Figure 4A IL - 6 secretion in HEI - OC1 cells treated with 100 pg / mL LPS or 20μM cisplatin ( n = 3 or 4 independent biological replicates ) . B IL - 6 secretion in TLR4 - / - cells transfected with empty vector ( EV ) or mouse Tlr4 ( mTlr4 ) following treatment with 20μM cisplatin ( n = 6 independent biological replicates ) . Data information : In all panels data are presented as mean and standard deviation . Statistical comparisons to 0 hr time point were assessed by 2 - way ANOVA . ns , not significant ; * , P < . 05 ; * * , P < . 01 ; * * * , P < . 001 ; * * * * , P < . 0001 ( Dunnett ' s test ) . C Il6 and Tlr4 transcript levels in HEI - OC1 cells following treatment with 20μM cisplatin for the indicated times ( n = 3 independent biological replicates ) . Data information : In all panels data are presented as mean and standard deviation . Statistical comparisons to 0 hr time point were assessed by 2 - way ANOVA . ns , not significant ; * , P < . 05 ; * * , P < . 01 ; * * * , P < . 001 ; * * * * , P < . 0001 ( Dunnett ' s test ) .

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Figure 4A IL-6 secretion in HEI-OC1 cells treated with 100 pg/mL LPS or 20μM cisplatin (n=3 or 4 independent biological replicates). B IL-6 secretion in TLR4-/- cells transfected with empty vector (EV) or mouse Tlr4 (mTlr4) following treatment with 20μM cisplatin (n=6 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test).C Il6 and Tlr4 transcript levels in HEI-OC1 cells following treatment with 20μM cisplatin for the indicated times (n=3 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test).


Figure 5B Hair cell viability in larval zebrafish pre - treated with control - , tlr4ba - and / or tlr4bb - targeting morpholino oligonucleotides ( MO ) and subsequently treated with 15 μM cisplatin . Data information : In both panels each data point represents a score of hair cell integrity in an individual animal ( taken from multiple samples per animal ) with lines representing mean and standard deviation . Statistical comparisons to control morpholino ( B , except as indicated in blue ) were assessed by one - way ANOVA . * , P < . 05 ; * * , P < . 01 ; * * * * , P < . 0001 ( Tukey test ) .

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Figure 5B Hair cell viability in larval zebrafish pre-treated with control-, tlr4ba- and/or tlr4bb-targeting morpholino oligonucleotides (MO) and subsequently treated with 15 μM cisplatin. Data information: In both panels each data point represents a score of hair cell integrity in an individual animal (taken from multiple samples per animal) with lines representing mean and standard deviation. Statistical comparisons to control morpholino (B, except as indicated in blue) were assessed by one-way ANOVA. *, P<.05; **, P<.01; ****, P<.0001 (Tukey test).


Figure 6A IL - 6 secretion in HEI - OC1 cells pretreated with DMF vehicle ( veh ) , 4μM TAK242 ( TAK ) or left untreated ( nil ) following treatment with 10 ng / mL LPS or 20μM cisplatin ( n = 5 - 7 independent biological replicates ) . Data Information : In all panels data are presented as mean and standard deviation . Statistical comparisons to nil treatment were assessed by 2 - way ( A ) or one - way * , P < . 05 ; * * * * , P < . 0001 ( Dunnett ' s test ) . B ROS generation in HEI - OC1 cells pretreated with DMF vehicle ( veh ) , 4μM TAK242 ( TAK ) or left untreated ( nil ) following treatment with 20μM cisplatin ( n = 3 independent biological replicates ) . Data Information : In all panels data are presented as mean and standard deviation . Statistical comparisons to nil treatment were assessed by one - way ( B ) ANOVA . ; * * , P < . 01 P < . 0001 ( Dunnett ' s test ) .

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

Figure 6A IL-6 secretion in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 10 ng/mL LPS or 20μM cisplatin (n=5-7 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by 2-way (A) or one-way *, P<.05 ; ****, P<.0001(Dunnett's test).B ROS generation in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 20μM cisplatin (n=3 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by one-way (B) ANOVA. ; **, P<.01 P<.0001(Dunnett's test).


Figure 1A - F Brain MRI of the proband at 23 years . ( A ) Sagittal fast spin echo ( FSE ) T1 - weighted image shows thinning of the corpus callosum , dilation of the IV ventricle and of the cisterna magna and reduced volume of vermis and brainstem ; two lacunar lesions are evident in the dorsal pons . ( B ) T2 - weighted axial fluid - attenuated inversion recovery ( FLAIR ) images show hyperintense periventricular white matter with several lacunar lesions also in the basal ganglia and thalami . ( C ) MR angiography 3D TOF ( time of flight ) reconstruction shows two saccular aneurisms in the right M1 ( ø 4 mm ) and left M2 ( ø 2 . 5 mm ) segments of middle cerebral arteries ( arrows ) . ( D ) A recent ischemic hyperintense lesion is detected on diffusion tensor imaging ( DTI ) in the right side of the dorsal pons . Severe cavitations and lateral ventricles dilation ( left > right ) on FLAIR images ( B , E ) and diffuse microbleeds as small hypointense foci on SWI are visible ( F ) in the same slices of ( E ) . R = right , L = left . G , H Brain MRI scans of the asymptomatic parents of the proband show multiple focal hyperintensities on T2 - weighted images in the periventricular and subcortical cerebral white matter , expression of gliosis secondary to chronic small vessel ischemic changes , more evident in the father ( G ) , 56 years , than in the mother ( H ) , 54 years .

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Figure 1A-F Brain MRI of the proband at 23 years. (A) Sagittal fast spin echo (FSE) T1-weighted image shows thinning of the corpus callosum, dilation of the IV ventricle and of the cisterna magna and reduced volume of vermis and brainstem; two lacunar lesions are evident in the dorsal pons. (B) T2-weighted axial fluid-attenuated inversion recovery (FLAIR) images show hyperintense periventricular white matter with several lacunar lesions also in the basal ganglia and thalami. (C) MR angiography 3D TOF (time of flight) reconstruction shows two saccular aneurisms in the right M1 (ø 4 mm) and left M2 (ø 2.5 mm) segments of middle cerebral arteries (arrows). (D) A recent ischemic hyperintense lesion is detected on diffusion tensor imaging (DTI) in the right side of the dorsal pons. Severe cavitations and lateral ventricles dilation (left > right) on FLAIR images (B, E) and diffuse microbleeds as small hypointense foci on SWI are visible (F) in the same slices of (E). R = right, L = left.G, H Brain MRI scans of the asymptomatic parents of the proband show multiple focal hyperintensities on T2-weighted images in the periventricular and subcortical cerebral white matter, expression of gliosis secondary to chronic small vessel ischemic changes, more evident in the father (G), 56 years, than in the mother (H), 54 years.


Figure 2Gene expression of NOTCH3 , HES1 , HEY1 , HEYL and the 17 recently identified targets were established by a real - time PCR panel assay in skeletal muscle of controls ( n = 6 ) , proband ( II : 1 ) , parents ( mother I : 1 , father I : 2 ) and CADASIL patients ( n = 3 ) . Graph shows gene expression fold changes relative to controls and normalized on GAPDH ( reference gene ) , expressed as mean of two experiments ± SEM . Among the 17 novel assayed target genes , RCAN2 , ANGPT4 , HP and SORBS were not detected in any sample and were therefore not reported . Green and red lines indicate 2 . 0 and 0 . 5 fold changes , respectively . Statistical significances and P - values are reported in Supplementary Table S1 .

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Figure 2Gene expression of NOTCH3, HES1, HEY1, HEYL and the 17 recently identified targets were established by a real-time PCR panel assay in skeletal muscle of controls (n = 6), proband (II:1), parents (mother I:1, father I:2) and CADASIL patients (n = 3). Graph shows gene expression fold changes relative to controls and normalized on GAPDH (reference gene), expressed as mean of two experiments ± SEM. Among the 17 novel assayed target genes, RCAN2, ANGPT4, HP and SORBS were not detected in any sample and were therefore not reported. Green and red lines indicate 2.0 and 0.5 fold changes, respectively. Statistical significances and P-values are reported in Supplementary Table S1.


