C | Quantification of intracellular Ca2 + influx The median area under the curve of the Ca2 + influx kinetics was calculated , single dots represent data from individual mice , mean ± SD . Statistical significance was calculated by applying the Mann - Whitney - U test n . s . = not significant , * * p ≤ 0 . 01 .

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C | Quantification of intracellular Ca2+ influx The median area under the curve of the Ca2+ influx kinetics was calculated, single dots represent data from individual mice, mean ±SD. Statistical significance was calculated by applying the Mann-Whitney-U test n. s. = not significant, ** p ≤ 0.01.


B | Analysis of GC B cells in splenic B220 + B cells from wild type ( WT , top ) and IgD - / - ( bottom ) animals after 4 , 7 and 10 days ( left to right ) post immunization . Percentages of follicular B cells ( Fo . B ; CD38 + / CD95 - ) and GC B cells ( CD38 - / CD95 + ) are depicted in the plots . GC B cells ( CD38 - / CD95 + ) were further analyzed for GL - 7 expression , compared to Fo . B cells and quantified .

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B | Analysis of GC B cells in splenic B220+ B cells from wild type (WT, top) and IgD-/- (bottom) animals after 4, 7 and 10 days (left to right) post immunization. Percentages of follicular B cells (Fo.B; CD38+/CD95-) and GC B cells (CD38-/CD95+) are depicted in the plots. GC B cells (CD38-/CD95+) were further analyzed for GL-7 expression, compared to Fo.B cells and quantified.


C | Quantification of Fo . B cells ( top , percentages ) and CD38 - / CD95 + / GL - 7 + GC cells ( percentages and absolute numbers , bottom panels ) in WT ( black ) and IgD - / - ( green ) animals after 4 , 7 , 10 and 14 days post immunization , mean ± SD ( numbers of animals indicated in the top plot ) . Statistical significance was analyzed by a two - tailed unpaired t - test .

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C | Quantification of Fo.B cells (top, percentages) and CD38-/CD95+/GL-7+ GC cells (percentages and absolute numbers, bottom panels) in WT (black) and IgD-/- (green) animals after 4, 7, 10 and 14 days post immunization, mean ±SD (numbers of animals indicated in the top plot). Statistical significance was analyzed by a two-tailed unpaired t-test.


D | Analysis of GC in spleen sections from WT and IgD - / - animals after 4 , 7 and 10 days post immunization . Representative images of 2 x 2 mm sections stained with DAPI ( nuclei ) and peanut agglutinin ( PNA ) are shown for the indicated time points . Scale bar represents 500 μm . Dashed white or yellow lines mark distinct GC foci in the merged images . E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within the images of D are shown as enlarged single GCs . As no distinct GC foci were observed in IgD - / - samples from days 4 and 7 , an identical 800 x 800 μm region is shown in each case . F | Quantification of number of GCs per mm2 area of spleen sections from WT ( black ) and IgD - / - ( green ) animals after 4 , 7 and 10 days post immunization . Data represent mean ± SD of two sections ( from 3 animals per group and time point ) and were analyzed by two - tailed unpaired t - test .

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D | Analysis of GC in spleen sections from WT and IgD-/- animals after 4, 7 and 10 days post immunization. Representative images of 2 x 2 mm sections stained with DAPI (nuclei) and peanut agglutinin (PNA) are shown for the indicated time points. Scale bar represents 500 μm. Dashed white or yellow lines mark distinct GC foci in the merged images. E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within the images of D are shown as enlarged single GCs. As no distinct GC foci were observed in IgD-/- samples from days 4 and 7, an identical 800 x 800 μm region is shown in each case. F | Quantification of number of GCs per mm2 area of spleen sections from WT (black) and IgD-/- (green) animals after 4, 7 and 10 days post immunization. Data represent mean ±SD of two sections (from 3 animals per group and time point) and were analyzed by two-tailed unpaired t-test.


G | Total serum α - TNP IgG . Sera from PBS - or TNP - Ova - immunized WT ( left ) and IgD - / - ( middle ) mice , collected at day 7 following immunization , were adjusted to an IgG concentration of 1 μg / ml and applied in duplicates and dilution steps of 1 : 3 to TNP - coated plates , respectively , mean ± SEM . Right : Overlay of α - TNP IgG titers from TNP - Ova - immunized WT and IgD - / - mice . Statistical significance was calculated by using the Mann - Whitney - U test ( see also Table S7 ) , n . s . = not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

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G | Total serum α-TNP IgG. Sera from PBS- or TNP-Ova-immunized WT (left) and IgD-/- (middle) mice, collected at day 7 following immunization, were adjusted to an IgG concentration of 1 μg/ml and applied in duplicates and dilution steps of 1 : 3 to TNP-coated plates, respectively, mean ±SEM. Right: Overlay of α-TNP IgG titers from TNP-Ova-immunized WT and IgD-/- mice. Statistical significance was calculated by using the Mann-Whitney-U test (see also Table S7), n. s. = not significant, * p ≤ 0.05, ** p ≤ 0.01.


C : Western blot analysis of proteins pulled down using the peptides input fraction corresponds to HeLa cell total protein extract . Bound proteins were analyzed using anti - VAP - A , anti VAP - B and anti - MOSPD2 antibodies . Actin was used as a loading control .

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C: Western blot analysis of proteins pulled down using the peptides input fraction corresponds to HeLa cell total protein extract. Bound proteins were analyzed using anti-VAP-A, anti VAP-B and anti-MOSPD2 antibodies. Actin was used as a loading control.


D : Western blot analysis of proteins pulled down using peptides comprising WT and mutant ( D478S and D478pS ) ORP1 FFAT sequence peptides Bound proteins were analyzed using anti VAP - A , anti - VAP - B and anti MOSPD2 antibodies . Actin was used as a loading control . Data information : Acidic ( D or phosphorylated residues ( pS alcoholic ( S residues are in red , green and blue , respectively ; the other residues are in black .

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D: Western blot analysis of proteins pulled down using peptides comprising WT and mutant (D478S and D478pS) ORP1 FFAT sequence peptides Bound proteins were analyzed using anti VAP-A, anti-VAP-B and anti MOSPD2 antibodies. Actin was used as a loading control. Data information: Acidic (D or phosphorylated residues (pS alcoholic (S residues are in red, green and blue, respectively; the other residues are in black.


B : Coomassie blue staining of the recombinant wild - type MSP domains of VAP - A and VAP - B , and of the KD / MD mutant of VAP - A , after SDS - PAGE .

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B: Coomassie blue staining of the recombinant wild-type MSP domains of VAP-A and VAP-B, and of the KD/MD mutant of VAP-A, after SDS-PAGE.


C : Recombinant MSP domains pulled down with peptides corresponding to the Phospho - FFAT motif of STARD3 ( unphosphorylated ; mono - phosphorylated on S209 or S213 ; bi - phosphorylated on S209 and S213 ) , and with the control peptides were revealed by Coomassie blue staining . The recombinant proteins subjected to the assay are showed in the input fraction .

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C: Recombinant MSP domains pulled down with peptides corresponding to the Phospho-FFAT motif of STARD3 (unphosphorylated; mono-phosphorylated on S209 or S213; bi-phosphorylated on S209 and S213), and with the control peptides were revealed by Coomassie blue staining. The recombinant proteins subjected to the assay are showed in the input fraction.


