A . Quantification of cortical thickness in the somatosensory ( SS ) , somatomotor ( SM ) and visual ( V ) area , in untreated Ppt1 - / - or Ppt1 + / + mice and in mice transplanted ( tx ) with hPPT1 - LV transduced HSPCs IV , ICV or ICV + IV or mock - transplanted , analyzed at study termination . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; Kruskal Wallis followed by Dunn ' s post - hoc test .

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A. Quantification of cortical thickness in the somatosensory (SS), somatomotor (SM) and visual (V) area, in untreated Ppt1-/- or Ppt1+/+ mice and in mice transplanted (tx) with hPPT1-LV transduced HSPCs IV, ICV or ICV+IV or mock-transplanted, analyzed at study termination. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; Kruskal Wallis followed by Dunn's post-hoc test.


B . Quantification of CD68 immunoreactivity in the cortex , hippocampus , thalamus and cerebellum of untreated ( UT ) or gene therapy treated Ppt1 - / - mice . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 00001 ; Kruskal Wallis followed by Dunn ' s post - hoc test .

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B. Quantification of CD68 immunoreactivity in the cortex, hippocampus, thalamus and cerebellum of untreated (UT) or gene therapy treated Ppt1-/- mice. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.00001; Kruskal Wallis followed by Dunn's post-hoc test.


A . Significant increase of the survival in Ppt1 - / - mice transplanted at the symptomatic stage with hPPT1 - LV transduced HSPCs administered IV + ICV .

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A. Significant increase of the survival in Ppt1-/- mice transplanted at the symptomatic stage with hPPT1-LV transduced HSPCs administered IV+ICV.


E . Representative fluorescence microscope photomicrographs showing autofluorescent storage material ( AF , green ) in different brain regions of Ppt1 - / - mice transplanted with hPPT1 - LV transduced HSPCs at 18 - 20 weeks of age , analyzed at study termination . Ppt1 - / - mice mock transplanted at 18 - 20 weeks of age and analyzed at about 250 days , i . e . humane end point , are shown as reference . Neurotrace fluorescent Nissl staining ( NT , red ) is used to highlight neurons in different brain regions . Scale bar = 100 μm . F . Top panel : quantification of autofluorescent ( AF ) storage material in different brain regions of untreated or transplanted mice analyzed at study termination . * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; 2 - way ANOVA followed by Tukey ' s post - hoc test . Bottom panel : histograms showing the quantification of cortical thickness . SM = somatomotor ; SS = somatosensory ; V = visual cortex . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ; Kruskal Wallis followed by Dunn ' s post - hoc test ( for SM and V area ) or ANOVA followed by Tukey ' s post - hoc test ( for SS area ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative fluorescence microscope photomicrographs showing autofluorescent storage material (AF, green) in different brain regions of Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs at 18-20 weeks of age, analyzed at study termination. Ppt1 -/- mice mock transplanted at 18-20 weeks of age and analyzed at about 250 days, i.e. humane end point, are shown as reference. Neurotrace fluorescent Nissl staining (NT, red) is used to highlight neurons in different brain regions. Scale bar = 100 μm. F. Top panel: quantification of autofluorescent (AF) storage material in different brain regions of untreated or transplanted mice analyzed at study termination. ** = p<0.01; *** = p< 0.001; **** = p< 0.0001; 2-way ANOVA followed by Tukey's post-hoc test. Bottom panel: histograms showing the quantification of cortical thickness. SM = somatomotor; SS = somatosensory; V = visual cortex. * = p <0.05; ** = p<0.01; **** = p<0.0001; Kruskal Wallis followed by Dunn's post-hoc test (for SM and V area) or ANOVA followed by Tukey's post-hoc test (for SS area). S


CD4 and CD8 T cells were stimulated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 beads as indicated . Cell lysates after 4 h of stimulation were analyzed by immunoblotting . Blots depict one representative donor out of three .

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CD4 and CD8 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28 beads as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. Blots depict one representative donor out of three.


CD4 T cells were stimulated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 ; and human IFNβ ( top ) and IP10 levels ( bottom ) in the supernatant were analyzed by ELISA at the indicated time points . Data panels depict mean ± SD of biological duplicates from three independent donors .

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CD4 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28; and human IFNβ (top) and IP10 levels (bottom) in the supernatant were analyzed by ELISA at the indicated time points. Data panels depict mean ± SD of biological duplicates from three independent donors.


CD4 T cells were pre - treated with cycloheximide ( 2 μg / ml ) for 2 h , then treated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 ( 3 / 28 ) for 4 h , then analyzed by quantitative PCR for IFNB1 and OASL expression . Data are depicted as mean + SEM of 3 independent donors ( for cGAMP and αCD3 / CD28 treated cells without CHX only 2 data points were obtained ) . Statistics were determined by a two - way ANOVA on log - transformed data using Šidák correction for multiple testing : * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ; ns , not significant .

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CD4 T cells were pre-treated with cycloheximide (2 μg/ml) for 2 h, then treated with cGAMP (20 μg/ml) and αCD3/CD28 (3/28) for 4 h, then analyzed by quantitative PCR for IFNB1 and OASL expression. Data are depicted as mean + SEM of 3 independent donors (for cGAMP and αCD3/CD28 treated cells without CHX only 2 data points were obtained). Statistics were determined by a two-way ANOVA on log-transformed data using Šidák correction for multiple testing: ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant.


CD4 T cells were stimulated with the indicated stimuli for 4 h . Lysates were analyzed by immunoblotting . One representative donor out of three is shown .

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CD4 T cells were stimulated with the indicated stimuli for 4 h. Lysates were analyzed by immunoblotting. One representative donor out of three is shown.


CRISPR / Cas9 targeted CD4 T cells for the indicated genes were treated with the indicated stimuli for 48 h . Human IFNβ levels were analyzed by ELISA . Data are depicted as mean + SEM of 3 independent donors . Statistics indicate significance by two - way ANOVA with Dunnett correction for multiple testing : * * * * p < 0 . 0001 ; ns , not significant .

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CRISPR/Cas9 targeted CD4 T cells for the indicated genes were treated with the indicated stimuli for 48 h. Human IFNβ levels were analyzed by ELISA. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ****p < 0.0001; ns, not significant.


CD4 T cells were pre - treated with Dasatinib ( 100 , 10 , 1 and 0 . 1 nM ) , Tacrolimus ( 1000 , 100 , 10 and 1 nM ) , TPCA - 1 ( 4 , 2 , 1 and 0 . 5 μM ) and Torin1 ( 50 , 5 , 0 . 5 and 0 . 05 nM ) for 30 min , then treated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 for 16 h . Ηuman IFNβ levels in the supernatant were analyzed by ELISA . Data panels depict mean of biological duplicates from three independent donors .

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CD4 T cells were pre-treated with Dasatinib (100, 10, 1 and 0.1 nM), Tacrolimus (1000, 100, 10 and 1 nM), TPCA-1 (4, 2, 1 and 0.5 μM) and Torin1 (50, 5, 0.5 and 0.05 nM) for 30 min, then treated with cGAMP (20 μg/ml) and αCD3/CD28 for 16 h. Ηuman IFNβ levels in the supernatant were analyzed by ELISA. Data panels depict mean of biological duplicates from three independent donors.


CD4 T cells were treated with cGAMP and indicated stimuli together with increasing concentrations of TPCA - 1 for 4 h . Cell lysates were analyzed by immunoblotting . One representative donor out of two is shown . CD4 T cells were stimulated with cGAMP ( 20 μg / ml ) and additional stimuli as indicated . Cell lysates after 4 h of stimulation were analyzed by immunoblotting . One representative donor out of two is shown . CD4 T cells were stimulated with cGAMP ( 20 μg / ml ) and additional stimuli as indicated . Cell lysates after 4 h of stimulation were analyzed by immunoblotting . One representative donor out of two is shown .

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CD4 T cells were treated with cGAMP and indicated stimuli together with increasing concentrations of TPCA-1 for 4 h. Cell lysates were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown.


Freshly isolated CD4 T cells were treated with cGAMP , αCD3 / CD28 beads and ABT737 / S63845 ( A / S ) for 48 h and analyzed by flow cytometry . Treatment induced cell death was assessed by Annexin V and propidium iodide staining . Data are depicted as mean + SEM of 3 independent donors . Statistics indicate significance by two - way ANOVA with Dunnett correction for multiple testing : * * * p < 0 . 001 ; * p < 0 . 05 ; ns , not significant .

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Freshly isolated CD4 T cells were treated with cGAMP, αCD3/CD28 beads and ABT737/S63845 (A/S) for 48 h and analyzed by flow cytometry. Treatment induced cell death was assessed by Annexin V and propidium iodide staining. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ***p < 0.001; *p < 0.05; ns, not significant.


CD4 T cells were treated with indicated concentrations of cGAMP for 18 h , and ABT737 / S63845 for 2 h . Lysates and supernatant precipitations were analyzed by immunoblotting . One representative donor out of two is shown .

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CD4 T cells were treated with indicated concentrations of cGAMP for 18 h, and ABT737/S63845 for 2 h. Lysates and supernatant precipitations were analyzed by immunoblotting. One representative donor out of two is shown.


CRISPR / Cas9 targeted CD4 T cells for indicated genes were treated with the indicated stimuli for 48 h and analyzed by flow cytometry . Each panel shows propidium iodide and Annexin V intensity for one representative donor out of three . Digits indicate the percentage of the parental population ( single events without subcellular debris ) .