Figure 3A - C Histopathological examination of skin ( A ) and skeletal muscle biopsies ( B , C ) of control ( left ) , proband ( middle ) and CADASIL patient ( right ) . ( A ) Collagen IV staining ( in green ) in skin vessels : Collagen wall appears compact in the control , while it is disorganized in the skin vessels of the proband and of the CADASIL patient . Derangement of the collagen wall in single collagen fibers is more evident in the proband than in the CADASIL patient , where collagen wall is quite compact . Vessel ' s endothelium is delineated by ULEX staining ( in red ) . Collagen IV staining in skeletal muscle vessels ( B ) recapitulates the skin picture . Smooth muscle actin immunostaining of skeletal muscle biopsies ( C ) shows attenuated SMCs in the tunica media of both the proband and the CADASIL patient , with foci characterized by a complete SMCs loss ( particularly in the CADASIL patient ) and thinning of the vessel wall ( arrows ) . D , E Ultrastructural analysis of skin biopsies . ( D ) Skin biopsy of the proband : ( left panel ) At low magnification , the tunica media of a vessel shows irregular SMCs surrounded by a markedly thickened basal membrane with a multilayered aspect ( arrows ) ; SMCs are identified by the presence of " focal adhesions " ( square boxes ) ; ( middle panel ) collagen fibrils appear organized in a parallel pathway along SMC ( arrow ) , note the " focal adhesions " ( square box ) ; ( right panel ) collagen fibrils ( " CF " ) are more represented in dense bundles than elastic ones ( " E " ) . No GOM is detected , in contrast to CADASIL patient ( E ) , showing deposits of granular osmiophilic material ( arrows ) located between SMC plasmalemma and basal lamina . F KCNA5 immunostaining of skeletal muscle biopsies of control ( left ) , proband ( middle ) and CADASIL patient ( right ) . A global decrease in reactivity is evident in the vessels of both the proband and the CADASIL patient . Note multiple areas in which KCNA5 is barely detectable ( arrows ) .

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

Figure 3A-C Histopathological examination of skin (A) and skeletal muscle biopsies (B, C) of control (left), proband (middle) and CADASIL patient (right). (A) Collagen IV staining (in green) in skin vessels: Collagen wall appears compact in the control, while it is disorganized in the skin vessels of the proband and of the CADASIL patient. Derangement of the collagen wall in single collagen fibers is more evident in the proband than in the CADASIL patient, where collagen wall is quite compact. Vessel's endothelium is delineated by ULEX staining (in red). Collagen IV staining in skeletal muscle vessels (B) recapitulates the skin picture. Smooth muscle actin immunostaining of skeletal muscle biopsies (C) shows attenuated SMCs in the tunica media of both the proband and the CADASIL patient, with foci characterized by a complete SMCs loss (particularly in the CADASIL patient) and thinning of the vessel wall (arrows).D, E Ultrastructural analysis of skin biopsies. (D) Skin biopsy of the proband: (left panel) At low magnification, the tunica media of a vessel shows irregular SMCs surrounded by a markedly thickened basal membrane with a multilayered aspect (arrows); SMCs are identified by the presence of "focal adhesions" (square boxes); (middle panel) collagen fibrils appear organized in a parallel pathway along SMC (arrow), note the "focal adhesions" (square box); (right panel) collagen fibrils ("CF") are more represented in dense bundles than elastic ones ("E"). No GOM is detected, in contrast to CADASIL patient (E), showing deposits of granular osmiophilic material (arrows) located between SMC plasmalemma and basal lamina.F KCNA5 immunostaining of skeletal muscle biopsies of control (left), proband (middle) and CADASIL patient (right). A global decrease in reactivity is evident in the vessels of both the proband and the CADASIL patient. Note multiple areas in which KCNA5 is barely detectable (arrows).


Figure 1Selected BioID REV7 results , shown as dot plots . The spectral counts for each indicated prey protein are shown as AvgSpec . Proteins were selected based on and iProphet probability of > 0 . 95 , BFDR of < 0 . 05 and ≥ 10 peptide count . The circle size represents the relative abundance of preys over baits . U2OS EJ5 - GFP cells were transfected with the indicated siRNAs . At 24hr post - transfection , cells were transfected with the I - SceI expression plasmid , and the GFP + population was analyzed 48 hr post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition ( siCTRL ) .

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Figure 1Selected BioID REV7 results, shown as dot plots. The spectral counts for each indicated prey protein are shown as AvgSpec. Proteins were selected based on and iProphet probability of >0.95, BFDR of <0.05 and ≥10 peptide count. The circle size represents the relative abundance of preys over baits.U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).


Figure 2Quantification of the Neutral Comet assay . U2OS cells stably expressing shCtrl , shREV7 or shSHLD2 were exposed to IR ( 10 Gy ) and run in low melting agarose under neutral conditions . Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment . U2OS cells were transfected with the indicated siRNA . 48h post - transfection the cells were treated with NCS to induce DNA damage and the cells were harvested at 0 , 1 , 2 , 4 and 24 hr post NCS treatment . Flow Cytometry analysis of phosphorylated - H2AX signal was used to measure γ - H2AX endogenous signal . Sensitivity to IR monitored by colony formation assay . MCF - 7 cells stably expressing the the indicated shRNAs were exposed to increasing doses of IR . 24 h post - irradiation , the cells were re - seeded to allow colony formation , fixed and stained with 0 . 4 % crystal violet . Shown is the quantification of colonies per condition which possessed more than 50 colonies . MCF - 7 cells stably expressing the the indicated shRNAs 24 h post - irradiation , the cells were re - seeded to allow colony formation , fixed and stained with 0 . 4 % crystal violet . Shown is the quantification of colonies per condition which possessed more than 50 colonies . cells were exposed to increasing doses of UV radiation .

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Figure 2Quantification of the Neutral Comet assay. U2OS cells stably expressing shCtrl, shREV7 or shSHLD2 were exposed to IR (10 Gy) and run in low melting agarose under neutral conditions. Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment.U2OS cells were transfected with the indicated siRNA. 48h post-transfection the cells were treated with NCS to induce DNA damage and the cells were harvested at 0, 1, 2, 4 and 24 hr post NCS treatment. Flow Cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal.Sensitivity to IR monitored by colony formation assay. MCF-7 cells stably expressing the the indicated shRNAs were exposed to increasing doses of IR. 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies.MCF-7 cells stably expressing the the indicated shRNAs 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies. cells were exposed to increasing doses of UV radiation.


Figure 3U2OS cells stably expressing HA - REV7 ( Top ) or HA - SHLD2 ( Bottom ) were pre - sensitized with 10ug / mL Hoescht 33342 before exposed to UV micro - irradiation . Immunofluorescence against endogenous HA and γ - H2AX epitope was subsequently performed to monitor REV7 and SHLD2 accumulation at sites of damage . Shown are representative micrographs . Scale bar = 5µm . U2OS LacR - Fok1 cells were transfected with GFP or GFP - SHLD2 and 24 h later DNA damage was induced using Shield - 1 and 4 - OHT . The cells were then processed for GFP and mCherry immunofluorescence . Shown are representative micrographs . Scale bar = 5µm . Quantification of the experiments shown in ( C ) . Data are represented as the mean ± SD ( n = 3 ) . At least 100 cells per condition were counted . Quantification of the experiments shown in ( C ) . Shown is the quantification of the GFP signal at the mCherry - LacR - Fok1 focus . 293T cell lines expressing ER - AsiSI with Flag - SHLD2 and treated with 1 µM of 4 - OHT . 6h later the cells were processed and immunoprecipitated with Anti - FLAG Magnetic Beads and anti - γ - H2AX . x / Protein A / G magnetic beads . DNA was purified and subjected to qPCR detection . Shown is the quantification of IP efficiency as the percentage of DNA precipitated from input ( Bottom ) . U2OS cells stably expressing HA - SHLD2 ∆ 720 - 904 ( Left ) or HA - SHLD2 ∆ 1 - 680 ( Right ) were processed as in ( B ) . Immunofluorescence against endogenous HA and RPA32 epitope was subsequently performed to monitor RPA32 and SHLD2 accumulation at sites of damage . Shown are representative micrographs . Scale bar = 5µm .

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Figure 3U2OS cells stably expressing HA-REV7 (Top) or HA-SHLD2 (Bottom) were pre-sensitized with 10ug/mL Hoescht 33342 before exposed to UV micro-irradiation. Immunofluorescence against endogenous HA and γ-H2AX epitope was subsequently performed to monitor REV7 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.U2OS LacR-Fok1 cells were transfected with GFP or GFP-SHLD2 and 24 h later DNA damage was induced using Shield-1 and 4-OHT. The cells were then processed for GFP and mCherry immunofluorescence. Shown are representative micrographs. Scale bar = 5µm. Quantification of the experiments shown in (C). Data are represented as the mean ± SD (n=3). At least 100 cells per condition were counted.Quantification of the experiments shown in (C). Shown is the quantification of the GFP signal at the mCherry-LacR-Fok1 focus.293T cell lines expressing ER-AsiSI with Flag-SHLD2 and treated with 1 µM of 4-OHT. 6h later the cells were processed and immunoprecipitated with Anti-FLAG Magnetic Beads and anti-γ-H2AX.x/Protein A/G magnetic beads. DNA was purified and subjected to qPCR detection. Shown is the quantification of IP efficiency as the percentage of DNA precipitated from input (Bottom).U2OS cells stably expressing HA-SHLD2∆720-904 (Left) or HA-SHLD2∆1-680 (Right) were processed as in (B). Immunofluorescence against endogenous HA and RPA32 epitope was subsequently performed to monitor RPA32 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.


