f8 ( a ) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide ( 48 - h cultures ) were immunoprecipitated with anti - ATG5 or antisurvivin antibodies . A physical interaction between ATG5 and survivin , absent in untreated or GFP - expressing control cells , was detected reciprocally . Full - length immunoblots are provided in Supplementary Fig . S12 . ( b ) Jurkat T cells overexpressing ATG5 - ΔNES exhibited no ATG5 / survivin molecular interaction . ( c ) The ATG5 - K130R mutant did show coprecipitation of survivin with ATG5 . ( d ) Calpain - mediated , truncated ATG5 ( tATG5 ) , that is , the N - terminal part of ATG5 , failed to bind survivin . The ATG5 - K130R mutant was the control . ( e ) Confocal microscopy . MDA - MA - 231 cells were etoposide treated or transduced as indicated ( 48 - h cultures ) and stained . In untreated cells , survivin and ATG5 were in the cytosol with no colocalization . ATG5 - transduced cells exhibited ATG5 / survivin colocalization in the nucleus . ATG5 - ΔNES - transduced cells showed survivin , but not ATG5 , in the nuclei of cycling cells . Numerical analysis was performed and Pearson ' s correlation coefficients are indicated in the images . Scale bar , 10 μm . Right : Statistical analysis ( analysis of variance ) is presented in which the results of ten representative cells within each group were integrated . Values are means ± s . d . ( f ) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5 . Neither ATG5 nor HSP90 were detectable in the Aurora B - containing immune precipitate , but ATG5 molecularly interacted with survivin . Results representative of three independent experiments . ( g ) Confocal microscopy . HeLa cells treated with etoposide or transduced as indicated ( 48 - h cultures ) were stained with centromere - specific antiserum and anti - Aurora B ( top panels ) or antisurvivin antibodies ( lower panels ) . In untreated or ATG5 - ΔNES transduced cells , both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres . In contrast , etoposide - treated or ATG5 - transduced cells recruited much less Aurora B to prometaphase centromeres . Such recruitment , however , was observed during anaphase . Compared with cells in normal mitosis , survivin , although reduced , was recruited to centromeres in prometaphase , but , similar to Aurora B , remained attached to centromeres during anaphase . Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects . Colocalization images were prepared using Imaris software . Numerical analysis was performed and correlation coefficients are indicated . Scale bar , 2 μm . ( h ) Confocal microscopy . Sections of lung ( non - small - cell lung cancer ) and oesophageal carcinoma stained for ATG5 and survivin expression . In untreated patients , ATG5 expression in cancer cells was slight and mainly cytosolic . Following radio - and / or chemotherapy , ATG5 was preferentially expressed in the nucleus and colocalized with survivin . Overlay images present the average Pearson ' s correlation coefficient as calculated with Imaris . Results are representative of three independent experiments . Scale bar , 10 μm .

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

f8(a) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide (48-h cultures) were immunoprecipitated with anti-ATG5 or antisurvivin antibodies. A physical interaction between ATG5 and survivin, absent in untreated or GFP-expressing control cells, was detected reciprocally. Full-length immunoblots are provided in Supplementary Fig. S12.(b) Jurkat T cells overexpressing ATG5-ΔNES exhibited no ATG5/survivin molecular interaction. (c) The ATG5-K130R mutant did show coprecipitation of survivin with ATG5.(d) Calpain-mediated, truncated ATG5 (tATG5), that is, the N-terminal part of ATG5, failed to bind survivin. The ATG5-K130R mutant was the control.(e) Confocal microscopy. MDA-MA-231 cells were etoposide treated or transduced as indicated (48-h cultures) and stained. In untreated cells, survivin and ATG5 were in the cytosol with no colocalization. ATG5-transduced cells exhibited ATG5/survivin colocalization in the nucleus. ATG5-ΔNES-transduced cells showed survivin, but not ATG5, in the nuclei of cycling cells. Numerical analysis was performed and Pearson's correlation coefficients are indicated in the images. Scale bar, 10 μm. Right: Statistical analysis (analysis of variance) is presented in which the results of ten representative cells within each group were integrated. Values are means±s.d.(f) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5. Neither ATG5 nor HSP90 were detectable in the Aurora B-containing immune precipitate, but ATG5 molecularly interacted with survivin. Results representative of three independent experiments.(g) Confocal microscopy. HeLa cells treated with etoposide or transduced as indicated (48-h cultures) were stained with centromere-specific antiserum and anti-Aurora B (top panels) or antisurvivin antibodies (lower panels). In untreated or ATG5-ΔNES transduced cells, both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres. In contrast, etoposide-treated or ATG5-transduced cells recruited much less Aurora B to prometaphase centromeres. Such recruitment, however, was observed during anaphase. Compared with cells in normal mitosis, survivin, although reduced, was recruited to centromeres in prometaphase, but, similar to Aurora B, remained attached to centromeres during anaphase. Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects. Colocalization images were prepared using Imaris software. Numerical analysis was performed and correlation coefficients are indicated. Scale bar, 2 μm.(h) Confocal microscopy. Sections of lung (non-small-cell lung cancer) and oesophageal carcinoma stained for ATG5 and survivin expression. In untreated patients, ATG5 expression in cancer cells was slight and mainly cytosolic. Following radio- and/or chemotherapy, ATG5 was preferentially expressed in the nucleus and colocalized with survivin. Overlay images present the average Pearson's correlation coefficient as calculated with Imaris. Results are representative of three independent experiments. Scale bar, 10 μm.


Figure 1 ( A ) Kaplan - Meier survival curves ( n = 12 - 16 ) . ( B ) Heart gross morphology . ( C ) Body weight ; ( D ) Heart weight and ( E ) Heart - to - body weight ratio ( n = 28 - 40 ) . ( F ) Relative expression levels of cardiac hypertrophy markers ( Nppa and Nppb ) ( n = 5 ) . Bars represent mean ± S . E . M . ( Student ' s t test ; * * p < 0 . 01 , * * * * p < 0 . 0001 ) . ( G ) Enzyme histochemistry double staining for COX and SDH activities . ( n = 4 ) . ( H ) Assessment of cardiac fibrosis by Masson ' s trichrome staining ( n = 4 ) . White scale bars , 100μm ; black scale bars , 1 mm .

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

Figure 1(A) Kaplan-Meier survival curves (n = 12 - 16).(B) Heart gross morphology.(C) Body weight; (D) Heart weight and (E) Heart-to-body weight ratio (n = 28-40).(F) Relative expression levels of cardiac hypertrophy markers (Nppa and Nppb) (n = 5). Bars represent mean ± S.E.M. (Student's t test; **p < 0.01, ****p < 0.0001).(G) Enzyme histochemistry double staining for COX and SDH activities. (n = 4).(H) Assessment of cardiac fibrosis by Masson's trichrome staining (n = 4). White scale bars, 100μm; black scale bars, 1 mm.


Figure 2 ( A ) Western blot analysis and ( B ) Relative quantification of UPRmt markers in heart extracts . HSC70 is used as a loading control ( CTRL ) , ( n = 3 ) . ( C and D ) Relative expression levels normalized to WT of ( C ) established UPRmt markers in 3 - 4 week old mice ( n = 3 - 4 ) . ( C and D ) Relative expression levels normalized to WT of ( D ) mitochondrial chaperones , proteases and TFAM in 3 - 4 week old mice ( n = 3 - 4 ) . ( E ) Western blot analysis of cellular stress markers . HSC70 is used as a loading control ( CTRL ) , ( n = 3 ) ( F ) Relative expression levels of transcription factors involved in UPRmt and ISR ( left ) and Fgf21 ( right ) ( n = 5 ) . Bars represent mean ± S . E . M . ( Student ' s t test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) .

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

Figure 2(A) Western blot analysis and (B) Relative quantification of UPRmt markers in heart extracts. HSC70 is used as a loading control (CTRL), (n = 3).(C and D) Relative expression levels normalized to WT of (C) established UPRmt markers in 3 - 4 week old mice (n = 3 - 4).(C and D) Relative expression levels normalized to WT of (D) mitochondrial chaperones, proteases and TFAM in 3 - 4 week old mice (n = 3 - 4).(E) Western blot analysis of cellular stress markers. HSC70 is used as a loading control (CTRL), (n = 3)(F) Relative expression levels of transcription factors involved in UPRmt and ISR (left) and Fgf21 (right) (n = 5). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).