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CRISPR/Cas9 targeted CD4 T cells for indicated genes were treated with the indicated stimuli for 48 h and analyzed by flow cytometry. Each panel shows propidium iodide and Annexin V intensity for one representative donor out of three. Digits indicate the percentage of the parental population (single events without subcellular debris).


CellTrace Violet ( CTV ) stained , freshly isolated CD4 and CD8 T cells were activated with αCD3 / CD28 beads , stimulated with cGAMP for 96 h and analyzed by flow cytometry . Each panel shows profiles from activated ( black ) , cGAMP - treated ( green ) and resting ( light gray ) single and live cells from one representative donor .

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CellTrace Violet (CTV) stained, freshly isolated CD4 and CD8 T cells were activated with αCD3/CD28 beads, stimulated with cGAMP for 96 h and analyzed by flow cytometry. Each panel shows profiles from activated (black), cGAMP-treated (green) and resting (light gray) single and live cells from one representative donor.


Freshly isolated CD4 T cells were treated with cGAMP ( 40 μg / ml ) and αCD3 / CD28 ( 3 / 28 ) for 48 h and analyzed by Seahorse XF mito stress test . Data are depicted as mean ± SEM of 3 independent donors . Statistics indicate significance by two - way ANOVA with Šidák correction for multiple testing : * * p < 0 . 01 ; * p < 0 . 05 ; ns , not significant .

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Freshly isolated CD4 T cells were treated with cGAMP (40 μg/ml) and αCD3/CD28 (3/28) for 48 h and analyzed by Seahorse XF mito stress test. Data are depicted as mean ± SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Šidák correction for multiple testing: **p < 0.01; *p < 0.05; ns, not significant.


D : Analytical ultracentrifugation absorbance data analysis of N - NTD . The clear peak at ~ 15 . 4 kDa molecular weight ( MW ) corresponds to the N - NTD monomer . E : Analytical ultracentrifugation absorbance data analysis of N - CTD . The clear peak at ~ 28 . 7 kDa MW corresponds to the N - CTD dimer .

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D: Analytical ultracentrifugation absorbance data analysis of N-NTD. The clear peak at ~15.4 kDa molecular weight (MW) corresponds to the N-NTD monomer. E: Analytical ultracentrifugation absorbance data analysis of N-CTD. The clear peak at ~28.7 kDa MW corresponds to the N-CTD dimer.


Immunofluorescence microscopy detection of Tmem117 in the mouse hypothalamus and pituitary : ( A , B ) Positive immunostaining was observed in the PVN , the SON and the posterior pituitary . ( C , D ) No immunostaining was detected in the hypothalamus and pituitary of mice with constitutive inactivation of Tmem117 in AVP neurons . Data information : 3V : 3rd ventricle , AP : Anterior pituitary , MZ : Medial zone , opt : optic tract , PP : Posterior pituitary , PVN : Paraventricular nucleus , SCh : Suprachiasmatic nucleus , SON : Supraoptic nucleus . Scale bar = 100μm for

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Immunofluorescence microscopy detection of Tmem117 in the mouse hypothalamus and pituitary: (A, B) Positive immunostaining was observed in the PVN, the SON and the posterior pituitary. (C, D) No immunostaining was detected in the hypothalamus and pituitary of mice with constitutive inactivation of Tmem117 in AVP neurons. Data information: 3V: 3rd ventricle, AP: Anterior pituitary, MZ: Medial zone, opt: optic tract, PP: Posterior pituitary, PVN: Paraventricular nucleus, SCh: Suprachiasmatic nucleus, SON: Supraoptic nucleus. Scale bar=100μm for panels


Immunofluorescence microscopy detection of Tmem117 in the mouse pituitary : ( E ) Co - staining for Tmem117 and AVP in the PVN . ( F ) Co - staining for Tmem117 and AVP in the SON . ( G ) Co - staining for Tmem117 and AVP in the posterior pituitary . ( H , I ) Quantification of AVP + / Tmem117 + cells in the PVN and SON , respectively . Data information : 3V : 3rd ventricle , opt : optic tract , PP : Posterior pituitary , PVN : Paraventricular nucleus , SON : Supraoptic nucleus . Scale bar = 100μm for

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Immunofluorescence microscopy detection of Tmem117 in the mouse pituitary: (E) Co-staining for Tmem117 and AVP in the PVN. (F) Co-staining for Tmem117 and AVP in the SON. (G) Co-staining for Tmem117 and AVP in the posterior pituitary. (H, I) Quantification of AVP+/Tmem117+ cells in the PVN and SON, respectively. Data information: 3V: 3rd ventricle, opt: optic tract, PP: Posterior pituitary, PVN: Paraventricular nucleus, SON: Supraoptic nucleus. Scale bar=100μm for panels


( J ) Higher resolution depiction of an AVP + / Tmem117 + neuron in the PVN captured with structured illumination microscopy . The white arrows highlight representative positions of signal co - localization . Data information : 3V : 3rd ventricle , PVN : Paraventricular nucleus , Scale bar = 10μm for panel J .

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(J) Higher resolution depiction of an AVP+/Tmem117+ neuron in the PVN captured with structured illumination microscopy. The white arrows highlight representative positions of signal co-localization. Data information: 3V: 3rd ventricle, PVN: Paraventricular nucleus, Scale bar= 10μm for panel J.


( B ) Glycemia one hour after saline ( black ) or insulin ( red ) i . p . injection [ n = 15 - 16 mice per group ] . ( C ) CPP plasma levels one hour after saline or insulin injection [ n = 13 mice per group ; mean ± SEM for INS : WT 89 ± 5 vs KO 121 ± 13 pg / ml ] . ( D ) GCG plasma levels one hour after saline or insulin injection [ n = 15 - 16 mice per group ; mean ± SEM for INS : WT 124 ± 19 vs KO 186 ± 21 pg / ml ] . ( E ) Glycemia one hour after saline ( black ) or insulin ( red ) i . p . injection [ n = 18 - 22 mice per group ] . ( F ) CPP plasma levels one hour after saline or insulin injection [ n = 18 - 19 mice per group ; mean ± SEM for INS : WT 78 ± 6 vs KO 92 ± 8 pg / ml ] . ( G ) GCG plasma levels one hour after saline or insulin injection [ n = 17 - 20 mice per group ; mean ± SEM for INS : WT 242 ± 14 vs KO 277 ± 18 pg / ml ] . ( H - J ) Analysis of glycemic levels and of CPP and GCG secretion following insulin injection in female mice at each stage of the estrus cycle [ n = 3 - 7 mice per group ] . ( H ) Glycemic levels . ( I ) CPP plasma levels . ( J ) GCG plasma levels . Data information : d : day , di : diestrus , est : estrus , INS : insulin , met : metestrus , pro : proestrus , For panels B - G , bars correspond to the mean value per group . For panels H - J , lines correspond to the mean value per group and error bars represent ± SEM . B - G : 2 - way ANOVA RM with Bonferroni post - hoc test ; H - J : unpaired t - test ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(B) Glycemia one hour after saline (black) or insulin (red) i.p. injection [n=15-16 mice per group]. (C) CPP plasma levels one hour after saline or insulin injection [n=13 mice per group; mean±SEM for INS: WT 89±5 vs KO 121±13 pg/ml]. (D) GCG plasma levels one hour after saline or insulin injection [n=15-16 mice per group; mean±SEM for INS: WT 124±19 vs KO 186±21 pg/ml]. (E) Glycemia one hour after saline (black) or insulin (red) i.p. injection [n=18-22 mice per group]. (F) CPP plasma levels one hour after saline or insulin injection [n=18-19 mice per group; mean±SEM for INS: WT 78±6 vs KO 92±8 pg/ml]. (G) GCG plasma levels one hour after saline or insulin injection [n=17-20 mice per group; mean±SEM for INS: WT 242±14 vs KO 277±18 pg/ml]. (H-J) Analysis of glycemic levels and of CPP and GCG secretion following insulin injection in female mice at each stage of the estrus cycle [n=3-7 mice per group]. (H) Glycemic levels. (I) CPP plasma levels. (J) GCG plasma levels. Data information: d: day, di: diestrus, est: estrus, INS: insulin, met: metestrus, pro: proestrus, For panels B-G, bars correspond to the mean value per group. For panels H-J, lines correspond to the mean value per group and error bars represent ± SEM. B-G: 2-way ANOVA RM with Bonferroni post-hoc test; H-J: unpaired t-test; *p<0.05, **p<0.01, ***p<0.001.


C57BL6 / N male mice were injected i . p . with saline or insulin and c - Fos expression ( green ) in AVP ( magenta ) neurons was quantitated by immunofluorescence microscopy 2 hours later . ( A ) C - Fos expression in the PVN after saline injection . ( B ) C - Fos expression in the PVN after insulin injection ( C ) Quantitation of c - Fos positive cells in the PVN after saline and insulin injections [ n = 4 mice per group ] . Data information : 3V : 3rd ventricle , AVP : Vasopressin , INS : insulin , PVN : Paraventricular nucleus , SAL : saline , Scale bar = 100μm . Lines correspond to the mean value per group and error bars represent ± SEM . Unpaired t - test ; ns : p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

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C57BL6/N male mice were injected i.p. with saline or insulin and c-Fos expression (green) in AVP (magenta) neurons was quantitated by immunofluorescence microscopy 2 hours later. (A) C-Fos expression in the PVN after saline injection. (B) C-Fos expression in the PVN after insulin injection (C) Quantitation of c-Fos positive cells in the PVN after saline and insulin injections [n=4 mice per group]. Data information: 3V: 3rd ventricle, AVP: Vasopressin, INS: insulin, PVN: Paraventricular nucleus, SAL: saline, Scale bar=100μm. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.


