Numbers of ASCL1 + in V - SVZ coronal sections 2 days after TAM treatment . n = 3 , * * p = 0 . 0087 . V - SVZ coronal sections stained for ASCL1 ( red ) nuclei ( blue ) . Scale bar : 50 μm .

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Numbers of ASCL1+ cells in V-SVZ coronal sections 2 days after TAM treatment. n=3, **p= 0.0087. V-SVZ coronal sections stained for ASCL1 (red) and nuclei (blue). Scale bar: 50 μm.


Densities ( / mm2 ) of BrdU + in OB granule layer ( GCL ) 2 days after TAM treatment . n = 4 ( Ctrl ) 3 ( Ccn1cKO ) , * * p = 0 . 0086 . OB sections stained for BrdU ( yellow ) nuclei ( blue ) . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB granule cell layer (GCL) 2 days after TAM treatment. n=4 (Ctrl) and 3 (Ccn1cKO), **p=0.0086. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.


Densities ( / mm2 ) of GFAP + Ki67 + in V - SVZ whole - mounts 4 after TAM treatment . Arrowheads point to dividing GFAP + . n = 4 , n . s . not significant . Scale bar : 10 μm .

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Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 4 weeks after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.


Numbers of ASCL1 + in V - SVZ coronal sections 4 after TAM treatment . n = 3 , n . s . not significant . Scale bar : 20 μm .

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Numbers of ASCL1+ cells in V-SVZ coronal sections 4 weeks after TAM treatment. n=3, n.s. not significant. Scale bar: 20 μm.


Densities ( / mm2 ) of BrdU + in OB GCL 4 after TAM treatment . n = 4 , n . s . not significant . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB GCL 4 weeks after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.


Densities ( / mm2 ) of GFAP + Ki67 + in V - SVZ whole - mounts 16 months after TAM treatment . Arrowheads point to dividing GFAP + . n = 4 , n . s . not significant . Scale bar : 10 μm .

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Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 16 months after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.


Densities ( / mm2 ) of BrdU + in OB GCL 16 months after TAM treatment . n = 4 , n . s . not significant . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB GCL 16 months after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.


Numbers of GFAP + BrdU + label - retaining NSCs in V - SVZ . Arrowheads point to label - retaining NSCs . n = 4 , * p = 0 . 039 . V - SVZ coronal sections stained for GFAP ( cyan ) BrdU ( red ) . Scale bar : 10 μm .

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Numbers of GFAP+BrdU+ label-retaining NSCs in the V-SVZ. Arrowheads point to label-retaining NSCs. n=4, *p= 0.039. V-SVZ coronal sections stained for GFAP (cyan) and BrdU (red). Scale bar: 10 μm.


Densities ( / mm2 ) of GFAP + Ki67 + in V - SVZ whole - mounts 24h after TMZ treatment . Arrowheads point to dividing NSCs under regenerative condition . n = 6 , * p = 0 . 0173 . Scale bar : 10 μm .

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Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=6, *p= 0.0173. Scale bar: 10 μm.


Densities ( / mm2 ) of DCX + neuroblasts in V - SVZ whole - mounts 3 days after TMZ treatment . n = 3 , * p = 0 . 0179 . V - SVZ whole - mounts stained for DCX . Scale bar : 500 μm .

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Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts 3 days after TMZ treatment. n=3, *p= 0.0179. V-SVZ whole-mounts stained for DCX. Scale bar: 500 μm.


Densities ( / mm2 ) of BrdU + in OB GCL labeled 24h after TMZ treatment . n = 5 ( Ctrl ) 6 ( Ccn1cKO ) , * p = 0 . 0484 . OB sections stained for BrdU ( yellow ) nuclei ( blue ) . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB GCL labeled 24h after TMZ treatment. n=5 (Ctrl) and 6 (Ccn1cKO), *p= 0.0484. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.


Densities ( / mm2 ) of GFAP + Ki67 + in V - SVZ whole - mounts of aged mice 24h after TMZ treatment . Arrowheads point to dividing NSCs under regenerative condition . n = 3 , * p = 0 . 0321 . Scale bar : 10 μm .

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Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts of aged mice 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=3, *p= 0.0321. Scale bar: 10 μm.


Densities ( / mm2 ) of DCX + neuroblasts in V - SVZ whole - mounts of aged mice 3 days after TMZ treatment . n = 3 , n . s . not significant . Scale bar : 500 μm .

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Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts of aged mice 3 days after TMZ treatment. n=3, n.s. not significant. Scale bar: 500 μm.


Densities ( / mm2 ) of BrdU + in OB GCL of aged mice labeled 3 days after TMZ treatment . n = 4 ( Ctrl ) 3 ( Ccn1cKO ) , n . s . not significant . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB GCL of aged mice labeled 3 days after TMZ treatment. n=4 (Ctrl) and 3 (Ccn1cKO), n.s. not significant. Scale bar: 50 μm.


Representative FACS plots showing gating strategy for of types in adult V - SVZ .

Representative FACS plots showing gating strategy for the analysis of cell types in the adult V-SVZ.


Total number of V - SVZ isolated from one mouse . n = 3 , n . s . not significant .

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Total number of V-SVZ cells isolated from one mouse. n=3, n.s. not significant.


Representative FACS plots showing aNSCs ( GFP + EGFR + ) , qNSCs ( GFP + EGFR - ) C ( GFP - EGFR + ) in adult V - SVZ .

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Representative FACS plots showing aNSCs (GFP+EGFR+), qNSCs (GFP+EGFR-) and C cells (GFP-EGFR+) in the adult V-SVZ.


of each population in adult V - SVZ . n = 3 , * * p = 0 . 0014 ( GFP + EGFR - ) , * * * p = 0 . 0002 ( GFP + EGFR + ) , * p = 0 . 0111 ( GFP - EGFR + ) , * * * p = 0 . 0006 ( GFP + ) , n . s . not significant .

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Percentage of each cell population in the adult V-SVZ. n=3, **p=0.0014 (GFP+EGFR-), ***p=0.0002 (GFP+EGFR+), *p=0.0111 (GFP-EGFR+), ***p=0.0006 (GFP+), n.s. not significant.