Figure 3 ( A ) Western blot analysis of individual OXPHOS subunits in heart extracts . HSC70 and Calnexin are used as loading controls , ( n = 3 ) . Antibodies against individual OXPHOS subunits ( on the right ) were used to detect OXPHOS complexes ( on the left ) . ( B ) BN - PAGE and subsequent Western blot analysis for OXPHOS complexes . Antibodies against individual OXPHOS subunits ( on the right ) were used to detect OXPHOS complexes ( on the left ) . ( C , D ) In gel activity of complexes I ( C ) and IV ( D ) performed after BN - PAGE . ( E ) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate - glutamate - malate ( C I ) and pyruvate - glutamate - malate + succinate ( C I + C II ) as substrates . State 3 ( substrates + ADP ) ; State 4 ( + oligomycin ) ; Uncoupled ( + FCCP ) . ( n = 3 ) . Bars represent mean ± S . E . M . ( Student ' s t test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

Figure 3(A) Western blot analysis of individual OXPHOS subunits in heart extracts. HSC70 and Calnexin are used as loading controls, (n = 3). Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left).(B) BN-PAGE and subsequent Western blot analysis for OXPHOS complexes. Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left).(C, D) In gel activity of complexes I (C) and IV (D) performed after BN-PAGE.(E) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates+ADP); State 4 (+oligomycin); Uncoupled (+FCCP). (n = 3). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001).


Figure 4 ( A ) Representative gel of the In organello translation analysis of heart mitochondria . De novo synthetized proteins are isolated after labeling with 35S - met ( 1 hour Pulse ) or after cold chase ( 3h Chase ) . Positions of individual proteins are indicated on the left . Position of full - length proteins ( Proficient ) and low molecular weight polypeptides ( Abortive ) are indicated on the right . Note that in WT and ClpP KO low molecular weight polypeptides include ATP8 / ND4L proteins . ( B ) Relative overall protein synthesis and turnover rate . ( C ) Western blot analysis of DARS2 levels . HSC70 is used as a loading control ( CTRL ) . ( D ) Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level . ( E ) Relative levels of protein synthesis of Proficient and Abortive polypeptides . ( F ) Quantification of the turnover rate of full - length ( Proficient ) de novo synthesized proteins . Bars represent mean ± S . E . M . ( Student ' s t test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) , ( n = 3 - 4 ) .

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

Figure 4(A) Representative gel of the In organello translation analysis of heart mitochondria. De novo synthetized proteins are isolated after labeling with 35S-met (1 hour Pulse) or after cold chase (3h Chase). Positions of individual proteins are indicated on the left. Position of full-length proteins (Proficient) and low molecular weight polypeptides (Abortive) are indicated on the right. Note that in WT and ClpP KO low molecular weight polypeptides include ATP8/ND4L proteins.(B) Relative overall protein synthesis and turnover rate.(C) Western blot analysis of DARS2 levels. HSC70 is used as a loading control (CTRL).(D) Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level.(E) Relative levels of protein synthesis of Proficient and Abortive polypeptides. (F) Quantification of the turnover rate of full-length (Proficient) de novo synthesized proteins. Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), (n=3-4).


Figure 2Three sex chromosome axes could be detected in the 2 - month - old XUsp26 + XUsp26 - Y spermatocytes . Immunofluorescence analysis of SYCP3 ( red ) and SYCP1 ( green ) in ( J ) , ATR ( red ) and SYCP3 ( green ) in ( K ) was performed in 2 - month - old WT and XUsp26 + XUsp26 - Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrows indicate the Y chromosome . The arrowheads indicate the X chromosome .

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

Figure 2Three sex chromosome axes could be detected in the 2-month-old XUsp26+XUsp26-Y spermatocytes. Immunofluorescence analysis of SYCP3 (red) and SYCP1 (green) in (J), ATR (red) and SYCP3 (green) in (K) was performed in 2-month-old WT and XUsp26+XUsp26-Y spermatocytes. Nuclei were stained with DAPI (blue).The arrows indicate the Y chromosome. The arrowheads indicate the X chromosome.


Figure 3D Histological analysis of the seminiferous tubules and caudal epididymis of the 6 - month - old Usp26 + / Y and Usp26 - / Y mice . F The deletion of Usp26 causes pachytene and meiotic division arrest . Representative PAS - hematoxylin staining in 6 - month - old Usp26 + / Y and Usp26 - / Y seminiferous tubules . Parafﬁn sections from Usp26 + / Y and Usp26 - / Y testes were stained with PAS - hematoxylin . P : pachytene spermatocyte , D : diplotene spermatocyte , rST : round spermatid , eST : elongating spermatid , M : meiotic spermatocyte , aP : apoptotic pachytene spermatocytes , aM : abnormal meiotic divisions .

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

Figure 3D Histological analysis of the seminiferous tubules and caudal epididymis of the 6-month-old Usp26+/Y and Usp26-/Y mice.F The deletion of Usp26 causes pachytene and meiotic division arrest. Representative PAS-hematoxylin staining in 6-month-old Usp26+/Y and Usp26-/Y seminiferous tubules. Parafﬁn sections from Usp26+/Y and Usp26-/Y testes were stained with PAS-hematoxylin. P: pachytene spermatocyte, D: diplotene spermatocyte, rST: round spermatid, eST: elongating spermatid, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions.


Figure 4E The X and Y chromosomes were unpaired in Usp26 - / Y spermatocytes . Immunofluorescence analysis of Chr X - FISH ( green ) , Chr Y - FISH ( red ) , and SYCP3 ( white ) was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . The arrowheads indicate the X chromosome . F Quantification of unpaired X and Y chromosomes in 6 - month - old Usp26 + / Y and Usp26 - / Y mice ( n = 5 independent experiments ) . Red dots indicate Usp26 + / Y mice and green dots indicate Usp26 - / Y mice . Sex chromosome recombination was perturbed in 6 - month - old Usp26 - / Y mice . Immunofluorescence analysis of SYCP3 ( green ) , MRE11 ( red ) ( G ) ; was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrows indicate the sex chromosomes . Sex chromosome recombination was perturbed in 6 - month - old Usp26 - / Y mice . Immunofluorescence analysis of ; SYCP3 ( green ) , γH2AX ( red ) ( H ) ; SYCP3 ( green ) , ATR ( red ) , p - ATM ( pink ) ( I ) was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrows indicate the sex chromosomes . L Crossover on sex chromosomes was impaired in 6 - month - old Usp26 - / Y mice . Immunofluorescence analysis of SYCP3 ( green ) , MLH1 ( red ) , and ATR ( white ) was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) .

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

Figure 4E The X and Y chromosomes were unpaired in Usp26-/Y spermatocytes. Immunofluorescence analysis of Chr X-FISH (green), Chr Y-FISH (red), and SYCP3 (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. The arrowheads indicate the X chromosome. F Quantification of unpaired X and Y chromosomes in 6-month-old Usp26+/Y and Usp26-/Y mice (n= 5 independent experiments). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MRE11 (red) (G); was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of ; SYCP3 (green), γH2AX (red) (H); SYCP3 (green), ATR (red), p-ATM (pink) (I) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.L Crossover on sex chromosomes was impaired in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MLH1 (red), and ATR (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue).


Figure 5D Representative TUNEL results in Usp26 + / Y and Usp26 - / Y testes . Parafﬁn sections from 6 - month - old Usp26 + / Y and Usp26 - / Y testes were stained with TUNEL ( green ) and DAPI ( blue ) to show dead cells in stage IV , VIII and XII tubules with pachytene and metaphase spermatocytes , respectively . The arrowheads indicate the laggard chromosomes . P : pachytene spermatocytes , rSt : round spermatid , spz : spermatozoa , M : meiotic spermatocyte , aP : apoptotic pachytene spermatocytes , aM : abnormal meiotic divisions , arSt : apoptotic round spermatid . E The protein level of three known SAC proteins was reduced in 2 - month - old and 6 - month - old Usp26 − / Y mouse testes . Immunoblotting of MAD2 , BUBR1 , PLK1 and USP26 was performed in 2 - month - old and 6 - month - old Usp26 + / Y and Usp26 - / Y testes . H3Ser10p and Tub served as loading control . F Usp26 - deficient mice produced XY aneuploid round spermatids . FISH analysis of Chr X ( green ) and Chr Y ( red ) was performed in 2 - month - old and 6 - month - old Usp26 + / Y and Usp26 − / Y round spermatids . Nuclei were stained with DAPI ( blue ) . The arrows indicate the Y chromosome , and the arrowheads indicate the X chromosome . G Quantification of different types of round spermatids in 2 - month - old Usp26 + / Y , Usp26 - / Y mice ( n = 3 independent experiments ) and 6 - month - old Usp26 + / Y ( n = 3 independent experiments ) , Usp26 - / Y mice ( n = 4 independent experiments ) . P = 0 . 0026 for XY spermatozoa in 6 - month - old Usp26 + / Y and Usp26 − / Y mice . P = 0 . 0216 for O spermatozoa in 6 - month - old Usp26 + / Y and Usp26 − / Y mice . H Usp26 - deficient mice produced XY aneuploid spermatozoa . FISH assay of Chr X ( green ) and Chr Y ( red ) was performed in 2 - month - old and 6 - month - old Usp26 + / Y and Usp26 − / Y spermatozoa . Nuclei were stained with DAPI ( blue ) . The arrows indicate the Y chromosome , and the arrowheads indicate the X chromosome . I Quantification of different types of spermatozoa in 2 - month - old Usp26 + / Y , Usp26 - / Y mice ( n = 3 independent experiments ) and 6 - month - old Usp26 + / Y , Usp26 - / Y mice ( n = 5 independent ex experiments ) . P = 0 . 0195 for XY spermatozoa in 6 - month - old Usp26 + / Y and Usp26 − / Y mice . Data are presented as means ± SD .