A - D : Surface Plasmon Resonance analysis of the MSP domain of VAP - A binding onto immobilized unphosphorylated ( A ) , monophosphorylated pS209 ( B ) and pS213 ( C ) , and bi - phosphorylated pS209 + pS213 ( D ) STARD3 FFAT peptides . Representative sensorgrams resulting from the interaction between the MSP domain of VAP - A injected at different concentrations and the different FFAT peptides are shown in ( A ) , ( B left ) , ( C ) , and ( D left ) . Binding curves display the SPR signal ( RU ) as a function of time . Some samples were measured twice ( concentrations printed in bold ) . Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted , as well as the bulk effect recorded with buffer only . Steady - state analysis of the interaction between pS209 ( B right ) and pS209 + pS213 ( D right ) STARD3 FFAT peptide , and the MSP domain of VAP - A . Equilibrium responses ( Req ) extracted from the left panel in association phase were plotted as a function of the dimeric MSP domain of VAP - A concentration , and fitted with a Langmuir binding model .

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A-D: Surface Plasmon Resonance analysis of the MSP domain of VAP-A binding onto immobilized unphosphorylated (A), monophosphorylated pS209 (B) and pS213 (C), and bi-phosphorylated pS209+pS213 (D) STARD3 FFAT peptides. Representative sensorgrams resulting from the interaction between the MSP domain of VAP-A injected at different concentrations and the different FFAT peptides are shown in (A), (B left), (C), and (D left). Binding curves display the SPR signal (RU) as a function of time. Some samples were measured twice (concentrations printed in bold). Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only. Steady-state analysis of the interaction between pS209 (B right) and pS209+pS213 (D right) STARD3 FFAT peptide, and the MSP domain of VAP-A. Equilibrium responses (Req) extracted from the left panel in association phase were plotted as a function of the dimeric MSP domain of VAP-A concentration, and fitted with a Langmuir binding model.


E : Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP - A and VAP - B . The kinetic experiments were performed at 25 ° C in Tris - HCl 50 mM , pH 7 . 5 , NaCl 75 mM buffer supplemented with 0 . 005 % ( v / v ) surfactant polysorbate 20 ( P20 , GE Healthcare ) . Mean of n independent experiments : n = 2 for pS209 and pS209 + pS213 ; 3 for pS213 ; and 4 for the non - phosphorylated FFAT . Uncertainties are obtained from the standard deviation considering a t - distribution coefficient for a risk factor of 32 % .

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E: Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP-A and VAP-B. The kinetic experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of n independent experiments: n=2 for pS209 and pS209+pS213; 3 for pS213; and 4 for the non-phosphorylated FFAT. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.


A , B : Binding assays . Immunoprecipitation ( GFP - Trap ) experiments between GFP - tagged VAP - A ( WT and KD / MD mutant ; A ) , VAP - B ( B ) and Flag - tagged STARD3 ( WT and S209A , S209D and S209D / P210A mutants ) . Please note that the mutations modify the FFAT and Phospho - FFAT scores of this region of STARD3 : compared to WT STARD3 ( Phospho - FFAT : 2 . 5 ; conventional FFAT : 6 . 5 ) , the STARD3 S209A , mutant has both low Phospho - FFAT and FFAT scores ( 6 . 5 ) , the S209D mutant has a high conventional FFAT score ( 2 . 5 ) but a low Phospho - FFAT score ( 6 . 5 ) , and the double mutant has a conventional FFAT score of 1 . 5 and a Phospho - FFAT score of 5 . 5 . Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag , anti - GFP and anti - Actin antibodies . Immunoprecipitated material was analyzed using anti - Flag and anti - GFP antibodies .

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A,B: Binding assays. Immunoprecipitation (GFP-Trap) experiments between GFP-tagged VAP-A (WT and KD/MD mutant; A), VAP-B (B) and Flag-tagged STARD3 (WT and S209A, S209D and S209D/P210A mutants). Please note that the mutations modify the FFAT and Phospho-FFAT scores of this region of STARD3: compared to WT STARD3 (Phospho-FFAT: 2.5; conventional FFAT: 6.5), the STARD3 S209A, mutant has both low Phospho-FFAT and FFAT scores (6.5), the S209D mutant has a high conventional FFAT score (2.5) but a low Phospho-FFAT score (6.5), and the double mutant has a conventional FFAT score of 1.5 and a Phospho-FFAT score of 5.5. Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag, anti-GFP and anti-Actin antibodies. Immunoprecipitated material was analyzed using anti-Flag and anti-GFP antibodies.


Recruitment assays . GFP - VAP - A ( C ; green ) were left untransfected ( C ) and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm . Inset scale bars : 2 µm .

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Recruitment assays. GFP-VAP-A (C ; green) were left untransfected (C) and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm.


: Recruitment assays . GFP - VAP - A D , E , G ; green ) and GFP - VAP - A KD / MD - expressing cells ( F ; green ) were transfected with Flag - STARD3 ( D , F ) , Flag - STARD3 S209A ( E ) , and Flag - STARD3 S209D / P210A ( G ) , and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm . Inset scale bars : 2 µm .

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: Recruitment assays. GFP-VAP-A D, E, G; green) and GFP-VAP-A KD/MD-expressing cells (F; green) were transfected with Flag-STARD3 (D, F), Flag-STARD3 S209A (E), and Flag-STARD3 S209D/P210A (G), and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm.


H : Pearson ' s correlation coefficients between VAP - A ( WT or KD / MD mutant ) and STARD3 ( WT or S209A , S209D , S209D / P210A , FA / YA mutants ) staining are shown . Each dot represents a single cell ( number of cells : VAP - A - STARD3 : 22 ; VAP - A - STARD3 S209A : 25 ; VAP - A - STARD3 S209D : 27 ; VAP - A - STARD3 S209D / P210A : 26 ; VAP - A - STARD3 FA / YA : 20 ; VAP - A KD / MD - STARD3 : 20 , from at least three independent experiments ) . Means and error bars ( SD ) are shown . Kruskal - Wallis with Dunn ' s multiple comparison test ( * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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H: Pearson's correlation coefficients between VAP-A (WT or KD/MD mutant) and STARD3 (WT or S209A, S209D, S209D/P210A, FA/YA mutants) staining are shown. Each dot represents a single cell (number of cells: VAP-A-STARD3: 22; VAP-A-STARD3 S209A: 25; VAP-A-STARD3 S209D: 27; VAP-A-STARD3 S209D/P210A: 26; VAP-A-STARD3 FA/YA: 20; VAP-A KD/MD-STARD3: 20, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn's multiple comparison test (**P < 0.01; ***P < 0.001).


B : SDS - PAGE gel and Western blot analysis of purified cSTD3 and pS209 cSTD3 proteins . Top : the gel was stained with Sypro Orange to visualize proteins and molecular weight markers . Bottom : two similar gels were blotted onto nitrocellulose and analysed for the presence of total and STARD3 - pS209 using specific antibodies .

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B: SDS-PAGE gel and Western blot analysis of purified cSTD3 and pS209 cSTD3 proteins. Top: the gel was stained with Sypro Orange to visualize proteins and molecular weight markers. Bottom: two similar gels were blotted onto nitrocellulose and analysed for the presence of total and STARD3-pS209 using specific antibodies.


C : Aggregation assays . LA liposomes ( 50 µM total lipids ) were incubated for 5 minutes with cSTD3 or pS209 cSTD3 ( 380 nM ) . Then , LB liposomes ( 50 µM total lipids ) and VAP - AHIS6 or VAP - A KD / MDHIS6 ( 700 nM ) were successively added . Left panels : mean radius ( black dots ) and polydispersity ( shaded area ) over time . Right panels : size distribution before ( grey bars ) and after the reaction ( black bars ) .

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C: Aggregation assays. LA liposomes (50 µM total lipids) were incubated for 5 minutes with cSTD3 or pS209 cSTD3 (380 nM). Then, LB liposomes (50 µM total lipids) and VAP-AHIS6 or VAP-A KD/MDHIS6 (700 nM) were successively added. Left panels: mean radius (black dots) and polydispersity (shaded area) over time. Right panels: size distribution before (grey bars) and after the reaction (black bars).