Figure 4U2OS mCherry - LacR - Fok1 cells were treated with the indicated siRNA and subsequently transfected with a GFP - SHLD2 construct . 24 h post - transfection DNA damage was induced using Shield - 1 and 4 - OHT . The cells were then fixed and analyzed for the intensity of the GFP - SHLD2 signal at mCherry - LacR - Fok1 focus . Shown is the quantification of the GFP - SHLD2 signal at the Fok1 focus . Data are represented as a box - and - whisker plot where the whiskers represent the 10 - 90 percentile . At least 75 cells were counted per condition . 293T cells were transfected with Flag - REV7 and GFP - SHLD2 expression vectors as indicated . 24h post - transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU - 60019 for 1 h prior to irradiation . 1h post - irradiation ( 10 Gy ) nuclear extracts were prepared and REV7 complexes were immunoprecipitated using anti - Flag ( M2 ) Resin and then analyzed by immunoblotting using GFP , REV7 and p - Chk1 antibodies . U2OS EJ5 - GFP cells were transfected with the indicated siRNAs . At 24hr post - transfection , cells were transfected with the I - SceI expression plasmid , and the GFP + population was analyzed 48 hr post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition ( siCTRL ) . CH12F3 - 2 cells stably expressing the indicated shRNAs were stimulated with a cocktail of cytokines ( CIT ) to induce class switching to IgA . The percentage of IgA + cells was monitored 24 and 48h post - stimulation by staining with an anti - IgA antibody followed by flow cytometry analysis . U2OS DR - GFP cells were transfected with the indicated siRNAs . At 24hr post - transfection , cells were transfected with the I - SceI expression plasmid , and the GFP + population was analyzed 48 hr post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition ( siCTRL ) . U2OS cells were transfected with the indicated siRNAs and then treated with CldU , IdU and NCS 48h post - transfection as indicated . The slides were stained , dehydrated , mounted and visualized and shown is the quantification of CldU / IdU tract length in order to visualize DNA end - resection ( Right ) . At least 500 DNA tracks were measured per condition .

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Figure 4U2OS mCherry-LacR-Fok1 cells were treated with the indicated siRNA and subsequently transfected with a GFP-SHLD2 construct. 24 h post-transfection DNA damage was induced using Shield-1 and 4-OHT. The cells were then fixed and analyzed for the intensity of the GFP-SHLD2 signal at mCherry-LacR-Fok1 focus. Shown is the quantification of the GFP-SHLD2 signal at the Fok1 focus. Data are represented as a box-and-whisker plot where the whiskers represent the 10-90 percentile. At least 75 cells were counted per condition.293T cells were transfected with Flag-REV7 and GFP-SHLD2 expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP, REV7 and p-Chk1 antibodies.U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).CH12F3-2 cells stably expressing the indicated shRNAs were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).U2OS cells were transfected with the indicated siRNAs and then treated with CldU, IdU and NCS 48h post-transfection as indicated. The slides were stained, dehydrated, mounted and visualized and shown is the quantification of CldU/IdU tract length in order to visualize DNA end-resection (Right). At least 500 DNA tracks were measured per condition.


Figure 5Genes significantly enriched or dropped out after a 14 - day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated . Ranking was determined based on the Log2 fold score ( Left ) . The top ten Doxorubicin sensitizers are indicated on the right with their respective fold change ( Fc ) in Log2 . 293T cells were transfected with Flag - REV7 and GFP - SHLD1 ( Left ) or Flag - SHLD2 and GFP - SHLD1 ( Right ) expression vectors as indicated . 24h post - transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU - 60019 for 1 h prior to irradiation . 1h post - irradiation ( 10 Gy ) nuclear extracts were prepared and REV7 or SHLD2 complexes were immunoprecipitated using anti - Flag ( M2 ) Resin and then analyzed by immunoblotting using GFP and REV7 antibodies . U2OS EJ5 - GFP cells were transfected with either siCTRL , siSHLD1 # 1 or siSHLD1 # 2 . At 24hr post - transfection , cells were transfected with the I - SceI expression plasmid , and the GFP + population was analyzed 48 hr post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition ( siCTRL ) . CH12F3 - 2 cells stably expressing either shCTRL , shSHLD1 # 1 or shSHLD1 # 2 were stimulated with a cocktail of cytokines ( CIT ) to induce class switching to IgA . The percentage of IgA + cells was monitored 24 and 48h post - stimulation by staining with an anti - IgA antibody followed by flow cytometry analysis . U2OS DR - GFP cells were transfected with the indicated siRNAs . At 24hr post - transfection , cells were transfected with the I - SceI expression plasmid , and the GFP + population was analyzed 48 hr post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition ( siCTRL ) .

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Figure 5Genes significantly enriched or dropped out after a 14-day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated. Ranking was determined based on the Log2 fold score (Left). The top ten Doxorubicin sensitizers are indicated on the right with their respective fold change (Fc) in Log2.293T cells were transfected with Flag-REV7 and GFP-SHLD1 (Left) or Flag-SHLD2 and GFP-SHLD1 (Right) expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 or SHLD2 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP and REV7 antibodies.U2OS EJ5-GFP cells were transfected with either siCTRL, siSHLD1 #1 or siSHLD1 #2. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).CH12F3-2 cells stably expressing either shCTRL, shSHLD1#1 or shSHLD1#2 were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).


Figure 6Survival analysis of low and high expressers of SHLD2 in a cohort of 24 patients affected by Triple Negative Breast cancer ( TNBC ) . Data are represented as Kaplan - Meyer curves with expression classified as low and high . Relapse - free Survival ( Basal Subtype ) of low and high expressers of SHLD2 obtained from the KM - plotter database . Data are represented as Kaplan - Meyer curves with expression classified as low and high .

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Figure 6Survival analysis of low and high expressers of SHLD2 in a cohort of 24 patients affected by Triple Negative Breast cancer (TNBC). Data are represented as Kaplan-Meyer curves with expression classified as low and high.Relapse-free Survival (Basal Subtype) of low and high expressers of SHLD2 obtained from the KM-plotter database. Data are represented as Kaplan-Meyer curves with expression classified as low and high.


Figure 1 ( A ) The transcript level of solo LTR in the wild type and the epcr1 mutant . Error bars are standard deviations of three technical replicates . The experiment was independently performed three times and similar results were obtained . ( B ) The developmental phenotype of the epcr1 and epcr2 single mutants and the epcr1 / 2 double mutant relative to the wild type . Two - week - old seedlings grown on vertical MS medium plates are shown . ( C ) The transcript levels of solo LTR , SDC , AtSN1 , AtGP1 and AtCOPIA28 in the wild type , epcr1 and epcr2 single mutants , epcr1 / 2 double mutant , and two individual EPCR1 transgenic lines in the epcr1 / 2 double mutant background . Error bars are standard deviations of three biological replicates .

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Figure 1(A) The transcript level of solo LTR in the wild type and the epcr1 mutant. Error bars are standard deviations of three technical replicates. The experiment was independently performed three times and similar results were obtained.(B) The developmental phenotype of the epcr1 and epcr2 single mutants and the epcr1/2 double mutant relative to the wild type. Two-week-old seedlings grown on vertical MS medium plates are shown.(C) The transcript levels of solo LTR, SDC, AtSN1, AtGP1 and AtCOPIA28 in the wild type, epcr1 and epcr2 single mutants, epcr1/2 double mutant, and two individual EPCR1 transgenic lines in the epcr1/2 double mutant background. Error bars are standard deviations of three biological replicates.


Figure 2 ( A ) The interaction between EPCR1 and ARID2 or ARID3 . Arabidopsis plants carrying EPCR1 - Flag and ARID2 - Myc or ARID3 - Myc transgenes were used for co - IP . ( B ) The interaction between TRB1 and ARID2 . Arabidopsis plants carrying TRB1 - Flag and / or ARID2 - Myc transgenes were used for co - IP . ( C ) The interaction between TRB1 and EPCR1 . Arabidopsis plants carrying TRB1 - Flag and / or EPCR1 - Myc transgenes were used for co - IP . ( E ) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull - down assay . ( F ) The interaction between EPCR1 , PWWP2 , ARID2 , and TRB1 was detected by an HIS pull - down assay . The full - length PWWP2 , ARID2 , and TRB1 , and the EPCR1 - N terminal ( 1 - 500 aa ) were used for the interaction assay .

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Figure 2(A) The interaction between EPCR1 and ARID2 or ARID3. Arabidopsis plants carrying EPCR1-Flag and ARID2-Myc or ARID3-Myc transgenes were used for co-IP.(B) The interaction between TRB1 and ARID2. Arabidopsis plants carrying TRB1-Flag and/or ARID2-Myc transgenes were used for co-IP.(C) The interaction between TRB1 and EPCR1. Arabidopsis plants carrying TRB1-Flag and/or EPCR1-Myc transgenes were used for co-IP.(E) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull-down assay.(F) The interaction between EPCR1, PWWP2, ARID2, and TRB1 was detected by an HIS pull-down assay. The full-length PWWP2, ARID2, and TRB1, and the EPCR1-N terminal (1-500 aa) were used for the interaction assay.