C57BL6 / N male mice were injected i . p . with saline or insulin and c - Fos expression ( green ) in AVP ( magenta ) neurons was quantitated by immunofluorescence microscopy 2 hours later . ( D ) C - Fos expression in the SON after saline injection . ( E ) C - Fos expression in the SON after insulin injection ( F ) Quantitation of c - Fos positive cells in the SON after saline and insulin injections [ n = 4 mice per group ] . Data information : AVP : Vasopressin , INS : insulin , opt : optic tract , SAL : saline , SON : Supraoptic nucleus . Scale bar = 100μm . Lines correspond to the mean value per group and error bars represent ± SEM . Unpaired t - test ; ns : p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

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

C57BL6/N male mice were injected i.p. with saline or insulin and c-Fos expression (green) in AVP (magenta) neurons was quantitated by immunofluorescence microscopy 2 hours later. (D) C-Fos expression in the SON after saline injection. (E) C-Fos expression in the SON after insulin injection (F) Quantitation of c-Fos positive cells in the SON after saline and insulin injections [n=4 mice per group]. Data information: AVP: Vasopressin, INS: insulin, opt: optic tract, SAL: saline, SON: Supraoptic nucleus. Scale bar=100μm. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.


( H ) Tmem117 mRNA levels in the PVN [ n = 4 mice per group ] . ( I ) Tmem117 mRNA levels in the SON [ n = 4 mice per group Data information : INS : insulin , PVN : Paraventricular nucleus , SAL : saline , SON : Supraoptic nucleus . Lines correspond to the mean value per group and error bars represent ± SEM . Unpaired t - test ; ns : p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tmem117 mRNA levels in the PVN [n=4 mice per group]. (I)Tmem117 mRNA levels in the SON [n=4 mice per group Data information: INS: insulin, PVN: Paraventricular nucleus, SAL: saline, SON: Supraoptic nucleus. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.


( B ) C - Fos expression in the SON Data information : AVP : Vasopressin INS : insulin opt : optic tract , SAL : saline , Scale bar = 100μm .

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(B) C-Fos expression in the SON Data information: AVP: Vasopressin INS: insulin opt: optic tract, SAL: saline, Scale bar=100μm.


( G ) Membrane potential was increased comparably in response to 0 . 1 mM glucose in both groups [ n = 7 - 9 cells per group ] . ( H ) No change in membrane potential in NR neurons of both genotypes [ n = 9 cells per group ] . ( I ) Membrane resistance was decreased in 6 out of 9 AVPTM117WT and 6 out of 7 AVPTM117KO cells in response to 0 . 1 mM glucose [ n = 7 - 9 cells per group ] . ( J ) Membrane resistance in NR neurons was stable for both genotypes [ n = 9 cells per group ] . Data information : AVP : Vasopressin , d : day , GI : glucose inhibited , NR : non - responding , Lines correspond to the mean value per group and error bars represent ± SEM . G - J : paired t - test for all graphs except Δ membrane potential ( unpaired ) ; nsp > 0 . 05 , * p < 0 . 05 , * * * p > 0 . 001 .

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(G) Membrane potential was increased comparably in response to 0.1 mM glucose in both groups [n=7-9 cells per group]. (H) No change in membrane potential in NR neurons of both genotypes [n=9 cells per group]. (I) Membrane resistance was decreased in 6 out of 9 AVPTM117WT and 6 out of 7 AVPTM117KO cells in response to 0.1 mM glucose [n=7-9 cells per group]. (J) Membrane resistance in NR neurons was stable for both genotypes [n=9 cells per group]. Data information: AVP: Vasopressin, d: day, GI: glucose inhibited, NR: non-responding, Lines correspond to the mean value per group and error bars represent ± SEM. G-J: paired t-test for all graphs except Δ membrane potential (unpaired); nsp>0.05, *p<0.05, ***p>0.001.


( A ) Quantification of BiP mRNA per cell in AVP - positive cells of the SON [ n = 483 - 704 cells per group ; each group consisted of 4 mice ] . ( B ) Quantification of BiP mRNA per cell in AVP - negative cells of the SON [ n = 347 - 584 cells per group ; each group consisted of 4 mice ] . Data information : AVP : Vasopressin For the violin plots dashed lines correspond to the median value and dotted lines to the quartile values . unpaired t - test ; * p < 0 . 05 , * * * p > 0 . 001 .

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(A) Quantification of BiP mRNA per cell in AVP-positive cells of the SON [n=483-704 cells per group; each group consisted of 4 mice]. (B) Quantification of BiP mRNA per cell in AVP-negative cells of the SON [n=347-584 cells per group; each group consisted of 4 mice]. Data information: AVP: Vasopressin For the violin plots dashed lines correspond to the median value and dotted lines to the quartile values. unpaired t-test ; *p<0.05, ***p>0.001.


( C ) Fluorescence microscopy detection of AVP mRNA ( green ) and BiP mRNA ( magenta ) in the SON . Data information : AVP : Vasopressin opt : optic tract Scale bar = 20μm .

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(C) Fluorescence microscopy detection of AVP mRNA (green) and BiP mRNA (magenta) in the SON. Data information: AVP: Vasopressin opt: optic tract Scale bar=20μm.


( F ) Fluorescence microscopy detection of DHE - derived fluorescence ( red ) in the SON . ( G ) Quantification of DHE - derived fluorescence in the SON [ n = 8 SON per group ; each group consisted of 4 mice ] . Data information : AVP : Vasopressin , d : day , DHE : dihydroethidium , opt : optic tract , SON : Supraoptic nucleus , . Scale bar = 20μm . For panels G data are represented as mean ± SEM . G : unpaired t - test ; ; * p < 0 . 05 , * * * p > 0 . 001 .

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(F) Fluorescence microscopy detection of DHE-derived fluorescence (red) in the SON. (G) Quantification of DHE-derived fluorescence in the SON [n=8 SON per group; each group consisted of 4 mice]. Data information: AVP: Vasopressin, d: day, DHE: dihydroethidium , opt: optic tract, SON: Supraoptic nucleus,. Scale bar=20μm. For panels G data are represented as mean ± SEM. G: unpaired t-test; ; *p<0.05, ***p>0.001.


( J ) Calcium recordings in AVP magnocellular terminals 10 minutes before and one hour after the i . p injection of insulin ( red and orange ) or saline ( black ) . The vertical arrow corresponds to the i . p . injection ( time = 0 ) . ( K ) Quantification of mean signal intensity in 10 - minute time bins . [ n = 6 - 8 mice per group ] . Data information : AUC : area under the curve , AVP : Vasopressin INS : insulin , SAL : saline For panels K , data are represented as mean ± SEM . K : 1 - way ANOVA with Tukey ' s post - hoc test ; * p < 0 . 05 , * * * p > 0 . 001 .

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(J) Calcium recordings in AVP magnocellular terminals 10 minutes before and one hour after the i.p injection of insulin (red and orange) or saline (black). The vertical arrow corresponds to the i.p. injection (time=0). (K) Quantification of mean signal intensity in 10-minute time bins. [n=6-8 mice per group]. Data information: AUC: area under the curve, AVP: Vasopressin INS: insulin, SAL: saline For panels K, data are represented as mean ± SEM. K: 1-way ANOVA with Tukey's post-hoc test; *p<0.05, ***p>0.001.


( M ) Fluorescence microscopy detection of GCaMP ( green ) in posterior pituitary . Immunofluorescent labelling was used to localize the AVP terminals ( magenta ) . Data information : AVP : Vasopressin , . Scale bar = 20μm .

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(M) Fluorescence microscopy detection of GCaMP (green) in posterior pituitary. Immunofluorescent labelling was used to localize the AVP terminals (magenta). Data information: AVP: Vasopressin ,. Scale bar=20μm.


( B ) Immunofluorescence microscopy detection of AVP in the PVN and the SON . Data information : 3V : 3rd ventricle , AVP : Vasopressin , KO : AVPTM117KO opt : optic tract , PVN : paraventricular nucleus , SON : Supraoptic nucleus , WT : AVPTM117WT . Scale bar = 100μm .

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(B) Immunofluorescence microscopy detection of AVP in the PVN and the SON. Data information: 3V: 3rd ventricle, AVP: Vasopressin, KO: AVPTM117KO opt: optic tract, PVN: paraventricular nucleus, SON: Supraoptic nucleus, WT: AVPTM117WT. Scale bar=100μm.


( C ) Quantitation of AVP positive cells in the PVN [ n = 3 - 4 mice per group ] . ( D ) Quantitation of AVP positive cells in the SON [ n = 3 - 4 mice per group ] . Data information : AVP : Vasopressin , KO : AVPTM117KO WT : AVPTM117WT . For panels C and D lines correspond to the mean value per group and error bars represent ± SEM . For panels C , D : unpaired t - test ; ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(C) Quantitation of AVP positive cells in the PVN [n=3-4 mice per group]. (D) Quantitation of AVP positive cells in the SON [n=3-4 mice per group]. Data information: AVP: Vasopressin, KO: AVPTM117KO WT: AVPTM117WT. For panels C and D lines correspond to the mean value per group and error bars represent ± SEM. For panels C,D: unpaired t-test; ; *p<0.05, **p<0.01, ***p<0.001.


