levels as proISG15CTD ( compare E , F ) , Experiments were performed in duplicate , see Source Data for uncropped gels .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

D-F) Gel based analysis showing hydrolysis time course of triubiquitin (D), proISG15CTD (E) and proISG15 (F) using wild-type PLpro (left, wild-type gels reproduced from Appendix Fig S1B-D to enable direct comparison) or PLpro F69S (right). PLpro F69S has a strong effect on triubiquitin hydrolysis (D), no marked effect on hydrolysis of proISG15CTD (E) and reduces proISG15 cleavage to the same levels as proISG15CTD (compare E, F), Experiments were performed in duplicate, see Source Data for uncropped gels.


B ) Hit compounds and compound rac5c ( see Fig 5 ) , were further assessed in 10 - point IC50 titrations using the Ub - Rhodamine assay , using a starting concentration of 100 µM serially diluted in 1 : 3 steps . Degree of inhibition is shown as a titration heat - map from dark ( full inhibition ) to light blue ( low / no inhibition ) . The catalytic domain of human USP21 ( Ye et al , 2011 ) was used as a counterscreen . Each PLpro hit compound showed either no activity in the titration analysis , or an identical inhibition profile against PLpro and USP21 , suggesting assay interference . Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM . IC50 assays were performed in technical triplicate in two independent experiments .

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

B) Hit compounds and compound rac5c (see Fig 5), were further assessed in 10-point IC50 titrations using the Ub-Rhodamine assay, using a starting concentration of 100 µM serially diluted in 1:3 steps. Degree of inhibition is shown as a titration heat-map from dark (full inhibition) to light blue (low/no inhibition). The catalytic domain of human USP21 (Ye et al, 2011) was used as a counterscreen. Each PLpro hit compound showed either no activity in the titration analysis, or an identical inhibition profile against PLpro and USP21, suggesting assay interference. Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM. IC50 assays were performed in technical triplicate in two independent experiments.


C ) In vitro inhibition ( IC50 ) for rac5c inhibiting SARS2 PLpro . Experiments were performed using the HTS assay ( Fig 4 ) , in technical triplicate in three independent experiments . A geometric mean was used to determine IC50 .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-SMALL_MOLECULE B-EXP_ASSAY B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

C) In vitro inhibition (IC50) for rac5c inhibiting SARS2 PLpro. Experiments were performed using the HTS assay (Fig 4), in technical triplicate in three independent experiments. A geometric mean was used to determine IC50.


D ) Full - length nsp3 was expressed from a C - terminally GFP - tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h . GFP is released from the C - terminus , presumably by nsp3 protease activity . Nsp3 can be detected by a SARS / SARS2 PLpro antibody ( see Fig EV5E for antibody validation ) . Lysates were blotted for Lys48 - linked polyubiquitin with a linkage specific antibody ( K48 ) . Experiments were performed in duplicate with similar results . Also see Fig EV5F , G and Source Data for uncropped blots .

O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O 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-EXP_ASSAY O

D) Full-length nsp3 was expressed from a C-terminally GFP-tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h. GFP is released from the C-terminus, presumably by nsp3 protease activity. Nsp3 can be detected by a SARS/SARS2 PLpro antibody (see Fig EV5E for antibody validation). Lysates were blotted for Lys48-linked polyubiquitin with a linkage specific antibody (K48). Experiments were performed in duplicate with similar results. Also see Fig EV5F, G and Source Data for uncropped blots.


B ) Reduction in SARS - CoV - 2 induced cytopathic effect with rac5c , Remdesivir ( RDV ) and hydroxychloroquine ( HCQ ) treatment . DMSO 0 . 3 % ( v / v ) was required to keep 33 µM rac5c in solution ( see Fig EV6A , B ) . Mean ( black line ) is provided for 18 samples in each group , representing 3 independent experiments with 6 biological replicates per experiment across the different concentrations of rac5c . HCQ data is pooled from 2 independent experiments and RDV from 1 experiment using 6 biological repeats . P values were calculated using a one - way ANOVA , with regular Dunnet ' s post - hoc test for multiple comparisons between treatment arms and infected / vehicle treated control using a single pooled variance .

O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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) Reduction in SARS-CoV-2 induced cytopathic effect with rac5c, Remdesivir (RDV) and hydroxychloroquine (HCQ) treatment. DMSO 0.3% (v/v) was required to keep 33 µM rac5c in solution (see Fig EV6A, B). Mean (black line) is provided for 18 samples in each group, representing 3 independent experiments with 6 biological replicates per experiment across the different concentrations of rac5c. HCQ data is pooled from 2 independent experiments and RDV from 1 experiment using 6 biological repeats. P values were calculated using a one-way ANOVA, with regular Dunnet's post-hoc test for multiple comparisons between treatment arms and infected/vehicle treated control using a single pooled variance.


