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

F 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.


The 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The 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.


A , 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 .

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

A, B 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C 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.


C , 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 .

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

C, D) 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 .

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

E) 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 .

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

F) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


TMR - 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 ) .

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

TMR-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

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

F) 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 brightness


H ) 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 .

H) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


B ) 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 O O O O O O O O O O O O O O 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) 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.


B ) Immunoblot of NDC80 levels following depletion of the Ndc80 complex by RNAi .

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

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.


A ) 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 .

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

A) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) 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.


A ) 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 .

A) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ( * ) .

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 (*).


Experimental 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Experimental 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.


A ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) 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.


WT 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 .

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

WT 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A , 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 .

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

A, B 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 .

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 .

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

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

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.


C , 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 .

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

C, D 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 .

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

E, F 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 O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

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.


D 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 .

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

D 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 .

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

H Lysates of Flag-tagged full-length SNX10 or its different truncated mutants transfected Caco-2 cells were subjected to immunoprecipitation.


E , 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 .

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

E, 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


C 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 .

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

C 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 .

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

D, 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 .

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

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 .

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

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

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.


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 ) . 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 .

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

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). 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 .

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

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 .

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

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

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.


( 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 .

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

(A) Western 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 .

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

(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 .

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

(C) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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.


( 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 ) .

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

(A) Western 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 .

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

(B) Quantification of 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 .

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

(C) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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.


A Western blot detection of ATM pS1981 , SMCHD , TRF2 , γH2AX , and hnRNPA1 in WT or SMCHD1 KO Hela cells transfected with shTRF2 and EV control .

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

A Western 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 :

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

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: 5µm


C 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C 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.


( 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 :

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

(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 .

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

(D) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) 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.


( 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 .

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

(A) Western 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 :

O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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.


( 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 .

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

(A) Western 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

(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.


A ) 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 .

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

A) 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 .

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

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

C) 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.


A ) 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 .

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

A) 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 .

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

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 .

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

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

(D) 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.


A ) 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 .

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

A) 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 ) .

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

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

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.


A ) MALDI imaging of key metabolites in IDHwt ( P3 ) and IDHm ( E478 ) glioma PDX , compared to control brain ( CB ) sections .

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

A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O

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.


A ) 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 .

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

A) 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 .

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

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 ) .

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

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

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.


A ) Comparative gene expression analysis of key enzymes involved in GSH and cysteine synthesis and supply in IDHwt ( n = 220 ) , IDHm with 1p / 19q co - deletion ( n = 151 ) or IDHm without 1p / 19q co - deletion ( n = 226 ) patients based on TCGA RNA - seq data from a cohort of 597 patients . CBS : Cystathionine beta synthase , key enzyme of the transsulfuration pathway . GCLC : catalytic subunit of glutamate - cysteine ligase . SLC7A11 : gene encoding xCT , the cystine - glutamate antiporter .

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

A) Comparative gene expression analysis of key enzymes involved in GSH and cysteine synthesis and supply in IDHwt (n=220), IDHm with 1p/19q co-deletion (n=151) or IDHm without 1p/19q co-deletion (n=226) patients based on TCGA RNA-seq data from a cohort of 597 patients. CBS: Cystathionine beta synthase, key enzyme of the transsulfuration pathway. GCLC: catalytic subunit of glutamate-cysteine ligase. SLC7A11: gene encoding xCT, the cystine-glutamate antiporter.


B ) Kaplan - Meier curves of CBS gene expression versus overall survival in patients with IDHwt , IDHm with or without 1p / 19q co - deletion . Statistical significance for the survival analysis is shown as : pv : p - value , ns = non significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier curves of CBS gene expression versus overall survival in patients with IDHwt, IDHm with or without 1p/19q co-deletion. Statistical significance for the survival analysis is shown as: pv: p-value, ns= non significant.