D : DHE transport assay . DOPC ( 1 . 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) liposomes ( 62 . 5 µM total lipids , LA ) containing 3 mol % MPB - PE were mixed with cSTD3 or pS209 cSTD3 ( 475 nM ) . After 5 minutes , liposomes ( DOPC / DOGS - NTA - Ni2 + / DNS - PE / DHE liposomes 77 . 5 / 10 / 2 . 5 / 10 mol / mol , 62 . 5 µM total lipids , LB ) , covered or not with 1 µM of VAPHIS6 or VAP - A KD / MDHIS6 , were added . FRET between DHE and DNS - PE in the LB liposomes diminishes as DHE is transported towards LA liposomes . The signal was converted into amount of DHE present in LB liposomes ( in mol % ) .

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D: DHE transport assay. DOPC (1.2-dioleoyl-sn-glycero-3-phosphocholine) liposomes (62.5 µM total lipids, LA) containing 3 mol% MPB-PE were mixed with cSTD3 or pS209 cSTD3 (475 nM). After 5 minutes, liposomes (DOPC/DOGS-NTA-Ni2+/DNS-PE/DHE liposomes 77.5/10/2.5/10 mol/mol, 62.5 µM total lipids, LB), covered or not with 1 µM of VAPHIS6 or VAP-A KD/MDHIS6, were added. FRET between DHE and DNS-PE in the LB liposomes diminishes as DHE is transported towards LA liposomes. The signal was converted into amount of DHE present in LB liposomes (in mol%).


E : Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP - A HIS6 or VAP - A KD / MDHIS6 . Data are represented as mean ± S . E . M . ( n = 3 for cSTD3 - VAP and n = 4 for all other data ) . Mann - Whitney test ( * P < 0 . 05 ) .

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E: Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP-A HIS6 or VAP-A KD/MDHIS6. Data are represented as mean ± S.E.M. (n = 3 for cSTD3-VAP and n=4 for all other data). Mann-Whitney test (*P < 0.05).


A - E : HeLa / Ctrl ( A ) , HeLa / STARD3 ( B ) , HeLa / STARD3 S209A ( C ) , HeLa / STARD3 S209D / P210A ( D ) and HeLa / STARD3 Conv - FFAT ( E ) cells were labeled with anti - STARD3 antibodies ( magenta ) , anti - Lamp1 antibodies ( green ) and with the fluorescent cholesterol probe filipin ( Cyan Hot ) . Nuclei are shown in gray ( TO - PRO - 3 ) . Higher magnifications images ( 2x ) of the area outlined in white are shown on the right . The filipin , STARD3 and Lamp1 merged image is labeled Overlay . Scale bars : 10 µm . Inset scale bars : 5 µm .

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A-E: HeLa/Ctrl (A), HeLa/STARD3 (B), HeLa/STARD3 S209A (C), HeLa/STARD3 S209D/P210A (D) and HeLa/STARD3 Conv-FFAT (E) cells were labeled with anti-STARD3 antibodies (magenta), anti-Lamp1 antibodies (green) and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are shown in gray (TO-PRO-3). Higher magnifications images (2x) of the area outlined in white are shown on the right. The filipin, STARD3 and Lamp1 merged image is labeled Overlay. Scale bars: 10 µm. Inset scale bars: 5 µm.


F : Pearson ' s correlation coefficients between STARD3 and Lamp1 staining in HeLa / STARD3 cells . Each dot represents a single cell ( 34 cells from 4 independent experiments ) . Mean and error bars ( SD ) are shown . Note that STARD3 and Lamp1 signals are highly correlated .

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F: Pearson's correlation coefficients between STARD3 and Lamp1 staining in HeLa/STARD3 cells. Each dot represents a single cell (34 cells from 4 independent experiments). Mean and error bars (SD) are shown. Note that STARD3 and Lamp1 signals are highly correlated.


G : Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa / Ctrl , HeLa / STARD3 , HeLa / STARD3 S209A , HeLa / STARD3 S209D / P210A and HeLa / STARD3 Conv - FFAT cells . Data are displayed as a Superplot ( Lord et al , 2020 ) showing the relative filipin fluorescence intensity in endosomes of individual cells ( small circles ) from 6 independent experiments ( mean of each experiment shown as a large circle ) . Independent experiments are color - coded . Means and error bars ( SD ) are shown as black bars . Kruskal - Wallis with Dunn ' s multiple comparison test ( * P < 0 . 05 ; * * P < 0 . 01 ; n = 6 independent experiments ) .

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G: Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 S209A, HeLa/STARD3 S209D/P210A and HeLa/STARD3 Conv-FFAT cells. Data are displayed as a Superplot (Lord et al, 2020) showing the relative filipin fluorescence intensity in endosomes of individual cells (small circles) from 6 independent experiments (mean of each experiment shown as a large circle). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. Kruskal-Wallis with Dunn's multiple comparison test (*P < 0.05; **P < 0.01; n=6 independent experiments).


A : Coomassie blue staining of the recombinant MSP domain of MOSPD2 after SDS - PAGE .

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A: Coomassie blue staining of the recombinant MSP domain of MOSPD2 after SDS-PAGE.


B : Recombinant MSP domains pulled down with STARD3 FFAT peptides ( unphosphorylated , mono - phosphorylated on S209 or S213 , bi - phosphorylated on S209 and S213 , or tri - phosphorylated on S203 , S209 and S213 . and control peptides were revealed by Coomassie blue staining . The recombinant proteins subjected to the assay are shown in the input fraction .

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B: Recombinant MSP domains pulled down with STARD3 FFAT peptides (unphosphorylated, mono-phosphorylated on S209 or S213, bi-phosphorylated on S209 and S213, or tri-phosphorylated on S203, S209 and S213.and control peptides were revealed by Coomassie blue staining. The recombinant proteins subjected to the assay are shown in the input fraction.


SPR analysis of the MSP domain of MOSPD2 binding onto immobilized unphosphorylated ( C ) STARD3 FFAT . Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in ( C ) Binding curves display the SPR signal ( RU ) as a function of time . Concentrations printed in bold indicate samples measured twice . Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted , as well as the bulk effect recorded with buffer only .

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SPR analysis of the MSP domain of MOSPD2 binding onto immobilized unphosphorylated (C) STARD3 FFAT. Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in (C) Binding curves display the SPR signal (RU) as a function of time. Concentrations printed in bold indicate samples measured twice. Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only.


SPR analysis of the MSP domain of MOSPD2 binding onto immobilized pS209 ( D ) STARD3 FFAT . Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in ( D left ) . Binding curves display the SPR signal ( RU ) as a function of time . Concentrations printed in bold indicate samples measured twice . Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted , as well as the bulk effect recorded with buffer only . ( D right ) steady - state analysis of the interaction between pS209 STARD3 FFAT peptide and the MSP domain of MOSPD2 . Equilibrium responses ( Req ) extracted from the left panel were plotted as a function of the monomeric concentration of the MSP domain of MOSPD2 , and fitted with a 1 : 1 binding model . The experiments were performed at 25 ° C in Tris - HCl 50 mM , pH 7 . 5 , NaCl 75 mM buffer supplemented with 0 . 005 % ( v / v ) surfactant polysorbate 20 ( P20 , GE Healthcare ) . Mean of 2 independent experiments . Uncertainties are obtained from the standard deviation considering a t - distribution coefficient for a risk factor of 32 % .

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SPR analysis of the MSP domain of MOSPD2 binding onto immobilized pS209 (D) STARD3 FFAT. Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in (D left). Binding curves display the SPR signal (RU) as a function of time. Concentrations printed in bold indicate samples measured twice. Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only. (D right) steady-state analysis of the interaction between pS209 STARD3 FFAT peptide and the MSP domain of MOSPD2. Equilibrium responses (Req) extracted from the left panel were plotted as a function of the monomeric concentration of the MSP domain of MOSPD2, and fitted with a 1:1 binding model. The experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of 2 independent experiments. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.