( E ) Glycemia one hour after saline ( black ) or insulin ( red ) i . p . injections in AVPTM117WT and AVPTM117KO male mice [ n = 8 - 9 mice per group ] . ( F ) CPP plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [ n = 8 mice per group ] . ( G ) GCG plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [ n = 8 - 9 mice per group ] . Data information : AVP : Vasopressin , d : day , INS : insulin , KO : AVPTM117KO , SAL : saline , WT : AVPTM117WT . For panels E - G bars correspond to the mean value per group and error bars represent ± SEM . E - G : 2 - way ANOVA for each timepoint in comparison to SAL baseline with Bonferroni post - hoc test ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Glycemia one hour after saline (black) or insulin (red) i.p. injections in AVPTM117WT and AVPTM117KO male mice [n=8-9 mice per group]. (F) CPP plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [n=8 mice per group]. (G) GCG plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [n=8-9 mice per group]. Data information: AVP: Vasopressin, d: day, INS: insulin, KO: AVPTM117KO, SAL: saline, WT: AVPTM117WT. For panels E-G bars correspond to the mean value per group and error bars represent ± SEM. E-G: 2-way ANOVA for each timepoint in comparison to SAL baseline with Bonferroni post-hoc test; *p<0.05, **p<0.01, ***p<0.001.


A Whole mount FISH for piwi - 1 and immunostaining for BrdU at 1 - day , 1 - week and 5 - weeks post - BrdU administration . White boxes denote the magnified regions shown in the bottom panels . Scale bars , 30 µm .

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A Whole mount FISH for piwi-1 and immunostaining for BrdU at 1-day, 1-week and 5-weeks post-BrdU administration. White boxes denote the magnified regions shown in the bottom panels. Scale bars, 30 µm.


B Quantification of the percentage of BrdU label - retaining neoblasts . Data are presented as mean ± s . d . ( n = 4 - 6 animals per time point ) .

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B Quantification of the percentage of BrdU label-retaining neoblasts. Data are presented as mean ± s.d. (n = 4-6 animals per time point).


C Double pulse experiment showing colocalization of BrdU after a 2 - week chase period with EdU after a 1 - day chase period . White boxes denote the magnified region shown on the left . Dashed lines mark the edges of the animal and white arrows mark colocalization . Scale bar , 50 µm .

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C Double pulse experiment showing colocalization of BrdU after a 2-week chase period with EdU after a 1-day chase period. White boxes denote the magnified region shown on the left. Dashed lines mark the edges of the animal and white arrows mark colocalization. Scale bar, 50 µm.


A Dissociated cells from unirradiated , 1 dpi or 7 dpi animals ( 60 Gy of irradiation ) were stained with Hoechst ( DNA ) and Pyronin Y ( RNA ) and sorted by FACS to reveal an RNAlow population . Percentages indicate the proportion of events within each gate .

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A Dissociated cells from unirradiated, 1 dpi or 7 dpi animals (60 Gy of irradiation) were stained with Hoechst (DNA) and Pyronin Y (RNA) and sorted by FACS to reveal an RNAlow population. Percentages indicate the proportion of events within each gate.


B Cell size and nucleus : cytoplasm ratio measured by CellMask ( cytoplasm ) and DAPI ( nucleus ) staining of FACS sorted cells at homeostasis ( n ≥ 47 per sample ) . Representative images are shown , with dashed lines to indicate the area measured . Scale bars , 5 µm .

B Cell size and nucleus:cytoplasm ratio measured by CellMask (cytoplasm) and DAPI (nucleus) staining of FACS sorted cells at homeostasis (n ≥ 47 per sample). Representative images are shown, with dashed lines to indicate the area measured. Scale bars, 5 µm.


C EdU pulse - chase time course of label - retention in sorted RNAlow and S / G2 / M neoblasts ( n ≥ 3 individual experiments per time point ) . Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S / G2 / M or RNAlow neoblasts , respectively . Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S / G2 / M neoblasts .

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C EdU pulse-chase time course of label-retention in sorted RNAlow and S/G2/M neoblasts (n ≥ 3 individual experiments per time point). Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S/G2/M or RNAlow neoblasts, respectively. Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S/G2/M neoblasts.


D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C . Red boxes denote examples of EdU - cells . Scale bars , 5 µm .

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D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C. Red boxes denote examples of EdU- cells. Scale bars, 5 µm.


A Area measurements of cells from the RNAlow , G1 and S / G2 / M gates in intact animals and at 6 and 48 hpa ( n ≥ 28 for each sample in the RNAlow plot , n ≥ 24 in the G1 plot , n ≥ 42 in the S / G2 / M plot ) .

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A Area measurements of cells from the RNAlow, G1 and S/G2/M gates in intact animals and at 6 and 48 hpa (n ≥ 28 for each sample in the RNAlow plot, n ≥ 24 in the G1 plot, n ≥ 42 in the S/G2/M plot).


B RNAlow neoblast size in intact animals and at 48 , 72 and 96 hpa ( n ≥ 54 ) .

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B RNAlow neoblast size in intact animals and at 48, 72 and 96 hpa (n ≥ 54).


C RNAlow neoblast size in intact animals and at 48 hours following amputation , poke or incision injuries ( n ≥ 61 ) .

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C RNAlow neoblast size in intact animals and at 48 hours following amputation, poke or incision injuries (n ≥ 61).


D RNAlow neoblast size in unirradiated animals and at 1 , 2 and 3 dpi ( 12 . 5 Gy . of irradiation , n ≥ 52 ) .

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D RNAlow neoblast size in unirradiated animals and at 1, 2 and 3 dpi (12.5 Gy. of irradiation, n ≥ 52).


A Representative images of RNAlow , G1 and S / G2 / M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa . All images shown are from samples processed in the same experiment . Intact images are duplicated from Figure 2B . White arrows mark projections . Percentage of cells displaying projections in each condition is noted ( n > 100 cells counted ) . Scale bars , 5 µm . B Average projection length in RNAlow , G1 and S / G2 / M cells from intact animals and at 6 and 48 hpa ( n ≥ 13 ) . C - E Quantification of the number of projections per cell ( blue data ) and the sum total projection length per cell ( orange data ) in RNAlow ( C ) , G1 ( D ) or S / G2 / M ( E ) cells isolated from intact animals and at 6 and 48 hpa ( n ≥ 23 for projections / cell , n ≥ 10 for Σ projection length / cell ) .

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A Representative images of RNAlow, G1 and S/G2/M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa. All images shown are from samples processed in the same experiment. Intact images are duplicated from Figure 2B. White arrows mark projections. Percentage of cells displaying projections in each condition is noted (n > 100 cells counted). Scale bars, 5 µm. B Average projection length in RNAlow, G1 and S/G2/M cells from intact animals and at 6 and 48 hpa (n ≥ 13). C-E Quantification of the number of projections per cell (blue data) and the sum total projection length per cell (orange data) in RNAlow (C), G1 (D) or S/G2/M (E) cells isolated from intact animals and at 6 and 48 hpa (n ≥ 23 for projections/cell, n ≥ 10 for Σ projection length/cell).


A Representative images of RNAlow , G1 and S / G2 / M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa . All images shown are from samples processed in the same experiment . Intact images are duplicated from Figure 2B . White arrows mark projections . Percentage of cells displaying projections in each condition is noted ( n > 100 cells counted ) . Scale bars , 5 µm .

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A Representative images of RNAlow, G1 and S/G2/M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa. All images shown are from samples processed in the same experiment. Intact images are duplicated from Figure 2B. White arrows mark projections. Percentage of cells displaying projections in each condition is noted (n > 100 cells counted). Scale bars, 5 µm.


B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment .

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B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment.


C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals ( n ≥ 55 ) .

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C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals (n ≥ 55).


D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals ( n = 5 - 10 ) .

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D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals (n = 5-10).


E RNAlow neoblast size in intact animals and at 48 hpa in control , raptor or rictor KD animals ( n ≥ 54 ) .

E RNAlow neoblast size in intact animals and at 48 hpa in control, raptor or rictor KD animals (n ≥ 54).


F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals . Percentages indicate the proportion of events within each gate .

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F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals. Percentages indicate the proportion of events within each gate.


A Representative WISH images for Lrig - 1 .

A Representative WISH images for Lrig-1.


B RNAlow neoblast size in intact animals following control or Lrig - 1 knockdown ( n ≥ 53 technical replicates ) .

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B RNAlow neoblast size in intact animals following control or Lrig-1 knockdown (n ≥ 53 technical replicates).


C Quantification of H3P immunostaining in intact animals following control or Lrig - 1 knockdown ( n = 9 - 10 technical replicates ) .

C Quantification of H3P immunostaining in intact animals following control or Lrig-1 knockdown (n = 9-10 technical replicates).


D Quantification of H3P immunostaining at homeostasis ( fixed at 7 days post - RNAi , white region ) and at 48 hpa ( amputated at 5 days post - RNAi and fixed at 7 days post - RNAi , yellow region ) during the first round of regeneration in control or Lrig - 1 KD animals ( n = 10 - 17 ) .

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D Quantification of H3P immunostaining at homeostasis (fixed at 7 days post-RNAi, white region) and at 48 hpa (amputated at 5 days post-RNAi and fixed at 7 days post-RNAi, yellow region) during the first round of regeneration in control or Lrig-1 KD animals (n = 10-17).