Densities ( / mm2 ) of VCAM1 + B1 in adult V - SVZ whole - mounts . n = 4 ( Ctrl + vehicle Ccn1cKO + vehicle ) 3 ( Ccn1cKO + erlotinib ) , * * p = 0 . 0021 ( Ccn1cKO + vehicle vs Ccn1cKO + erlotinib ) , * * p = 0 . 0014 ( Ccn1cKO + erlotinib vs Ctrl + vehicle ) , * * * * p < 0 . 0001 .

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Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), **p=0.0021 (Ccn1cKO + vehicle vs Ccn1cKO + erlotinib), **p=0.0014 (Ccn1cKO + erlotinib vs Ctrl + vehicle), ****p<0.0001.


Densities ( units / mm2 ) of NSC units in adult V - SVZ whole - mounts . n = 4 ( Ctrl + vehicle Ccn1cKO + vehicle ) 3 ( Ccn1cKO + erlotinib ) , * p = 0 . 017 , * * p = 0 . 0061 , * * * p = 0 . 0002 .

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Densities (units/mm2) of NSC units in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), *p=0.017, **p=0.0061, ***p=0.0002.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) VCAM1 ( ) . Scale bar : 50 μm .

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V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Scale bar: 50 μm.


A Neonatal rat ventricular myocytes ( NRVMs ) were infected with 3xMEF2 - Luc reporter , serum starved for 20 hours stimulated for 24 hours with different agonists : 100 nM endothelin - 1 ( ET1 ) , 1 μM sphingosine - 1 - phosphate ( S1P ) , 10 μM lisophosphatidic acid ( LPA ) , 1 μM WIN55 , 212 - 2 ( WIN55 , cannabinoid receptor agonist ) , 1 μM isoproterenol ( ISO , β - adrenergic receptor agonist ) , 100 nM angiotensin II ( AngII ) , 10 μM prostaglandin E1 ( PGE1 ) , 10 μM prostaglandin E2 ( PGE2 ) or 100 nM prostanoid F receptor agonist fluprostenol ( Flupro ) , 100 nM treprostinil ( Trepro , prostacyclin receptor agonist ) Data information : values are mean ± s . e . m . In ( A ) , experiment was performed in triplicates , similar results were obtained in 3 different experiments . Student ' s two - t - , * , P < 0 . 05 vs . control ( ET1 , P = 0 . 0013 ; S1P , P = 0 . 0144 ; LPA , P = 0 . 026 ; WIN55 , P = 0 . 6205 ; ISO = 0 . 0399 ; AngII , P = 0 . 5812 ; PGE1 , P = 0 . 001 ; PGE2 , P < 0 . 0001 ; Flupro , P = 0 . 0846 ; Trepro , P = 0 . 1958 )

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

A Neonatal rat ventricular myocytes (NRVMs) were infected with the 3xMEF2-Luc reporter, serum starved for 20 hours and stimulated for 24 hours with different agonists: 100 nM endothelin-1 (ET1), 1 μM sphingosine-1-phosphate (S1P), 10 μM lisophosphatidic acid (LPA), 1 μM WIN55,212-2 (WIN55, cannabinoid receptor agonist), 1 μM isoproterenol (ISO, β-adrenergic receptor agonist), 100 nM angiotensin II (AngII), 10 μM prostaglandin E1 (PGE1), 10 μM prostaglandin E2 (PGE2) or 100 nM prostanoid F receptor agonist fluprostenol (Flupro), 100 nM treprostinil (Trepro, prostacyclin receptor agonist) Data information: values are mean±s.e.m. In (A), the experiment was performed in triplicates, similar results were obtained in 3 different experiments. Student's two-tailed t-test, *,P<0.05 vs. control (ET1, P=0.0013; S1P, P=0.0144; LPA, P=0.026; WIN55, P=0.6205; ISO=0.0399; AngII, P=0.5812; PGE1, P=0.001; PGE2, P<0.0001; Flupro, P=0.0846; Trepro, P=0.1958)


B PGE2 induces expression of MEF2 target genes . NRVMs were serum - starved for 24 hours , then were stimulated with 1 μM PGE2 for 2 hours mRNA of indicated genes were determined Data information : values are mean ± s . e . m In ( B ) , n = 5 , technical replicates , Student ' s two - t - , * , P < 0 . 05 vs . control ( Nur77 , P = 0 . 0148 ; Myomaxin , P = 0 . 0417 ; Adamts1 , P = 0 . 0112 ; BNP , P = 0 . 0065 )

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

B PGE2 induces the expression of MEF2 target genes. NRVMs were serum-starved for 24 hours, then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information: values are mean±s.e.m In (B), n=5, technical replicates, Student's two-tailed t-test, *,P<0.05 vs. control (Nur77, P=0.0148; Myomaxin, P=0.0417; Adamts1, P=0.0112; BNP, P=0.0065)


C Correlation between MEF2 activation NRVM hypertrophy . MEF2 activity was measured as in ( A ) upon increasing of PGE2 . of NRVMs was determined after 48 - hour stimulation with indicated concentrations of PGE2 . Left : representative of α - actinin stained NRVMs stimulated with DMSO , 300 nM PGE2 , or 1 nM ET1 . Scale bar is 10 µm Data information : values are mean ± s . e . m In ( C ) , NRVM was determined from a minimum of 100 of 3 technical replicates upon different concentrations of PGE2 , MEF2 - activity was parallel assessed from 6 technical replicates . Correlation was statistically analyzed by calculating Pearson correlation coefficient ( Pearson r = 0 . 9626 , P = 0 . 0021 )

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

C Correlation between MEF2 activation and NRVM hypertrophy. MEF2 activity was measured as in (A) upon increasing concentration of PGE2. Cell size of NRVMs was determined after 48-hour stimulation with the indicated concentrations of PGE2. Left: representative images of α-actinin stained NRVMs stimulated with DMSO, 300 nM PGE2, or 1 nM ET1. Scale bar is 10 µm Data information: values are mean±s.e.m In (C), NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2, MEF2-activity was parallel assessed from 6 technical replicates. Correlation was statistically analyzed by calculating Pearson correlation coefficient (Pearson r=0.9626, P=0.0021)