E : Interaction dissociation constants between the different STARD3 FFAT peptides and the MSP domains of MOSPD2 .

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E: Interaction dissociation constants between the different STARD3 FFAT peptides and the MSP domains of MOSPD2.


F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - tagged MOSPD2 and Flag - tagged STARD3 ( WT and S209A , S209D and S209D / P210A mutants ) . Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag , anti - GFP and anti - Actin antibodies . Immunoprecipitated material was analyzed using anti - Flag and anti - GFP antibodies .

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F: Immunoprecipitation (GFP-Trap) experiments between GFP-tagged MOSPD2 and Flag-tagged STARD3 (WT and S209A, S209D and S209D/P210A mutants). Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag, anti-GFP and anti-Actin antibodies. Immunoprecipitated material was analyzed using anti-Flag and anti-GFP antibodies.


G - I : GFP - MOSPD2 - expressing cells ( green ) were left untransfected ( G ) or transfected with Flag - STARD3 ( H ) and Flag - STARD3 S209A ( I ) , and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm .

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G-I: GFP-MOSPD2-expressing cells (green) were left untransfected (G) or transfected with Flag-STARD3 (H) and Flag-STARD3 S209A (I), and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm.


J : Pearson ' s correlation coefficients between MOSPD2 ( WT or RD / LD ) and STARD3 ( WT , S209A or FA / YA ) staining are shown . Each dot represents a single cell ( number of cells : MOSPD2 - STARD3 : 22 ; MOSPD2 - STARD3 S209A : 21 ; MOSPD2 - STARD3 FA / YA : 20 ; MOSPD2 RD / LD - STARD3 : 17 , from at least three independent experiments ) . Means and error bars ( SD ) are shown . Kruskal - Wallis with Dunn ' s multiple comparison test ( * * * P < 0 . 001 ) .

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J: Pearson's correlation coefficients between MOSPD2 (WT or RD/LD) and STARD3 (WT, S209A or FA/YA) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-STARD3: 22; MOSPD2- STARD3 S209A: 21; MOSPD2-STARD3 FA/YA: 20; MOSPD2 RD/LD-STARD3: 17, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn's multiple comparison test (***P < 0.001).


Schematic of single ‐ plane acquisition FRAP on endogenously tagged Swi6 ‐ EGFP . Representative still images of a FRAP experiment are shown . See also Movie EV1 . Green sphere : yeast nucleus containing Swi6 ‐ EGFP ; green dots : heterochromatic Swi6 ‐ EGFP ; red circle : bleached area .

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Schematic of single‐plane acquisition FRAP on endogenously tagged Swi6‐EGFP. Representative still images of a FRAP experiment are shown. See also Movie EV1. Green sphere: yeast nucleus containing Swi6‐EGFP; green dots: heterochromatic Swi6‐EGFP; red circle: bleached area.


Normalized average intensities ( black dots ) of FRAP on Swi6 ‐ EGFP heterochromatic loci ( n = 31 ) fitted to a one ‐ component model ( blue line ) . The dashed lines indicate the final relative intensity that is set to 1 and the fluorescence half ‐ recovery time ( t 1 / 2 ) .

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Normalized average intensities (black dots) of FRAP on Swi6‐EGFP heterochromatic loci (n = 31) fitted to a one‐component model (blue line). The dashed lines indicate the final relative intensity that is set to 1 and the fluorescence half‐recovery time (t 1/2).


Fluorescence t 1 / 2 values of FRAP on Swi6 ‐ EGFP heterochromatic loci . The box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box .

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Fluorescence t 1/2 values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.


Normalized fluorescence recovery values of FRAP on Swi6 ‐ EGFP heterochromatic loci . The box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box .

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Normalized fluorescence recovery values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.


FLIP experiment showing a bleached ( FRAP , red circle ) and unbleached , adjacent locus ( FLIP , blue circle ) in the same nucleus . The graph displays average relative fluorescence of FLIP ( blue ) , FRAP ( red ) , and control loci ( no bleach , green ) in other non ‐ bleached cells .

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FLIP experiment showing a bleached (FRAP, red circle) and unbleached, adjacent locus (FLIP, blue circle) in the same nucleus. The graph displays average relative fluorescence of FLIP (blue), FRAP (red), and control loci (no bleach, green) in other non‐bleached cells.


Schematic of a line ‐ scan FRAP experiment . Fluorescence intensities are measured along a line of a non ‐ bleached nucleus ( Ctrl nucleus ) and a bleached nucleus ( FRAP nucleus ) in Swi6 ‐ EGFP clr4 ∆ cells . Red box : bleached region . Right : raw data example .

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Schematic of a line‐scan FRAP experiment. Fluorescence intensities are measured along a line of a non‐bleached nucleus (Ctrl nucleus) and a bleached nucleus (FRAP nucleus) in Swi6‐EGFP clr4∆ cells. Red box: bleached region. Right: raw data example.


Average relative intensities of a line ‐ scan FRAP experiment on Swi6 ‐ EGFP in heterochromatin ( blue line ) , euchromatin ( green line ) , and clr4 ∆ ( red line ) . The curves have a gliding average of 40 .

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Average relative intensities of a line‐scan FRAP experiment on Swi6‐EGFP in heterochromatin (blue line), euchromatin (green line), and clr4∆ (red line). The curves have a gliding average of 40.


A , BAverage relative intensities over time and corresponding fluorescence t 1 / 2 values of heterochromatic Swi6 obtained from FRAP experiments performed with cells expressing NLS ‐ Swi6 ‐ EGFP ( blue ) or NLS ‐ Swi6 ‐ KR25A ‐ EGFP ( red ) .

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A, BAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with cells expressing NLS‐Swi6‐EGFP (blue) or NLS‐Swi6‐KR25A‐EGFP (red).


C , DAverage relative intensities over time and corresponding fluorescence t 1 / 2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild ‐ type ( brown ) or cid14 ∆ ( dark green ) cells expressing Swi6 ‐ EGFP .

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C, DAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild‐type (brown) or cid14∆ (dark green) cells expressing Swi6‐EGFP.


E , FAverage relative intensities over time and corresponding fluorescence t 1 / 2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild ‐ type cells expressing heterochromatic NLS ‐ Swi6 ‐ EGFP ( blue ) or NLS ‐ Swi6 KR25A ‐ EGFP ( red ) , or cid14 ∆ cells expressing NLS ‐ Swi6 ‐ EGFP ( green ) or NLS ‐ Swi6 ‐ KR25A ‐ EGFP ( yellow ) . Data information : In ( B ) , ( D ) and ( F ) , the box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box .

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E, FAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild‐type cells expressing heterochromatic NLS‐Swi6‐EGFP (blue) or NLS‐Swi6 KR25A‐EGFP (red), or cid14∆ cells expressing NLS‐Swi6‐EGFP (green) or NLS‐Swi6‐KR25A‐EGFP (yellow).Data information: In (B), (D) and (F), the box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.


Schematic of a chromosome indicating Swi6 ‐ EGFP heterochromatic domains ( green ) . Cnp1 ‐ mCherry and Taz1 ‐ mCherry ( red ) mark centromeres and telomeres , respectively . Representative images demonstrating Swi6 ‐ EGFP co ‐ localization with centromeres ( Cnp1 ‐ mCherry ) and telomeres ( Taz1 ‐ mCherry ) are shown .

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Schematic of a chromosome indicating Swi6‐EGFP heterochromatic domains (green). Cnp1‐mCherry and Taz1‐mCherry (red) mark centromeres and telomeres, respectively. Representative images demonstrating Swi6‐EGFP co‐localization with centromeres (Cnp1‐mCherry) and telomeres (Taz1‐mCherry) are shown.


Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing NLS ‐ Swi6 ‐ EGFP .

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Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing NLS‐Swi6‐EGFP.


Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA ‐ binding mutant NLS ‐ Swi6 ‐ KR25A ‐ EGFP . Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing Swi6 ‐ EGFP .

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Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP.Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing Swi6‐EGFP.


Fluorescence t 1 / 2 values of centromeric Swi6 obtained from FRAP experiments performed after release of cells expressing NLS ‐ Swi6 ‐ EGFP ( black dots ) or the RNA ‐ binding mutant NLS ‐ Swi6 ‐ KR25A ‐ EGFP ( red dots ) from G1 / S cell cycle arrest . Each dot represents one FRAP experiment at the respective time after release from cell cycle arrest . Swi6 is dispersed in M phase due to H3S10 phosphorylation , and M phase was therefore not included in the FRAP analysis .

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Fluorescence t 1/2 values of centromeric Swi6 obtained from FRAP experiments performed after release of cells expressing NLS‐Swi6‐EGFP (black dots) or the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP (red dots) from G1/S cell cycle arrest. Each dot represents one FRAP experiment at the respective time after release from cell cycle arrest. Swi6 is dispersed in M phase due to H3S10 phosphorylation, and M phase was therefore not included in the FRAP analysis.


Fluorescence t 1 / 2 values obtained from FRAP experiments performed on centromeric Swi6 . A total of 40 , 66 , and 104 individual cells residing in the G1 , S , and G2 phases of the cell cycle were analyzed , respectively . Representative bright ‐ field images of cells in G1 , S , or G2 are shown . Scale bars = 2 μm .

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Fluorescence t 1/2 values obtained from FRAP experiments performed on centromeric Swi6. A total of 40, 66, and 104 individual cells residing in the G1, S, and G2 phases of the cell cycle were analyzed, respectively. Representative bright‐field images of cells in G1, S, or G2 are shown. Scale bars = 2 μm.


Fluorescence t 1 / 2 values obtained from FRAP experiments performed on centromeric Swi6 of cells expressing NLS ‐ Swi6 ‐ EGFP and NLS ‐ Swi6 ‐ KR25A ‐ EGFP that reside in the G2 of the cell cycle .

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Fluorescence t 1/2 values obtained from FRAP experiments performed on centromeric Swi6 of cells expressing NLS‐Swi6‐EGFP and NLS‐Swi6‐KR25A‐EGFP that reside in the G2 of the cell cycle.


Fluorescence t 1 / 2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA ‐ binding mutant NLS ‐ Swi6 ‐ KR25A ‐ EGFP .

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Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP.


H3K9me2 ChIP ‐ seq enrichment profiles for swi6 + and swi6 ∆ cells on centromere 1 . Centromeric repeat elements ( dg / dh ) are indicated . Inverted repeats ( IRC1 ) constitute heterochromatin boundaries in wild ‐ type cells ( Cam et al , 2005 ) . The y ‐ axes represent log2 ChIP ‐ seq enrichments in 200 ‐ bp genomically tiled windows calculated over clr4Δ cells . Two independent biological replicates for each genotype were performed ( replicates 1 and 2 ) . Positions of genomic elements on the plus and minus strands are indicated . Blue , protein ‐ coding genes ; white , repeats ; green , long terminal repeat ( LTR ) .

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H3K9me2 ChIP‐seq enrichment profiles for swi6+ and swi6∆ cells on centromere 1. Centromeric repeat elements (dg/dh) are indicated. Inverted repeats (IRC1) constitute heterochromatin boundaries in wild‐type cells (Cam et al, 2005). The y‐axes represent log2 ChIP‐seq enrichments in 200‐bp genomically tiled windows calculated over clr4Δ cells. Two independent biological replicates for each genotype were performed (replicates 1 and 2). Positions of genomic elements on the plus and minus strands are indicated. Blue, protein‐coding genes; white, repeats; green, long terminal repeat (LTR).


H3K9me2 ChIP ‐ seq enrichment profiles for swi6 + and swi6 ∆ cells on the telomere of the right arm of chromosome 1 . Two independent biological replicates for each genotype were performed ( replicates 1 and 2 ) .

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H3K9me2 ChIP‐seq enrichment profiles for swi6+ and swi6∆ cells on the telomere of the right arm of chromosome 1. Two independent biological replicates for each genotype were performed (replicates 1 and 2).


Differential expression of transcripts coming from centromere 1 assessed by tiling microarray gene expression analysis . Expression data for swi6 + and swi6 ∆ cells were taken from a previous publication ( Woolcock et al , 2012 ) .

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Differential expression of transcripts coming from centromere 1 assessed by tiling microarray gene expression analysis. Expression data for swi6+ and swi6∆ cells were taken from a previous publication (Woolcock et al, 2012).


C - EH3K9me2 levels on heterochromatin ‐ adjacent genes assessed by ChIP - PCR in swi6 + , swi6 ∆ , and swi6 ‐ L315E ( CSD dimerization mutant ) ( C ) , swi6 ‐ W104A ( H3K9me2 binding mutant ) ( D ) , and epe1 ∆ ( E ) cells . Enrichments over clr4 ∆ are normalized to adh1 + . Average fold enrichment with s . d . is shown for three ( C and E ) or four ( D ) independent experiments . P ‐ values were generated by the Student ' s t ‐ test ( two ‐ tailed distribution , two ‐ sample , unequal variance ) .

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C-EH3K9me2 levels on heterochromatin‐adjacent genes assessed by ChIP-PCR in swi6+, swi6∆, and swi6‐L315E (CSD dimerization mutant) (C), swi6‐W104A (H3K9me2 binding mutant) (D), and epe1∆ (E) cells. Enrichments over clr4∆ are normalized to adh1+. Average fold enrichment with s.d. is shown for three (C and E) or four (D) independent experiments. P‐values were generated by the Student's t‐test (two‐tailed distribution, two‐sample, unequal variance).


sRNA reads mapping to centromere 1 in swi6 + ( green ) and swi6 ∆ ( blue ) cells . The dashed red box highlights the loss of brdrRNAs at the IRC1R in the absence of Swi6 . Centromeric repeat elements and the central core are indicated . Counts were normalized to the library size . Asterisks denote tRNA fragments . Note that tRNA genes flank IRC3 ( Fig EV4 ) but not IRC1R .

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sRNA reads mapping to centromere 1 in swi6+ (green) and swi6∆ (blue) cells. The dashed red box highlights the loss of brdrRNAs at the IRC1R in the absence of Swi6. Centromeric repeat elements and the central core are indicated. Counts were normalized to the library size. Asterisks denote tRNA fragments. Note that tRNA genes flank IRC3 (Fig EV4) but not IRC1R.


H3K9me2 levels on heterochromatin ‐ adjacent genes assessed by ChIP - PCR in swi6 + , swi6 ∆ , tas3 L479E , and tas3 L479E swi6 ∆ cells . Enrichments over clr4 ∆ are normalized to adh1 + . Average fold enrichment with s . d . is shown for three independent experiments . P ‐ values were generated by Student ' s t ‐ test ( two ‐ tailed distribution , two ‐ sample , unequal variance ) .

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H3K9me2 levels on heterochromatin‐adjacent genes assessed by ChIP-PCR in swi6+, swi6∆, tas3 L479E, and tas3 L479E swi6∆ cells. Enrichments over clr4∆ are normalized to adh1+. Average fold enrichment with s.d. is shown for three independent experiments. P‐values were generated by Student's t‐test (two‐tailed distribution, two‐sample, unequal variance).


B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young ( 2 months ) and old ( 22 months ) mice ( n = 3 mice per group ) . Scale bar 200 µm .

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B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young (2 months) and old (22 months) mice (n = 3 mice per group). Scale bar 200 µm.


C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young ( 20 - 35 years old ) and old ( 60 - 80 years old ) humans ( n = 4 - 7 individuals per group ) . Scale bar , 200 µm .

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C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young (20-35 years old) and old (60-80 years old) humans (n = 4-7 individuals per group). Scale bar, 200 µm.


D Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive and CD31 - positive endothelia in lungs from young and old mice ( n = 3 - 6 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs from young and old mice (n = 3-6 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


E Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1 - positive and CD31 - positive endothelia in lungs from young and old humans ( n = 4 - 7 individuals per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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E Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1-positive and CD31-positive endothelia in lungs from young and old humans (n = 4-7 individuals per group). Scale bar, 100 µm. a.u. = arbitrary units.


C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age - matched wild type and G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 200 µm .

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C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 200 µm.


D Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive and CD31 - positive endothelia in lungs of age - matched wild type and G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F / F at the indicated time points following 4OHT treatment and consequent TRF2 knockout . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

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A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F/F at the indicated time points following 4OHT treatment and consequent TRF2 knockout. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown . Scale bar , 25 µm . D RT - qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C ( n = 6 independent experiments ) .

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C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown. Scale bar, 25 µm. D RT-qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C (n = 6 independent experiments).


E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F / F mice treated with tamoxifen ( to induce TRF2 loss and telomere uncapping ) or vehicle . The animals as been injected also with PBS . Scale bar , 10 µm . F RT - qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E ( n = 5 - 7 mice per group ) .

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E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce TRF2 loss and telomere uncapping) or vehicle. The animals as been injected also with PBS. Scale bar, 10 µm. F RT-qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E (n = 5-7 mice per group).


B Relative luciferase activity in HeLa shTRF2 following ionizing radiation ( 5 Gy ) or TRF2 knockdown upon doxycycline - induced shTRF2 expression ( n = 3 independent experiments ) . Error bars represent the s . e . m . * P < 0 . 05 . Two - way paired ANOVA .

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B Relative luciferase activity in HeLa shTRF2 following ionizing radiation (5 Gy) or TRF2 knockdown upon doxycycline-induced shTRF2 expression (n=3 independent experiments). Error bars represent the s.e.m. *P<0.05. Two-way paired ANOVA.


A Immunofluorescence showing gammaH2AX foci in Trf2F / F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

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A Immunofluorescence showing gammaH2AX foci in Trf2F/F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F / F mice treated with tamoxifen ( to induce telomere uncapping ) or vehicle and injected with the indicated ASOs or PBS as control . Scale bar , 10 µm . D RT - qPCR detection of Ace2 mRNA levels in livers of mice treated as in C ( n = 5 - 8 mice per group ) .

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C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce telomere uncapping) or vehicle and injected with the indicated ASOs or PBS as control. Scale bar, 10 µm. D RT-qPCR detection of Ace2 mRNA levels in livers of mice treated as in C (n = 5-8 mice per group).


E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age - matched wild type and G3 Terc - / - mice , treated with the indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 200 µm .

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E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 200 µm.


F Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive cells in lungs of age - matched wild type and G3 Terc - / - mice , treated with the indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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F Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive cells in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


A Atf7ip KO cells show increased expression of SETDB1 - regulated ERVs and the provirus reporter , MSCV - GFP as evidenced by RT - qPCR analysis . RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells . Data are mean ± SD ; n = 3 , technical replicates .

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A Atf7ip KO cells show increased expression of SETDB1-regulated ERVs and the provirus reporter, MSCV-GFP as evidenced by RT-qPCR analysis. RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells. Data are mean ± SD; n=3, technical replicates.


B Atf7ip KO mESCs ( TT # 2 - 12 ) show decreased H3K9me3 at the LTR of the SETDB1 - regulated reporter and the ERVs , as evidenced by Native ChIP followed by qPCR analysis . Gapdh was used as a negative control . Data are mean ± SEM ; n = 5 , independent experiments . * P < 0 . 05 and * * P < 0 . 01 by paired t - test .

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B Atf7ip KO mESCs (TT#2-12) show decreased H3K9me3 at the LTR of the SETDB1-regulated reporter and the ERVs, as evidenced by Native ChIP followed by qPCR analysis. Gapdh was used as a negative control. Data are mean ± SEM; n=5, independent experiments. *P < 0.05 and **P < 0.01 by paired t-test.


C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs . Endogenous SETDB1 - ATF7IP interaction is validated by anti - ATF7IP antibody co - IP experiment with anti - ATF7IP antibody .

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C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs. Endogenous SETDB1-ATF7IP interaction is validated by anti-ATF7IP antibody co-IP experiment with anti-ATF7IP antibody.


A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs . Representative projected images are shown , and the quantitative analyses are shown in B - D . Scale bar : 10 µm . B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 100 cells were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - test . C , D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated . Violin plot for SETDB1 ' s foci numbers from all the cells analysed in three independent experiments is shown in c . The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D . Over 100 cells were analysed per sample per experiment . * P < 0 . 05 by unpaired Student ' s t - test .

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A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown, and the quantitative analyses are shown in B-D. Scale bar: 10 µm. B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test. C, D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated. Violin plot for SETDB1's foci numbers from all the cells analysed in three independent experiments is shown in c. The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D. Over 100 cells were analysed per sample per experiment. *P < 0.05 by unpaired Student's t-test.


E , IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs . Representative projected images are shown . Arrows indicate the region used for line - plot analysis . Cells were treated with 10 ng / mL LMB or mock for 5 h before analysis . Scale bar : 10 µm . F The line - plot analysis for E . Relative intensities of SETDB1 signal were measured by Image J . G The quantitative analysis for E is shown . SETDB1 signal in the nucleus that was determined by DAPI staining was calculated . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 100 cells were analysed per sample per experiment . NS : P > 0 . 05 , * * P < 0 . 01 versus " Mock , WT " group by Dunnett ' s test .

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E, IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown. Arrows indicate the region used for line-plot analysis. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis. Scale bar: 10 µm. F The line-plot analysis for E. Relative intensities of SETDB1 signal were measured by Image J. G The quantitative analysis for E is shown. SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. NS: P > 0.05, **P < 0.01 versus "Mock, WT" group by Dunnett's test.


A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype . Endogenous SETDB1 in HEK293T cells shows cytoplasm + nuclear localization profile . Scale bar : 10 µm .

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A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype. Endogenous SETDB1 in HEK293T cells shows cytoplasm+nuclear localization profile. Scale bar: 10 µm.


B Over - production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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B Over-production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells. The representative data of two independent experiments is shown. Scale bar: 10 µm.


C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells , but no significant impact for ATF7IP KO HEK293T cells . Cells were treated with 10ng / ml of LMB for 5 h and then analysed with immunofluorescent staining . The representative data of three independent experiments is shown . Scale bar : 10 µm . D SETDB1 signal in the nucleus shown in C was calculated . The mean from three independent experiments is shown as a bar - graph ± SEM ; n = 3 . Over 50 cells were analysed per sample per experiment . * * P < 0 . 01 by Student ' s t - test ( two - sided test ) .

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C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells, but no significant impact for ATF7IP KO HEK293T cells. Cells were treated with 10ng/ml of LMB for 5 h and then analysed with immunofluorescent staining. The representative data of three independent experiments is shown. Scale bar: 10 µm. D SETDB1 signal in the nucleus shown in C was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. **P < 0.01 by Student's t-test (two-sided test).


E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells . The representative data of three independent experiments is shown . Scale bar : 10 µm . F SETDB1 signal in the nucleus shown in E was calculated . The mean from three independent experiments is shown as a bar - graph ± SEM ; n = 3 . Over 50 cells were analysed per sample per experiment . Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells . * P < 0 . 05 and * * P < 0 . 01 by Dunnett ' s method .

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E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells. The representative data of three independent experiments is shown. Scale bar: 10 µm. F SETDB1 signal in the nucleus shown in E was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells. *P < 0.05 and **P < 0.01 by Dunnett's method.