E Quantification of unpigmented blastema area ( as % total tissue area ) in regenerating control or Lrig - 1 KD animals from 2 dpa to 7 dpa during the first round of regeneration ( n = 10 animals per condition per time point ) . The same animals were used for all timepoints . Representative images from 7 dpa are shown . Scale bars , 500 µm .

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E Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the first round of regeneration (n = 10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.


F Quantification of H3P immunostaining at homeostasis ( fixed at 14 days post - RNAi , white region ) and at 48 hpa ( amputated at 12 days post - RNAi and fixed at 14 days post - RNAi , yellow region ) during a second round of regeneration in control or Lrig - 1 KD animals ( n = 10 - 18 ) .

F Quantification of H3P immunostaining at homeostasis (fixed at 14 days post-RNAi, white region) and at 48 hpa (amputated at 12 days post-RNAi and fixed at 14 days post-RNAi, yellow region) during a second round of regeneration in control or Lrig-1 KD animals (n = 10-18).


G Quantification of unpigmented blastema area ( as % total tissue area ) in regenerating control or Lrig - 1 KD animals from 2 dpa to 7 dpa during the second round of regeneration ( n = 9 - 10 animals per condition per time point ) . The same animals were used for all timepoints . Representative images from 7 dpa are shown . Scale bars , 500 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

G Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the second round of regeneration (n = 9-10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.


( A - D ) Images of a representative wild - type adult eye ( A ) or adult eyes containing mtm mutant clones ( B and C ) . Quantification of relative eye sizes ( n = 10 ) ( D ) .

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(A-D) Images of a representative wild-type adult eye (A) or adult eyes containing mtm mutant clones (B and C). Quantification of relative eye sizes (n=10) (D).


( F - G ) A pupal eye disc containing mtm mutant clones ( marked by the absence of GFP ) was stained for Dlg . Note the increased interommatidial cells in the mtm mutant clone . Quantification of numbers of interommatidial cells per ommatidium ( n = 10 ) ( G ) .

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(F-G) A pupal eye disc containing mtm mutant clones (marked by the absence of GFP) was stained for Dlg. Note the increased interommatidial cells in the mtm mutant clone. Quantification of numbers of interommatidial cells per ommatidium (n=10) (G).


( K ) RNAi of Mtm reduces Yki phosphorylation . Kc167 cells were incubated with dsRNA of gfp or mtm for three days before western blot analysis . Total cell lysates were probed with anti - phospho - Yki ( p - Yki ) antibody . Note that RNAi of mtm decreased Yki phosphorylation .

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

(K) RNAi of Mtm reduces Yki phosphorylation. Kc167 cells were incubated with dsRNA of gfp or mtm for three days before western blot analysis. Total cell lysates were probed with anti-phospho-Yki (p-Yki) antibody. Note that RNAi of mtm decreased Yki phosphorylation.


PI ( 3 ) P expression levels detected by a 2xFYVE - GFP reporter in a third instar larval wing disc ( A ) and eye disc ( B ) containing mtm mutant clones ( marked by the absence of RFP ) . Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis . Note the strong increases of PI ( 3 ) P levels in the mtm mutant clones .

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

PI(3)P expression levels detected by a 2xFYVE-GFP reporter in a third instar larval wing disc (A) and eye disc (B) containing mtm mutant clones (marked by the absence of RFP). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.


Quantification of the mean FYVE - GFP fluorescence intensity of mtm mutant clones and surrounding wild - type ( WT ) cells are shown ( C ) . Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis . Note the strong increases of PI ( 3 ) P levels in the mtm mutant clones .

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

Quantification of the mean FYVE-GFP fluorescence intensity of mtm mutant clones and surrounding wild-type (WT) cells are shown (C). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.


Display of differentially expressed genes in NeuNT tumors ( WT , TNCKO ) as volcano plot representing all associated genes in WT tumors . N = 3 tumors per genotype . ( A ) Log2 Fold Change ( X - axis ) and the negative log10 ( P . Value ) ( Y - axis ) is displayed , the blue line indicating p = 0 . 05 ( Y - axis ) , points above the line , p < 0 . 05 . The dashed red lines indicate the log 2 Fold Change cutoff of - 0 . 25 and 0 . 25 , respectively ( X - axis ) . Green dots represent genes that display both statistical significance ( p < 0 . 05 ) and fold changes - 0 . 25 and 0 . 25 , respectively . Selected genes regulated by TNC are marked in red ( type I interferon response ) or pink ( Cxcl12 ) dot .

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

Display of differentially expressed genes in NeuNT tumors (WT, TNCKO) as volcano plot representing all associated genes in WT tumors. N = 3 tumors per genotype. (A) Log2 Fold Change (X-axis) and the negative log10 (P.Value) (Y-axis) is displayed, the blue line indicating p = 0.05 (Y-axis), points above the line, p < 0.05. The dashed red lines indicate the log 2 Fold Change cutoff of -0.25 and 0.25, respectively (X-axis). Green dots represent genes that display both statistical significance (p < 0.05) and fold changes -0.25 and 0.25, respectively. Selected genes regulated by TNC are marked in red (type I interferon response) or pink (Cxcl12) dot.


( A ) Representative staining images ( N = 5 ) of tissue from TNC high ( WT / shC ) and TNC low ( KO / shTNC ) tumors ( N = 5 per condition ) with the indicated antibodies . Arrows point at the respective immune subtype . Scale bar , 50 µm .

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(A) Representative staining images (N = 5) of tissue from TNC high (WT/shC) and TNC low (KO/shTNC) tumors (N = 5 per condition) with the indicated antibodies. Arrows point at the respective immune subtype. Scale bar, 50 µm.


( D , E ) Apoptosis measurement upon tumor staining for cl . casp3 ( D ) and signal quantification per field ( E ) in TNC high and TNC low tumors ( N = 7 per condition ) , 2 sections per tumor , 5 random fields . * * * p = 0 . 0006 , Mann - Whitney test , Mean ± SEM . Scale bar , 50 µm .

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(D, E) Apoptosis measurement upon tumor staining for cl. casp3 (D) and signal quantification per field (E) in TNC high and TNC low tumors (N = 7 per condition), 2 sections per tumor, 5 random fields. *** p = 0.0006, Mann-Whitney test, Mean ± SEM. Scale bar, 50 µm.


( I , J ) Detection ( I ) and quantification ( J ) of CD8 TIL in lung metastasis of NeuNT WT and TNCKO mice ( N = 3 mice , n = 7 and 12 lung metastases , respectively ) . Scale bar , 100 µm . * * p = 0 . 0097 , Mann Whitney test . Mean ± SEM .

(I, J) Detection (I) and quantification (J) of CD8 TIL in lung metastasis of NeuNT WT and TNCKO mice (N = 3 mice, n = 7 and 12 lung metastases, respectively). Scale bar, 100 µm. ** p = 0.0097, Mann Whitney test. Mean ± SEM.


( E ) Analysis of Cxcl12 ( qRT - PCR ) in tumor cells upon pretreatment with the indicated inhibitors for 45 minutes ( GW , BOP , DMSO and PBS as control respectively ) or 60 minutes ( Sunitinib ) and 6 hours ( Cli95 ) ( 6 hours DMSO as control ) before incubation with TNC for 24 hours . N = 6 independent experiments , * * p = 0 . 0022 , Mann Whitney test . Mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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) Analysis of Cxcl12 (qRT-PCR) in tumor cells upon pretreatment with the indicated inhibitors for 45 minutes (GW, BOP, DMSO and PBS as control respectively) or 60 minutes (Sunitinib) and 6 hours (Cli95) (6 hours DMSO as control) before incubation with TNC for 24 hours. N = 6 independent experiments, ** p = 0.0022, Mann Whitney test. Mean ± SEM.


CXCL12 binding to TNC as determined by negative EM imaging ( G ) , followed by quantification ( H ) . ( G ) scale bars , 100 nm ( left ) , 50 nm ( right ) , the arrowheads point at gold labeled CXCL12 and the asterisk points at the fibrinogen globe of the murine TNC monomer . ( H ) Representation of TNC monomer , oligomerization domain ( triangle ) to form hexamers as seen in ( G ) , FNIII repeats ( grey boxes , contant domains , white boxes , alternative domains ) , fibrinogen like domain ( circle ) . Representative result ( 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O

CXCL12 binding to TNC as determined by negative EM imaging (G), followed by quantification (H). (G) scale bars, 100 nm (left), 50 nm (right), the arrowheads point at gold labeled CXCL12 and the asterisk points at the fibrinogen globe of the murine TNC monomer. (H) Representation of TNC monomer, oligomerization domain (triangle) to form hexamers as seen in (G), FNIII repeats (grey boxes, contant domains, white boxes, alternative domains), fibrinogen like domain (circle). Representative result (3 independent experiments).


Attraction and retention were determined towards coatings with collagen I ( COL ) , fibronectin ( FN ) or TNC in the presence of CXCL10 , CCL21 , CXCL12 ( B , C ) 3 independent experiments in duplicates , Mann - Whitney test , ns > 0 . 05 ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 005 . Mean ± SEM .

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Attraction and retention were determined towards coatings with collagen I (COL), fibronectin (FN) or TNC in the presence of CXCL10, CCL21, CXCL12 (B, C) 3 independent experiments in duplicates, Mann-Whitney test, ns > 0.05; * p <0.05; **p < 0.01; ***p < 0.005. Mean ± SEM.