D , PGE2 activates MEF2 via EP3 receptor . NRVMs were infected with 3xMEF2 - Luc reporter serum - starved for 20 hours . were stimulated with DMSO or 1 μM PGE2 for 24 hours in presence or absence of different EP receptor antagonists : AH6809 ( 10 μM , EP1 - EP2 - antagonist ) or 798106 ( 200 nM , EP3 - antagonist ) or L161 , 982 ( 2 μM , EP4 - antagonist ) Data information : values are mean ± s . e . m In ( D ) , n = 3 , independent experiments , * represents significant interaction between two treatments ( P < 0 . 05 , Two - Way ANOVA , AH6809 , P = 0 . 1092 ; L798106 , P = 0 . 0002 ; L161 , 982 , P = 0 . 5579 ) . exact n P values can be also found in Source Data excel file for Figure

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D, PGE2 activates MEF2 via EP3 receptor. NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists: AH6809 (10 μM, EP1- and EP2-antagonist) or 798106 (200 nM, EP3-antagonist) or L161,982 (2 μM, EP4-antagonist) Data information: values are mean±s.e.m In (D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, AH6809, P=0.1092; L798106, P=0.0002; L161,982, P=0.5579). The exact n and P values can be also found in the Source Data excel file for Figure 1


B - D ( B , D ) NRVMs were infected with 3xMEF2 - Luciferase reporter with recombinant adenoviruses encoding EGFP ( AdGFP ) , RGS16 , RGS2 , p63ΔN , RGS - LSCII , or Gαt , as indicated . were serum starved for 20 hours stimulated with DMSO or 1 μM PGE2 for 24 hours . ( C ) NRVMs were infected with 3xMEF2 - Luciferase reporter pretreated with adenylyl cyclase inhibitor SQ22536 ( 100 μM ) for 20 minutes or Gi / o - inhibitor pertussis toxin ( PTX , 100 ng / ml ) for 20 hours in serum - starved conditions treated with DMSO or 1 μM PGE2 for 24 hours . Data information : In ( B - D ) , n = 3 , independent experiments , * represents significant interaction between two treatments ( P < 0 . 05 , Two - Way ANOVA , RGS16 , P < 0 . 0001 ; RGS2 , P = 0 . 1725 ; p63ΔN , P = 0 . 0666 ; RGS - LSCII , P = 0 . 0818 , SQ22536 , P = 0 , 385 ; PTX , P < 0 . 0001 ; Gαt , P < 0 , 0001 ) , values are mean ± s . e . m . exact n P values can be also found in Source Data excel file for Figure 2

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

B-D (B, D) NRVMs were infected with the 3xMEF2-Luciferase reporter and with recombinant adenoviruses encoding EGFP (AdGFP), RGS16, RGS2, p63ΔN, RGS-LSCII, or Gαt, as indicated. The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours. (C) NRVMs were infected with the 3xMEF2-Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 (100 μM) for 20 minutes or the Gi/o-protein inhibitor pertussis toxin (PTX, 100 ng/ml) for 20 hours in serum-starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours. Data information: In (B-D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, RGS16, P<0.0001; RGS2, P=0.1725; p63ΔN, P=0.0666; RGS-LSCII, P=0.0818, SQ22536, P=0,385; PTX, P<0.0001; Gαt, P<0,0001), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 2


A , B NRVMs were infected with recombinant adenovirus encoding Flag - HDAC5 ( A ) or not infected ( B ) serum starved for 20 hours . were pretreated with PKC - inhibitor bisindolylmaleimide I ( 2 μM ) or PKD - inhibitor BPKDi ( 3 μM ) for 20 minutes stimulated with 100 nM endothelin - 1 ( ET1 ) or 100 μM phenylephrine ( PE ) or 1 μM PGE2 for 4 hours , as indicated . HDAC phosphorylation was detected by immunoblotting with anti - HDAC5 phospho - Ser - 498 antibody , phosphorylation of PKD was assessed using anti - phospho - PKD antibodies ( Ser - 744 / Ser - 748 Ser - 916 ) , representative are shown from 3 independent experiments

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

A,B NRVMs were infected with recombinant adenovirus encoding Flag-HDAC5 (A) or not infected (B) and serum starved for 20 hours. The cells were pretreated with the PKC-inhibitor bisindolylmaleimide I (2 μM) or the PKD-inhibitor BPKDi (3 μM) for 20 minutes and stimulated with 100 nM endothelin-1 (ET1) or 100 μM phenylephrine (PE) or 1 μM PGE2 for 4 hours, as indicated. HDAC phosphorylation was detected by immunoblotting with the anti-HDAC5 phospho-Ser-498 antibody, phosphorylation of PKD was assessed using anti-phospho-PKD antibodies (Ser-744/Ser-748 and Ser-916), representative images are shown from 3 independent experiments


C NRVMs were infected with 3xMEF2 - Luc reporter serum - starved for 20 hours . were pretreated with PKD - inhibitor BPKDi ( 3 μM ) or CamKII - inhibitor AIP ( 1 μM ) for 20 minutes stimulated with DMSO or 1 μM PGE2 for 24 hours . N = 3 , * represents significant interaction between two treatments ( P < 0 . 05 , Two - Way ANOVA , BPKDi , P < 0 . 0001 ; AIP , P = 0 . 2197 ) , values are mean ± s . e . m . exact n P values can be also found in Source Data excel file for Figure

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C NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were pretreated with the PKD-inhibitor BPKDi (3 μM) or the CamKII-inhibitor AIP (1 μM) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours. N=3, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, BPKDi, P<0.0001; AIP, P=0.2197), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 3


A NRVMs were serum - starved for 24 hours stimulated for 2 minutes with 10 μM PGE2 . Rac1 RhoA GTPase activation was determined by effector pulldown analyzed by immunoblot Data information : In ( A - C ) representative from at least 3 independent experiments are shown

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A NRVMs were serum-starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2. Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot Data information: In (A-C) representative images from at least 3 independent experiments are shown