C ) TCID50 data , mean and SD , for one representative experiment from B with 6 technical replicates .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

C) TCID50 data, mean and SD, for one representative experiment from B with 6 technical replicates.


( A - F ) TNF - α , Cxcl1 and Cxcl2 ( A , C , and E respectively ) were measured at 24 h after stimulation of bone - marrow - derived neutrophils ( 5 x 105 cells ) with increasing concentrations of bacterial nucleic acids ( 1 , 5 , 10 and 20 µg of DNA + RNA in equal proportions ) , HK - bacteria ( 1 , 5 , 10 or 20 μg / ml ) or live bacteria ( MOIs of 2 , 5 , 10 and 20 ) . LPS ( 1 , 10 , 100 and 1000 ng / ml ) was used as a positive control stimulus . ( B , D and F ) , Effect of FPR antagonist Boc - 2 on GBS - induced TNF - α , Cxcl1 and Cxcl2 production , respectively . Bone - marrow - derived neutrophils were exposed to Boc - 2 ( 50 µM ) for 1 hour and then stimulated with HK - GBS ( 10 µg / ml ) or live GBS ( MOI of 5 ) . Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test ; ns , not significant .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O 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-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) TNF-α, Cxcl1 and Cxcl2 (A, C, and E respectively) were measured at 24 h after stimulation of bone-marrow-derived neutrophils (5 x 105 cells) with increasing concentrations of bacterial nucleic acids (1, 5, 10 and 20 µg of DNA + RNA in equal proportions), HK-bacteria (1, 5, 10 or 20 μg/ml) or live bacteria (MOIs of 2, 5, 10 and 20). LPS (1, 10, 100 and 1000 ng/ml) was used as a positive control stimulus. (B, D and F), Effect of FPR antagonist Boc-2 on GBS-induced TNF-α, Cxcl1 and Cxcl2 production, respectively. Bone-marrow-derived neutrophils were exposed to Boc-2 (50 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.


Bone - marrow - derived neutrophils were exposed to Boc - 2 ( 10 µM ) or WRW4 ( 1 , 5 and 10 µM ) for 1 hour and then stimulated with HK - GBS ( 10 µg / ml ) or live GBS ( MOI of 5 ) . Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test ; ns , not significant .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bone-marrow-derived neutrophils were exposed to Boc-2 (10 µM) or WRW4 (1, 5 and 10 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.


( A - F ) Cxcl1 and Cxcl2 concentrations in 24 - h culture supernatants of neutrophils ( A and B ) , CSF - polarized ( C and D ) or GM - CSF - polarized ( E and F ) BMDMs ( ΜΦ ) from WT or Fpr - deficient mice stimulated with live GBS ( MOI of 2 , 5 , and 10 ) or HK - GBS ( 10 µg / ml ) . LPS ( 100 ng / ml ) was used as a positive control stimulus . Means + SD of data from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 vs WT mice , determined by unpaired t - test .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O

(A-F) Cxcl1 and Cxcl2 concentrations in 24-h culture supernatants of neutrophils (A and B), CSF-polarized (C and D) or GM-CSF-polarized (E and F) BMDMs (ΜΦ) from WT or Fpr-deficient mice stimulated with live GBS (MOI of 2, 5, and 10) or HK-GBS (10 µg/ml). LPS (100 ng/ml) was used as a positive control stimulus. Means + SD of data from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs WT mice, determined by unpaired t-test.


( E ) , Kaplan - Meier analysis of survival rates in WT , Fpr1 and Fpr2 KO mice after i . p . challenge with 2x105 bacteria . Data are the cumulative results from two experiments , each involving 8 animals per group . * * P < 0 . 01 , * * * * P < 0 . 0001 vs WT mice , as determined by Kaplan - Meier analysis . Data information : means + SDs of three duplicate determinations , each conducted in a different animal .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(E), Kaplan-Meier analysis of survival rates in WT, Fpr1 and Fpr2 KO mice after i.p. challenge with 2x105 bacteria. Data are the cumulative results from two experiments, each involving 8 animals per group. **P < 0.01, ****P < 0.0001 vs WT mice, as determined by Kaplan-Meier analysis. Data information: means + SDs of three duplicate determinations, each conducted in a different animal.