Figure 3 ( A ) The developmental phenotype of the arid2 / 3 / 4 mutant as compared to the wild type . Two - week - old seedlings grown on MS medium are shown . ( B ) The developmental phenotype of the pwwp1 / 2 / 3 + / - mutant as compared to the wild type . One - month - old plants grown in soil are shown . The effect of the arid2 , arid3 , and arid4 mutations on the silencing of solo LTR , SDC , and FWA as determined by qPCR . Showing are results of three biological replicates with standard deviations . The effect of the pwwp1 , pwwp2 , and pwwp3 mutations on the silencing of solo LTR , SDC , and FWA as determined by qPCR . Showing are results of three biological replicates with standard deviations . ( E ) The effect of the trb1 mutation and the TRB2 knockdown ( TRB2 - KD ) on the silencing of solo LTR , as determined by qPCR ( right panel ) . The transcript level of TRB2 was evaluated to confirm the TRB2 knockdown ( left panel ) . Error bars represent standard deviations of three biological replicates .

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Figure 3(A) The developmental phenotype of the arid2/3/4 mutant as compared to the wild type. Two-week-old seedlings grown on MS medium are shown.(B) The developmental phenotype of the pwwp1/2/3+/- mutant as compared to the wild type. One-month-old plants grown in soil are shown.The effect of the arid2, arid3, and arid4 mutations on the silencing of solo LTR, SDC, and FWA as determined by qPCR. Showing are results of three biological replicates with standard deviations.The effect of the pwwp1, pwwp2, and pwwp3 mutations on the silencing of solo LTR, SDC, and FWA as determined by qPCR. Showing are results of three biological replicates with standard deviations.(E) The effect of the trb1 mutation and the TRB2 knockdown (TRB2-KD) on the silencing of solo LTR, as determined by qPCR (right panel). The transcript level of TRB2 was evaluated to confirm the TRB2 knockdown (left panel). Error bars represent standard deviations of three biological replicates.


Figure 4 ( B ) Heat maps showing differentially transcribed genes and TEs in the arid2 / 3 / 4 and epcr1 / 2 mutants relative to the wild type . ( D ) Genome browser snapshots showing the transcript patterns of ROS1 and IBM1 in arid2 / 3 / 4 and epcr1 / 2 relative to the wild type . ( E ) Effects of arid2 / 3 / 4 epcr1 / 2 on the transcript levels of ROS1 and IBM1 - L as determined by qPCR . The expression of ROS1 and the longer version of IBM1 ( IBM1 - L ) was evaluated by qPCR . ACT7 was amplified as an internal control . Bars represent SD from two independent experiments , each with three technical replications .

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Figure 4(B) Heat maps showing differentially transcribed genes and TEs in the arid2/3/4 and epcr1/2 mutants relative to the wild type.(D) Genome browser snapshots showing the transcript patterns of ROS1 and IBM1 in arid2/3/4 and epcr1/2 relative to the wild type.(E) Effects of arid2/3/4 epcr1/2 on the transcript levels of ROS1 and IBM1-L as determined by qPCR. The expression of ROS1 and the longer version of IBM1 (IBM1-L) was evaluated by qPCR. ACT7 was amplified as an internal control. Bars represent SD from two independent experiments, each with three technical replications.


Figure 5 ( A ) The interaction of EPCR1 with HAM1 and HAM2 . EPCR1 - Flag transgenic plants were crossed to HAM1 - Myc and HAM2 - Myc transgenic plants . The progeny were used to evaluate the interaction between EPCR1 with HAM1 and HAM2 by co - IP . ( B ) The interaction of ARID2 , ARID3 , and ARID4 with HAM1 and HAM2 . ARID2 - Myc , ARID3 - Myc , and ARID4 - Myc transgenic plants were crossed to HAM1 - Flag and HAM2 - Flag transgenic plants and their progeny were used for co - IP . ( C ) The interaction of TRB1 with HAM1 and HAM2 . TRB1 - Flag transgenic plants were crossed to HAM1 - Myc and HAM2 - Myc transgenic plants and their progeny were used for co - IP . ( D ) The interaction of HDA9 with ARID2 , ARID3 , and ARID4 . ARID2 - Myc , ARID3 - Myc , and ARID4 - Myc transgenic plants were crossed to HDA9 - Flag transgenic plants and their progeny were used for co - IP . ( E ) The interaction of HDA6 with EPCR1 and ARID2 . EPCR1 - Flag and ARID2 - Flag transgenic plants were crossed to HDA6 - Myc transgenic plants and their progeny were used for co - IP .

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Figure 5(A) The interaction of EPCR1 with HAM1 and HAM2. EPCR1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants. The progeny were used to evaluate the interaction between EPCR1 with HAM1 and HAM2 by co-IP.(B) The interaction of ARID2, ARID3, and ARID4 with HAM1 and HAM2. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HAM1-Flag and HAM2-Flag transgenic plants and their progeny were used for co-IP.(C) The interaction of TRB1 with HAM1 and HAM2. TRB1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants and their progeny were used for co-IP.(D) The interaction of HDA9 with ARID2, ARID3, and ARID4. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HDA9-Flag transgenic plants and their progeny were used for co-IP.(E) The interaction of HDA6 with EPCR1 and ARID2. EPCR1-Flag and ARID2-Flag transgenic plants were crossed to HDA6-Myc transgenic plants and their progeny were used for co-IP.


Figure 6 ( A ) Overlap among H4K5 hyperacetylated regions in the arid2 / 3 / 4 and epcr1 / 2 mutants relative to the wild type . The overlap is significant ( P = 4 . 410 - 78 ) as determined by hypergeometric test . ( B ) Metaplot of histone H4K5 acetylation levels of TEs in wild type , arid2 / 3 / 4 , and epcr1 / 2 plants . Histone H4K5 acetylation levels are represented by normalized reads that obtained in the H4K5Ac ChIP - seq analysis . TSS represents transcription start site and TTS represents transcription termination site . ( C ) Boxplot of histone H4K5 acetylation levels of TEs in wild type , arid2 / 3 / 4 , and epcr1 / 2 plants . Asterisks indicate that H4K5Ac levels is significantly higher in the arid2 / 3 / 4 ( P = 2 . 4210 - 13 ) and epcr1 / 2 ( P = 1 . 5210 - 8 ) mutants than in the wild type as determined by Welch ' s Two - Sample t - test . Horizontal lines represent the median , and the bottom and top of the box represent the 25th and 75th percentile . The whiskers represent data range within 1 . 5 × of the interquantile range . ( D ) The nuclei with condensed , intermediate , or decondensed heterochromatin status , as marked by DAPI staining and H3K27me signals . ( E ) Percentages of nuclei with condensed , intermediate , or decondensed heterochromatin signals in the wild type and the arid2 / 3 / 4 mutant . n = 113 . ( F ) Abundance of Pol IV - dependent siRNAs in the epcr1 / 2 , arid2 / 3 / 4 , nrpd1 , nrpe1 , and hda6 mutants relative to the wild type [ ( Mut - WT ) / ( Mut + WT ) ] from chromosome 3 of Arabidopsis . ( H ) Boxplot showing levels of Pol IV - dependent siRNAs that are up - regulated in the hda6 mutant . Asterisks indicate that siRNA levels in the mutants are significantly ( P → 0 ) up - regulated compared to the wild type as determined by paired student t - test . Horizontal lines represent the median , and the bottom and top of the box represent the 25th and 75th percentile . The whiskers represent data range within 1 . 5 × of the interquantile range .

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Figure 6(A) Overlap among H4K5 hyperacetylated regions in the arid2/3/4 and epcr1/2 mutants relative to the wild type. The overlap is significant (P=4.410-78) as determined by hypergeometric test. (B) Metaplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Histone H4K5 acetylation levels are represented by normalized reads that obtained in the H4K5Ac ChIP-seq analysis. TSS represents transcription start site and TTS represents transcription termination site. (C) Boxplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Asterisks indicate that H4K5Ac levels is significantly higher in the arid2/3/4 (P=2.4210-13) and epcr1/2 (P=1.5210-8) mutants than in the wild type as determined by Welch's Two-Sample t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.(D) The nuclei with condensed, intermediate, or decondensed heterochromatin status, as marked by DAPI staining and H3K27me signals. (E) Percentages of nuclei with condensed, intermediate, or decondensed heterochromatin signals in the wild type and the arid2/3/4 mutant. n=113.(F) Abundance of Pol IV-dependent siRNAs in the epcr1/2, arid2/3/4, nrpd1, nrpe1, and hda6 mutants relative to the wild type [(Mut-WT)/(Mut+WT)] from chromosome 3 of Arabidopsis.(H) Boxplot showing levels of Pol IV-dependent siRNAs that are up-regulated in the hda6 mutant. Asterisks indicate that siRNA levels in the mutants are significantly (P→0) up-regulated compared to the wild type as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.