B NRVMs were infected with recombinant adenoviruses encoding EGFP ( AdGFP ) or RGS16 24 hours prior to stimulation , then Rac1 activity was measure Data information : In ( A - C ) representative from at least 3 independent experiments are shown . In ( B - D ) values are mean ± s . e . m . , ( B , C ) n = 4 3 ; * , P < 0 . 05 ( Two - Way ANOVA , RGS16 , P = 0 . 0415 ( B ) , NSC effect on Rac1 activation , P = 0 . 0412 ( C ) ; NSC effect on MEF2 induction , P < 0 . 0001 . PGE2 effect was significant in Rac1 activation , P < 0 . 0001 ) . exact n P values can be also found in Source Data excel file for Figure

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B NRVMs were infected with recombinant adenoviruses encoding EGFP (AdGFP) or RGS16 24 hours prior to stimulation, then Rac1 activity was measure Data information: In (A-C) representative images from at least 3 independent experiments are shown. In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS16, P=0.0415 (B ), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4


C were pretreated with 100 ng / ml pertussis toxin ( PTX , Gi / o - inhibitor ) for 24 hours or with 50 μM NSC23766 ( NSC , Tiam1 - Rac1 interaction inhibitor ) for 2 hours . Thereafter Rac1 activation was determined by effector pulldown Data information : In ( A - C ) representative from at least 3 independent experiments are shown In ( B - D ) values are mean ± s . e . m . , ( B , C ) n = 4 3 ; * , P < 0 . 05 ( Two - Way ANOVA , RGS1 PTX , P = 0 . 039 ( C ) , NSC effect on Rac1 activation , P = 0 . 0412 ( C ) ; NSC effect on MEF2 induction , P < 0 . 0001 . PGE2 effect was significant in Rac1 activation , P < 0 . 0001 ) . exact n P values can be also found in Source Data excel file for Figure

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

C Cells were pretreated with 100 ng/ml pertussis toxin (PTX, Gi/o-inhibitor) for 24 hours or with 50 μM NSC23766 (NSC, Tiam1-Rac1 interaction inhibitor) for 2 hours. Thereafter Rac1 activation was determined by effector pulldown assay Data information: In (A-C) representative images from at least 3 independent experiments are shown In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS1 PTX, P=0.039 (C), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4


D NRVMs were transduced with 3xMEF2 - Luc reporter were serum - starved for 20 hours . After 2 - hour of 50 μM NSC23766 ( A ) pretreatments , were stimulated with DMSO or 1 μM PGE2 for 24 hours , then MEF2 activity was determined . NSC abolished MEF2 activation by PGE2 Data information In ( B - D ) values are mean ± s . e . m . ( D ) , n = 3 ; * , P < 0 . 05 ( Two - Way ANOVA ,

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D NRVMs were transduced with the 3xMEF2-Luc reporter and were serum-starved for 20 hours. After 2-hour of 50 μM NSC23766 (A) pretreatments, the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours, then MEF2 activity was determined. NSC abolished the MEF2 activation by PGE2 Data information In (B-D) values are mean±s.e.m. (D), n=3; *,P<0.05 (Two-Way ANOVA, RGS1


A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours stimulated with 1 μM PGE2 for 1 hour . Total phosphorylated PKD were analyzed by immunoblot . PKD was activated by PGE2 , but it was not affected by NSC Data information : In ( A - C ) representative from 3 independent experiments are shown

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A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour. Total and phosphorylated PKD levels were analyzed by immunoblot. PKD was activated by PGE2, but it was not affected by NSC Data information: In (A-C) representative images from 3 independent experiments are shown


B After infection with Flag - HDAC5 serum starvation , NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation . were stimulated with 1 μM PGE2 for 1 hour . HDAC5 phosphorylation at Ser - 498 was detected by immunoblot . phosphorylation of HDAC5 at this site was not prevented by NSC Data information : In ( A - C ) representative from 3 independent experiments are shown

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B After infection with Flag-HDAC5 and serum starvation, NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation. The cells were stimulated with 1 μM PGE2 for 1 hour. HDAC5 phosphorylation at Ser-498 was detected by immunoblot. The phosphorylation of HDAC5 at this site was not prevented by NSC Data information: In (A-C) representative images from 3 independent experiments are shown


C NRVMs neonatal cardiac fibroblasts ( CFs ) were serum - starved , pretreated with vehicle or 200 nM L798106 ( EP3 receptor antagonist ) for 20 minutes , were stimulated with 1 μM PGE2 for 10 minutes . PAK2 PKD phosphorylation was assessed by immunoblot . Calsequestrin was used as a myocyte - marker Data information : In ( A - C ) representative from 3 independent experiments are shown

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C NRVMs and neonatal cardiac fibroblasts (CFs) were serum-starved, pretreated with vehicle or 200 nM L798106 (EP3 receptor antagonist) for 20 minutes, and were stimulated with 1 μM PGE2 for 10 minutes. PAK2 and PKD phosphorylation was assessed by immunoblot. Calsequestrin was used as a myocyte-marker Data information: In (A-C) representative images from 3 independent experiments are shown


D MEF2 activity was determined in infected with 3xMEF2 - Luc reporter after serum starvation . were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1 - hour of 30 μM IPA - 3 pretreatment . IPA - 3 inhibited MEF2 activation by PGE2 Data information In ( D ) values are mean ± s . e . m . , n = 3 , biological replicates , * , P < 0 . 05 ( two - way ANOVA , P < 0 . 0001 ) . exact n P values can be also found in Source Data excel file for Figure

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D MEF2 activity was determined in cells infected with the 3xMEF2-Luc reporter after serum starvation. Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1-hour of 30 μM IPA-3 pretreatment. IPA-3 inhibited the MEF2 activation by PGE2 Data information In (D) values are mean±s.e.m., n=3, biological replicates, *,P<0.05 (two-way ANOVA, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 5


Top : GFP - HDAC5 infected NRVMs were serum - starved for a day , pretreated for two hours with indicated compounds ( BPKDi , 3 µM , IPA - 3 , 30 µM ) alone or in combination , then were stimulated for 4 hours . Representative . Scale bar is 10 µm . Bottom : Statistical of localization of HDAC5 . GFP - HDAC5 localization ( or ) was assessed in > 100 of each sample ( n = 3 , technical replicates ) . * , P < 0 , 05 ( one - way ANOVA with Bonferroni post - hoc , vehicle vs . ET1 or vs . PGE2 , P < 0 . 0001 ; PGE2 vs . PGE2 + BPKDi , P = 0 . 0004 ; PGE2 vs . PGE2 + BPKDi , P = 0 . 0114 ; PGE2 vs . PGE2 + BPKDi + IPA - 3 , P < 0 . 0001 ) . exact n P values can be also found in Source Data excel file for Figure 6