( F - G ) Neutrophil influx ( F ) and bacterial burden ( G ) in peritoneal lavage fluid samples collected from Fpr2 - / - mice at 3 h after i . p . challenge with live GBS ( 2x107 CFU ) . At 1h post - challenge mice were inoculated i . p . with 1 μg of recombinant Cxcl1 or Cxcl2 or with vehicle . Data information : , individual data points and means . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle treated mice determined by unpaired t test or Mann - Whitney U

O O O O O B-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE I-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O

(F-G) Neutrophil influx (F) and bacterial burden (G) in peritoneal lavage fluid samples collected from Fpr2-/- mice at 3 h after i.p. challenge with live GBS (2x107 CFU). At 1h post-challenge mice were inoculated i.p. with 1 μg of recombinant Cxcl1 or Cxcl2 or with vehicle. Data information: , individual data points and means. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle treated mice determined by unpaired t test or Mann-Whitney U test


( C and D ) , Cxcl2 release in neutrophil cultures stimulated with combinations of HK - bacteria , GBS fMet peptides or TLR agonists . Neutrophils were exposed to a 50 µM concentration of Fpr agonists ( fMIFL , WKYMVM or fPep8 and fPep10 ) for 1 h before the addition of the indicated TLR agonists , which included HK - GBS ( 10 μg / ml ) , LPS ( 1 µg / ml ) and RNA ( 1 µg / ml ) . In some cases , neutrophils were exposed to graded doses ( 10 , 20 and 50 µM ) of the indicated Fpr agonists alone . Data information : Data are expressed as means + SD from three independent experiments , each conducted in duplicate . * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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 and D), Cxcl2 release in neutrophil cultures stimulated with combinations of HK-bacteria, GBS fMet peptides or TLR agonists. Neutrophils were exposed to a 50 µM concentration of Fpr agonists (fMIFL, WKYMVM or fPep8 and fPep10) for 1 h before the addition of the indicated TLR agonists, which included HK-GBS (10 μg/ml), LPS (1 µg/ml) and RNA (1 µg /ml). In some cases, neutrophils were exposed to graded doses (10, 20 and 50 µM) of the indicated Fpr agonists alone. Data information: Data are expressed as means + SD from three independent experiments, each conducted in duplicate. **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( A ) , Effect of actinonin on Cxcl2 production by neutrophils . Neutrophils were stimulated with live GBS ( MOI 5 ) in the presence or absence of the formyl peptidase inhibitor actinonin ( 32µg / ml ) . Data information : Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-CELL_TYPE O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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), Effect of actinonin on Cxcl2 production by neutrophils. Neutrophils were stimulated with live GBS (MOI 5) in the presence or absence of the formyl peptidase inhibitor actinonin (32µg/ml). Data information: Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( D ) , Effect of treatment with ROS scavengers / inhibitors on Cxcl2 production . Neutrophils were treated with the singlet oxygen scavenger MnTBAP ( 100 μM ) , the hydrogen peroxide scavenger DMTU ( 10 mM ) , the NADPH oxidase inhibitor DPI ( 20 μM ) or the ASK1 inhibitor GS - 444217 ( 1 μM ) for 1 h before the addition of live GBS ( MOI of 5 ) . Data information : Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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), Effect of treatment with ROS scavengers/inhibitors on Cxcl2 production. Neutrophils were treated with the singlet oxygen scavenger MnTBAP (100 μM), the hydrogen peroxide scavenger DMTU (10 mM), the NADPH oxidase inhibitor DPI (20 μM) or the ASK1 inhibitor GS-444217 (1 μM) for 1 h before the addition of live GBS (MOI of 5). Data information: Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( A - B ) Bone - marrow - derived neutrophils were exposed to different concentrations of signaling molecule ( A ) or transcription factors inhibitors ( B ) for 1 hour and then stimulated with live GBS ( MOI 5 ) . Target molecules are indicated in parentheses . Data are means + SD from three independent experiments conducted in duplicate . * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Bone-marrow-derived neutrophils were exposed to different concentrations of signaling molecule (A) or transcription factors inhibitors (B) for 1 hour and then stimulated with live GBS (MOI 5). Target molecules are indicated in parentheses. Data are means + SD from three independent experiments conducted in duplicate. **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( C ) , Effect of p38MAPK inhibition on the activation of AP - 1 family members in neutrophils stimulated with live GBS . Neutrophils were pretreated with SB202190 ( 5μM ) , stimulated for 90min with live GBS ( MOI 5 ) , lysed and assayed for AP - 1 activation . Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * * P < 0 . 001 , as determined by unpaired t - test .

O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-CELL_TYPE O O O B-ORGANISM O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O 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), Effect of p38MAPK inhibition on the activation of AP-1 family members in neutrophils stimulated with live GBS. Neutrophils were pretreated with SB202190 (5μM), stimulated for 90min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001, as determined by unpaired t-test.


( D ) , Effect of the absence of Fpr1 , Fpr2 or the TLR adaptor MyD88 on the activation of AP - 1 family members in neutrophils . Neutrophils from WT , Fpr - or MyD88 - deficient mice were stimulated for 90 min with live GBS ( MOI 5 ) , lysed and assayed for AP - 1 activation . Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * * P < 0 . 001 vs WT mice , as determined by unpaired t - test .