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

Figure 5D Representative TUNEL results in Usp26+/Y and Usp26-/Y testes. Parafﬁn sections from 6-month-old Usp26+/Y and Usp26-/Y testes were stained with TUNEL (green) and DAPI (blue) to show dead cells in stage IV, VIII and XII tubules with pachytene and metaphase spermatocytes, respectively. The arrowheads indicate the laggard chromosomes. P: pachytene spermatocytes, rSt: round spermatid, spz: spermatozoa, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions, arSt: apoptotic round spermatid.E The protein level of three known SAC proteins was reduced in 2-month-old and 6-month-old Usp26−/Y mouse testes. Immunoblotting of MAD2, BUBR1, PLK1 and USP26 was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26-/Y testes. H3Ser10p and Tub served as loading control.F Usp26-deficient mice produced XY aneuploid round spermatids. FISH analysis of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y round spermatids. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. G Quantification of different types of round spermatids in 2-month-old Usp26 +/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y (n= 3 independent experiments), Usp26-/Y mice (n= 4 independent experiments). P=0.0026 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. P=0.0216 for O spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. H Usp26-deficient mice produced XY aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y spermatozoa. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. I Quantification of different types of spermatozoa in 2-month-old Usp26+/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y, Usp26-/Y mice (n= 5 independent ex experiments). P=0.0195 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. Data are presented as means ± SD.


Figure 6The X and Y chromosomes were unpaired in Tex11 - / Y spermatocytes . Immunofluorescence analysis of SYCP3 ( green ) , TRF1 ( red ) , ATR ( white ) ( D ) ; SYCP3 ( green ) , MLH1 ( red ) , ATR ( white ) ( E ) was performed in in Tex11 + / Y and Tex11 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrowheads indicate the X chromosome .

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

Figure 6The X and Y chromosomes were unpaired in Tex11-/Y spermatocytes. Immunofluorescence analysis of SYCP3 (green), TRF1 (red), ATR (white) (D); SYCP3 (green), MLH1 (red), ATR (white) (E) was performed in in Tex11+/Y and Tex11-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrowheads indicate the X chromosome.


Figure 7A , B Quantification of different types of spermatozoa in fertile men with or without USP26 mutations . The spermatozoa from fertile men without USP26 mutations ( n = 10 independent experiments ) ; fertile men contained the USP26 c . 463C > T mutation ( The sample was analyzed in three repeated experiments ) , the USP26 c . 125T > C mutation ( n = 2 independent experiments , one sample was analyzed in two repeated experiments ) , the USP26 c . 1044T > A mutation ( n = 5 independent experiments ) , and the USP26 c . 370 _ 371insACA / 494T > C / 1423C > T mutation ( n = 10 independent experiments ) were collected to perform Chr X and Chr Y FISH . P - values are shown for comparison to fertile men without USP26 mutations . C The fertile men with the USP26 mutated haplotype produced XY and O aneuploid spermatozoa . FISH assay of Chr X ( green ) and Chr Y ( red ) was performed in fertile men with or without the USP26 mutated haplotype . Nuclei were stained with DAPI ( blue ) . The arrows indicate the Y chromosome , and the arrowheads indicate the X chromosome .

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

Figure 7A, B Quantification of different types of spermatozoa in fertile men with or without USP26 mutations. The spermatozoa from fertile men without USP26 mutations (n = 10 independent experiments); fertile men contained the USP26 c.463C>T mutation (The sample was analyzed in three repeated experiments), the USP26 c.125T>C mutation (n = 2 independent experiments, one sample was analyzed in two repeated experiments), the USP26 c.1044T>A mutation (n = 5 independent experiments), and the USP26 c.370_371insACA/494T>C/1423C>T mutation (n = 10 independent experiments) were collected to perform Chr X and Chr Y FISH. P-values are shown for comparison to fertile men without USP26 mutations.C The fertile men with the USP26 mutated haplotype produced XY and O aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in fertile men with or without the USP26 mutated haplotype. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.


Figure 1C , D ) Low - angle Pt / C shadowing of Mis12 : Ndc80 ( panel C ) and Mis12 : Ndc80Δloop ( Δ431 - 463 ) ( panel D ) complexes . The Mis12 complex appears as a 20 nm rod - like extension and marks the SPC24 : SPC25 side of the Ndc80 complex . E ) Size exclusion chromatography coupled with multi - angle light scattering ( SEC - MALS ) profiles of fluorescently labelled . Ndc80 and Ndc80Δloop . Calculated and ( theoretical ) masses are indicated . F ) Total Internal Reflection Fluorescence ( TIRF ) microscopy was used to investigate Ndc80Alexa488 complexes on taxol - stabilized microtubules that were attached to a passivated glass surface . Kymographs show Ndc80 complexes at a concentration of 0 . 2 nM with ( FL , blue ) or without ( ΔL , orange ) the loop . Scale bars : vertical ( 5 µm ) , horizontal ( 5 s ) . G ) Quantification of Ndc80 residence times for data as in panel F . Solid line represents a single exponential fit . J ) Typical fields of view showing decoration of taxol - stabilized microtubules ( cyan ) incubated with full - length or loopless Ndc80 ( yellow ) at the indicated concentration . Images show an average projection of 200 frames . The contrast between individual fluorescent channels ( inverted grayscale ) was fixed . Auto - contrast was used for the composite images to highlight the differences in the uniformity of Ndc80 decoration .

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

Figure 1C, D) Low-angle Pt/C shadowing of Mis12:Ndc80 (panel C) and Mis12:Ndc80Δloop (Δ431-463) (panel D) complexes. The Mis12 complex appears as a 20 nm rod-like extension and marks the SPC24:SPC25 side of the Ndc80 complex.E) Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) profiles of fluorescently labelled. Ndc80 and Ndc80Δloop. Calculated and (theoretical) masses are indicated.F) Total Internal Reflection Fluorescence (TIRF) microscopy was used to investigate Ndc80Alexa488 complexes on taxol-stabilized microtubules that were attached to a passivated glass surface. Kymographs show Ndc80 complexes at a concentration of 0.2 nM with (FL, blue) or without (ΔL, orange) the loop. Scale bars: vertical (5 µm), horizontal (5 s). G) Quantification of Ndc80 residence times for data as in panel F. Solid line represents a single exponential fit.J) Typical fields of view showing decoration of taxol-stabilized microtubules (cyan) incubated with full-length or loopless Ndc80 (yellow) at the indicated concentration. Images show an average projection of 200 frames. The contrast between individual fluorescent channels (inverted grayscale) was fixed. Auto-contrast was used for the composite images to highlight the differences in the uniformity of Ndc80 decoration.


Figure 2TMR - labelled Ndc80 trimers with ( FL , blue ) or without ( ΔL , orange ) the loop were added to taxol - stabilized microtubules to measure their residence time ( panel C ) and one - dimensional diffusion ( panel D ) . Ndc80 trimers with and without the loop are shown . Scale bars : vertical ( 1000 s ) , horizontal ( 5 µm ) . F ) Kymographs of full - length and loopless Ndc80 trimers ( 10 pM ) that reside on dynamically growing and shortening microtubules . Trimers remain bound to the ends of shortening microtubules . Scale bars : vertical ( 100 s ) , horizontal ( 5 µm ) . G ) Distribution of initial brightnessH ) Fraction of Ndc80 trimers that switches from lateral microtubule binding to tracking shortening ends . Data from four repeats ( total 140 events ) for full - length Ndc80 trimers ( blue ) , and two repeats ( total 158 events ) for loopless Ndc80 trimers ( orange ) . Horizontal lines indicate average values . I ) End - tracking speed of full - length ( blue ) and loopless ( orange ) Ndc80 trimers that follow shortening microtubule ends . Compared to Ndc80 - free shortening ends in the same field of views ( gray ) . Horizontal lines indicate median values .