A HEK293T cells were transfected with V5 - SETDB1 and either empty or 3xF - ATF7IP expressing vector . Over 50 transfected cells analysed per sample were analyzed . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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A HEK293T cells were transfected with V5-SETDB1 and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells analysed per sample were analyzed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


B HEK293T cells were transfected with V5 - SETDB1 d1 mutant and either empty or 3xF - ATF7IP expressing vector . Over 50 transfected cells per sample were analysed . The representative data of two independent experiments was shown . Scale bar : 10 µm . The yellow arrowheads indicate the cells that show nuclear ( and cytoplasmic ) - localization of introduced exogenous mSETDB1 .

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B HEK293T cells were transfected with V5-SETDB1 d1 mutant and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments was shown. Scale bar: 10 µm. The yellow arrowheads indicate the cells that show nuclear (and cytoplasmic)-localization of introduced exogenous mSETDB1.


A Co - transfection with ATF7IP augments the K885 ubiquitination of SETDB1 . HEK293T cells were transfected After the transfection , V5 - SETDB1 was immune - purified with anti - V5 antibody and used for WB analysis .

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A Co-transfection with ATF7IP augments the K885 ubiquitination of SETDB1. HEK293T cells were transfected After the transfection, V5-SETDB1 was immune-purified with anti-V5 antibody and used for WB analysis.


B ATF7IP regulates the ubiquitination of SETDB1 in mESCs . Endogenous SETDB1 was immune - purified from WT or Atf7ip KO mESCs cells and used for WB analysis .

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B ATF7IP regulates the ubiquitination of SETDB1 in mESCs. Endogenous SETDB1 was immune-purified from WT or Atf7ip KO mESCs cells and used for WB analysis.


C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs . Cells were treated with 10 ng / mL LMB or mock for 5 h before analysis .

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C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis.


D HEK293T cells were transfected with either 3xF - or 3xF - NLS - SETDB1 . Over 50 transfected cells per sample were analysed . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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D HEK293T cells were transfected with either 3xF- or 3xF-NLS-SETDB1. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


E Nuclear localization itself promotes the ubiquitination of SETDB1 . HEK293T cells were transfected with 3xFLAG - or 3xFLAG - NLS - SETDB1 . After FLAG - IP , the purified SETDB1s were analyzed by WB .

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E Nuclear localization itself promotes the ubiquitination of SETDB1. HEK293T cells were transfected with 3xFLAG- or 3xFLAG-NLS-SETDB1. After FLAG-IP, the purified SETDB1s were analyzed by WB.


F , G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF - or 3xF - NLS - SETDB1 . Representative images are shown in F , and the quantitative analysis is shown in G . Scale bar : 10 µm . Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 50 cells were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - test .

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F, G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF- or 3xF-NLS-SETDB1. Representative images are shown in F, and the quantitative analysis is shown in G. Scale bar: 10 µm. Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 50 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test.


H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs . Atf7ip KO mESCs expressing 3xF - or 3xF - NLS - SETDB were used for FLAG - IP , and the purified SETDB1s were analyzed by WB .

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H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs. Atf7ip KO mESCs expressing 3xF- or 3xF-NLS-SETDB were used for FLAG-IP, and the purified SETDB1s were analyzed by WB.


A ) Double fluorescence in situ hybridization using Nrg3 ( green ) and Gad1 ( red ) specific probes ; DAPI was used as a counter stain . Nrg3 is present in Gad1 - positive ( filled arrowheads ) and - negative neurons . ( A ' ) and ( A ' ' ) show higher magnifications of boxed areas indicated in ( A ) . Nrg3 - negative neurons are indicated by open arrowheads ; note that many but not all neurons express Nrg3 .

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A) Double fluorescence in situ hybridization using Nrg3 (green) and Gad1 (red) specific probes; DAPI was used as a counter stain. Nrg3 is present in Gad1-positive (filled arrowheads) and -negative neurons. (A') and (A'') show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3.


B ) Comparison of mRNA levels of Nrg3 , Nrg1β type III ( Nrg1β − III ) , Nrg2β , Nrg1β type II ( Nrg1β − II ) , Nrg2α and Nrg1β type I ( Nrg1β − I ) in the cortex ( Cx ) , hippocampus ( HC ) and striatum ( Str ) of juvenile mice ( P30 - 60 ) by quantitative RT - PCR . Data represent means ± SD of 4 biological replicates . 2 - way - ANOVA with Bonferroni ' s multiple comparisons test was performed to assess statistical significance ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , ns = not significant ) .

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B) Comparison of mRNA levels of Nrg3, Nrg1β type III (Nrg1β−III), Nrg2β, Nrg1β type II (Nrg1β−II), Nrg2α and Nrg1β type I (Nrg1β−I) in the cortex (Cx), hippocampus (HC) and striatum (Str) of juvenile mice (P30-60) by quantitative RT-PCR. Data represent means ± SD of 4 biological replicates. 2-way-ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (****P<0.0001, ***P<0.001, ns=not significant).


D ) Detection of Nrg3 by western blot analysis in fractions from brain lysates : S1 , crude lysate ; S2 , cytosol and light membranes ; S2 ' , cytosol ; LM , light membranes ; P2 ' , crude synaptosomes ; S3 ' , synaptosomal cytoplasm ; SV , synaptic vesicles ; P3 , synaptosomal membranes ; SPM , synaptic plasma membranes .

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D) Detection of Nrg3 by western blot analysis in fractions from brain lysates: S1, crude lysate; S2, cytosol and light membranes; S2', cytosol; LM, light membranes; P2', crude synaptosomes; S3', synaptosomal cytoplasm; SV, synaptic vesicles; P3, synaptosomal membranes; SPM, synaptic plasma membranes.


E , F ) Immunohistochemical analysis in the hippocampus of wildtype ( E , E ' , E ' ' ) and ErbB4 mutant mice ( F ) at 2 month of age using Nrg3 - , ErbB4 - and parvalbumin ( Parv ) - specific antibodies . The white box in ( E ) is displayed magnified in ( E ' , E ' ' ) . In control mice , Nrg3 is enriched on dendrites of ErbB4 + PV interneurons , compared to the surrounding neuropil ( E ' , E ' ' ) . In ErbB4 mutants , the Nrg3 enrichment on PV interneurons is not apparent ( F ) .

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E, F) Immunohistochemical analysis in the hippocampus of wildtype (E,E',E'') and ErbB4 mutant mice (F) at 2 month of age using Nrg3-, ErbB4- and parvalbumin (Parv)-specific antibodies. The white box in (E) is displayed magnified in (E',E''). In control mice, Nrg3 is enriched on dendrites of ErbB4+ PV interneurons, compared to the surrounding neuropil (E',E''). In ErbB4 mutants, the Nrg3 enrichment on PV interneurons is not apparent (F).


G ) Nrg3 , ErbB4 and GluA4 were identified by immunohistology and co - localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1 . Note that ( G ) displays Nrg3 / ErbB4 , ( G ' ) Nrg3 / GluA4 , and ( G ' ' ) ErbB4 / GluA4 signals of the same triple - stained image ; false colors were assigned for better signal visualization .

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G) Nrg3, ErbB4 and GluA4 were identified by immunohistology and co-localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1. Note that (G) displays Nrg3/ErbB4, (G') Nrg3/GluA4, and (G'') ErbB4/GluA4 signals of the same triple-stained image; false colors were assigned for better signal visualization.


H ) Quantification of GluA4 puncta present on ErbB4 + dendrites in the CA1 stratum radiatum of adult wildtype ( wt ) and Nrg3 - / - mice ( age P90 - 120 ) . Data are presented as box plots with Tukey ' s whiskers and outliers ; means are indicated by a Plus symbols . n = 65 ( wt ) and n = 62 ( Nrg3 - / - ) from 5 animals each . Unpaired t - test ( 2 - tailed ) with Welch ' s correction was performed to assess statistical significance ( * * * * P < 0 . 0001 ) .