O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O 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-ORGANISM O O O O O O O O O

(D), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on the activation of AP-1 family members in neutrophils. Neutrophils from WT, Fpr- or MyD88-deficient mice were stimulated for 90 min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001 vs WT mice, as determined by unpaired t-test.


( E ) , Effect of TAK1 inhibition on p38 MAPK activation in neutrophils stimulated with live GBS ( MOI 5 ) .

O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O O O O O

(E), Effect of TAK1 inhibition on p38 MAPK activation in neutrophils stimulated with live GBS (MOI 5).


( F ) , Effect of the absence of Fpr1 , Fpr2 or the TLR adaptor MyD88 on p38MAPK activation in neutrophils stimulated with live GBS ( MOI 5 ) . Shown is a blot representative of three separate experiments .

O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O O O O

(F), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on p38MAPK activation in neutrophils stimulated with live GBS (MOI 5). Shown is a blot representative of three separate experiments.


b ) Histograms of GFP expression ( x - axis ) are presented from A549 cells transduced with the different viral vectors .

O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O O O

b) Histograms of GFP expression (x-axis) are presented from A549 cells transduced with the different viral vectors.


c ) Percentage of GFP positive cells from the experiment described in ( b ) .

O O O O B-GENEPROD O O O O O O O O O O O

c) Percentage of GFP positive cells from the experiment described in (b).


. d ) A PCR assay along with primers , as illustrated in Appendix Figure S2a , was used to detect formation of LTR junction and thereby circularization of the vector . The expected size of PCR product is 552 bp from a 1 - LTR circle and 1029 bp from a 2 - LTR circle .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

. d) A PCR assay along with primers, as illustrated in Appendix Figure S2a, was used to detect formation of LTR junction and thereby circularization of the vector. The expected size of PCR product is 552 bp from a 1-LTR circle and 1029 bp from a 2-LTR circle.


. e ) Southern Blot analysis to detect integration events in LV - and NILV - S / MAR - transduced single cell clones with DNA probed against the viral genome . A 6 . 8 kb positive control from a linearized plasmid ( lane 3 ) was included for indication of the size of DNA circles formed by non - integrated NILV - S / MAR vector .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR 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-SMALL_MOLECULE O O O O O O O O O O O O O

. e) Southern Blot analysis to detect integration events in LV- and NILV-S/MAR-transduced single cell clones with DNA probed against the viral genome. A 6.8 kb positive control from a linearized plasmid (lane 3) was included for indication of the size of DNA circles formed by non-integrated NILV-S/MAR vector.


f - h ) A padlock probe assay , as illustrated in Appendix Figure S4 , was used to demonstrate persistence of proviral DNA either in a circular or integrated form . Representative images show DNA persistence in ( f ) LV - , ( g ) NILV - or ( h ) NILV - S / MAR - transduced cells . Each red dot ( Cy3 ) represents a single DNA molecule and cell nuclei were stained with Hoechst 33342 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM 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-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O

f-h) A padlock probe assay, as illustrated in Appendix Figure S4, was used to demonstrate persistence of proviral DNA either in a circular or integrated form. Representative images show DNA persistence in (f) LV-, (g) NILV- or (h) NILV-S/MAR-transduced cells. Each red dot (Cy3) represents a single DNA molecule and cell nuclei were stained with Hoechst 33342.


c ) Flow cytometry histograms of RFP expression ( x - axis ) of A549 / RFP + Luc + cells at day 20 and 27 after NILV - S / MAR ( GFP , shNT ) or NILV - S / MAR ( GFP , shRFP ) transduction .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O

c) Flow cytometry histograms of RFP expression (x-axis) of A549/RFP+Luc+ cells at day 20 and 27 after NILV-S/MAR(GFP, shNT) or NILV-S/MAR(GFP, shRFP) transduction.


d ) Luciferase activity of the A549 / RFP + Luc + cells at day 20 and day 44 after NILV - S / MAR ( GFP , shNT ) - or NILV - S / MAR ( GFP , shRFP ) - transduction .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O

d) Luciferase activity of the A549/RFP+Luc+ cells at day 20 and day 44 after NILV-S/MAR(GFP, shNT)- or NILV-S/MAR(GFP, shRFP)-transduction.


e - h ) Representative fluorescence microscopy images show the expression of RFP from either NILV - S / MAR - transduced or un - transduced A549 / RFP + Luc + cells with cell nuclei stained with Hoechst 33342 ( blue ) . RFP signal ( orange ) were collected with a fixed exposure time ( 300 ms ) for equal comparison .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

e-h) Representative fluorescence microscopy images show the expression of RFP from either NILV-S/MAR-transduced or un-transduced A549/RFP+Luc+ cells with cell nuclei stained with Hoechst 33342 (blue). RFP signal (orange) were collected with a fixed exposure time (300 ms) for equal comparison.