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

Figure 2TMR-labelled Ndc80 trimers with (FL, blue) or without (ΔL, orange) the loop were added to taxol-stabilized microtubules to measure their residence time (panel C) and one-dimensional diffusion (panel D). Ndc80 trimers with and without the loop are shown. Scale bars: vertical (1000 s), horizontal (5 µm).F) Kymographs of full-length and loopless Ndc80 trimers (10 pM) that reside on dynamically growing and shortening microtubules. Trimers remain bound to the ends of shortening microtubules. Scale bars: vertical (100 s), horizontal (5 µm). G) Distribution of initial brightnessH) Fraction of Ndc80 trimers that switches from lateral microtubule binding to tracking shortening ends. Data from four repeats (total 140 events) for full-length Ndc80 trimers (blue), and two repeats (total 158 events) for loopless Ndc80 trimers (orange). Horizontal lines indicate average values.I) End-tracking speed of full-length (blue) and loopless (orange) Ndc80 trimers that follow shortening microtubule ends. Compared to Ndc80-free shortening ends in the same field of views (gray). Horizontal lines indicate median values.


Figure 3B ) Correlations of stall duration and stall force . Each datapoint represents a single stall event . Filled squares : stalls resulting in microtubule rescue . Open squares : stalls resulting in bead detachment from the microtubule .

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

Figure 3B) Correlations of stall duration and stall force. Each datapoint represents a single stall event. Filled squares: stalls resulting in microtubule rescue. Open squares: stalls resulting in bead detachment from the microtubule.


Figure 4B ) Immunoblot of NDC80 levels following depletion of the Ndc80 complex by RNAi . C ) Quantification of the time that cells spent in mitosis following various treatments . Each dot represents a cell and the red lines indicate median values . Nocodazole was added 17 hours after electroporation and 1 hour before microscopy . A minimum of 30 cells were analysed for each condition .

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

Figure 4B) Immunoblot of NDC80 levels following depletion of the Ndc80 complex by RNAi.C) Quantification of the time that cells spent in mitosis following various treatments. Each dot represents a cell and the red lines indicate median values. Nocodazole was added 17 hours after electroporation and 1 hour before microscopy. A minimum of 30 cells were analysed for each condition.


Figure 5A ) Schematic of a cold - shock assay following an electroporation experiment . Immunofluorescence images showing the attachment status of kinetochores to microtubules in cells electroporated with recombinant Ndc80 - wt , Ndc80 - ∆ L , or Ndc80 - M5 complexes . The number of cells with multipolar spindles and the total number of analysed cells are shown . Some signal from the tubulin channel is visible in the CENP - C channel . Scale bar : 5 µm . B ) Total Internal Reflection Fluorescence ( TIRF ) microscopy was used to investigate Ndc80Alexa488 complexes ( 0 . 6 nM ) added to trimeric Ndc80TMR ( 10 pM ) on fluorescent taxol - stabilized microtubules that were attached to a passivated glass surface . Typical kymographs showing virtually motionless Ndc80 trimers ( magenta ) and transiently binding Ndc80 monomers ( yellow ) . Wild - type ( wt ) monomers associate with wt trimers ( left ) , but not with M5 trimers ( right ) . Scale bars : vertical ( 100 s ) , horizontal ( 5 µm ) . C ) Quantification of the intensity of the monomeric Ndc80 associating with microtubule - bound trimeric Ndc80 . A threshold for binding was set at an intensity equivalent to one Alexa488 copy . Intensities well above 1 ( yellow ) could thus reflects multiple monomers binding simultaneously .

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

Figure 5A) Schematic of a cold-shock assay following an electroporation experiment. Immunofluorescence images showing the attachment status of kinetochores to microtubules in cells electroporated with recombinant Ndc80-wt, Ndc80-∆L, or Ndc80-M5 complexes. The number of cells with multipolar spindles and the total number of analysed cells are shown. Some signal from the tubulin channel is visible in the CENP-C channel. Scale bar: 5 µm.B) Total Internal Reflection Fluorescence (TIRF) microscopy was used to investigate Ndc80Alexa488 complexes (0.6 nM) added to trimeric Ndc80TMR (10 pM) on fluorescent taxol-stabilized microtubules that were attached to a passivated glass surface. Typical kymographs showing virtually motionless Ndc80 trimers (magenta) and transiently binding Ndc80 monomers (yellow). Wild-type (wt) monomers associate with wt trimers (left), but not with M5 trimers (right). Scale bars: vertical (100 s), horizontal (5 µm).C) Quantification of the intensity of the monomeric Ndc80 associating with microtubule-bound trimeric Ndc80. A threshold for binding was set at an intensity equivalent to one Alexa488 copy. Intensities well above 1 (yellow) could thus reflects multiple monomers binding simultaneously.


Figure 6A ) Representation of the peptides used to raise AB - 849 and AB - 850 and immunoblots showing their recognition of wild - type , M5 and loopless Ndc80 complexes . 9G3 is a commercially available monoclonal antibody raised against NDC8056 - 642 , later shown to recognize NDC80200 - 215 . Asterisk shows the non - specific recognition of another protein , presumably NUF2 . B ) Diffusion of full - length and loopless Ndc80 trimers in absence and presence of AB - 849 . The primary rabbit polyclonal AB - 849 was detected using a Alexa650 - labelled anti - rabbit secondary IgG antibody . Scale bars : vertical ( 100 s ) , horizontal ( 5 µm ) . D ) Summary showing diffusion coefficients ( µm2 / minute ) that follow from the data shown in panel C . Mean values , SEM , and number of diffusion traces ( n ) are indicated . Statistically significant differences were determined using a two - tailed t - test . P - values : no AB , FL vs ΔL : 0 . 0180 ( * ) ; ΔL , no AB vs AB - 849 : 0 . 0108 ( * ) ; M5 , no AB vs AB - 849 : 0 . 0458 ( * ) ; ΔL , AB849 vs AB - 850 : 0 . 0156 ( * ) ; AB - 850 , ΔL vs FL : 0 . 0461 ( * ) ; AB - 850 , ΔL vs M5 : 0 . 0494 ( * ) .

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

Figure 6A) Representation of the peptides used to raise AB-849 and AB-850 and immunoblots showing their recognition of wild-type, M5 and loopless Ndc80 complexes. 9G3 is a commercially available monoclonal antibody raised against NDC8056-642, later shown to recognize NDC80200-215. Asterisk shows the non-specific recognition of another protein, presumably NUF2.B) Diffusion of full-length and loopless Ndc80 trimers in absence and presence of AB-849. The primary rabbit polyclonal AB-849 was detected using a Alexa650-labelled anti-rabbit secondary IgG antibody. Scale bars: vertical (100 s), horizontal (5 µm).D) Summary showing diffusion coefficients (µm2/minute) that follow from the data shown in panel C. Mean values, SEM, and number of diffusion traces (n) are indicated. Statistically significant differences were determined using a two-tailed t-test. P-values: no AB, FL vs ΔL: 0.0180 (*); ΔL, no AB vs AB-849: 0.0108 (*); M5, no AB vs AB-849: 0.0458 (*); ΔL, AB849 vs AB-850: 0.0156 (*); AB-850, ΔL vs FL: 0.0461 (*); AB-850, ΔL vs M5: 0.0494 (*).


Figure 7Experimental workflow to investigate the time that cells , electroporated with recombinant Ndc80 complexes , spent in mitosis in the presence and absence of mitotic kinases inhibitors . Every dot represents a cell and red lines indicate median values . At least 30 cells were analysed for each condition .

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

Figure 7Experimental workflow to investigate the time that cells, electroporated with recombinant Ndc80 complexes, spent in mitosis in the presence and absence of mitotic kinases inhibitors. Every dot represents a cell and red lines indicate median values. At least 30 cells were analysed for each condition.


Figure 8A ) Effects of M13 and M14 on the time spent in mitosis following the experimental setup outlined in Figure 4 . All other datapoints are also shown in Figure 4E . Colors indicate whether cells divided normally , sometimes with delayed chromosome congression ( green ) , or showed long arrests followed by a catastrophic division ( orange ) . Each dot represents a cell and the red lines indicate median values . A minimum of 30 cells were analysed for each condition .

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

Figure 8A) Effects of M13 and M14 on the time spent in mitosis following the experimental setup outlined in Figure 4. All other datapoints are also shown in Figure 4E. Colors indicate whether cells divided normally, sometimes with delayed chromosome congression (green), or showed long arrests followed by a catastrophic division (orange). Each dot represents a cell and the red lines indicate median values. A minimum of 30 cells were analysed for each condition.