Figure 7 ( A ) Metaplot of CG , CHG , and CHH methylation of genes and TEs in the genomes of wild type , epcr1 / 2 , and arid2 / 3 / 4 plants . H indicates C , A , or T . ( B ) Metaplot of CG , CHG , and CHH methylation on chromosome 3 of Arabidopsis in wild type , epcr1 / 2 , and arid2 / 3 / 4 plants . ( D ) Boxplot of DNA methylation in the wild type , nrpe1 , epcr1 / 2 , and arid2 / 3 / 4 mutants . The hypo - DMRs identified in the nrpe1 mutant was defined as RdDM target loci . Asterisks indicate that DNA methylation is significantly ( P < 0 . 001 ) decreased in the mutants as determined by paired student t - test . Horizontal lines represent the median , and the bottom and top of the box represent the 25th and 75th percentile . The whiskers represent data range within 1 . 5 × of the interquantile range . ( E ) Heat maps of CG , CHG , and CHH methylation at RdDM target loci . The hypo - DMRs identified in the nrpe1 mutant were defined as RdDM target loci and analyzed in the wild type , nrpe1 , eprc1 / 2 , and arid2 / 3 / 4 mutants . Black and light yellow indicate low methylation and high methylation , respectively .

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

Figure 7(A) Metaplot of CG, CHG, and CHH methylation of genes and TEs in the genomes of wild type, epcr1/2, and arid2/3/4 plants. H indicates C, A, or T.(B) Metaplot of CG, CHG, and CHH methylation on chromosome 3 of Arabidopsis in wild type, epcr1/2, and arid2/3/4 plants.(D) Boxplot of DNA methylation in the wild type, nrpe1, epcr1/2, and arid2/3/4 mutants. The hypo-DMRs identified in the nrpe1 mutant was defined as RdDM target loci. Asterisks indicate that DNA methylation is significantly (P<0.001) decreased in the mutants as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.(E) Heat maps of CG, CHG, and CHH methylation at RdDM target loci. The hypo-DMRs identified in the nrpe1 mutant were defined as RdDM target loci and analyzed in the wild type, nrpe1, eprc1/2, and arid2/3/4 mutants. Black and light yellow indicate low methylation and high methylation, respectively.


Figure 8 ( A ) Scatter plots showing the effect of the epcr1 / 2 and arid2 / 3 / 4 mutations on CG , CHG , and CHH methylation at the co - upregulated TEs and genes in the epcr1 / 2 and arid2 / 3 / 4 mutants . TE methylation and gene DNA methylation refer , respectively , to methylation of the TE body and methylation of the 1 - kb - gene - promoter region . ( B ) Box plots of CG , CHG , and CHH methylation in the wild type , arid2 / 3 / 4 , and epcr1 / 2 mutants at the co - upregulated TEs and genes in the arid2 / 3 / 4 and epcr1 / 2 mutants . Asterisks indicate statistical significance as determined by paired student t test . CHG methylation of co - upregulated TEs is significantly increased in arid2 / 3 / 4 ( P = 1 . 2610 - 4 ) and epcr1 / 2 ( P = 6 . 310 - 9 ) ; CHH methylation of co - upregulated genes is significantly decreased in arid2 / 3 / 4 ( P = 3 . 510 - 6 ) and epcr1 / 2 ( P = 6 . 410 - 7 ) . Horizontal lines represent the median , the bottom and top of the box represent the 25th and 75th percentile . The whiskers represent data range within 1 . 5 × of the interquantile range . ( C ) Overexpression of DRD1 fails to restore the silencing of EPCR1 / 2 target loci in the epcr1 / 2 mutant background .

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

Figure 8(A) Scatter plots showing the effect of the epcr1/2 and arid2/3/4 mutations on CG, CHG, and CHH methylation at the co-upregulated TEs and genes in the epcr1/2 and arid2/3/4 mutants. TE methylation and gene DNA methylation refer, respectively, to methylation of the TE body and methylation of the 1-kb-gene-promoter region.(B) Box plots of CG, CHG, and CHH methylation in the wild type, arid2/3/4, and epcr1/2 mutants at the co-upregulated TEs and genes in the arid2/3/4 and epcr1/2 mutants. Asterisks indicate statistical significance as determined by paired student t test. CHG methylation of co-upregulated TEs is significantly increased in arid2/3/4 (P=1.2610-4) and epcr1/2 (P=6.310-9); CHH methylation of co-upregulated genes is significantly decreased in arid2/3/4 (P=3.510-6) and epcr1/2 (P=6.410-7). Horizontal lines represent the median, the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.(C) Overexpression of DRD1 fails to restore the silencing of EPCR1/2 target loci in the epcr1/2 mutant background.


Figure 4 ( F - G ) Thermal shift assay traces of human Teneurin4WT ( F ) and human Teneurin4Mut ( G ) in the presence of physiological calcium concentrations ( + Ca2 + ; black ) , low calcium concentration ( + / - Ca2 + ; blue and red , respectively ) and no calcium ( - Ca2 + ; grey ) .

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

Figure 4(F-G) Thermal shift assay traces of human Teneurin4WT (F) and human Teneurin4Mut (G) in the presence of physiological calcium concentrations (+ Ca2+; black), low calcium concentration (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey).


Figure 5 ( A ) Dimensionless Kratky plots for Teneurin4WT ( left ) and Teneurin4Mut ( right ) in physiological calcium concentrations ( + Ca2 + ; black ) , low calcium concentrations ( + / - Ca2 + ; blue and red , respectively ) and no calcium ( - Ca2 + ; grey ) . I ( 0 ) and Rg were obtained from Guinier analysis ( Fig EV5A , B ) . Crosshairs indicate the maximum position for a fully folded globular protein . ( B ) Pair - distance distribution plots for Teneurin4 proteins colored as in A .

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

Figure 5(A) Dimensionless Kratky plots for Teneurin4WT (left) and Teneurin4Mut (right) in physiological calcium concentrations (+ Ca2+; black), low calcium concentrations (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey). I(0) and Rg were obtained from Guinier analysis (Fig EV5A, B). Crosshairs indicate the maximum position for a fully folded globular protein.(B) Pair-distance distribution plots for Teneurin4 proteins colored as in A.


Figure 6 ( A ) Representative images of HEK293 - T cells transfected with GFP ( top ) , GFP - Teneurin4WT ( GFP - Ten4WT ; middle ) or GFP - Teneurin4Mut ( GFP - Ten4Mut ; bottom ) for 24h and stained with Alexa 568 Phalloidin ( magenta ) and Dapi ( blue ) . Images are maximum intensity projections of 14 - 16 stacks . Scale bar , 10 µm . ( B ) Zoom shows the regions indicated in A . White arrows indicate the tip of the filopodium from the transfected cell . Scale bar , 2 µm . ( ( C ) Average number of filopodia per cell in cells transfected with GFP , GFP - Teneurin4WT ( Ten4 WT ; red ) and GFP - Teneurin4Mut ( Ten4Mut ; blue ) . ( D ) Fraction of filopodia enriched with GFP per cell in GFP - , GFP - Teneurin4WT ( Ten4WT ; blue ) - and GFP - Teneurin4Mut ( Ten4Mut ; red ) - transfected cells . (

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

Figure 6(A) Representative images of HEK293-T cells transfected with GFP (top), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP- Teneurin4Mut (GFP-Ten4Mut; bottom) for 24h and stained with Alexa 568 Phalloidin (magenta) and Dapi (blue). Images are maximum intensity projections of 14-16 stacks. Scale bar, 10 µm. (B) Zoom shows the regions indicated in A. White arrows indicate the tip of the filopodium from the transfected cell. Scale bar, 2 µm. ((C) Average number of filopodia per cell in cells transfected with GFP, GFP-Teneurin4WT (Ten4 WT; red) and GFP- Teneurin4Mut (Ten4Mut; blue). (D) Fraction of filopodia enriched with GFP per cell in GFP-, GFP-Teneurin4WT (Ten4WT; blue)- and GFP-Teneurin4Mut (Ten4Mut; red)-transfected cells. (


Figure 7 ( A ) Representative images of non - adherent K562 cells electroporated with GFP ( left ) , GFP - Teneurin4WT ( GFP - Ten4WT ; middle ) or GFP - Teneurin4Mut ( GFP - Ten4Mut ; right ) . Zoomed images show the formation of cellular clusters in cells expressing GFP - Ten4WT and GFP - Ten4Mut , but not in GFP - expressing cells . Scale bar top panel , 50 µm . Scale bar bottom panel , 20 µm . ( C ) Fraction of cell clusters detected per image in GFP - ( black ) , GFP - Teneurin4WT ( blue ) - and GFP - Teneurin4Mut ( red ) - electroporated cells .