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H) Quantification of GluA4 puncta present on ErbB4+ dendrites in the CA1 stratum radiatum of adult wildtype (wt) and Nrg3-/- mice (age P90-120). Data are presented as box plots with Tukey's whiskers and outliers; means are indicated by a Plus symbols. n=65 (wt) and n=62 (Nrg3-/-) from 5 animals each. Unpaired t-test (2-tailed) with Welch's correction was performed to assess statistical significance (****P<0.0001).


A ) His6 - tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations . Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho - tyrosine ( pY ) and ErbB4 , and of whole lysates using antibodies against pErk1 / 2 and pAkt

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A) His6-tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho-tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt


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C , D ) Neurons from Nrg3 - / - ; ErbB4 - / - ; vGAT - Cre mice were transduced with a lentivirus expressing either ( C ) ErbB4wt or ( D ) ErbB4kd ( kinase dead ErbB4 ) in a Cre - dependent manner , and with a second virus at a low titer expressing Nrg3 / SypCFP . Immunocytochemical analysis of ErbB4 and SypCFP ; ( C , C ' ) and ( D , D ' ) each show the same image , ( C ' , D ' ) show the SypCFP signal

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C, D) Neurons from Nrg3-/-;ErbB4-/-;vGAT-Cre mice were transduced with a lentivirus expressing either (C) ErbB4wt or (D) ErbB4kd (kinase dead ErbB4) in a Cre-dependent manner, and with a second virus at a low titer expressing Nrg3/SypCFP. Immunocytochemical analysis of ErbB4 and SypCFP; (C,C') and (D,D') each show the same image, (C',D') show the SypCFP signal


E - G ) Quantification of Synapse preference towards ErbB4 + neurons ( E ) , ErbB4 enrichment ( F ) and SypCFP enrichment ( G ) in synapses on neurons expressing wildtype ErbB4 ( wt ) or ErbB4kd ( kd )

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E-G) Quantification of Synapse preference towards ErbB4+ neurons (E), ErbB4 enrichment (F) and SypCFP enrichment (G) in synapses on neurons expressing wildtype ErbB4 (wt) or ErbB4kd (kd)


H ) Nrg3 - / - neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4 , GFP and GluA ; ( H ) shows the triple stained image , while ( H ' ) and ( H ' ' ) only show ErbB4 and GluA signals , respectively

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H) Nrg3-/- neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4, GFP and GluA; (H) shows the triple stained image, while (H') and (H'') only show ErbB4 and GluA signals, respectively


I ) Co - cultures of Nrg3 - / - neurons with HEK293 cells expressing Nrg3 . Cultures were stained with antibodies against Nrg3 , ErbB4 and GluA . Note that Nrg3 , ErbB4 and GluA are enriched at the contact sites between Nrg3 - expressing HEK293 cells and ErbB4 + neurons

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I) Co-cultures of Nrg3-/- neurons with HEK293 cells expressing Nrg3. Cultures were stained with antibodies against Nrg3, ErbB4 and GluA. Note that Nrg3, ErbB4 and GluA are enriched at the contact sites between Nrg3-expressing HEK293 cells and ErbB4+ neurons


C ) Example traces of evoked excitatory postsynaptic currents ( eEPSCs ) averaged over 50 sweeps . D , E ) Quantification of the peak amplitude of the first eEPSC ( D ) and of the paired pulse ratio ( E )

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C) Example traces of evoked excitatory postsynaptic currents (eEPSCs) averaged over 50 sweeps. D, E) Quantification of the peak amplitude of the first eEPSC (D) and of the paired pulse ratio (E)


F - I ) sEPSC recordings from CA1 pyramidal ( F , H ) and PV neurons ( G , I ) in acute slices . sEPSC frequencies ( F , G ) and amplitudes ( H , I ) are

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F-I) sEPSC recordings from CA1 pyramidal (F,H) and PV neurons (G, I) in acute slices. sEPSC frequencies (F,G) and amplitudes (H,I) are shown


( B - C ) Fluorescent dye release from the mitochondria - mimic liposomes by the indicated wild - type ( wt ) or mutant Bax in the presence ( B ) or absence ( C ) of tBid or by tBid alone ( C ) was measured by quenching the fluorescence of the released dyes by the dye - specific antibodies outside the liposomes during a time course . The fraction of dye release was normalized to that by detergent . The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes .

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(B-C) Fluorescent dye release from the mitochondria-mimic liposomes by the indicated wild-type (wt) or mutant Bax in the presence (B) or absence (C) of tBid or by tBid alone (C) was measured by quenching the fluorescence of the released dyes by the dye-specific antibodies outside the liposomes during a time course. The fraction of dye release was normalized to that by detergent. The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes.


( D ) Digitonin permeabilized BMK Bax - / - / Bak - / - cells expressing SMAC - mCherry in the mitochondria intermembrane space ( 50 μL , 500 , 000 total cells ) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37 ° C in a 96 - well plate . The samples were centrifuged for 10 min and separated into supernatant and pellet fractions . SMAC - mCherry release was calculated as the fraction of total ( supernatant + pellet ) mCherry fluorescence coming from the supernatant fraction . The data was normalized to the percent SMAC - mCherry release of wt Bax and cBid at 60 min . Each symbol represents the normalized SMAC - mCherry release for one single replicate ( n = 3 or more independent replicates ) . Where applicable the data was fit with the [ Agonist ] vs . response - Variable slope equation in Graphpad Prism 8 . 0 . 1 . The dotted lines represent the 95 % confidence interval of the fit .

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(D) Digitonin permeabilized BMK Bax-/-/Bak-/- cells expressing SMAC-mCherry in the mitochondria intermembrane space (50 μL, 500,000 total cells) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37°C in a 96-well plate. The samples were centrifuged for 10 min and separated into supernatant and pellet fractions. SMAC-mCherry release was calculated as the fraction of total (supernatant + pellet) mCherry fluorescence coming from the supernatant fraction. The data was normalized to the percent SMAC-mCherry release of wt Bax and cBid at 60 min. Each symbol represents the normalized SMAC-mCherry release for one single replicate (n = 3 or more independent replicates). Where applicable the data was fit with the [Agonist] vs. response - Variable slope equation in Graphpad Prism 8.0.1. The dotted lines represent the 95% confidence interval of the fit.


Fig . 2 Identification of protein complexes and communities in the cellular extracts . ( A ) Elution of selected protein complexes as a function of their retention times ( see Appendix Fig . S4 for their corresponding subunit elutions ) .

Fig. 2 Identification of protein complexes and communities in the cellular extracts. (A) Elution of selected protein complexes as a function of their retention times (see Appendix Fig. S4 for their corresponding subunit elutions).


( B ) Scatter plot indicating discrepancies in the expected and measured molecular weights of 102 protein complexes that elute as a single peak ; 50 % of protein complexes are observed to have higher molecular weights than structurally characterized , indicating that they are organized in higher - order assemblies .

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(B) Scatter plot indicating discrepancies in the expected and measured molecular weights of 102 protein complexes that elute as a single peak; 50% of protein complexes are observed to have higher molecular weights than structurally characterized, indicating that they are organized in higher-order assemblies.


Fig . 4 Higher - order assemblies identified by proteome - wide cross - linking mass spectrometry , biomolecular modeling and negative - stain electron microscopy . ( A ) Distance distributions of identified cross - links on top of the modeled protein complexes and identification of novel interactions . Satisfied distances are shown in blue and over - length cross - links are shown in red .

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Fig. 4 Higher-order assemblies identified by proteome-wide cross-linking mass spectrometry, biomolecular modeling and negative-stain electron microscopy. (A) Distance distributions of identified cross-links on top of the modeled protein complexes and identification of novel interactions. Satisfied distances are shown in blue and over-length cross-links are shown in red.