c ) Percentage of CD19CAR expression on CD3 + T cells from each independent donor . d ) Histograms of CD19 CAR expression on T cells form one representative donor before and after expansion . e ) CD107a expression on transduced T cells ( CD3 + ) after exposure to CD19 + target cells ( Daudi ) for 24 h ( n = 6 ) . f ) IFN - γ production in the supernatant was analyzed by ELISA after 24 h of exposure of transduced T cells to target cells ( n = 6 ) . g ) Relative viability of target cells ( CD19 + Daudi ) , as an indication of T cell killing efficacy , were measured at different effector T cell to target cell ratio ( E : T ratio ) and normalized to the signal from target cells alone ( n = 6 ) . h ) Transduced T cells were labeled with a fluorescent cell membrane dye and co - cultured with CD19 + Daudi cells for 4 days . Dye dilution upon T cell division was monitored by flow cytometry and representative histograms are shown .

O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-CELL_LINE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O 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-CELL_TYPE I-CELL_TYPE O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

c) Percentage of CD19CAR expression on CD3+ T cells from each independent donor. d) Histograms of CD19 CAR expression on T cells form one representative donor before and after expansion. e) CD107a expression on transduced T cells (CD3+) after exposure to CD19+ target cells (Daudi) for 24 h (n=6). f) IFN-γ production in the supernatant was analyzed by ELISA after 24 h of exposure of transduced T cells to target cells (n=6). g) Relative viability of target cells (CD19+ Daudi), as an indication of T cell killing efficacy, were measured at different effector T cell to target cell ratio (E:T ratio) and normalized to the signal from target cells alone (n=6). h) Transduced T cells were labeled with a fluorescent cell membrane dye and co-cultured with CD19+ Daudi cells for 4 days. Dye dilution upon T cell division was monitored by flow cytometry and representative histograms are shown.


i - m ) Mice were treated by intravenous injection of NILV - S / MAR ( Mock ) , LV ( CD19CAR ) and NILV - S / MAR ( CD19CAR ) - transduced T cells ( 8x106 cells / injection ) at days 18 , 21 , 24 after tumor implantation . Tumor growth was monitored by caliper measurements . i - k ) Tumor volume of individual mice in each treatment groups are presented . l ) The average tumor volume of each treatment group is presented and the values on day 37 were statistically compared ( n = 12 in LV group , n = 9 in NILV - S / MAR group and n = 11 in Mock control group ) . m ) The survival of mice is shown as Kaplan - Meier curves .

O O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-ORGANISM O 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-EXP_ASSAY O B-ORGANISM O O O O O O O O

i-m) Mice were treated by intravenous injection of NILV-S/MAR(Mock), LV(CD19CAR) and NILV-S/MAR(CD19CAR)-transduced T cells (8x106 cells/injection) at days 18, 21, 24 after tumor implantation. Tumor growth was monitored by caliper measurements. i-k) Tumor volume of individual mice in each treatment groups are presented. l) The average tumor volume of each treatment group is presented and the values on day 37 were statistically compared (n=12 in LV group, n=9 in NILV-S/MAR group and n=11 in Mock control group). m) The survival of mice is shown as Kaplan-Meier curves.


n - p ) Representative images showing infiltration of adoptively transferred human T cells into tumors . T cells were visualized by staining for human CD3 ( red ) and nucleus ( blue ) . q ) Quantification of CD3 + T cells from each treatment group is presented ( n = 5 ) .

O O O O O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-ORGANISM B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O

n-p) Representative images showing infiltration of adoptively transferred human T cells into tumors. T cells were visualized by staining for human CD3 (red) and nucleus (blue). q) Quantification of CD3+ T cells from each treatment group is presented (n=5).


B Antiproliferative activities of XMU - MP - 5 and crizotinib against EML4 - ALK Ba / F3 and parental Ba / F3 cells , respectively . Cells were treated with increasing concentration of each drug for 48 hours and then analyzed by MTS assay . Each concentration was performed in triplicate . Data were presented as Mean ± SEM from three independent experiments ( n = 3 ) .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O 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 Antiproliferative activities of XMU-MP-5 and crizotinib against EML4-ALK Ba/F3 and parental Ba/F3 cells, respectively. Cells were treated with increasing concentration of each drug for 48 hours and then analyzed by MTS assay. Each concentration was performed in triplicate. Data were presented as Mean ± SEM from three independent experiments (n = 3) .


C XMU - MP - 5 downregulates ALK signaling pathways in EML4 - ALK Ba / F3 cells . Cells were treated with XMU - MP - 5 or crizotinib for 4 h , and then analyzed by immunobloting .