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

Top: GFP-HDAC5 infected NRVMs were serum-starved for a day, pretreated for two hours with the indicated compounds (BPKDi, 3 µM, IPA-3, 30 µM) alone or in combination, then were stimulated for 4 hours. Representative images. Scale bar is 10 µm. Bottom : Statistical analysis of cellular localization of HDAC5. GFP-HDAC5 localization (nuclear or cytosolic) was assessed in >100 cells of each sample (n=3, technical replicates). *, P<0,05 (one-way ANOVA with Bonferroni post-hoc test, vehicle vs. ET1 or vs. PGE2, P<0.0001; PGE2 vs. PGE2+BPKDi, P=0.0004; PGE2 vs. PGE2+BPKDi, P=0.0114; PGE2 vs. PGE2+BPKDi+IPA-3, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 6


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally were sacrificed after 24 hours A mRNA - of different inflammatory cytokines , as indicated . graphs show relative mRNA - , fold increase compared to saline - treated controls , normalized to 18s - content Data information : values are mean ± s . e . m . exact n values are shown in bottom of bar graphs . * , P < 0 . 05 , Welch ' s two - unpaired t - was used for statistical , n . s . = not significant P values : IL6 , P = 0 . 0007 ( A ) ; TNFα , P < 0 . 0001 ( A )

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MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours A mRNA-levels of different inflammatory cytokines, as indicated. The graphs show relative mRNA-levels, fold increase compared to saline-treated controls, normalized to 18s-content Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values: IL6, P=0.0007 (A); TNFα, P<0.0001 (A)


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally were sacrificed after 24 hours B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano - liquid chromatography tandem mass spectrometry Data information : values are mean ± s . e . m . exact n values are shown in bottom of bar graphs . * , P < 0 . 05 , Welch ' s two - unpaired t - was used for statistical , n . s . = not significant P values PGE2 , P = 0 . 0016 (

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MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano-liquid chromatography tandem mass spectrometry Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values PGE2, P=0.0016 (B


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally were sacrificed after 24 hours C Histologic macroscopic stainings of β - galactosidase - activity in MEF2 - lacZ reporter mice show MEF2 - activation ( blue precipitates , respectively ) in myocardium upon LPS - treatment . Saline - treated served as control . Scale bar of histological stainings is 100 µm . of whole - heart stainings is shown in right panel ( pixel in blue channel normalized to total ) Data information : values are mean ± s . e . m . exact n values are shown in bottom of bar graphs . * , P < 0 . 05 , Welch ' s two - unpaired t - was used for statistical , n . s . = not significant . In ( C ) representative of myocardial sections are shown from 3 independent experiments representative whole hearts of 6 independent experiments . P values blue pixel , P = 0 . 0083 ( C )

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MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours C Histologic and macroscopic stainings of β-galactosidase-activity in MEF2-lacZ reporter mice show MEF2-activation (blue cells and precipitates, respectively) in the myocardium upon LPS-treatment. Saline-treated littermates served as control. Scale bar of histological stainings is 100 µm. Quantification of whole-heart stainings is shown in the right panel (pixel intensity in blue channel normalized to total intensity) Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant. In (C) representative images of myocardial sections are shown from 3 independent experiments and representative whole hearts of 6 independent experiments. P values blue pixel intensity, P=0.0083 (C)


MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally were sacrificed after 24 hours D Induction of PKD , HDAC5 PAK2 in myocardial inflammations . Immunoblots were performed on extracts of hearts of saline - LPS - treated mice . Representative blots are shown in left panel , is in right panel Data information : values are mean ± s . e . m exact n values are shown in bottom of bar graphs . * , P < 0 . 05 , Welch ' s two - unpaired t - was used for statistical , n . s . = not significant P values ) ; phospho - PKD - Ser - 744 / Ser - 748 , P = 0 . 0049 ( D ) ; phospho - PKD - Ser - 916 , P = 0 . 2684 ( D ) ; phospho - HDAC5 - Ser - 498 ( D ) , P = 0 . 0019 ; phospho - HDAC5 - Ser - 259 , P = 0 . 0003 ( D ) ; phospho - PAK2 - Ser - 192 / Ser - 197 , P = 0 . 1962 ( D ) ; phospho - PAK2 - Thr - 402 , P = 0 . 0446 ( D ) . exact n P values can be also found in Source Data excel file for Figure

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

MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours D Induction of PKD, HDAC5 and PAK2 in myocardial inflammations. Immunoblots were performed on extracts of hearts of saline- and LPS-treated mice. Representative blots are shown in the left panel, the quantification is in the right panel Data information: values are mean±s.e.m The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values ); phospho-PKD-Ser-744/Ser-748, P=0.0049 (D); phospho-PKD-Ser-916, P=0.2684 (D); phospho-HDAC5-Ser-498 (D), P=0.0019; phospho-HDAC5-Ser-259, P=0.0003 (D); phospho-PAK2-Ser-192/Ser-197, P=0.1962 (D); phospho-PAK2-Thr-402, P=0.0446 (D). The exact n and P values can be also found in the Source Data excel file for Figure 7


A Isothermal calorimetry ( ITC ) assays using ADP - ribose ligand purified macrodomains of human macroH2A1 . 1 macroH2A2 .

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A Isothermal calorimetry (ITC) assays using ADP-ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2.


B In vitro PARP1 activity in presence of increasing concentrations ( 10 , 25 50 μM ) of purified macrodomains of human macroH2A1 . 1 , macroH2A1 . 1 G224E mutant macroH2A2 . PARP1 activity is assessed by its auto - PARylation detected by an anti - PAR immunoblot . naphtol blue shows increasing amounts of purified macrodomain added to each reaction . A representative blot of one of three independent experiments is shown .

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B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.


A diameter ( d ) of signaling of H2B tagged with photo - activatable GFP ( PAGFP - H2B ) is used to quantify expansion across 120 seconds after induction of damage using intercalating dye local irradiation with a .