Figure 1WT and SNX10 KO Caco - 2 cells were treated with indicated doses of OMVs for 24h . Protein expression of E - cadherin and SNX10 was determined by immunoblots ( A ) and quantified by ImageJ software ( B ) ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . G , H Confocal images of E - cadherin staining in Caco - 2 cells treated with OMVs ( 100 μg / mL ) for the indicated time were captured ( G ) , and the fluorescence intensity of E - cadherin staining was quantified by ImageJ software ( H ) ( n = 3 independent experiments , n = 18 fields analysed ) . Scale bar : 20 μm . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

Figure 1WT and SNX10 KO Caco-2 cells were treated with indicated doses of OMVs for 24h. Protein expression of E-cadherin and SNX10 was determined by immunoblots (A) and quantified by ImageJ software (B) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.G, H Confocal images of E-cadherin staining in Caco-2 cells treated with OMVs (100 μg/mL) for the indicated time were captured (G), and the fluorescence intensity of E-cadherin staining was quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analysed). Scale bar: 20 μm. Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


Figure 2A , B WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24 h . The fold changes in mRNA levels of SNAI1 ( encoding Snail ) ( A ) and SNAI2 ( encoding Slug ) ( B ) in Caco - 2 cells were determined by RT - qPCR . n = 6 independent experiments . Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . ns , not significant . C WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24 h . Cytoplasmic and nuclear proteins were extracted for Snail and Slug determination by immunoblots . WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24 h . quantiﬁed the percentage of Snail ( E ) or Slug ( F ) - positive nuclei respectively . Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 . H WT and SNX10 KO Caco - 2 cells were transfected with Ad - vector or Ad - SNX10 , followed by treatment with or without 100 μg / mL OMVs for 24 h . Proteins from nucleus , cytoplasm and total cell lysates were subjected to immunoblots with the indicated antibodies .

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

Figure 2A, B WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. The fold changes in mRNA levels of SNAI1 (encoding Snail) (A) and SNAI2 (encoding Slug) (B) in Caco-2 cells were determined by RT-qPCR. n = 6 independent experiments. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ns, not significant.C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. Cytoplasmic and nuclear proteins were extracted for Snail and Slug determination by immunoblots.WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. quantiﬁed the percentage of Snail (E) or Slug (F)-positive nuclei respectively. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.H WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without 100 μg/mL OMVs for 24 h. Proteins from nucleus, cytoplasm and total cell lysates were subjected to immunoblots with the indicated antibodies.


Figure 3C , D Levels of p - Lyn , Lyn , E - cadherin and SNX10 in WT and SNX10 KO Caco - 2 cells treated with or without OMVs ( 100 μg / mL ) for 24h were determined by immunoblots ( C ) and quantified by ImageJ software ( D ) ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . E , F Caco - 2 cells were transfected with Ad - vector or Ad - SNX10 , followed by treatment with or without OMVs ( 100 μg / mL ) for 24h . Lysates were extracted and subjected to immunoblots with the indicated antibodies ( E ) and quantified by ImageJ software ( F ) ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . I Cytoplasmic and nuclear proteins were extracted from WT and SNX10 KO Caco - 2 cells treated with or without Tolimidone ( 10 μM ) , and the indicated proteins were analyzed by immunoblots .

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

Figure 3C, D Levels of p-Lyn, Lyn, E-cadherin and SNX10 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24h were determined by immunoblots (C) and quantified by ImageJ software (D) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.E, F Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/mL) for 24h. Lysates were extracted and subjected to immunoblots with the indicated antibodies (E) and quantified by ImageJ software (F) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.I Cytoplasmic and nuclear proteins were extracted from WT and SNX10 KO Caco-2 cells treated with or without Tolimidone (10 μM), and the indicated proteins were analyzed by immunoblots.


Figure 4D WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24 h and then subjected to IP with anti - caspase - 5 antibody . E Representative images of co - staining of Lyn and caspase - 5 in WT and SNX10 KO Caco - 2 cells treated with or without OMVs ( 100 μg / mL ) for 24 h . Scale bar : 20 μm . F The co - localization of Lyn and caspase - 5 was quantified by ImageJ software . n = 3 independent experiments , n = 18 fields analysed . Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 . H Lysates of Flag - tagged full - length SNX10 or its different truncated mutants transfected Caco - 2 cells were subjected to immunoprecipitation .

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

Figure 4D WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h and then subjected to IP with anti-caspase-5 antibody.E Representative images of co-staining of Lyn and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h. Scale bar: 20 μm. F The co-localization of Lyn and caspase-5 was quantified by ImageJ software. n = 3 independent experiments, n = 18 fields analysed. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001. H Lysates of Flag-tagged full-length SNX10 or its different truncated mutants transfected Caco-2 cells were subjected to immunoprecipitation.


Figure 5E , WT and SNX10 KO Caco - 2 cells were treated with OMVs ( 100 μg / mL ) for the indicated time and stained with antibodies against LPS and Rab5 ( E ) . Scale bar : 20 μm . G LPS levels in the cell culture supernatants of WT and SNX10 KO Caco - 2 cells stimulated by OMVs ( 100 μg / mL ) for the indicated time were detected by LAL assay ( n = 6 independent experiments ) . H LPS levels in the cytosolic and residual fractions of Caco - 2 cells stimulated by OMVs ( 100 μg / mL ) with or without chloroquine ( CQ ) ( 200 nM ) or bafilomycin A1 ( Baf A1 ) ( 50 nM ) for the indicated time were detected by LAL assay ( n = 6 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not significant .

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

Figure 5E, WT and SNX10 KO Caco-2 cells were treated with OMVs (100 μg/mL) for the indicated time and stained with antibodies against LPS and Rab5 (E). Scale bar: 20 μm.G LPS levels in the cell culture supernatants of WT and SNX10 KO Caco-2 cells stimulated by OMVs (100 μg/mL) for the indicated time were detected by LAL assay (n = 6 independent experiments). H LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without chloroquine (CQ) (200 nM) or bafilomycin A1 (Baf A1) (50 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.


Figure 6C WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24h and then subjected to IP with anti - PIKfyve antibody . D , E Co - staining of PIKfyve and caspase - 5 in WT and SNX10 KO Caco - 2 cells treated with or without OMVs ( 100 μg / mL ) for 24 h ( D ) . Anti - PIKfyve and anti - caspase - 5 antibodies were used to detect endogenous PIKfyve and caspase - 5 respectively . Scale bar : 20 μm . Colocalized PIKfyve and caspase - 5 were quantified by ImageJ software ( E ) ( n = 3 independent experiments , n = 18 fields analysed ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 . F Caco - 2 cells were stimulated by OMVs ( 100 μg / mL ) with or without apilimod ( 30 nM ) for 24h . Proteins of lysates extracted from Caco - 2 cells were subjected to immunoblots with the indicated antibodies . G , H Co - staining of PIKfyve and Rab5 in WT and SNX10 KO Caco - 2 cells treated with or without OMVs ( 100 μg / mL ) for 24 h ( G ) . Anti - PIKfyve and anti - Rab5 antibodies were used to detect endogenous PIKfyve and Rab5 respectively . Scale bar : 20 μm . Colocalized PIKfyve and caspase - 5 were quantified by ImageJ software ( H ) ( n = 3 independent experiments , n = 18 fields analysed ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 . I LPS levels in the cytosolic and residual fractions of Caco - 2 cells stimulated by OMVs ( 100 μg / mL ) with or without apilimod ( 30 nM ) for the indicated time were detected by LAL assay ( n = 6 independent experiments ) . J Caco - 2 cells transfected with control siRNA or CASP5 siRNA were treated with OMVs ( 100 μg / mL ) for the indicated time . LPS levels in the cytosolic and residual fractions of Caco - 2 cells were detected by LAL assay ( n = 6 independent experiments ) . K LPS levels in the cytosolic and residual fractions of Caco - 2 cells stimulated by OMVs ( 100μg / mL ) with or without bafetinib ( 1μM ) for the indicated time were detected by LAL assay ( n = 6 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 .