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

Figure 7(A) Representative images of non-adherent K562 cells electroporated with GFP (left), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP-Teneurin4Mut (GFP-Ten4Mut; right). Zoomed images show the formation of cellular clusters in cells expressing GFP-Ten4WT and GFP-Ten4Mut, but not in GFP-expressing cells. Scale bar top panel, 50 µm. Scale bar bottom panel, 20 µm.(C) Fraction of cell clusters detected per image in GFP- (black), GFP-Teneurin4WT (blue)- and GFP-Teneurin4Mut (red)-electroporated cells.


f1 ( a ) TbMCU - GFP co - localization with MitoTracker ( MT ) in mitochondria of PCF trypanosomes as detected by immunofluorescence analyses with antibodies against GFP ( Pearson ' s correlation coefficient ( PCC ) : 0 . 912 ) . ( b , c ) TbMCU co - localization with MitoTracker in the mitochondria of PCF ( b ) or BSF ( c ) trypanosomes , as detected by immunofluorescence analyses with antibodies against TbMCU . PCC : 0 . 930 and 0 . 920 , respectively . Merge images show the co - localization in yellow . DIC , differential interference contrast microscopy . Scale bar , 10 μm ( a - c ) . ( d , e ) Growth of BSF ( d ) and PCF ( e ) trypanosomes in the absence ( black lines , − Tet ) or presence ( red lines , + Tet ) of 1 μg ml − 1 tetracycline for the indicated number of days . Values are mean ± s . d . ( n = 3 ) . ( f , g ) Northern blot analyses of TbMCU RNAi of BSF ( f ) and PCF ( g ) trypanosomes grown in the absence ( 0 ) and presence ( 2 - 8 ; 2 - 10 ) of tetracycline ( top ) . Tubulin is shown as a loading control ( bottom ) . Markers are shown on the right . The transcript of TbMCU , including the 5 ′ - and 3 ′ - UTR was ~ 1 . 5 kb in length . ( h , i ) Western blot analyses of TbMCU RNAi of BSF ( h ) and PCF ( i ) trypanosomes grown in the absence ( 0 ) or presence ( 2 - 8 ; 2 - 10 ) of tetracycline . Total lysates ( 30 μg ) were subjected to 10 % SDS - polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against TbMCU ( top ) . One band of ~ 30 kDa was detected in T . brucei homogenates . Membranes were stripped and re - incubated with antibody against the voltage - dependent anion channel ( TbVDAC ) as a loading control ( bottom ) .

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

f1(a) TbMCU-GFP co-localization with MitoTracker (MT) in mitochondria of PCF trypanosomes as detected by immunofluorescence analyses with antibodies against GFP (Pearson's correlation coefficient (PCC): 0.912). (b,c) TbMCU co-localization with MitoTracker in the mitochondria of PCF (b) or BSF (c) trypanosomes, as detected by immunofluorescence analyses with antibodies against TbMCU. PCC: 0.930 and 0.920, respectively. Merge images show the co-localization in yellow. DIC, differential interference contrast microscopy. Scale bar, 10 μm (a-c).(d,e) Growth of BSF (d) and PCF (e) trypanosomes in the absence (black lines, −Tet) or presence (red lines, +Tet) of 1 μg ml−1 tetracycline for the indicated number of days. Values are mean±s.d. (n=3).(f,g) Northern blot analyses of TbMCU RNAi of BSF (f) and PCF (g) trypanosomes grown in the absence (0) and presence (2-8; 2-10) of tetracycline (top). Tubulin is shown as a loading control (bottom). Markers are shown on the right. The transcript of TbMCU, including the 5′- and 3′-UTR was ~1.5 kb in length.(h,i) Western blot analyses of TbMCU RNAi of BSF (h) and PCF (i) trypanosomes grown in the absence (0) or presence (2-8; 2-10) of tetracycline. Total lysates (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against TbMCU (top). One band of ~30 kDa was detected in T. brucei homogenates. Membranes were stripped and re-incubated with antibody against the voltage-dependent anion channel (TbVDAC) as a loading control (bottom).


f2 ( a ) The reaction buffer contained 2 mM succinate and 1 μM Calcium Green 5 - N . After several pulses of Ca2 + ( 8 μM final concentration ) cells ( 5 × 107 trypanosomes grown in the absence ( black tracing ) or presence ( green tracing ) of tetracycline ) were added to the reaction medium ( 2 . 45 ml ) and the reaction was started adding 50 μM digitonin . Ruthenium red ( RR , 40 μM , yellow and red tracing ) , and FCCP ( 5 μM ) were added where indicated . A decrease in fluorescence indicates decreasing medium Ca2 + or increasing vesicular Ca2 + . ( b ) Relative Ca2 + uptake at 700 s as compared with that of control trypanosomes grown in the absence of tetracycline considered as 1 . 0 ( − Tet ) ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( c ) Similar conditions as in a except that further CaCl2 additions ( 8 μM each time ) were added to − Tet trypanosomes to show the high mitochondrial capacity to take up Ca2 + . EGTA ( 8 μM ) and CaCl2 ( Ca2 + ) were added where indicated to show the presence of medium Ca2 + . ( d ) Relative initial rate of Ca2 + uptake by + Tet as compared with − Tet trypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( e ) Representative traces of Ca2 + uptake kinetics in digitonin - permeabilized TbMCU BSF trypanosomes cultured in the absence ( − Tet ) or presence ( + Tet ) of tetracycline . The reactions were started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate . Ca2 + uptake was monitored over time using 1 μM Calcium Green - 5N . Ruthenium red ( RR , 40 μM , yellow and red tracings ) , oligomycin ( Oligo , 2 . 5 μg ml − 1 ) , EGTA ( 0 . 4 μM ) and CaCl2 ( pulses of 0 . 4 μM ) were added where indicated . ( f ) Relative Ca2 + uptake at 200 s as compared with that of control BSF trypanosomes grown in the absence of tetracycline considered as 1 ( − Tet ) ( means ± s . d . , n = 3 , * ( + Tet ± RR ) , * * ( − Tet ± RR ) , * * * ( − Tet / + Tet ) , P0 . 001 , Student ' s t - test ) .

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

f2(a) The reaction buffer contained 2 mM succinate and 1 μM Calcium Green 5-N. After several pulses of Ca2+ (8 μM final concentration) cells (5 × 107 trypanosomes grown in the absence (black tracing) or presence (green tracing) of tetracycline) were added to the reaction medium (2.45 ml) and the reaction was started adding 50 μM digitonin. Ruthenium red (RR, 40 μM, yellow and red tracing), and FCCP (5 μM) were added where indicated. A decrease in fluorescence indicates decreasing medium Ca2+ or increasing vesicular Ca2+. (b) Relative Ca2+ uptake at 700 s as compared with that of control trypanosomes grown in the absence of tetracycline considered as 1.0 (−Tet) (means±s.d., n=3, **P0.001, Student's t-test).(c) Similar conditions as in a except that further CaCl2 additions (8 μM each time) were added to −Tet trypanosomes to show the high mitochondrial capacity to take up Ca2+. EGTA (8 μM) and CaCl2 (Ca2+) were added where indicated to show the presence of medium Ca2+. (d) Relative initial rate of Ca2+ uptake by +Tet as compared with −Tet trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).(e) Representative traces of Ca2+ uptake kinetics in digitonin-permeabilized TbMCU BSF trypanosomes cultured in the absence (−Tet) or presence (+Tet) of tetracycline. The reactions were started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate. Ca2+ uptake was monitored over time using 1 μM Calcium Green-5N. Ruthenium red (RR, 40 μM, yellow and red tracings), oligomycin (Oligo, 2.5 μg ml−1), EGTA (0.4 μM) and CaCl2 (pulses of 0.4 μM) were added where indicated. (f) Relative Ca2+ uptake at 200 s as compared with that of control BSF trypanosomes grown in the absence of tetracycline considered as 1 (−Tet) (means±s.d., n=3, *(+Tet±RR), **(−Tet±RR), ***(−Tet/+Tet) , P0.001, Student's t-test).


f3 ( a ) PCFtrypanosomes ( 5 × 107 cells ) were added to the reaction buffer ( 2 . 45 ml ) containing 2 mM succinate , and 5 μM safranine , and the reaction was started with digitonin ( 50 μM ) . ADP ( 10 μM ) , oligomycin ( Oligo , 2 μg ml − 1 ) , CaCl2 ( 40 μM ) , EGTA ( 200 μM ) and FCCP ( 10 μM ) were added where indicated . ( b ) Changes in safranine fluorescence after addition of ADP or Ca2 + to − Tet and + Tet PCFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) . ( c ) BSFtrypanosomes ( 2 × 108 cells ) were added to the reaction buffer ( 2 . 45 ml ) containing EGTA ( 20 μM ) , ATP ( 1 mM ) , sodium orthovanadate ( 500 μM ) and 12 . 5 μM safranine . The reaction was started with digitonin ( 40 μM ) . CaCl2 ( 50 μM ) , EGTA ( 200 μM ) and FCCP ( 10 μM ) were added where indicated . ( d , e ) Changes in safranine fluorescence after addition of Ca2 + ( d ) or FCCP ( e ) to − Tet and + Tet BSFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) .