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A The diameter (d) of the fluorescence signaling of H2B tagged with photo-activatable GFP (PAGFP-H2B) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser.


B expansion in absence presence of intercalating dye ( - + damage ) pretreatment of 1h with 1μM of Olaparib PARP inhibitor . Boxplots represent single measurement of expansion at 120 sec . post damage from 3 biological replicates with n > 30 each . box limits correspond to 25th 75th percentiles , bold indicates median cross indicates average values ( * , p < 0 . 05 using unpaired , two - Student ' s T - ) .

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B Chromatin expansion in absence and presence of intercalating DNA dye (- and + DNA damage) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage from 3 biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values (*, p < 0.05 using unpaired, two-tailed Student's T-test).


C Quantified expansion in not transfected ( control ) transfected with each mCherry - tagged histone ( as indicated ) . Data is represented as in B ( n > 30 , 3 bio . repl . ; * , p < 0 . 05 compared to mCherry - H2A - expressing using unpaired , two - Student ' s T - ) .

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C Quantified chromatin expansion in cells not transfected (control) and cells transfected with each mCherry-tagged histone (as indicated). Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 compared to mCherry-H2A-expressing cells using unpaired, two-tailed Student's T-test).


D expansion in Hela containing a doxocyclin ( doxo . ) - inducible macroH2A1 . 1 transgene . expansion was performed 48 hours after induction expression was controlled by anti - macroH2A1 . 1 - immunoblotting . Data is represented as in B ( n > 30 , 3 bio . repl . ; * , p < 0 . 05 using unpaired , two - Student ' s T - ) .

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D Chromatin expansion in Hela cells containing a doxocyclin (doxo.)-inducible macroH2A1.1 transgene. Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti-macroH2A1.1-immunoblotting. Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 using unpaired, two-tailed Student's T-test).


Quantified expansion in transfected with indicated histone constructs . Boxplots represent single measurement of expansion at 120 sec . post damage ( n > 30 , 3 bio . repl . ) . box limits correspond to 25th 75th percentiles , bold indicates median cross indicates average values . ( * , p < 0 . 05 using unpaired two - Student ' s T - comparing to H2A ; n . s . , not significant ) .

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Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant).


A Western blot showing expression of macroH2A in control HepG2 , knock - down for macroH2A1 macroH2A2 ( DKD ) DKD with ectopic expression of indicated YFP - tagged constructs . H3 is used as a loading control .

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A Western blot showing the expression of macroH2A proteins in control HepG2 cells, knock-down for macroH2A1 and macroH2A2 (DKD) and DKD with ectopic expression of the indicated YFP-tagged constructs. H3 is used as a loading control.


B H3K9me3 immunostaining in indicated HepG2 lines with counterstaining in DAPI . indicated areas are zoomed - in for a better observation of H3K9me3 signal profile . are maximum Z - projections of confocal stacks .

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B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed-in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z-projections of confocal stacks.


of box limits correspond to 25th 75th percentiles , bold indicates median cross indicates average values . ( * , p < 0 . 05 using unpaired two - Student ' s T - ; n . s . , not significant ) .

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Quantification of the nuclear area The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).


of H3K9me3 foci per nucleus , box limits correspond to 25th 75th percentiles , bold indicates median cross indicates average values . ( * , p < 0 . 05 using unpaired two - Student ' s T - ; n . s . , not significant ) .

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Quantification of H3K9me3 foci per nucleus, The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).


Wild - type , apg12 - 1 mutant Δapg12 were cultured in nitrogen - starvation medium containing 1 mM PMSF . After incubation for 6 h , were observed under a phase - contrast microscope . Arrows indicate autophagic bodies .

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Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies.


c , Wild - type ( squares ) Δapg12 ( circles ) were cultured in nitrogen - starvation medium their viability was determined by phloxine B .

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c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining.


d , of autophagic activity of wild - type Δapg12 by alkaline phosphatase ( ALP ) before ( black bars ) after ( bars ) nitrogen starvation for 4 h . Error bars indicate s . d . of three independent experiments .

d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments.


a , b , Lysates from Δapg12 carrying only vector or 3 × HA - APG12 ( a ) , Δapg5 , apg7 - 1 , apg10 - 1 Δapg1 carrying 3 × HA - Apg12 plasmid ( b ) were immunoblotted using anti - HA antibody . positions of 3 × HA - Apg12 larger product ( asterisks ) are indicated .

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a, b, Lysates from Δapg12 cells carrying only vector or 3 × HA-APG12 (a), and Δapg5, apg7-1, apg10-1 and Δapg1 cells carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody. The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated.


c , Immunoblot of wild - type Δapg12 harbouring HA - APG5 plasmid .

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c, Immunoblot analysis of wild-type and Δapg12 cells harbouring HA-APG5 plasmid.


d , Δapg5 Δapg12 were co - transformed with Myc - APG12 HA - APG5 . Their lysates were immunoprecipitated with anti - Myc or anti - HA antibodies detected by immunoblotting using anti - Myc antibody . position of crossreacting IgG heavy chain is

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d, Δapg5 Δapg12 cells were co-transformed with Myc-APG12 and HA-APG5. Their lysates were immunoprecipitated with anti-Myc or anti-HA antibodies and detected by immunoblotting using anti-Myc antibody. The position of the crossreacting IgG heavy chain is indicated


b , Δapg12 were transformed with mutant plasmids their lysates were immunoblotted with anti - HA antibody .

b, Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti-HA antibody.


c , Autophagic activity was measured as described for Fig . 1d .

c, Autophagic activity was measured as described for Fig. 1d.


d , Transport of pro - API to vacuole was examined by immunoblotting with anti - API antiserum . positions of pro - API mature API are indicated .

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d, Transport of pro-API to the vacuole was examined by immunoblotting with anti-API antiserum. The positions of pro-API and mature API are indicated.


Δapg5 were transformed with vector alone , wild - type APG5 or APG5K149R , then immunoblotted with anti - HA ( b )

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted with anti-HA (b)


Autophagic activity was determined by alkaline phosphatase ( c ) .

Autophagic activity was determined by alkaline phosphatase assay (c).


Δapg5 were transformed with vector alone , wild - type APG5 or APG5K149R , then immunoblotted anti - API ( d ) .