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

Figure 6C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24h and then subjected to IP with anti-PIKfyve antibody.D, E Co-staining of PIKfyve and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (D). Anti-PIKfyve and anti-caspase-5 antibodies were used to detect endogenous PIKfyve and caspase-5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (E) (n = 3 independent experiments, n = 18 fields analysed). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.F Caco-2 cells were stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for 24h. Proteins of lysates extracted from Caco-2 cells were subjected to immunoblots with the indicated antibodies.G, H Co-staining of PIKfyve and Rab5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (G). Anti-PIKfyve and anti-Rab5 antibodies were used to detect endogenous PIKfyve and Rab5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analysed). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.I LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments). J Caco-2 cells transfected with control siRNA or CASP5 siRNA were treated with OMVs (100 μg/mL) for the indicated time. LPS levels in the cytosolic and residual fractions of Caco-2 cells were detected by LAL assay (n = 6 independent experiments). K LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100μg/mL) with or without bafetinib (1μM) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


Figure 7SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background and littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 animals , each group ) . B , C Length of colons from WT , SNX10 cKO , IL - 10 KO and SNX10 cKO IL - 10 KO mice was measured ( B ) and analysed ( C ) at the 16th week . Data information : data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background and littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 animals , each group ) . E , F Representative H & E images of colon tissues of WT , SNX10 cKO , IL - 10 KO and SNX10 cKO IL - 10 KO mice at the 16th week were shown ( E ) and analyzed ( F ) . Scale bar : 100 μm . Data information : data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background and littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 animals , each group ) . G The content of TNF - α , IL - 6 , IL - 12 / IL - 23 p40 and IL - 17A in the serum of WT , SNX10 cKO , IL - 10 KO and SNX10 cKO IL - 10 KO mice at the 16th week were measured by ELISA . Data information : data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background and littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 animals , each group ) . J Protein expression of E - cadherin , p - Lyn and Lyn in colon epithelial tissues was determined by immunoblots .

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

Figure 7SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). B, C Length of colons from WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice was measured (B) and analysed (C) at the 16th week. Data information: data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). E, F Representative H&E images of colon tissues of WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were shown (E) and analyzed (F). Scale bar: 100 μm. Data information: data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). G The content of TNF-α, IL-6, IL-12/IL-23 p40 and IL-17A in the serum of WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were measured by ELISA. Data information: data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). J Protein expression of E-cadherin, p-Lyn and Lyn in colon epithelial tissues was determined by immunoblots.


Figure 1 ( A ) Western Blot detection of ATM pS1981 , SMCHD1 , TRF2 and hnRNPA1 loading control in wild - type and two SMCHD1 knockout ( KO1 and KO2 ) HeLa cells transfected with shTRF2 plasmid or EV control . ( B ) Representative metaphase spreads from wild - type and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid or EV control . Telomeric signals were detected with Cy3 - OO - ( CCCTAA ) 3 and are false colored in red , DNA is stained with DAPI and is false colored in cyan . Scale bar : 5µm . ( C ) Quantification of telomere fusions from experiment Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 3 , 000 telomeres counted / condition / experiment ) . ( * ) P < 0 . 05 ; unpaired two - tailed Student ' s t - test . ( D ) Representative cell cycle profiles from two biological replicates of WT and SMCHD1 KO HeLa cells transfected with shTRF2 plasmid or EV control . Cells were stained with propidium iodide and analyzed by Flow cytometry .

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

Figure 1(A) Western Blot detection of ATM pS1981, SMCHD1, TRF2 and hnRNPA1 loading control in wild-type and two SMCHD1 knockout (KO1 and KO2) HeLa cells transfected with shTRF2 plasmid or EV control.(B) Representative metaphase spreads from wild-type and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid or EV control. Telomeric signals were detected with Cy3-OO-(CCCTAA)3 and are false colored in red, DNA is stained with DAPI and is false colored in cyan. Scale bar: 5µm.(C) Quantification of telomere fusions from experiment Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>3,000 telomeres counted/condition/experiment). (*) P < 0.05; unpaired two-tailed Student's t-test.(D) Representative cell cycle profiles from two biological replicates of WT and SMCHD1 KO HeLa cells transfected with shTRF2 plasmid or EV control. Cells were stained with propidium iodide and analyzed by Flow cytometry.


Figure 2 ( A ) Western Blot detection of SMCHD1 , TRF2 and hnRNPA1 in SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days ( d7 , d8 , d11 ) . ( B ) Quantification of telomere fusions in SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days ( d7 , d8 , d11 ) . Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 1 , 900 telomeres counted / condition / experiment ) . ( * * * ) P < 0 . 001 , ( * * ) P < 0 . 01 ; ( * ) P < 0 . 05 ; unpaired two - tailed Student ' s t - test . ( C ) Terminal Restriction Fragment ( TRF ) analysis of telomeric DNA to detect 3 ' overhang processing of genomic DNA isolated from SMCHD1 wild - type or SMCHD1 knockout HeLa inducible shTRF2 cells treated as in the experiment in ( B ) . ( Left ) Radiolabeled ( CCCTAA ) n probe was hybridized with a short run ( upper panel ) and long run ( lower panel ) native DNA gel to detect the signal of the telomeric 3 ' overhang . Samples used for the short and the long run were from the same digestion split into two . Exo I treatment ( + Exo ) was used as a control that single stranded telomeric signal was terminal . ( Right ) The total TTAGGG signal in the same lane was deteced upon denaturation and hybridization with the same probe . ( D ) Quantification of the telomeric overhang signal at d11 after doxycyclin addition to SMCHD1 wild - type and SMCHD1 knockout HeLa shTRF2 inducible cells . The bar graph represents the average overhang signal intensity from two biological replicates as percentage of the signal in the cells untreated with doxycycline with error bars representing the standard deviation ( SD ) of the sample .

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

Figure 2(A) Western Blot detection of SMCHD1, TRF2 and hnRNPA1 in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11).(B) Quantification of telomere fusions in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11). Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>1,900 telomeres counted/condition/experiment). (***) P < 0.001, (**) P< 0.01; (*) P< 0.05; unpaired two-tailed Student's t-test.(C) Terminal Restriction Fragment (TRF) analysis of telomeric DNA to detect 3'overhang processing of genomic DNA isolated from SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated as in the experiment in (B). (Left) Radiolabeled (CCCTAA)n probe was hybridized with a short run (upper panel) and long run (lower panel) native DNA gel to detect the signal of the telomeric 3' overhang. Samples used for the short and the long run were from the same digestion split into two. Exo I treatment (+ Exo) was used as a control that single stranded telomeric signal was terminal. (Right) The total TTAGGG signal in the same lane was deteced upon denaturation and hybridization with the same probe.(D) Quantification of the telomeric overhang signal at d11 after doxycyclin addition to SMCHD1 wild-type and SMCHD1 knockout HeLa shTRF2 inducible cells. The bar graph represents the average overhang signal intensity from two biological replicates as percentage of the signal in the cells untreated with doxycycline with error bars representing the standard deviation (SD) of the sample.


Figure 3A Western blot detection of ATM pS1981 , SMCHD , TRF2 , γH2AX , and hnRNPA1 in WT or SMCHD1 KO Hela cells transfected with shTRF2 and EV control . B Representative images for CO - FISH on metaphase spreads in wild type ( WT ) Hela and U2OS cells . The leading strand telomeres were labeled by PNA - FISH with a ( TTAGGG ) 3 - Alexa488 probe ( green ) , the lagging strand with a ( CCCTAA ) 3 - Cy5 probe ( pink ) , and the DNA was stained with DAPI . Examples of telomere sister chromatid exchange ( t - SCEs ) events are marked with white arrows . Scale bar : 5µmC Quantification of chromosome ends with t - SCEs detected by CO - FISH . Data represent means of 2 independent experiments ± SD ( > 700 telomeres / condition / experiment ) with individual average values indicated .

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

Figure 3A Western blot detection of ATM pS1981, SMCHD, TRF2, γH2AX, and hnRNPA1 in WT or SMCHD1 KO Hela cells transfected with shTRF2 and EV control.B Representative images for CO-FISH on metaphase spreads in wild type (WT) Hela and U2OS cells. The leading strand telomeres were labeled by PNA-FISH with a (TTAGGG)3-Alexa488 probe (green), the lagging strand with a (CCCTAA)3-Cy5 probe (pink), and the DNA was stained with DAPI. Examples of telomere sister chromatid exchange (t-SCEs) events are marked with white arrows. Scale bar: 5µmC Quantification of chromosome ends with t-SCEs detected by CO-FISH. Data represent means of 2 independent experiments± SD (>700 telomeres/condition/experiment) with individual average values indicated.