( B , C ) Imaging and signal quantification upon staining for cl . casp3 and DAPI in PBS and AMD treated tumors , N = 5 tumors per condition , 2 sections per tumor , 5 random fields , * * p = 0 . 0079 , Mann - Whitney test . Mean ± SEM . Scale bar , 50µm , arrows point at cl . Casp3 .

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, C) Imaging and signal quantification upon staining for cl. casp3 and DAPI in PBS and AMD treated tumors, N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50µm, arrows point at cl. Casp3.


( D , E ) CD8 TIL abundance and localization in PBS and AMD treated tumors , arrow points at CD8 TIL , scale bar , 50µm ( D ) . ( E ) Quantification of CD8 T cells per randomly chosen fields . N = 5 tumors per condition , 2 sections per tumor , 5 random fields , * * p = 0 . 0079 , Mann - Whitney test . Mean ± SEM . ( F ) Gzmb levels in PBS and AMD treated tumors . N = 10 tumors , * * * * p < 0 . 0001 , unpaired t - test . Mean ± SEM . (

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

(D, E) CD8 TIL abundance and localization in PBS and AMD treated tumors, arrow points at CD8 TIL, scale bar, 50µm (D). (E) Quantification of CD8 T cells per randomly chosen fields. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. (F) Gzmb levels in PBS and AMD treated tumors. N = 10 tumors, **** p < 0.0001, unpaired t-test. Mean ± SEM. (


( I , J ) Abundance and quantification of F4 / 80 + macrophages in PBS and AMD treated tumors , ( I ) staining for the indicated molecules , ( arrow , macrophages in the stroma ; asterisk , macrophages in the tumor nest ) , ( J ) quantification of F4 / 80 + cells inside the tumor nest . N = 5 tumors per condition , 2 sections per tumor , 5 random fields , * * p = 0 . 0079 , Mann - Whitney test . Mean ± SEM . Scale bar , 50 µm .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, J) Abundance and quantification of F4/80+ macrophages in PBS and AMD treated tumors, (I) staining for the indicated molecules, (arrow, macrophages in the stroma; asterisk, macrophages in the tumor nest), (J) quantification of F4/80+ cells inside the tumor nest. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50 µm.


( I - L ) qRT - PCR assessment of Neu ( I ) , Cd8a ( J ) , Ifnγ ( K ) and Gzmb ( L ) by qRT - PCR . PBS ( N = 16 ) and AMD ( N = 19 ) treated mice , * , p = 0 . 0364 , Mann whitney test ( I ) , * * , p = 0 . 0086 unpaired t - test ( J ) , * * , p = 0 . 0055 , unpaired t - test ( K ) , * * * , p = 0 . 0003 unpaired t - test ( L ) , respectively . The central band represents the median , the ends of the box the first and the third quartiles , and the whiskers reach from each quartile to the minimum or maximum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L) qRT-PCR assessment of Neu (I), Cd8a (J), Ifnγ (K) and Gzmb (L) by qRT-PCR. PBS (N = 16) and AMD (N = 19) treated mice, *, p = 0.0364, Mann whitney test (I), **, p = 0.0086 unpaired t-test (J), **, p = 0.0055, unpaired t-test (K), ***, p = 0.0003 unpaired t-test (L), respectively. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum.


( A - C ) Adjacent human breast cancer tissue microarray ( TMA ) sections were stained with the indicated antibodies , scale bar , 50 μm , arrows point at CD8 TIL ( A ) and , CD8 TIL were counted per field ( B ) , or in the stroma and nest , respectively ( C ) , N = 103 TNC + tumors , N = 116 TNC - tumors . . ( B ) * * p = 0 . 0055 , Fisher ' s exact test . The central band represents the median , the ends of the box the first and the third quartiles , and the whiskers reach from each quartile to the minimum or maximum . ( C ) * * p = 0 , 0055 , Mann - Whitney 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 O O O O O O O O O O O O O O O O O O O O

(A-C) Adjacent human breast cancer tissue microarray (TMA) sections were stained with the indicated antibodies, scale bar, 50 μm, arrows point at CD8 TIL (A) and, CD8 TIL were counted per field (B), or in the stroma and nest, respectively (C), N = 103 TNC+ tumors, N = 116 TNC- tumors.. (B) ** p = 0.0055, Fisher's exact test. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum. (C) ** p = 0,0055, Mann-Whitney test.


( D - G ) Kaplan Meier analysis to address correlation of TNC expression ( D ) , total numbers of CD8 TIL ( E ) , combined high ( above median ) CD8 TIL and high or low ( above / below median ) TNC ( F ) and stromal CD8 cells ( G ) with metastasis - free survival ( MFS ) of breast cancer patients . Stromal CD8 - or CD8 + is defined as below or above median stromal CD8 TIL per area . Log - rank 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

(D-G) Kaplan Meier analysis to address correlation of TNC expression (D), total numbers of CD8 TIL (E), combined high (above median) CD8 TIL and high or low (above/below median) TNC (F) and stromal CD8 cells (G) with metastasis-free survival (MFS) of breast cancer patients. Stromal CD8- or CD8+ is defined as below or above median stromal CD8 TIL per area. Log-rank test.


( H , I ) Correlation of TNC and CD8a levels with overall survival ( OS ) using survival analysis on breast cancer patient cohort GSE 19783 - GPL6580 . Hazard ratio ( HR ) and p values are indicated in Appendix Fig . S5N . Log - rank test .

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(H, I) Correlation of TNC and CD8a levels with overall survival (OS) using survival analysis on breast cancer patient cohort GSE 19783-GPL6580. Hazard ratio (HR) and p values are indicated in Appendix Fig. S5N. Log-rank test.


( A ) Representative IHC images of CXCL12 intensity and staining pattern in the TMA . N = 219 tumors . Upper panel , scale bar , 400 μm ; lower panel , scale bar , 100 μm . Arrows point at punctate staining in the stroma .

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(A) Representative IHC images of CXCL12 intensity and staining pattern in the TMA. N = 219 tumors. Upper panel, scale bar, 400 μm; lower panel, scale bar, 100 μm. Arrows point at punctate staining in the stroma.


( D ) Kaplan Meier survival analysis of breast cancer patients upon stratification into tumors with a CXCL12 Hscore below or above the median , p = 0 . 002 . Mann - Whitney test . ( E ) Survival analysis of breast cancer patients in the GSE 19783 - GPL6580 cohort upon stratification into tumors with high or low expression of TNC and CXCL12 . Log - rank test . HR and p values are indicated ,

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(D) Kaplan Meier survival analysis of breast cancer patients upon stratification into tumors with a CXCL12 Hscore below or above the median, p = 0.002. Mann-Whitney test. (E) Survival analysis of breast cancer patients in the GSE 19783-GPL6580 cohort upon stratification into tumors with high or low expression of TNC and CXCL12. Log-rank test. HR and p values are indicated,


B Body weight of Uqcrh - / - mice and wild - type littermates at 1 - 2 weeks ( n = 5 to 10 ) , during the post - weaning period from 5 - 6 weeks of age ( n = 3 to 7 ) and weekly from 6 to 10 weeks ( n = 13 to 66 ) , data are shown as mean ± SD . * * * * p < 0 . 0001 , 2 - Way ANOVA , from 7 . 5wk of age for Uqcrh - / - males and females versus sex - matched controls . Data are shown as single data points and median , or categorical data , males and females pooled , since genotype - related effects were comparable for both sexes from different cohorts . * p < 0 . 05 , * * * p < 0 . 001 , Wilcoxon rank - sum test .

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B Body weight of Uqcrh -/- mice and wild-type littermates at 1-2 weeks (n = 5 to 10), during the post-weaning period from 5-6 weeks of age (n = 3 to 7) and weekly from 6 to 10 weeks (n = 13 to 66), data are shown as mean ± SD. **** p < 0.0001, 2-Way ANOVA, from 7.5wk of age for Uqcrh-/- males and females versus sex-matched controls. Data are shown as single data points and median, or categorical data, males and females pooled, since genotype-related effects were comparable for both sexes from different cohorts. * p < 0.05, *** p <0.001, Wilcoxon rank-sum test.


C , D Clinical chemistry plasma concentrations of parameters related to acid - base balance ( Lactate and Ammonia ) and metabolic state ( Glucose ) n = 15 Uqcrh - / - and 16 WT pups 1 - 2 - week - old ( C ) ; n = 15 f , 15 m Uqcrh - / - and 15 f , 15 m WT mice aged 8 - 9 weeks ( D ) ; sex not differentiated . Data shown as scatter dot plots with line at median . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , Wilcoxon rank sum test .

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 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 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 Clinical chemistry plasma concentrations of parameters related to acid-base balance (Lactate and Ammonia) and metabolic state (Glucose) n = 15 Uqcrh-/- and 16 WT pups 1-2-week-old (C); n = 15 f, 15 m Uqcrh-/- and 15 f,15 m WT mice aged 8-9 weeks (D); sex not differentiated. Data shown as scatter dot plots with line at median. * p < 0.05, ** p < 0.01, **** p < 0.0001, Wilcoxon rank sum test.