A . Pull - down experiments using as a bait the biotinylated peptides mimicking the N - terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates ( top blot ) or purified His - tagged GOLPH3 protein ( bottom blot ) . The N - terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot ( LxxR motif in red , biotin at the C - terminus ) . Immunoblotting shows affinity - captured GOLPH3 ( endogenous and recombinant ) .

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

A. Pull-down experiments using as a bait the biotinylated peptides mimicking the N-terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot (LxxR motif in red, biotin at the C-terminus). Immunoblotting shows affinity-captured GOLPH3 (endogenous and recombinant).


B . Control or GOLPH3 - KD HeLa cells expressing luminal SI - GFP or the chimeras of sucrose - isomaltase containing the N - terminal cytosolic tails of LCS , Gb3S , GM3S and GD3S were fixed and labelled for surface localized GFP ( red ) . Scale bar , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Control or GOLPH3-KD HeLa cells expressing luminal SI-GFP or the chimeras of sucrose-isomaltase containing the N-terminal cytosolic tails of LCS, Gb3S, GM3S and GD3S were fixed and labelled for surface localized GFP (red). Scale bar, 20 μm.


C . Pull - down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates ( top blot ) or purified His - tagged GOLPH3 protein ( bottom blot ) . The N - terminal cytosolic tail of LCS WT and LCS mutants ( mut . 1 , 2 and 3 ) is shown on the top of the blot ( mutations are in blue , LxxR motif in red , biotin is at the C - terminus ) .

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

C. Pull-down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail of LCS WT and LCS mutants (mut. 1, 2 and 3) is shown on the top of the blot (mutations are in blue, LxxR motif in red, biotin is at the C-terminus).


D . HeLa cells expressing luminal SI - GFP chimeras of sucrose - isomaltase ( SI - GFP , green ) containing the WT and mutant N - terminal cytosolic tails of LCS , were fixed and labelled for surface localized GFP ( red ) . Cells expressing LCS - SI - RxR and LCS - SI - LxxR chimeras shown PM staining . Scale bar , 20 μm , ( left ) . PM ( surface ) fluorescence intensity ( in Table S10 are listed the software used in this study ) of each chimera is normalised on the LCS - SI - GFP surface intensity ( data are means ± SEM derived from two biological replicates ; n is indicated in the graph , * * * p < 0 . 001 [ Student ' s t - test ] ) ( right ) .

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

D. HeLa cells expressing luminal SI-GFP chimeras of sucrose-isomaltase (SI-GFP, green) containing the WT and mutant N-terminal cytosolic tails of LCS, were fixed and labelled for surface localized GFP (red). Cells expressing LCS-SI-RxR and LCS-SI-LxxR chimeras shown PM staining. Scale bar, 20 μm, (left). PM (surface) fluorescence intensity (in Table S10 are listed the software used in this study) of each chimera is normalised on the LCS-SI-GFP surface intensity (data are means ± SEM derived from two biological replicates; n is indicated in the graph, ***p <0.001 [Student's t-test]) (right).


E . Quantitative flow cytometry - based analysis of the experiment reported in D ; n is indicated in the graph ( left ) . Percentage of cells with plasma membrane staining ( surface ) is reported for each chimera ( right ) .

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

E. Quantitative flow cytometry-based analysis of the experiment reported in D; n is indicated in the graph (left). Percentage of cells with plasma membrane staining (surface) is reported for each chimera (right).


F . GOLPH3 - KD or OE HeLa cells were processed for ShTxB ( upper panels ) or ChTxB ( lower panels ) staining followed by flow cytometry analysis . Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition .

F. GOLPH3-KD or OE HeLa cells were processed for ShTxB (upper panels) or ChTxB (lower panels) staining followed by flow cytometry analysis. Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition.