Figure 4 ( A - C ) Representative images for detection of ATM pS9181 , γH2AX , and 53BP1 at telomeres in wild - type ( WT ) and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid and empty vector ( EV ) control . Immunofluoresence ( IF ) for ATM pS1981 ( gray ) , γH2AX ( green ) and 53BP1 ( yellow ) was combined with telomeric ( CCCTAA ) 3 - FISH ( red ) and DAPI staining total DNA . Scale bars : 5µm ( D ) Quantification of the number of cells containing > 5 Telomere dysfunction Induced Foci ( TIFs ) detected Data represent the mean of 4 independent experiments ± SD ( > 200 cells / condition / experiment ) for ATM pS1981 and 3 independent experiments ± SD ( > 200 cells / condition / experiment ) for γH2AX and 53BP1 . ( * * * ) P < 0 . 001 ; unpaired two - tailed Student ' s t - test . ( E ) Quantification of the number of 53BP1 TIFs per cell detected from three independent experiments . Data represent individual values per cell with the bar representing mean ± SD . ( * * * ) P < 0 . 001 ; unpaired two - tailed Student ' s t - test .

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

Figure 4(A-C) Representative images for detection of ATM pS9181, γH2AX, and 53BP1 at telomeres in wild-type (WT) and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid and empty vector (EV) control. Immunofluoresence (IF) for ATM pS1981 (gray), γH2AX (green) and 53BP1 (yellow) was combined with telomeric (CCCTAA)3-FISH (red) and DAPI staining total DNA. Scale bars: 5µm(D) Quantification of the number of cells containing >5 Telomere dysfunction Induced Foci (TIFs) detected Data represent the mean of 4 independent experiments ± SD (>200 cells/condition/experiment) for ATM pS1981 and 3 independent experiments ± SD (>200 cells/condition/experiment) for γH2AX and 53BP1. (***) P < 0.001; unpaired two-tailed Student's t-test.(E) Quantification of the number of 53BP1 TIFs per cell detected from three independent experiments. Data represent individual values per cell with the bar representing mean ± SD. (***) P < 0.001; unpaired two-tailed Student's t-test.


Figure 5 ( A ) Western blot detection of SMCHD1 , ATMpS1981 , CHK2 pT68 , CHK1 pS345 , RPA pS33 , RPA pS4 / 8 , γH2AX and Vinculin in wild - type or SMCHD1 KO HeLa cells γ - irradiated with 3Gy dose and harvested at the indicated times ( 1h , 12h , 24h ) after the treatment . ( B ) Representative images for Immunofluoresence ( IF ) detection of ATMpS1981 in wild - type HeLa cells γ - irradiated with a dose of 3Gy and harvested after the indicated times . Scale bar : 5µm ( C ) Quantification of the average number of ATMpS1981 foci per cell detected as in ( B ) in wild - type and SMCHD1 KO γ - irradiated HeLa cells . Data represent mean of 3 independent experiments ± SD ( > 200 cells / condition / experiment ) with individual average values indicated .

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

Figure 5(A) Western blot detection of SMCHD1, ATMpS1981, CHK2 pT68, CHK1 pS345, RPA pS33, RPA pS4/8, γH2AX and Vinculin in wild-type or SMCHD1 KO HeLa cells γ-irradiated with 3Gy dose and harvested at the indicated times (1h, 12h, 24h) after the treatment.(B) Representative images for Immunofluoresence (IF) detection of ATMpS1981 in wild-type HeLa cells γ-irradiated with a dose of 3Gy and harvested after the indicated times. Scale bar: 5µm(C) Quantification of the average number of ATMpS1981 foci per cell detected as in (B) in wild-type and SMCHD1 KO γ-irradiated HeLa cells. Data represent mean of 3 independent experiments ± SD (>200 cells/condition/experiment) with individual average values indicated.


Figure 6 ( A ) Western Blot detection of SMCHD1 , TRF2 , TPP1 and hnRNPA1 in wild - type or SMCHD1 knockout HeLa cells transfected with the indicated shRNAs ( shTRF2 , shTPP1 , shTRF2 / shTPP1 ) or EV control . ( B ) Representative metaphase spreads ( Scale bar : 5µm ) from HeLa cells transfected with indicated shRNAs or EV control and quantification of telomere fusions . Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 2 , 500 telomeres counted / condition / experiment ) . ( * * * ) P < 0 . 001 ; ( * * ) P < 0 . 01 unpaired two - tailed Student ' s t - test . ( C ) Representative metaphase spreads ( Scale bar : 5µm ) from HeLa cells transfected with indicated shRNAs or EV control treated for 4 days with ATRi ( VE - 821 ) and quantification of telomere fusions . Bars represent average number of fused chromosome ends . SDs were obtained from 3 independent experiments ( > 2 , 000 telomeres counted / condition / experiment ) . ( * * * ) P < 0 . 001 ; ( * * ) P < 0 . 01 ; ( * ) P < 0 . 05 unpaired two - tailed Student ' s t - test .

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

Figure 6(A) Western Blot detection of SMCHD1, TRF2, TPP1 and hnRNPA1 in wild-type or SMCHD1 knockout HeLa cells transfected with the indicated shRNAs (shTRF2, shTPP1, shTRF2/shTPP1) or EV control.(B) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,500 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01 unpaired two-tailed Student's t-test.(C) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control treated for 4 days with ATRi (VE-821) and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,000 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01; (*) P < 0.05 unpaired two-tailed Student's t-test.


Figure 1A ) MALDI imaging was performed on tumor - containing brain sections at a lateral resolution of 100µm and ( M - H ) ions were analysed in negative mode with a FTICR Mass Spectrometer ( analytical range 100 - 1000Da ) . Hematoxylin - eosin ( HE ) staining of the same sections was performed after MALDI analysis . Patient - derived glioma xenografts ( PDXs ) with IDH wildtype ( IDHwt ) , IDH mutant ( IDHm ) , and control brain tissue ( CB ) were analyzed in a non - targeted and targeted approach using regions of interest ( ROI ) of different sizes to compare tumor ( T , yellow line ) and contralateral control brain ( CB , blue line ) . The non - targeted approach was used for statistical pair - wise comparisons between samples , while the targeted approach on a selected set of metabolites allowed distribution and quantification analyses . B ) Histological sections of 3 IDHwt PDXs ( P3 , T434 , P8 ) and 3 IDHm PDXs ( ( T394 , E478 , T186 ) showing a large ROI ( boxed rectangle , > 500 pixels ) within the tumor area applied for targeted quantification of selected metabolites . Left rectangle in P3 PDX was used as control contralateral brain ( CB ) for quantifications . Middle panel shows tissue distribution of D - 2 - hydroxyglutarate ( D2HG ) by MSI , which is exclusively detected in IDHm PDXs ( D2HG in IDHwt tumours is below detection limit ) . Right panel shows tissue distribution of a key metabolite ( m / z 778 . 51 ) presenting strong accumulation in IDHm lower grade gliomas ( LGG ) versus glioblastomas ( GBM ) independent of IDH status . An intensity - dependent color code indicates the relative amount of a specific compound ( defined by m / z value ) throughout the tissue section . C ) Quantification of several high mass metabolites differentially present in IDHm PDXs ( IDHwt glioblastomas ( GBM ) in blue , IDHm GBM in orange , IDHm lower grade gliomas ( LGG ) in red , contralateral control brain ( CB ) in grey ) . Box plots represent log values of metabolite intensities measured within a large ROI ( > 500 pixels ) . Many metabolites in this mass range ( m / z 700 - 900 ) have been putatively assigned to phospholipids ( Tables EV2 - 3 ) . m / z represents the mass over charge ratio of ionized metabolites , as measured by the mass spectrometer . Except where otherwise indicated , statistical significance was calculated for IDHm ( n = 3 ) versus IDHwt ( n = 3 ) PDXs using T test . pv : p - values .

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

Figure 1A) MALDI imaging was performed on tumor-containing brain sections at a lateral resolution of 100µm and (M-H) ions were analysed in negative mode with a FTICR Mass Spectrometer (analytical range 100-1000Da). Hematoxylin-eosin (HE) staining of the same sections was performed after MALDI analysis. Patient-derived glioma xenografts (PDXs) with IDH wildtype (IDHwt), IDH mutant (IDHm), and control brain tissue (CB) were analyzed in a non-targeted and targeted approach using regions of interest (ROI) of different sizes to compare tumor (T, yellow line) and contralateral control brain (CB, blue line). The non-targeted approach was used for statistical pair-wise comparisons between samples, while the targeted approach on a selected set of metabolites allowed distribution and quantification analyses.B) Histological sections of 3 IDHwt PDXs (P3, T434, P8) and 3 IDHm PDXs ((T394, E478, T186) showing a large ROI (boxed rectangle, >500 pixels) within the tumor area applied for targeted quantification of selected metabolites. Left rectangle in P3 PDX was used as control contralateral brain (CB) for quantifications. Middle panel shows tissue distribution of D-2-hydroxyglutarate (D2HG) by MSI, which is exclusively detected in IDHm PDXs (D2HG in IDHwt tumours is below detection limit). Right panel shows tissue distribution of a key metabolite (m/z 778.51) presenting strong accumulation in IDHm lower grade gliomas (LGG) versus glioblastomas (GBM) independent of IDH status. An intensity-dependent color code indicates the relative amount of a specific compound (defined by m/z value) throughout the tissue section.C) Quantification of several high mass metabolites differentially present in IDHm PDXs (IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey). Box plots represent log values of metabolite intensities measured within a large ROI (>500 pixels). Many metabolites in this mass range (m/z 700-900) have been putatively assigned to phospholipids (Tables EV2-3). m/z represents the mass over charge ratio of ionized metabolites, as measured by the mass spectrometer. Except where otherwise indicated, statistical significance was calculated for IDHm (n=3) versus IDHwt (n=3) PDXs using T test. pv: p-values.