F Measures related to behaviour and neurological function , data generated by the SHIRPA ( 9 weeks old , F ) , n = 7f , 8m Uqcrh - / - and 8f , 9m WT animals . Prepulse inhibition of the acoustic startle as a parameter associated with sensorimotor gating ( 10 weeks , E right panel ) , n = 7 f / 5 m Uqcrh - / - and 8 f / 9 m WT mice . Sex is not differentiated . Data are shown as scatter dot plots with a line representing the median . * * * p < 0 . 001 , * * * * p < 0 . 0001 , Wilcoxon rank - sum 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 O O O O

F Measures related to behaviour and neurological function, data generated by the SHIRPA (9 weeks old, F), n = 7f, 8m Uqcrh-/- and 8f, 9m WT animals. Prepulse inhibition of the acoustic startle as a parameter associated with sensorimotor gating (10 weeks, E right panel), n = 7 f/5 m Uqcrh-/- and 8 f/ 9 m WT mice. Sex is not differentiated. Data are shown as scatter dot plots with a line representing the median. *** p < 0.001, **** p < 0.0001, Wilcoxon rank-sum test.


G Parameters related to heart function generated by Echocardiography ( ECHO ) and Electrocardiography ( ECG ) , n = 15 f , 15 m Uqcrh - / - and 15 f , 15 m WT mice , 6 weeks old .

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G Parameters related to heart function generated by Echocardiography (ECHO) and Electrocardiography (ECG), n = 15 f, 15 m Uqcrh-/- and 15 f, 15 m WT mice, 6 weeks old.


A Respiratory chain enzyme activities from human control ( green ) and patient ( red ) fibroblasts . Mean activities of controls ( n = 8 ) are set to 100 % and error bars represent 1 standard deviation . Data are normalised to citrate synthase ( CS ) activity . B Respiratory chain enzyme activities in heart , brain and liver , of wild - type and Uqcrh - / - mice . Data are normalised to citrate synthase ( CS ) activity . Values are given as mean ± SD , n = 4 WT , 5 Uqcrh - / - ( Heart ) , 3 WT , 3 Uqcrh - / - ( Brain ) , 1 WT and 2 Uqcrh - / - ( Liver ) . * p < 0 . 05 , * * * * p < 0 . 0001 , 2 - way ANOVA ( Multiple comparison ) .

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A Respiratory chain enzyme activities from human control (green) and patient (red) fibroblasts. Mean activities of controls (n = 8) are set to 100% and error bars represent 1 standard deviation. Data are normalised to citrate synthase (CS) activity. B Respiratory chain enzyme activities in heart, brain and liver, of wild-type and Uqcrh-/- mice. Data are normalised to citrate synthase (CS) activity. Values are given as mean ± SD, n = 4 WT, 5 Uqcrh-/- (Heart), 3 WT, 3 Uqcrh-/- (Brain), 1 WT and 2 Uqcrh-/- (Liver). * p < 0.05, **** p < 0.0001, 2-way ANOVA (Multiple comparison).


C Western blot analysis of OXPHOS components in the patient ( P ) and control ( n = 2 , C1 - 2 ) fibroblasts . D BN - PAGE analysis of OXPHOS complex assembly in enriched mitochondria from patient and control fibroblasts ( n = 2 , C1 - 2 ) solubilised with DDM . Immunoblotting was performed using antibodies to a subunit of each complex ( CI [ NDUFB8 ] , CII [ SDHA ] , CIII [ UQCRC2 ] , CIV [ MT - CO1 ] , and CV [ ATP5A ] )

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C Western blot analysis of OXPHOS components in the patient (P) and control (n = 2, C1-2) fibroblasts. D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from patient and control fibroblasts (n = 2, C1-2) solubilised with DDM. Immunoblotting was performed using antibodies to a subunit of each complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A])


F Immunohistochemical staining of UQCRC2 was performed on liver , heart and kidney derived from wild - type and Uqcrh - / - mice at 9 weeks of age . Staining of VDAC1 / porin was also carried out G Graphs indicate score values of immunohistochemical staining intensity of both VDAC1 / porin and UQCRC2 . Data are given as mean ± SEM . * p < 0 . 05 ; n = 3 , Student ' s t - test ( unpaired samples ) .

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F Immunohistochemical staining of UQCRC2 was performed on liver, heart and kidney derived from wild-type and Uqcrh-/- mice at 9 weeks of age. Staining of VDAC1/porin was also carried out G Graphs indicate score values of immunohistochemical staining intensity of both VDAC1/porin and UQCRC2. Data are given as mean ± SEM. * p < 0.05; n = 3, Student's t-test (unpaired samples).


A Western blot analysis of control ( n = 2 , C1 - 2 ) and patient fibroblasts transduced with a lentiviral vector ( pLVX ) containing wild - type UQCRH . Expression of wild - type UQCRH was induced using various concentrations of doxycycline ( dox ) up to 20 ng / ml for 72 hours . B BN - PAGE analysis of control ( n = 2 , C1 - 2 ) and patient fibroblasts transduced with a lentiviral vector ( pLVX ) containing wild - type UQCRH . Transduced fibroblasts were either uninduced or induced with 20 ng / ml doxycycline ( dox ) for 72 hours .

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A Western blot analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Expression of wild-type UQCRH was induced using various concentrations of doxycycline (dox) up to 20 ng/ml for 72 hours. B BN-PAGE analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Transduced fibroblasts were either uninduced or induced with 20 ng/ml doxycycline (dox) for 72 hours.


C Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in control ( NHDF ) fibroblasts . D Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in patient fibroblasts . E Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in control ( NHDF ) fibroblasts transduced with wild - type UQCRH . F Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in patient fibroblasts transduced with wild - type ( WT ) UQCRH . G Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in control ( NHDF ) fibroblasts transduced with the pseudogene UQCRHL . H Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in patient fibroblasts transduced with the pseudogene UQCRHL . I Graph indicates the staining intensity of UQCRC2 in control ( NHDF ) fibroblasts ( green ) and patient fibroblasts ( red ) , those respective cell lines transduced ( T ) with wild - type UQCRH ( diagonal lines ) and those cell lines transduced with the pseudogene UQCRHL ( vertical lines ) measured using Image J , Data are given as mean ± SEM . One - way ANOVA ( Kruskal - Wallis test ) , * p < 0 . 05 ; n = 3 macroscopic field ( 10 - 14 cells for macroscopic field were measured ) .

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C Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts. D Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts. E Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with wild-type UQCRH. F Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with wild-type (WT) UQCRH. G Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with the pseudogene UQCRHL. H Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with the pseudogene UQCRHL. I Graph indicates the staining intensity of UQCRC2 in control (NHDF) fibroblasts (green) and patient fibroblasts (red), those respective cell lines transduced (T) with wild-type UQCRH (diagonal lines) and those cell lines transduced with the pseudogene UQCRHL (vertical lines) measured using Image J, Data are given as mean ± SEM. One-way ANOVA (Kruskal-Wallis test), * p < 0.05; n = 3 macroscopic field (10-14 cells for macroscopic field were measured).


Mitochondrial membranes from fibroblasts of control ( WT ) and patient fibroblasts ( i , immortalised ) were solubilised with digitonin and separated on native gradient gels . A BN - PAGE gel stained with Coomassie showing molecular weight marker ( BHM ) and samples from control and patient ( primary fibroblasts , ( I ) = immortalised fibroblasts ) B BN - PAGE gel stained with NADH : NTB reductase activity stain showing molecular weight marker ( BHM ) and samples from control and patient ( primary fibroblasts , ( I ) = immortalised fibroblasts ) C Further complexome analysis of the BN - PAGE gels shown in A and B . Each native lane was cut into even fractions from high to low molecular mass . Proteins in each slice were digested with trypsin and analysed by quantitative mass spectrometry . Shown here are average IBAQ values of all identified subunits from complexes III , I and IV

Mitochondrial membranes from fibroblasts of control (WT) and patient fibroblasts (i, immortalised) were solubilised with digitonin and separated on native gradient gels. A BN-PAGE gel stained with Coomassie showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts) B BN-PAGE gel stained with NADH:NTB reductase activity stain showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts) C Further complexome analysis of the BN-PAGE gels shown in A and B. Each native lane was cut into even fractions from high to low molecular mass. Proteins in each slice were digested with trypsin and analysed by quantitative mass spectrometry. Shown here are average IBAQ values of all identified subunits from complexes III, I and IV


D Profile of complex III ( averaged IBAQ values ) in the patient ( dashed line ) and control ( solid line ) fibroblasts . E Profile of complexes III ( red ) , I ( yellow ) and IV ( green ) to visualise and assess supercomplex composition and stability in control ( solid lines ) and patient ( dashed lines ) fibroblasts .

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D Profile of complex III (averaged IBAQ values) in the patient (dashed line) and control (solid line) fibroblasts. E Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in control (solid lines) and patient (dashed lines) fibroblasts.


heart tissue from wild - type ( WT ) and Uqcrh - / - mice were solubilised with digitonin and separated on native gradient gels . F BN - PAGE gel stained with Coomassie ( left ) and NADH : NTB reductase activity stain ( right ) showing molecular weight marker ( BHM ) and heart tissue samples from wild - type ( WT ) and Uqcrh - / - mice . G BN - PAGE and immunoblot using the antibody to CIII subunit UQCRC2 in heart tissue samples from wild - type ( WT ) and Uqcrh - / - mice . H Further complexome analysis of the BN - PAGE gels shown in F , shown here are average IBAQ values of subunits from complexes III , I and IV

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heart tissue from wild-type (WT) and Uqcrh-/- mice were solubilised with digitonin and separated on native gradient gels. F BN-PAGE gel stained with Coomassie (left) and NADH:NTB reductase activity stain (right) showing molecular weight marker (BHM) and heart tissue samples from wild-type (WT) and Uqcrh-/- mice. G BN-PAGE and immunoblot using the antibody to CIII subunit UQCRC2 in heart tissue samples from wild-type (WT) and Uqcrh-/- mice. H Further complexome analysis of the BN-PAGE gels shown in F, shown here are average IBAQ values of subunits from complexes III, I and IV


I Profile of complex III ( averaged IBAQ values ) in wild - type ( + / + , solid line ) and Uqcrh - / - ( - / - , dashed line ) heart tissue . J Profile of complexes III ( red ) , I ( yellow ) and IV ( green ) to visualise and assess supercomplex composition and stability in wild - type ( + / + , solid line ) and Uqcrh - / - ( - / - , dashed line ) heart tissue .