C XMU-MP-5 downregulates ALK signaling pathways in EML4-ALK Ba/F3 cells. Cells were treated with XMU-MP-5 or crizotinib for 4 h, and then analyzed by immunobloting.


C XMU - MP - 5 suppressed ALK signaling pathway in H3122 cells .

C XMU-MP-5 suppressed ALK signaling pathway in H3122 cells.


E Comparison of tumor volume of different treatment groups in 11 days . BALB / c nude mice bearing H3122 xenograft tumors were treated with vehicle , XMU - MP - 5 and crizotinib by tail vein injection once daily . Data were shown as Mean ± SEM ( n = 6 ) . Statistical comparasions were performed using a two - tailed , unpaired Student ' s t test . N . S . , P = 0 . 1063 ; * * P = 0 . 0097 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O B-CELL_LINE B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Comparison of tumor volume of different treatment groups in 11 days. BALB/c nude mice bearing H3122 xenograft tumors were treated with vehicle, XMU-MP-5 and crizotinib by tail vein injection once daily. Data were shown as Mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test. N.S., P = 0.1063; **P = 0.0097.


G Representative images of tumors analyzed by H & E and IHC staining . Scale bars , 100 μm .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

G Representative images of tumors analyzed by H&E and IHC staining. Scale bars, 100 μm.


A Cell viability was measured by MTS assay . Each concentration was performed in triplicate . The exact IC50 value was presented as Mean ± SEM from three independent experiments ( n = 3 ) and shown in Appendix Table S6 . IC50 fold changes of wild type and mutant EML4 - ALK Ba / F3 cell lines to parental Ba / F3 cell line of XMU - MP - 5 and crizotinib were determined .

O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O

A Cell viability was measured by MTS assay. Each concentration was performed in triplicate. The exact IC50 value was presented as Mean ± SEM from three independent experiments (n = 3) and shown in Appendix Table S6. IC50 fold changes of wild type and mutant EML4-ALK Ba/F3 cell lines to parental Ba/F3 cell line of XMU-MP-5 and crizotinib were determined.


B Heatmap of the anti - proliferative activity of ALK inhibitors across Ba / F3 cell lines with different ALK mutations .

O O O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O

B Heatmap of the anti-proliferative activity of ALK inhibitors across Ba/F3 cell lines with different ALK mutations.


C , D In vivo activity of XMU - MP - 5 against L1196M xenograft mouse model . BALB / c nude mice bearing L1196M Ba / F3 xenograft tumors were treated with vehicle , 30 mg / kg or 60 mg / kg XMU - MP - 5 by tail vein injection once daily . Tumor volume ( C ) and tumor weight ( D ) were shown as mean ± SEM ( n = 6 ) . Statistical comparasions were performed using a two - tailed , unpaired Student ' s t test for ( C ) , N . S . , P = 0 . 1482 ; * P = 0 . 037 . Statistical comparasions were performed using a one - way ANOVA with Dunnett test for ( D ) , N . S . , P = 0 . 2162 ; * P = 0 . 0122 .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY B-ORGANISM O O O O O O B-ORGANISM O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE I-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D In vivo activity of XMU-MP-5 against L1196M xenograft mouse model. BALB/c nude mice bearing L1196M Ba/F3 xenograft tumors were treated with vehicle, 30 mg/kg or 60 mg/kg XMU-MP-5 by tail vein injection once daily. Tumor volume (C) and tumor weight (D) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (C), N.S., P = 0.1482; *P = 0.037. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (D), N.S., P = 0.2162; *P = 0.0122.


E , F Efficacy of XMU - MP - 5 in G1202R Ba / F3 xenograft mouse model . BALB / c nude mice bearing G1202R Ba / F3 cells xenograft tumors were administrated with vehicle or XMU - MP - 5 by tail vein injection twice daily . Lorlatinib was used as a positive control by oral administration . Tumor volume ( E ) and tumor weight ( F ) were shown as mean ± SEM ( n = 6 ) . Statistical comparasions were performed using a two - tailed , unpaired Student ' s t test for ( E ) , Vehicle vs XMU - MP - 5 30 mg / kg , * P = 0 . 0433 ; Vehicle vs XMU - MP - 5 30 mg / kg BID , * * P = 0 . 0093 ; Vehicle vs Lorlatinib , * * P = 0 . 0029 . Statistical comparasions were performed using a one - way ANOVA with Dunnett test for ( F ) . Vehicle vs XMU - MP - 5 30 mg / kg , * * * P = 0 . 0008 ; Vehicle vs XMU - MP - 5 30 mg / kg BID , * * * P = 0 . 0003 ; Vehicle vs Lorlatinib , * * * P = 0 . 0002 .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-EXP_ASSAY B-ORGANISM O O O O O O B-ORGANISM O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