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted anti-API (d).


c , In vitro conjugation of Apg5 Apg12 . A lysate of Δapg12 carrying HA - APG5 ( lane 1 ) was incubated with an equal amount of lysate from Δapg5 carrying HA - APG12 ( 2 µ plasmid ) ( lane 2 ) at 30 ° C with ( lane 4 - 6 ) or without ( lane 7 ) 5 mM ATP . Samples were mixed with SDS - sample buffer at times indicated .

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c, In vitro conjugation of Apg5 and Apg12. A cell lysate of Δapg12 carrying HA-APG5 (lane 1) was incubated with an equal amount of lysate from Δapg5 carrying HA-APG12 (2 µ plasmid) (lane 2) at 30 °C with (lane 4-6) or without (lane 7) 5 mM ATP. Samples were mixed with SDS-sample buffer at the times indicated.


Spheroplasts were generated from expressing either HA - Apg12 or HA - Apg5 . Their lysates were mixed layered on top of a 10 - step ( 18 - 54 % w / w ) sucrose gradient , centrifuged at 174 , 000g for 2 . 5 h ( ref . 29 ) . Fifteen fractions were collected positions of free Apg5 , free Apg12 Apg5 / Apg12 conjugate were examined by western blotting . peak fractions of alcohol dehydrogenase ( ADH ) ( cytosol ) , ALP ( vacuole ) , Kex2 ( Golgi ) Sec12 ( endoplasmic reticulum ) are indicated by arrows .

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Spheroplasts were generated from cells expressing either HA-Apg12 or HA-Apg5. Their lysates were mixed and layered on top of a 10-step (18-54 % w/w) sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12 conjugate were examined by western blotting. The peak fractions of alcohol dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endoplasmic reticulum) are indicated by arrows.


A ) We identified previously unknown telomere regulators in fission yeast using haploid S . pombe whole - genome gene deletion library including ssu72 + ( SPAC3G9 . 04 ) . B ) Telomere of wt , ssu72Δ ssu72 - C13S ( point mutation at phosphatase active site ) strains were measured by Southern blot in ApaI digested genomic using a telomeric probe .

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A) We identified previously unknown telomere regulators in fission yeast using the haploid S. pombe whole-genome gene deletion library including ssu72+ (SPAC3G9.04). B) Telomere length of wt, ssu72Δ and ssu72-C13S (point mutation at the phosphatase active site) strains were measured by Southern blot in ApaI digested genomic DNA using a telomeric probe.


C ) Ssu72 recruitment to telomeres is cycle regulated . Ssu72 was myc - tagged at 3 ' end ChIP was carried out in cycle synchronized populations of cdc25ts strains . Septa formation was used proxy for S - phase . n ≥ 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to ssu72 + control samples . Error bars represent standard error of mean ( ) .

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C) Ssu72 recruitment to telomeres is cell cycle regulated. Ssu72 was myc-tagged at the 3' end and ChIP analysis was carried out in cell cycle synchronized populations of cdc25ts strains. Septa formation was used proxy for S-phase. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).


D ) Telomere of ssu72Δ is dependent on telomerase . Diploid strains with appropriate genotypes were sporulated trt1 ∆ ssu72 ∆ double mutants were streaked for multiple passages ( triangle indicates increased number of generations ) .

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D) Telomere length of ssu72Δ is dependent on telomerase. Diploid strains with the appropriate genotypes were sporulated and trt1∆ ssu72∆ double mutants were streaked for multiple passages (triangle indicates increased number of generations).


E ) Telomerase is recruited to telomeres in absence of Ssu72 . ChIP for Trt1 - myc in wt ssu72 ∆ was performed as described in material methods using a non - tagged as a control . n ≥ 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

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E) Telomerase is recruited to telomeres in the absence of Ssu72. ChIP analysis for Trt1-myc in wt and ssu72∆ was performed as described in material and methods using a non-tagged strain as a control. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


F ) Telomerase activator Ccq1 is hyperphosphorylated in ssu72 ∆ . rap1 ∆ were used as positive control for Ccq1 phosphorylation status . Western blots were performed using Ccq1 - flag tagged strains .

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F) Telomerase activator Ccq1 is hyperphosphorylated in ssu72∆ cells. rap1∆ cells were used as positive control for Ccq1 phosphorylation status. Western blots were performed using Ccq1-flag tagged strains.


A ) ssu72Δ telomeres present longer G - rich overhangs than wt rif1 ∆ . In - gel hybridization in native denaturing conditions was labelled with a radiolabelled C - rich telomere probe quantified for ssDNA at telomeres . n = 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

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A) ssu72Δ telomeres present longer G-rich overhangs than wt and rif1∆. In-gel hybridization in native and denaturing conditions was labelled with a radiolabelled C-rich telomere probe and quantified for ssDNA at the telomeres. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


B ) RPA ( Rad11 - GFP ) is enriched at ssu72 ∆ telomeres . Colocalization of Rad11 - GFP with Taz1 - mCherry , used as a telomere marker was performed in wt ssu72 ∆ ; n = 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) . More than 1000 were analyzed for each

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B) RPA (Rad11-GFP) is enriched at ssu72∆ telomeres. Colocalization of Rad11-GFP with Taz1-mCherry, used as a telomere marker was performed in wt and ssu72∆ cells; n =3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). More than 1000 cells were analyzed for each genotype


C ) ssu72 + controls telomere independently of rif1 + . Epistasis of telomere of ssu72 ∆ ssu72 - C13S ( catalytically inactive mutant ) with rif1 ∆ was performed by Southern blotting of ApaI digested using a telomeric probe . D ) ssu72 + stn1 + regulate telomere in same genetic pathway . Epistasis of ssu72 ∆ stn1 - 75 performed by Southern blotting of ApaI digested using a telomeric probe . Two independently generated ssu72 ∆ stn1 - 75 double mutants are shown .

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C) ssu72+ controls telomere length independently of rif1+. Epistasis analysis of telomere length of ssu72∆ and ssu72-C13S (catalytically inactive mutant) with rif1∆ was performed by Southern blotting of ApaI digested DNA using a telomeric probe. D) ssu72+ and stn1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and stn1-75 performed by Southern blotting of ApaI digested DNA using a telomeric probe. Two independently generated ssu72∆ stn1-75 double mutants are shown.