Figure 2A ) MALDI imaging of indicated metabolites in IDHwt PDX ( P3 ) , IDHm PDX ( E478 ) and control brain ( CB ) . Hex - P : hexose - phosphate ; AMP , ADP , ATP : adenosine mono - , di - and triphosphate respectively . B ) LC - MS quantification of indicated metabolites in 6 PDXs . Relative quantities of metabolites are expressed in log scale and normalized to tissue quantity used for extract preparation . IDHwt glioblastomas ( GBM ) in blue , IDHm GBM in orange , IDHm lower grade gliomas ( LGG ) in red , contralateral control brain ( CB ) in grey . Sdh7 - P : sedoheptulose - 7 - phosphate . C ) Energetic charge defined as the ratio of ( ATP + 1 / 2ADP ) / ( ATP + ADP + AMP ) was calculated at each pixel to present its distribution throughout the tumor . ( D ) The energetic charge ratio was confirmed by the LC - MS data performed on separate tumor extracts ( n = 3 / sample / group ) . Indicated statistical significant group differences are based on T test , pv : p - values .

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

Figure 2A) MALDI imaging of indicated metabolites in IDHwt PDX (P3), IDHm PDX (E478) and control brain (CB). Hex-P: hexose-phosphate; AMP, ADP, ATP: adenosine mono-, di- and triphosphate respectively.B) LC-MS quantification of indicated metabolites in 6 PDXs. Relative quantities of metabolites are expressed in log scale and normalized to tissue quantity used for extract preparation. IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey. Sdh7-P: sedoheptulose-7-phosphate.C) Energetic charge defined as the ratio of (ATP+1/2ADP)/(ATP+ADP+AMP) was calculated at each pixel to present its distribution throughout the tumor.(D) The energetic charge ratio was confirmed by the LC-MS data performed on separate tumor extracts (n=3/ sample/ group). Indicated statistical significant group differences are based on T test, pv: p-values.


Figure 3A ) LC - MS quantification of steady - state level of glycolysis and TCA cycle metabolites in three IDH1wt and three IDH1m PDXs and contralateral control brain ( CB ) ( n = 3 / sample / group ) . Metabolites showing statistically significant group differences between IDH1wt and IDH1m are indicated ( pvalue : * < 0 . 05 ) . Color code as in Fig . 1 - 2 . C ) In vivo flux analysis of 13C - labelled glucose injected 20 minutes prior to sacrifice in one IDH1wt PDX ( P3 ) and one IDH1m PDX ( E478 ) ( n = 3 / sample ) . The percentage of glucose - derived 13C label is shown in selected metabolites containing 0 to 6 13C atoms ( 13C0 - 13C6 as indicated by color code ) . Note the absence of 13C2 label of αKG and D2HG in IDHm tumours , while the label was detectable in IDHwt PDX and in contralateral brain of both tumors ( lower panel ) . D ) Quantities of 13C3 and 13C2 labeled metabolites from glucose tracer experiment in ( C ) . D2HG in IDH1m PDX shows a detectable 13C2 signature here , but represents a minor fraction of the total cellular D2HG pool in this tumor . Hex - P : hexose phosphate , PEP : phosphoenolpyruvate , Pyr : pyruvate , Cit : citrate , Mal : malate , Asp : aspartate , Glu : glutamate , Gln : glutamine , α - KG : α - Ketoglutarate , D2HG : D2 - Hydroxyglutarate . Indicated statistical significant group differences are based on T test , pv : p - values .

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

Figure 3A) LC-MS quantification of steady-state level of glycolysis and TCA cycle metabolites in three IDH1wt and three IDH1m PDXs and contralateral control brain (CB) (n=3/ sample/ group). Metabolites showing statistically significant group differences between IDH1wt and IDH1m are indicated (pvalue: * <0.05). Color code as in Fig. 1-2.C) In vivo flux analysis of 13C-labelled glucose injected 20 minutes prior to sacrifice in one IDH1wt PDX (P3) and one IDH1m PDX (E478) (n=3/ sample). The percentage of glucose-derived 13C label is shown in selected metabolites containing 0 to 6 13C atoms (13C0 - 13C6 as indicated by color code). Note the absence of 13C2 label of αKG and D2HG in IDHm tumours, while the label was detectable in IDHwt PDX and in contralateral brain of both tumors (lower panel).D) Quantities of 13C3 and 13C2 labeled metabolites from glucose tracer experiment in (C). D2HG in IDH1m PDX shows a detectable 13C2 signature here, but represents a minor fraction of the total cellular D2HG pool in this tumor. Hex-P: hexose phosphate, PEP: phosphoenolpyruvate, Pyr: pyruvate, Cit: citrate, Mal: malate, Asp: aspartate, Glu: glutamate, Gln: glutamine, α-KG: α-Ketoglutarate, D2HG: D2-Hydroxyglutarate. Indicated statistical significant group differences are based on T test, pv: p-values.


Figure 4A ) MALDI imaging of key metabolites in IDHwt ( P3 ) and IDHm ( E478 ) glioma PDX , compared to control brain ( CB ) sections . B ) Quantification of metabolite distribution by MSI in 6 PDX with IDH1wt and IDH1m status ( n = 3 / group ) and in contralateral control brain ( CB ) . No significant differences between IDH groups were detected , although significant differences are found between tumors and control brain ( glutamate , apsartate ) . Color code as in Fig . 1 , NAA : N - acetylaspartic acid ; NAAG : N - acetylaspartylglutamic acid .

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

Figure 4A) MALDI imaging of key metabolites in IDHwt (P3) and IDHm (E478) glioma PDX, compared to control brain (CB) sections.B) Quantification of metabolite distribution by MSI in 6 PDX with IDH1wt and IDH1m status (n=3 / group) and in contralateral control brain (CB). No significant differences between IDH groups were detected, although significant differences are found between tumors and control brain (glutamate, apsartate). Color code as in Fig. 1, NAA: N-acetylaspartic acid; NAAG: N-acetylaspartylglutamic acid.


Figure 5A ) NADPH levels are reduced in IDHm compared to wildtype tumors , resulting in a reduced NADPH / NAD + ratio ( n = 3 / sample ) . p - value from T test : * < 0 . 05 . NADPH , NADP : reduced , oxydized nicotinamide adenine dinucleotide phosphate , NADH , NAD : reduced , oxydized nicotinamide adenine dinucleotide . B ) MALDI images for glutathione ( GSH ) and cystathionine on IDHwt ( P3 ) and IDHm ( E478 ) PDXs . C ) Quantification of MSI signals for GSH and cystathionine from 6 PDXs ( n = 3 / group ) . D ) LC - MS quantification of independent samples for GSH , GSSG ( oxidized GSH ) , GSSG / GSH ratio and cystathionine from 6 PDXs ( n = 3 / sample / group ) . Statistical significance between IDH groups calculated by T test , pv : p - value .

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

Figure 5A) NADPH levels are reduced in IDHm compared to wildtype tumors, resulting in a reduced NADPH/NAD+ ratio (n=3/ sample). p-value from T test: *<0.05. NADPH, NADP: reduced, oxydized nicotinamide adenine dinucleotide phosphate, NADH, NAD: reduced, oxydized nicotinamide adenine dinucleotide.B) MALDI images for glutathione (GSH) and cystathionine on IDHwt (P3) and IDHm (E478) PDXs.C) Quantification of MSI signals for GSH and cystathionine from 6 PDXs (n=3 / group).D) LC-MS quantification of independent samples for GSH, GSSG (oxidized GSH), GSSG/GSH ratio and cystathionine from 6 PDXs (n=3/ sample / group). Statistical significance between IDH groups calculated by T test, pv: p-value.