E, F Efficacy of XMU-MP-5 in G1202R Ba/F3 xenograft mouse model. BALB/c nude mice bearing G1202R Ba/F3 cells xenograft tumors were administrated with vehicle or XMU-MP-5 by tail vein injection twice daily. Lorlatinib was used as a positive control by oral administration. Tumor volume (E) and tumor weight (F) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (E), Vehicle vs XMU-MP-5 30 mg/kg, *P = 0.0433; Vehicle vs XMU-MP-5 30 mg/kg BID, **P = 0.0093; Vehicle vs Lorlatinib, **P = 0.0029. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (F). Vehicle vs XMU-MP-5 30 mg/kg, ***P = 0.0008; Vehicle vs XMU-MP-5 30 mg/kg BID, ***P = 0.0003; Vehicle vs Lorlatinib, ***P = 0.0002.


A Representative CT images ( upper panel ) and H & E staining ( lower panel ) of lungs from EML4 - ALK tumor - bearing mice are shown . The complete set of CT images refers to Appendix Figure S10A . Mice were treated with 30 mg / kg XMU - MP - 5 twice daily for 3 weeks . H : heart ; arrow indicated tumor . Scale bars , 200 μm . B IHC analyses of lungs from EML4 - ALK transgenic mice . pALK and Cl - caspase 3 were probed . Scale bars , 100 μm .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O

A Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10A. Mice were treated with 30 mg/kg XMU-MP-5 twice daily for 3 weeks. H: heart; arrow indicated tumor. Scale bars, 200 μm. B IHC analyses of lungs from EML4-ALK transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.


C Representative CT images ( upper panel ) and H & E staining ( lower panel ) of lungs from EML4 - ALK ( L1196M ) tumor - bearing mice are shown . The complete set of CT images refers to Appendix Figure S10B . Mice were treated with 40 mg / kg XMU - MP - 5 twice daily for 1 week . H : heart ; arrow indicated tumor . Scale bars , 200 μm . D IHC analyses of lungs from EML4 - ALK ( L1196M ) transgenic mice . pALK and Cl - caspase 3 were probed . Scale bars , 100 μm .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O

C Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK(L1196M) tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10B. Mice were treated with 40 mg/kg XMU-MP-5 twice daily for 1 week. H: heart; arrow indicated tumor. Scale bars, 200 μm. D IHC analyses of lungs from EML4-ALK(L1196M) transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.


B . ARPI ( CSS , ENZA , ODM - 201 , 14449 ) decreased , while R1881 increased , SDHA and SDHB subunit protein levels in both LNCaP and LAPC4 cells . PSA is used as a positive control .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-CELL_LINE O O B-GENEPROD O O O O O O O

B. ARPI (CSS, ENZA, ODM-201, 14449) decreased, while R1881 increased, SDHA and SDHB subunit protein levels in both LNCaP and LAPC4 cells. PSA is used as a positive control.


D . SDH repression during acute phase of ENZA stress increased transcription of different AR - regulated genes without altering AR mRNAs levels in LNCaP cells . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical analysis was performed using two tailed unpaired Student ' s t - test * , p < 0 . 05 and * * , p < 0 . 01 compared between groups .

O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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. SDH repression during acute phase of ENZA stress increased transcription of different AR-regulated genes without altering AR mRNAs levels in LNCaP cells. Data information: ENZA: enzalutamide.Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using two tailed unpaired Student's t-test *, p<0.05 and **, p<0.01 compared between groups.


E - F . Overexpression of SDHA and SDHB subunits decreased AR protein levels ( E ) and AR transactivation ( F ) in LNCaP cells . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s test * , p < 0 . 05 and * * , p < 0 . 01 compared between groups .

O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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-F. Overexpression of SDHA and SDHB subunits decreased AR protein levels (E) and AR transactivation (F) in LNCaP cells. Data information: ENZA: enzalutamide.Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's test *, p<0.05 and **, p<0.01 compared between groups.


C . SDH - repressed LNCaP cells were able to maintain higher proliferation rate particularly during ENZA stress with or without Hsp27 silencing in real time cell proliferation assay . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical analysis was performed using one - way ANOVA following Tukey ' s test for panels C ( end points ) . * , p < 0 . 05 , * * , p < 0 . 01 and * * * , p < 0 . 001 compared between

O O O O O B-CELL_LINE O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SDH-repressed LNCaP cells were able to maintain higher proliferation rate particularly during ENZA stress with or without Hsp27 silencing in real time cell proliferation assay. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels C (end points) . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups


E . Treatment with p - p38 inhibitors : SB203580 ( 10 µM ) or Genistein ( 20 µM ) for 24 h reduced AR upregulation induced by SDH repression in LNCaP cells ( left panel ) . Bar graph in the right panel shows band intensity normalized to loading control . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical analysis was performed using one - way ANOVA following Tukey ' s test for panels E . * , p < 0 . 05 , * * , p < 0 . 01 and * * * , p < 0 . 001 compared between