A . HeLa cells mock - treated or GOLPH3 - KD were transiently transfected with LCS _ GFP _ RUSH ( for simplicity , referred in the text as LCS - GR ) and subjected to the synchronisation protocol described in Materials and Methods . Cells were then subjected to IF at each indicated time point : LCS - GR ( green ) , cis Golgi marker GM130 ( red ) , and TGN marker TGN46 ( blue ) . Representative individual Golgi mini - stacks are shown . Scale bar , 1 µm . LCS - GR intra - Golgi trafficking in mock or GOLPH3 - KD conditions it has been quantified ( right graph ) by computational coalescence of line scans , and was performed by normalized line scan analysis ( normalisation of the distances was performed considering the GM130 peak as 0 , and the TGN46 peak as 1 ) . The n indicated in the graph refers to the number of Golgi stacks analysed in each condition .

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

A. HeLa cells mock-treated or GOLPH3-KD were transiently transfected with LCS_GFP_RUSH (for simplicity, referred in the text as LCS-GR) and subjected to the synchronisation protocol described in Materials and Methods. Cells were then subjected to IF at each indicated time point: LCS-GR (green), cis Golgi marker GM130 (red), and TGN marker TGN46 (blue). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm. LCS-GR intra-Golgi trafficking in mock or GOLPH3-KD conditions it has been quantified (right graph) by computational coalescence of line scans, and was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1). The n indicated in the graph refers to the number of Golgi stacks analysed in each condition.


B . Mock and GOLPH3 - KD HeLa cells co - expressing LCS - GR and VSV - G were subjected to the co - synchronisation protocol described in Materials and Methods . Cells were then subjected to IF : LCS - GR or VSV - G ( green ) , GM130 ( blue ) , and TGN46 ( red ) . Representative individual Golgi mini - stacks are shown . Scale bar , 1 µm . C . Quantification of the experiment in ( B ) was performed by normalized line scan analysis ( normalisation of the distances was performed considering the GM130 peak as 0 , and the TGN46 peak as 1 ) ; n indicated in the graph refers to the number of Golgi stacks analysed in each condition , individual data points and means ± SEM are shown , * * * p < 0 . 001 ( Student ' s t test ) ; ns , not significant . D

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Mock and GOLPH3-KD HeLa cells co-expressing LCS-GR and VSV-G were subjected to the co-synchronisation protocol described in Materials and Methods. Cells were then subjected to IF: LCS-GR or VSV-G (green), GM130 (blue), and TGN46 (red). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm. C. Quantification of the experiment in (B) was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1); n indicated in the graph refers to the number of Golgi stacks analysed in each condition, individual data points and means ± SEM are shown, ***p < 0.001 (Student's t test); ns, not significant. D


D . HeLa cells were transfected with LCS - GR , subjected to the synchronisation protocol ( see Materials and Methods ) , fixed and processed for cryoimmunolabeling with an anti - GFP antibody ( 15 nm gold particles ) and anti - GM130 antibody ( 10 nm gold particles , black arrowheads ) peri - Golgi vesicles containing LCS - GR are marked by red arrowheads . Scale bar , 150 nm .

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

D. HeLa cells were transfected with LCS-GR, subjected to the synchronisation protocol (see Materials and Methods), fixed and processed for cryoimmunolabeling with an anti-GFP antibody (15 nm gold particles) and anti-GM130 antibody (10 nm gold particles, black arrowheads) peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. Scale bar, 150 nm.


E , F . Quantification of the distribution of LCS - GR as measured by Frequency Distribution Analysis ( E ) and linear density ( LD , in F ) ( data are means ± SEM , number of Golgi stacks is n = 15 at 0 min , n = 14 at 10 min , n = 17 at 20 min . ( E ) The red arrows indicate the fraction of LCS - GR gold particles ( % ) in vesicles at each indicated time - point .

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

E, F. Quantification of the distribution of LCS-GR as measured by Frequency Distribution Analysis (E) and linear density (LD, in F) (data are means± SEM, number of Golgi stacks is n=15 at 0 min, n=14 at 10 min, n=17 at 20 min. (E) The red arrows indicate the fraction of LCS-GR gold particles (%) in vesicles at each indicated time-point.