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

f3(a) PCFtrypanosomes (5 × 107 cells) were added to the reaction buffer (2.45 ml) containing 2 mM succinate, and 5 μM safranine, and the reaction was started with digitonin (50 μM). ADP (10 μM), oligomycin (Oligo, 2 μg ml−1), CaCl2 (40 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (b) Changes in safranine fluorescence after addition of ADP or Ca2+ to −Tet and +Tet PCFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).(c) BSFtrypanosomes (2 × 108 cells) were added to the reaction buffer (2.45 ml) containing EGTA (20 μM), ATP (1 mM), sodium orthovanadate (500 μM) and 12.5 μM safranine. The reaction was started with digitonin (40 μM). CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (d,e) Changes in safranine fluorescence after addition of Ca2+ (d) or FCCP (e) to −Tet and +Tet BSFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).


f4 ( a ) Comparison of AMP / ATP ratios between PCF trypanosomes grown in the absence ( − Tet ) or presence ( + Tet ) of tetracycline to induce RNAi , and with or without glucose ( Gl ) , expressed as fold increase ( means ± s . d . , n = 3 ; * P0 . 05 ; * * P0 . 01 , Student ' s t - test ) . ( b ) Comparison of AMP / ATP ratios between ( − Tet ) and ( + Tet ) PCF trypanosomes grown in a glucose - depleted medium ( SDM - 80 ) containing 5 . 2 mM L - proline for 2 days after 2 - day growth in SDM - 79 ± tetracycline , expressed as fold increase ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) . ( c ) Fluorescence microscopic images of TbATG8 . 2 in PCF trypanosomes cultured in SDM - 79 in the absence ( WT ) or presence ( RNAi ) of tetracycline to induce RNAi ; scale bars , 10 μm . ( d ) Quantification of autophagosome formation in RNAi PCF trypanosomes grown in SDM - 79 or SDM - 80 medium in the presence ( + Tet ) or absence ( − Tet ) of tetracycline . Over 500 cells from three experiments with 20 random fields / experiment were analysed ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( e ) Western blot analysis of ATG8 . 2 or tubulin in PCFtrypanosomes cultured in SDM - 79 or SDM - 80 in the absence ( − Tet ) or presence ( + Tet ) of tetracycline to induce RNAi . Cell lysates ( 30 μg ) were fractionated in 12 % urea - SDS - PAGE gel . Immunoblots were labelled with anti - ATG8 . 2 or anti - tubulin antibody . ( f ) Quantification of ATG8 . 2 - II / ( ATG8 . 2 - II + ATG8 . 2 - I ) presented as mean ratios ± s . d . ( n = 3 , * * P0 . 01 , Student ' s t - test ) . ( g ) Groups of five mice were infected with WT T . brucei ( black line ) or trypanosomes transfected with the construct for RNAi of TbMCU ( red line ) . Doxycycline ( green line ; 200 μg ml − 1 ) was given in the drinking water throughout a 30 - day period . ( h ) Parasitemia levels in surviving mice were monitored at 2 - day intervals after infection . The results are means ± s . d . ( n = 2 ) .

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

f4(a) Comparison of AMP/ATP ratios between PCF trypanosomes grown in the absence (−Tet) or presence (+Tet) of tetracycline to induce RNAi, and with or without glucose (Gl), expressed as fold increase (means±s.d., n=3; *P0.05; **P0.01, Student's t-test).(b) Comparison of AMP/ATP ratios between (−Tet) and (+Tet) PCF trypanosomes grown in a glucose-depleted medium (SDM-80) containing 5.2 mM L-proline for 2 days after 2-day growth in SDM-79±tetracycline, expressed as fold increase (means±s.d., n=3, **P0.01, Student's t-test).(c) Fluorescence microscopic images of TbATG8.2 in PCF trypanosomes cultured in SDM-79 in the absence (WT) or presence (RNAi) of tetracycline to induce RNAi; scale bars, 10 μm. (d) Quantification of autophagosome formation in RNAi PCF trypanosomes grown in SDM-79 or SDM-80 medium in the presence (+Tet) or absence (−Tet) of tetracycline. Over 500 cells from three experiments with 20 random fields/experiment were analysed (means±s.d., n=3, **P0.001, Student's t-test).(e) Western blot analysis of ATG8.2 or tubulin in PCFtrypanosomes cultured in SDM-79 or SDM-80 in the absence (−Tet) or presence (+ Tet) of tetracycline to induce RNAi. Cell lysates (30 μg) were fractionated in 12% urea-SDS-PAGE gel. Immunoblots were labelled with anti-ATG8.2 or anti-tubulin antibody. (f) Quantification of ATG8.2-II/(ATG8.2-II+ATG8.2-I) presented as mean ratios±s.d. (n=3, **P0.01, Student's t-test).(g) Groups of five mice were infected with WT T. brucei (black line) or trypanosomes transfected with the construct for RNAi of TbMCU (red line). Doxycycline (green line; 200 μg ml−1) was given in the drinking water throughout a 30-day period.(h) Parasitemia levels in surviving mice were monitored at 2-day intervals after infection. The results are means±s.d. (n=2).


f5 ( a ) Growth of TbMCU - KO BSF in the absence ( black line ) or presence ( red line ) of 1 μg ml − 1 tetracycline for the indicated number of days . Addition of 10 mM threonine to the medium ( green line ) partially rescued the mutant BSF trypanosomes but had no effect on + Tet cells ( yellow line ; means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( b ) Southern blot analysis of the BSF conditional KOs . Genomic DNA from the T . brucei SM parental strain ( + / + ) , single - ( + / − ) or double - allele ( − / − ) KO cells . ( c ) Northern blot analysis of wild - type ( WT ) and TbMCU - KO BSF cultured in the presence ( + Tet ) or absence ( − Tet ) of tetracycline for 2 days . Tubulin is shown as a loading control ( bottom panel ) . ( d ) Western blot analysis of total cell lysates . Proteins from BSF trypanosomes cultured in the presence ( + Tet ) or absence ( − Tet ) of tetracycline were detected using anti - HA antibody . TbVDAC was used as a loading control . ( e ) Immunofluorescence analysis of control ( upper panels ) and TbMCU - KO ( lower panels ) trypanosomes . TbMCU co - localized with MitoTracker ( MT ) in the control parasites ( + Tet ; PCC = 0 . 808 ) . Scale bar , 10 μm . ( f ) Conditional TbMCU - KO BSF trypanosomes were grown in the presence ( + Tet ) or absence ( − Tet ) of tetracycline for 2 days . BSF trypanosomes ( 2 × 108 cells ) were incubated as in Fig . 2e . Multiple pulses of CaCl2 ( arrowheads ) were added to a final concentration of 20 μM . A representative trace ( red or black ) of Ca2 + uptake from one of three independent experiments is shown . ( g ) Representative traces of mitochondrial membrane potential of digitonin - permeabilized BSF trypanosomes grown in the presence ( + Tet ) or absence ( − Tet ) of tetracycline . The reactions were incubated as in Fig . 3c . CaCl2 ( 50 μM ) , EGTA ( 200 μM ) and FCCP ( 10 μM ) were added where indicated . ( h , i ) Representative traces of mitochondrial membrane potential of digitonin - permeabilized BSFtrypanosomes grown in the presence ( + Tet ) or absence ( − Tet ) of tetracycline . The reactions were incubated as in Fig . 3c . CaCl2 25 , 50 and 100 μM CaCl2 ) or FCCP ( i ) to + Tet and − Tet BSFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) .

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

f5(a) Growth of TbMCU-KO BSF in the absence (black line) or presence (red line) of 1 μg ml−1 tetracycline for the indicated number of days. Addition of 10 mM threonine to the medium (green line) partially rescued the mutant BSF trypanosomes but had no effect on +Tet cells (yellow line; means±s.d., n=3, **P0.001, Student's t-test).(b) Southern blot analysis of the BSF conditional KOs. Genomic DNA from the T. brucei SM parental strain (+/+), single- (+/−) or double-allele (−/−) KO cells.(c) Northern blot analysis of wild-type (WT) and TbMCU-KO BSF cultured in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. Tubulin is shown as a loading control (bottom panel).(d) Western blot analysis of total cell lysates. Proteins from BSF trypanosomes cultured in the presence (+Tet) or absence (−Tet) of tetracycline were detected using anti-HA antibody. TbVDAC was used as a loading control.(e) Immunofluorescence analysis of control (upper panels) and TbMCU-KO (lower panels) trypanosomes. TbMCU co-localized with MitoTracker (MT) in the control parasites (+Tet; PCC=0.808). Scale bar, 10 μm.(f) Conditional TbMCU-KO BSF trypanosomes were grown in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. BSF trypanosomes (2 × 108 cells) were incubated as in Fig. 2e. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 20 μM. A representative trace (red or black) of Ca2+ uptake from one of three independent experiments is shown.(g) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSF trypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated.(h,i) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSFtrypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 25, 50 and 100 μM CaCl2) or FCCP (i) to +Tet and −Tet BSFtrypanosomes (means±s.d., n=3, **P0.001, Student's t-test).