O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Treatment with p-p38 inhibitors: SB203580 (10 µM) or Genistein (20 µM) for 24 h reduced AR upregulation induced by SDH repression in LNCaP cells (left panel). Bar graph in the right panel shows band intensity normalized to loading control. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels E . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups


F . . Treatment with 5 µM STO - 609 for 24 h in LNCaP cells inhibited AR upregulation induced by SDHA / SDHB subunit silencing .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O

F.. Treatment with 5 µM STO-609 for 24 h in LNCaP cells inhibited AR upregulation induced by SDHA/SDHB subunit silencing.


G . Thapsigargin , DMM , DMS and ENZA treatment for the indicated time points increased intracellular calcium flux in LNCaP cells as visualized with calcium sensitive Fluo4 - AM probe under confocal microscope .

G. Thapsigargin, DMM, DMS and ENZA treatment for the indicated time points increased intracellular calcium flux in LNCaP cells as visualized with calcium sensitive Fluo4-AM probe under confocal microscope.


A - C . Human patient samples demonstrate relative differences in the immunoexpression of AR , SDHA , p - CaMKK2 / p - AMPK , p - p38 , and p - Hsp27 in untreated ( n = 70 ) , neoadjuvant - treated ( NHT ) ( n = 130 ) and CRPC ( n = 24 ) samples . Representative images ( A ) , dot plots ( B ) , compiled bar graph ( C ) support the AR - SDH loop where inhibition of AR axis in NHT samples reduced SDHA levels along with upregulation of p - CaMKK2 / p - AMPK / p - p38 / p - Hsp27 axis . AR - positive CRPC samples restored SDHA as well as the whole axis . Data information : Data shown as mean ± SEM . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s test . * * , p < 0 . 01 ; * * * , p < 0 . 001 and * * * * , p < 0 . 0001 . Scale bar : 100µm . All the TMAs were constructed from minimum 2 cores per patient .

O O O O B-ORGANISM I-ORGANISM O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-DISEASE O O O 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-GENEPROD O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-DISEASE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O

A-C. Human patient samples demonstrate relative differences in the immunoexpression of AR, SDHA, p-CaMKK2/p-AMPK, p-p38, and p-Hsp27 in untreated (n=70), neoadjuvant-treated (NHT) (n=130) and CRPC (n=24) samples. Representative images (A), dot plots (B), compiled bar graph (C) support the AR-SDH loop where inhibition of AR axis in NHT samples reduced SDHA levels along with upregulation of p-CaMKK2/p-AMPK/p-p38/p-Hsp27 axis. AR-positive CRPC samples restored SDHA as well as the whole axis. Data information: Data shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. **, p<0.01; ***, p<0.001 and ****, p<0.0001. Scale bar: 100µm. All the TMAs were constructed from minimum 2 cores per patient.


D . IVM reversed the cell proliferation benefit mediated by SDH repression in LNCaP cells . Data shown as means ± SEM In vitro data represent three independent experiments . Statistical analysis was performed for the last data point * , p < 0 . 05 , * * , p < 0 . 01 and * * * , p < 0 . 001 compared between groups , one - way ANOVA followed by Tukey ' s test .

O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. IVM reversed the cell proliferation benefit mediated by SDH repression in LNCaP cells. Data shown as means ± SEM In vitro data represent three independent experiments. Statistical analysis was performed for the last data point *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups, one-way ANOVA followed by Tukey's test.


E . LNCaP xenografts treated with IVM ( n = 10 ) ( 10mg / kg ) demonstrated lower serum PSA values ( upper panel ) and growth of tumours after castration ( lower panel ) compared to vehicle ( n = 10 ) . PSA was measured every week . Data shown as means ±

O O B-CELL_LINE B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD 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-GENEPROD O O O O O O O O O O

E. LNCaP xenografts treated with IVM (n=10) (10mg/kg) demonstrated lower serum PSA values (upper panel) and growth of tumours after castration (lower panel) compared to vehicle (n=10). PSA was measured every week. Data shown as means ± SEM


F . IVM treatment reduced p - Hsp27 levels and therefore AR protein level , with subsequent p - CAMKK2 axis activation in castrated LNCaP xenografts . Bar graph in the right panel shows quantification of data from western blot normalized to loading control . Data shown as means ±

O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-CELL_LINE B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

F. IVM treatment reduced p-Hsp27 levels and therefore AR protein level, with subsequent p-CAMKK2 axis activation in castrated LNCaP xenografts. Bar graph in the right panel shows quantification of data from western blot normalized to loading control. Data shown as means ± SD