G . HeLa cells expressing LCS - GR mock or GOLPH3 - KD were treated and stained as in D . Black arrowheads indicate GM130 ; peri - Golgi vesicles containing LCS - GR are marked by red arrowheads . LCS - GR in the TGN is marked by blue arrowheads . Scale bar , 150 nm .

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

G. HeLa cells expressing LCS-GR mock or GOLPH3-KD were treated and stained as in D. Black arrowheads indicate GM130; peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. LCS-GR in the TGN is marked by blue arrowheads. Scale bar, 150 nm.


H . Quantification of LCS - GR distribution ( linear density , LD ) across the stack and in peri - Golgi vesicles ( data are means ± SEM , number of Golgi stacks is n = 17 at 20 min for the CTRL , n = 13 at 20 min for the GOLPH3 KD ; * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

H. Quantification of LCS-GR distribution (linear density, LD) across the stack and in peri-Golgi vesicles (data are means ± SEM, number of Golgi stacks is n=17 at 20 min for the CTRL, n=13 at 20 min for the GOLPH3 KD; *p <0.05, **p < 0.01; ***p <0.001 [Student's t-test]).


A . PHFs GOLPH3 OE ( left ) , or KD ( right ) , were fixed and processed for IF : Endogenous LCS ( green ) , GOLPH3 ( red ) , TGN46 or DAPI ( blue ) . Dashed lines indicate cells overexpressing or interfered for GOLPH3 . Scale bar , 20 µm . Quantification of the amount of endogenous LCS ( right graph ) at the Golgi ; data are means ± SEM derived from two biological replicates , n = 42 in CTRL , n = 49 in GOLPH3 KD and n = 27 in GOLPH3 OE ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

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

A. PHFs GOLPH3 OE (left), or KD (right), were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), TGN46 or DAPI (blue). Dashed lines indicate cells overexpressing or interfered for GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi; data are means ± SEM derived from two biological replicates, n=42 in CTRL, n=49 in GOLPH3 KD and n=27 in GOLPH3 OE; ***p <0.001 [Student's t-test]).


B . GOLPH3 - OE HeLa cells were fixed and processed for IF : Endogenous LCS ( green ) , GOLPH3 ( red ) , and TGN46 ( blue ) . Dashed lines indicate cells overexpressing GOLPH3 . Scale bar , 20 µm . Quantification of the amount of endogenous LCS ( right graph ) at the Golgi ( data are means ± SEM derived from two biological replicates , n = 21 in CTRL , n = 11 in GOLPH3 OE ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GOLPH3-OE HeLa cells were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), and TGN46 (blue). Dashed lines indicate cells overexpressing GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi (data are means ± SEM derived from two biological replicates, n=21 in CTRL, n=11 in GOLPH3 OE; ***p <0.001 [Student's t-test]).


C . GOLPH3 OE or KD HeLa cells expressing LCS - HA were fixed and processed for IF : HA ( green ) . Asterisks indicate GOLPH3 OE cells . Scale bar , 20 µm .

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

C. GOLPH3 OE or KD HeLa cells expressing LCS-HA were fixed and processed for IF: HA (green). Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.


D . Assessment of LCS - GR synchronized transport in GOLPH3 OE or KD HeLa cells . Micrographs show cells at the indicated trafficking time points . Scale bar , 20 µm .

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

D. Assessment of LCS-GR synchronized transport in GOLPH3 OE or KD HeLa cells. Micrographs show cells at the indicated trafficking time points. Scale bar, 20 µm.


E . Representative WB of cells treated as in ( D ) ( n = 3 ) .

E. Representative WB of cells treated as in (D) (n = 3).


F . Plots of the densitometric quantification of the amount of LCS - GR ( Golgi mature band ) over time , starting from 2 hr chase ( data are means of at least 3 experiments ± SD ) .

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

F. Plots of the densitometric quantification of the amount of LCS-GR (Golgi mature band) over time, starting from 2 hr chase (data are means of at least 3 experiments ± SD).