f6 ( a ) Rhod - 2 fluorescence in control or overexpressing TbMCU ( TbMCU - OE ) PCF . Scale bar , 10 μm . ( b , c ) Rhod - 2 ( mitochondrial ) ( b ) and Fluo - 4 ( cytosolic ) ( c ) fluorescence in control ( − Tet ) and overexpressing TbMCU ( + Tet ) PCF ( means ± s . d . , n = 3 , * * P0 . 05 , Student ' s t - test ) ; NS , not significant . ( d ) Ca2 + uptake by digitonin - permeabilized PCF overexpressing TbMCU . Trypanosomes were incubated as in Fig . 2c . Multiple pulses of CaCl2 ( arrowheads ) were added to a final concentration of 8 μM . ( e ) Ca2 + uptake in TbMCU - OE PCF ( + Tet ) 200 s after addition of Ca2 + , as compared with uninduced trypanosomes considered as 1 ( − Tet ) ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) . ( f ) Mitochondrial membrane potential of digitonin - permeabilized control ( − Tet ) and TbMCU - OE ( + Tet ) PCF . The reactions were incubated as in Fig . 3a . ( g ) Changes in safranine fluorescence after addition of ADP , Ca2 + or FCCP to - Tet and + Tet PCF trypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( h ) Mitochondrial oxidative stress as measured with MitoSOX Red in control ( C ) and TbMCU - overexpressing ( OE ) PCF permeabilized with 50 μM digitonin in the presence of 0 . 4 mM Ca2 + . Controls were treated with 2 μM antimycin A ( C + AA ) , or were trypanosomes exposed to 0 . 4 mM Ca2 + in the absence of digitonin ( − DIG + Ca2 + ) , and trypanosomes permeabilized with digitonin in the absence of Ca2 + ( C , yellow line ) . ( i ) Quantification of change at 900 s . Results are expressed as mean fluorescence ± s . d . ( n = 3 , * * P0 . 001 , Student ' s t - test ) . ( j ) Growth of trypanosomes in the absence ( control , black lines ) or presence ( TbMCU - OE , red lines ) of 1 μg ml − 1 tetracycline ( means ± s . d . , n = 3 ) . ( k ) Dead cells in representative fields of control ( − Tet ) and TbMCU - OE ( + Tet ) PCF are indicated ( white arrows ) . Scale bar , 10 μm . ( l ) Cell viability on apoptotic challenge of control ( − Tet ) or TbMCU - OE ( + Tet ) PCF trypanosomes with H2O2 and C2 - ceramide . The percentage of cell death was obtained from means ± s . d . by analysing > 60 fields ( including > 400 cells ) ( n = 3 ) . * * P0 . 01 , Student ' s t - test .

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

f6(a) Rhod-2 fluorescence in control or overexpressing TbMCU (TbMCU-OE) PCF. Scale bar, 10 μm. (b,c) Rhod-2 (mitochondrial) (b) and Fluo-4 (cytosolic) (c) fluorescence in control (−Tet) and overexpressing TbMCU (+Tet) PCF (means±s.d., n=3, **P0.05, Student's t-test); NS, not significant.(d) Ca2+ uptake by digitonin-permeabilized PCF overexpressing TbMCU. Trypanosomes were incubated as in Fig. 2c. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 8 μM. (e) Ca2+ uptake in TbMCU-OE PCF (+Tet) 200 s after addition of Ca2+, as compared with uninduced trypanosomes considered as 1 (−Tet) (means±s.d., n=3, **P0.01, Student's t-test).(f) Mitochondrial membrane potential of digitonin-permeabilized control (−Tet) and TbMCU-OE (+Tet) PCF. The reactions were incubated as in Fig. 3a.(g) Changes in safranine fluorescence after addition of ADP, Ca2+ or FCCP to -Tet and +Tet PCF trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).(h) Mitochondrial oxidative stress as measured with MitoSOX Red in control (C) and TbMCU-overexpressing (OE) PCF permeabilized with 50 μM digitonin in the presence of 0.4 mM Ca2+. Controls were treated with 2 μM antimycin A (C+AA), or were trypanosomes exposed to 0.4 mM Ca2+ in the absence of digitonin (−DIG+Ca2+), and trypanosomes permeabilized with digitonin in the absence of Ca2+ (C, yellow line). (i) Quantification of change at 900 s. Results are expressed as mean fluorescence±s.d. (n=3, **P0.001, Student's t-test).(j) Growth of trypanosomes in the absence (control, black lines) or presence (TbMCU-OE, red lines) of 1 μg ml−1 tetracycline (means±s.d., n=3). (k) Dead cells in representative fields of control (−Tet) and TbMCU-OE (+Tet) PCF are indicated (white arrows). Scale bar, 10 μm.(l) Cell viability on apoptotic challenge of control (−Tet) or TbMCU-OE (+Tet) PCF trypanosomes with H2O2 and C2-ceramide. The percentage of cell death was obtained from means±s.d. by analysing >60 fields (including >400 cells) (n=3). **P0.01, Student's t-test.


Figure 1 ( A ) Confocal images of representative eye cryosections immunostained with anti - Lamp1 antibody from 2 - month - old miR - 211 - / - and control mice sacrificed 3h after light on , at 10 AM ( diurnal condition ) . Enlarged boxes highlight Lamp1 - positive structures ( white arrowheads ) in the RPE . Autofluorescence from lipofuscin granules are visible in all cryosections of miR - 211 - / - compared to control littermates . Nuclei were counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer . ( B ) RPE were isolated at 10 AM ( diurnal condition ) from 2 - month - old WT and miR - 211 - / - mice . Representative Western blot analysis of the Lamp1 , Cln5 , Trpml1 , Sqstm1 / p62 , Cathepsin D ( CtsD ) and LC3 proteins from WT and miR - 211 - / - mice . Note a decrease of both proCtsD and its maturation CtsD heavy chain ( hc ) . The plots show the quantification of the indicated proteins normalized to the Gapdh loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 6 mice ) Mann and Whitney test ( miR - 211 - / - vs WT ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 . Data information : ( RPE ) Retinal Pigment Epithelium ( C ) Cathepsin B ( CtsB ) activity in RPE lysates from 2 - month - old miR - 211 - / - and control mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . CtsB was reduced in miR - 211 - / - compared to control mice . Bar graphs represent percentage of CtsB activity ± s . e . m . of independent experiments ( n = 3 mice ) Mann and Whitney test ( miR - 211 - / - vs WT ) , * p ≤ 0 . 05 . Data information : ( RPE ) Retinal Pigment Epithelium ( D ) Representative images of conventional TEM analysis of RPE of both 2 - month - old and 3 - month - old WT and miR - 211 - / - mice . miR - 211 - / - mice show accumulation of phagolysosomes ( red arrows ) in the RPE . Scale bar 2 μm . Enlarged boxes highlight phagolysosome - like structures containing poorly processed POS . Scale bar 1 . 5 μm . Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin - like granules ( blue arrows ) in 3 - month - old miR - 211 - / - compared to control WT mice . Melanosomes are indicated by white asterisks . Scale bar 500 nm ( n = 3 mice ) . ( E ) Graphs showing number of lipofuscin - like granules ( n / 100 μm2 ) from the RPE of 3 - month - old miR - 211 - / - mice as in ( D ) . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( miR - 211 - / - vs WT ) * * * p ≤ 0 . 005 ( n = 6 mice ) . Data information : ( RPE ) Retinal Pigment

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

Figure 1(A) Confocal images of representative eye cryosections immunostained with anti-Lamp1 antibody from 2-month-old miR-211-/- and control mice sacrificed 3h after light on, at 10 AM (diurnal condition). Enlarged boxes highlight Lamp1-positive structures (white arrowheads) in the RPE. Autofluorescence from lipofuscin granules are visible in all cryosections of miR-211-/- compared to control littermates. Nuclei were counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.(B) RPE were isolated at 10 AM (diurnal condition) from 2-month-old WT and miR-211-/- mice. Representative Western blot analysis of the Lamp1, Cln5, Trpml1, Sqstm1/p62, Cathepsin D (CtsD) and LC3 proteins from WT and miR-211-/- mice. Note a decrease of both proCtsD and its maturation CtsD heavy chain (hc). The plots show the quantification of the indicated proteins normalized to the Gapdh loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice) Mann and Whitney test (miR-211-/- vs WT), *p ≤ 0.05, **p ≤ 0.01. Data information: (RPE) Retinal Pigment Epithelium(C) Cathepsin B (CtsB) activity in RPE lysates from 2-month-old miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). CtsB was reduced in miR-211-/- compared to control mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (miR-211-/- vs WT), *p ≤ 0.05. Data information: (RPE) Retinal Pigment Epithelium(D) Representative images of conventional TEM analysis of RPE of both 2-month-old and 3-month-old WT and miR-211-/- mice. miR-211-/- mice show accumulation of phagolysosomes (red arrows) in the RPE. Scale bar 2 μm. Enlarged boxes highlight phagolysosome-like structures containing poorly processed POS. Scale bar 1.5 μm. Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin-like granules (blue arrows) in 3-month-old miR-211-/- compared to control WT mice. Melanosomes are indicated by white asterisks. Scale bar 500 nm (n=3 mice). (E) Graphs showing number of lipofuscin-like granules (n/100 μm2) from the RPE of 3-month-old miR-211-/- mice as in (D). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT) ***p ≤ 0.005 (n=6 mice). Data information: (RPE) Retinal Pigment Epithelium