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I Profile of complex III (averaged IBAQ values) in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue. J Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue.


( A ) Heatmap of differentially enriched metabolites in heart , liver , skeletal muscle and serum of young ( Y ) and aged ( A ) killifish ( n = 4 ) .

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(A) Heatmap of differentially enriched metabolites in heart, liver, skeletal muscle and serum of young (Y) and aged (A) killifish (n = 4).


( B - C ) Ageing leads to significant cardiac - specific accumulation of sphinganine .

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(B-C) Ageing leads to significant cardiac-specific accumulation of sphinganine.


( D ) Targeted lipidomics confirm age - dependent accumulation of sphinganine in killifish ( n = 4 ; per condition ) , zebrafish ( n = 4 , per condition ) , mouse ( n = 4 for young and n = 6 for aged samples ) and human hearts ( n = 1 for young and 2 for aged ) . In the case of human heart tissue , statistical significance was computed from technical replicates .

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(D) Targeted lipidomics confirm age-dependent accumulation of sphinganine in killifish (n = 4; per condition), zebrafish (n = 4, per condition), mouse (n = 4 for young and n=6 for aged samples) and human hearts (n = 1 for young and 2 for aged). In the case of human heart tissue, statistical significance was computed from technical replicates.


( E ) Heatmap of differentially expressed transcripts ( Log2 fold change + / - 1 , adjusted p - value cut off < 0 . 05 ) between young and aged killifish hearts ( n = 3 ) . Significantly enriched relevant GO categories depicted as bar graph .

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(E) Heatmap of differentially expressed transcripts (Log2 fold change +/- 1, adjusted p-value cut off <0.05) between young and aged killifish hearts (n = 3). Significantly enriched relevant GO categories depicted as bar graph.


( F ) Transcriptional drift variance of genes involved in genome integrity , epigenetic regulation and sphingolipid metabolism .

(F) Transcriptional drift variance of genes involved in genome integrity, epigenetic regulation and sphingolipid metabolism.


( G ) γH2A . X staining of young , mid - aged and aged killifish hearts show signs of DNA damage , increasing with age ( n = 4 , # of sections per animal = 10 alternate sections , 10 µm thickness ) .

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(G) γH2A.X staining of young, mid-aged and aged killifish hearts show signs of DNA damage, increasing with age (n = 4, # of sections per animal = 10 alternate sections, 10 µm thickness).


( H ) Quantification of γH2A . X + cardiomyocyte nuclei .

(H) Quantification of γH2A.X+ cardiomyocyte nuclei.


( I ) The aged killifish heart shows significant signs of genome instability shown here by neutral comet assays . Representative images of single nuclei from young and old hearts along with comet length on the right ( n = 4 ) .

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(I) The aged killifish heart shows significant signs of genome instability shown here by neutral comet assays. Representative images of single nuclei from young and old hearts along with comet length on the right (n = 4).


( J ) γH2A . X staining of young and aged human ventricles show signs of DNA damage accumulating with age ( n = 2 , # of sections per sample = 5 alternate sections , 10 µm thickness ) .

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(J) γH2A.X staining of young and aged human ventricles show signs of DNA damage accumulating with age (n = 2, # of sections per sample = 5 alternate sections, 10 µm thickness).


( K ) Quantification of γH2A . X + cardiomyocyte nuclei is on the right . Scale bar = 50 µm .

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(K) Quantification of γH2A.X+ cardiomyocyte nuclei is on the right. Scale bar = 50 µm.


Sphinganine induces DNA damage in human cardiomyocytes depicted here ( A - C ) by γH2A . X + nuclei in cardiac - specific ACTN2 + cells ( n = 3 biological replicates ; # of cells quantified = 147 per condition )

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Sphinganine induces DNA damage in human cardiomyocytes depicted here (A-C) by γH2A.X+ nuclei in cardiac-specific ACTN2+ cells (n = 3 biological replicates; # of cells quantified = 147 per condition)


Sphinganine induces DNA damage in human cardiomyocytes depicted here ( D ) by neutral comet assay of DHS treated hCMs . ( E ) Quantification of comet length in the indicated conditions .

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Sphinganine induces DNA damage in human cardiomyocytes depicted here (D) by neutral comet assay of DHS treated hCMs. (E) Quantification of comet length in the indicated conditions.


Elevated DHS levels lead to ageing signatures in hCMs shown here by ( F - G ) SA - β - GAL activity , Respective graphs depict the quantification of each of the ageing signatures are on the right .

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Elevated DHS levels lead to ageing signatures in hCMs shown here by (F-G) SA-β-GAL activity, Respective graphs depict the quantification of each of the ageing signatures are on the right.


Elevated DHS levels lead to ageing signatures in hCMs shown here by ( H ) p21 activation ( # cells quantified per replicate = 150 - 200 ) , ( I ) HP1 - α + nuclear foci ( # of cells quantified per replicate = 120 - 200 ) and ( J ) disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining ( # of cells quantified per replicate = 100 - 150 per condition ) . Respective graphs depict the quantification of each of the ageing signatures are on the right .

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Elevated DHS levels lead to ageing signatures in hCMs shown here by (H) p21 activation (# cells quantified per replicate = 150-200), (I) HP1-α+ nuclear foci (# of cells quantified per replicate = 120-200) and (J) disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining (# of cells quantified per replicate = 100-150 per condition). Respective graphs depict the quantification of each of the ageing signatures are on the right.


High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs ( L ) and ( M ) in aged human hearts . ( N ) Violin plot depicting the distributions of the grey - scale nuclear intensity of the indicated markers . ( When not specified , the experiments were conducted in at least 3 biological replicates ) . Scale bar = 10 µm .

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High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs (L) and (M) in aged human hearts. (N) Violin plot depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (When not specified, the experiments were conducted in at least 3 biological replicates). Scale bar = 10 µm.


( A ) Pharmacological inhibition of HDAC1 using romidepsin , MS - 275 and pyroxamide in human cardiomyocytes cause extensive DNA damage , shown here by γH2A . X staining .

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(A) Pharmacological inhibition of HDAC1 using romidepsin, MS-275 and pyroxamide in human cardiomyocytes cause extensive DNA damage, shown here by γH2A.X staining.


( B ) Bar - graphs depict quantification of γH2A . X + hCM nuclei in the indicated conditions .

(B) Bar-graphs depict quantification of γH2A.X+ hCM nuclei in the indicated conditions.


( E ) Sphinganine and its derivative DHS1P inhibits class 1 HDACs in the human cardiomyocytes as inferred from the in cellulo HDAC - activity assay , shown here as bar graph .

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(E) Sphinganine and its derivative DHS1P inhibits class 1 HDACs in the human cardiomyocytes as inferred from the in cellulo HDAC-activity assay, shown here as bar graph.


( F ) In vitro HDAC activity assay revealed inhibition of nuclear HDAC ' s and purified HDAC1 activity by DHS1P and S1P , shown here as bar graphs .

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(F) In vitro HDAC activity assay revealed inhibition of nuclear HDAC's and purified HDAC1 activity by DHS1P and S1P, shown here as bar graphs.


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Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs


Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs and respective quantifications .

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Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs and respective quantifications.


( I ) Representative micrographs of hCMs indicating the rescue of the DHS induced DNA damage by co - incubation with RNA Pol II inhibitor , triptolide and ( J ) the respective quantifications of γH2A . X + CM nuclei represented as bar graph . Scale bars for A , I = 10 µm and G = 50 µm . ( When not specified , the experiments were conducted in at least 3 biological replicates ) .

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(I) Representative micrographs of hCMs indicating the rescue of the DHS induced DNA damage by co-incubation with RNA Pol II inhibitor, triptolide and (J) the respective quantifications of γH2A.X+ CM nuclei represented as bar graph. Scale bars for A,I=10 µm and G = 50 µm. (When not specified, the experiments were conducted in at least 3 biological replicates).


( A - B ) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS - induced DNA damage as shown here by staining for γH2A . X ( green ) in hCMs .

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(A-B) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) in hCMs.


( C ) Bar graphs representing the percentage of γH2A . X + hCMs in the indicated conditions ( # cells quantified per replicate ~ 200 ) .

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(C) Bar graphs representing the percentage of γH2A.X+ hCMs in the indicated conditions (# cells quantified per replicate ~200).


( D - E ) Treatment of hCMs with ABC294640 , a specific SPHK2 inhibitor prevents DHS - induced epigenetic aberrations in hCMs visualized here by H3K56ac and H3K27ac staining ( ~ 100 cells quantified / condition ) .

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(D-E) Treatment of hCMs with ABC294640, a specific SPHK2 inhibitor prevents DHS-induced epigenetic aberrations in hCMs visualized here by H3K56ac and H3K27ac staining (~100 cells quantified/ condition).


( F ) Violin plots depicting the distributions of the grey - scale nuclear intensity of the indicated markers .

(F) Violin plots depicting the distributions of the grey-scale nuclear intensity of the indicated markers.