G . GOLPH3 OE or KD HeLa cells expressing LCS - HA were treated with bafilomycin A1 ( BafA1 , 10 nM ) for 16 hr , fixed , and processed for IF labelling : LCS ( green ) , LAMP1 ( red ) , and DAPI ( blue ) . Arrowheads indicate LCS and LAMP1 co - localisation . Scale bar , 20 µm .

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

G. GOLPH3 OE or KD HeLa cells expressing LCS-HA were treated with bafilomycin A1 (BafA1, 10 nM) for 16 hr, fixed, and processed for IF labelling: LCS (green), LAMP1 (red), and DAPI (blue). Arrowheads indicate LCS and LAMP1 co-localisation. Scale bar, 20 µm.


H . WB of cells treated as in ( G ) . Densitometric quantification of the blot is plotted in the top panel ( data are means of at least 3 experiments ± SD ) .

H. WB of cells treated as in (G). Densitometric quantification of the blot is plotted in the top panel (data are means of at least 3 experiments ± SD).


I . GOLPH3 OE or KD HeLa cells expressing the indicated HA - tagged glycoenzymes were fixed and processed for IF . Asterisks indicate GOLPH3 OE cells . Scale bar , 20 µm .

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

I. GOLPH3 OE or KD HeLa cells expressing the indicated HA-tagged glycoenzymes were fixed and processed for IF. Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.


J . Number of glycoenzymes - HA positive spots per cell in GOLPH3 - KD cells ( left panel ) and percentage of cells with glycoenzymes - HA in the ER in GOLPH3 - OE cells ( right panel ) are plotted ( n is indicated in the graph ; data are means ± SEM ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

J. Number of glycoenzymes-HA positive spots per cell in GOLPH3-KD cells (left panel) and percentage of cells with glycoenzymes-HA in the ER in GOLPH3-OE cells (right panel) are plotted (n is indicated in the graph; data are means ± SEM; ***p <0.001 [Student's t-test]).


A . Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [ 3H ] - sphingosine pulse ( 2 hr ) and chase ( indicated times ) , lipid extraction and HPTLC separation and radioactive counting ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) .

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

A. Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [3H]-sphingosine pulse (2 hr) and chase (indicated times), lipid extraction and HPTLC separation and radioactive counting (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]).


B . Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC / MS of HeLa cells ( top ) and PHFs ( middle ) . Effects of LCS KD or OE on sphingolipid levels as assessed by LC / MS of HeLa cells ( bottom ) ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs : ST001673 for HeLa cells , and ST001672 for PHFs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (bottom) (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.


C . Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI - MS after lipid extraction . Profiles are representative of 3 independent experiments . Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID : ST001674 .

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

C. Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI-MS after lipid extraction. Profiles are representative of 3 independent experiments. Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID: ST001674.


D . Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig . 1F ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * * p < 0 . 001 [ Student ' s t - test ] ) .

D. Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig. 1F (data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001 [Student's t-test]).


E . Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC / MS of HeLa cells ( top ) and PHFs ( middle ) . Effects of LCS KD or OE on ceramide levels as assessed by LC / MS of HeLa cells ( bottom ) . The dotted black box highlights the effects on C16 : 0 Cer ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs : ST001673 for HeLa cells , and ST001672 for PHFs .

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

E. Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (bottom). The dotted black box highlights the effects on C16:0 Cer (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.


A . Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS - PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway . Data are representative of at least 3 independent experiments .

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

A. Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS-PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway. Data are representative of at least 3 independent experiments.


B . Clonogenic assay of HeLa cells in adherence . 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days . Afterward , colonies were fixed and stained as detailed in Materials and Methods . The graph on right reports the number of colonies per well in each indicated condition ( data are means ± SEM of 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 [ Student ' s t - test ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Clonogenic assay of HeLa cells in adherence. 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days. Afterward, colonies were fixed and stained as detailed in Materials and Methods. The graph on right reports the number of colonies per well in each indicated condition (data are means ± SEM of 3 independent experiments; *p <0.05, **p <0.01 [Student's t-test]).