A ) Ssu72 is required for telomere recruitment of Stn1 in late S phase . ChIP of stn1 - myc in wt ssu72 ∆ was performed in synchronized cdc25ts . n ≥ 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to ssu72 + control samples . Error bars represent standard error of mean ( ) .

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A) Ssu72 is required for telomere recruitment of Stn1 in late S phase. ChIP analysis of stn1-myc in wt and ssu72∆ cells was performed in synchronized cdc25ts cells. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).


B ) 2D - gel of NsiI telomeric fragments of wt ssu72 ∆ strains . Smart ladder from Eurogentec was used for measurement .

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B) 2D-gel analysis of NsiI telomeric fragments of wt and ssu72∆ strains. Smart ladder from Eurogentec was used for DNA size measurement.


C ) Serine 74 substitution to a phosphomimetic aspartate acid ( stn1 - S74D ) is sufficient to confer ssu72 ∆ telomere defects . Telomere epistasis of ssu72 ∆ stn1 - S74D mutants were performed by Southern blotting of ApaI digested genomic using a telomeric probe .

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C) Serine 74 substitution to a phosphomimetic aspartate amino acid (stn1-S74D) is sufficient to confer ssu72∆ telomere defects. Telomere length epistasis analysis of ssu72∆ and stn1-S74D mutants were performed by Southern blotting of ApaI digested genomic DNA using a telomeric probe.


E ) Similar to ssu72 ∆ mutants , stn1 - S74D is defective in telomere recruitment . ChIP of stn1 - myc stn1 - S74D - myc using a non - tagged as a control . n = 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

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E) Similar to ssu72∆ mutants, stn1-S74D is defective in telomere recruitment. ChIP analysis of stn1-myc and stn1-S74D-myc using a non-tagged strain as a control. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


A ) ssu72 + pol1 + regulate telomere in same genetic pathway . Epistasis of ssu72 ∆ pol1 - 13 mutants was performed by Southern blotting of ApaI digested using a telomeric probe .

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A) ssu72+ and pol1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and pol1-13 mutants was performed by Southern blotting of ApaI digested DNA using a telomeric probe.


B ) Stn1 - Pol1 interaction requires Ssu72 phosphatase . Immunoprecipitation experiments of Pol1 - Flag with Stn1 - Myc was preformed both in wt ssu72 ∆ mutants . C ) Immunoprecipitation of Ten1 - Flag with Stn1 - Myc in either wt or ssu72 ∆ was performed as control for Stn1 - Ten1 integrity .

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B) Stn1-Pol1 interaction requires Ssu72 phosphatase. Immunoprecipitation experiments of Pol1-Flag with Stn1-Myc was preformed both in wt and ssu72∆ mutants. C) Immunoprecipitation of Ten1-Flag with Stn1-Myc in either wt or ssu72∆ was performed as control for Stn1-Ten1 complex integrity.


D ) Overexpression of polymerase alpha partially rescues telomere defect of ssu72Δ mutants . Multi - copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ .

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D) Overexpression of polymerase alpha partially rescues telomere length defect of ssu72Δ mutants. Multi-copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ cells.


A ) Telomere elongation of SSU72 down - regulated is telomerase dependent . Telomere Southern blot was carried out with HT1080 infected for 4 with lentiviral particles carrying two independent shRNAs against SSU72 ( CDS UTR regions ) Luciferase ( Luc ) as control for shRNA . Knockdown efficiencies were determined by RT - qPCR using specific primers against hSSU72 .

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A) Telomere elongation of SSU72 down-regulated cells is telomerase dependent. Telomere Southern blot was carried out with HT1080 cells infected for 4 weeks with lentiviral particles carrying two independent shRNAs against SSU72 (CDS and UTR regions) and Luciferase (Luc) as control for shRNA. Knockdown efficiencies were determined by RT-qPCR using specific primers against hSSU72.


B ) Telomere elongation of SSU72 down - regulated is epistatic with STN1 downregulation . TRF of HT1080 infected with appropriate retroviral or lentiviral particles carrying either GFP shRNA , SSU72 shRNA ( against UTR ) , STN1 shRNA or double SSU72 shRNA / STN1 shRNA . were grown for 4 was isolated to carry out Southern Blot .

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B) Telomere elongation of SSU72 down-regulated cells is epistatic with STN1 downregulation. TRF analysis of HT1080 cells infected with the appropriate retroviral or lentiviral particles carrying either GFP shRNA, SSU72 shRNA (against UTR), STN1 shRNA or double SSU72 shRNA/STN1 shRNA. Cells were grown for 4 weeks and DNA was isolated to carry out Southern Blot analysis.


C ) hSSU72 down - regulation results in multi - telomeric signals ( MTS ) that are dependent on replication . Visualization of mitotic spreads of HT1080 hSSU72 shRNA treated with aphidicolin colcemid . Telomere FISH was carried out using a PNA - telomere probe . MTS are represented by arrows . of MTS per metaphase : n = 4 ; * * p ≤ 0 . 01 * * * * p ≤ 0 . 0001 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

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C) hSSU72 down-regulation results in multi-telomeric signals (MTS) that are dependent on DNA replication. Visualization of mitotic spreads of HT1080 hSSU72 shRNA cells treated with aphidicolin and colcemid. Telomere FISH was carried out using a PNA-telomere probe. MTS are represented by arrows. Quantification of MTS per metaphase: n=4; **p ≤0.01 ****p ≤0.0001 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


A ) hSSU72 downregulation results in telomere damage foci ( TIF ) . with indicated shRNAs were fixed IF - FISH was performed using a 53BP1 antibody a PNA - telomere probe . ( Right ) of with more than 5 telomeric 53BP1 foci : n = 3 ; * * p ≤ 0 . 01 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

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A) hSSU72 downregulation results in telomere DNA damage foci (TIF). Cells with indicated shRNAs were fixed and IF-FISH analysis was performed using a 53BP1 antibody and a PNA-telomere probe. (Right) Quantification of cells with more than 5 telomeric 53BP1 foci: n=3; **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


B ) hSSU72 is required for efficient loading of hSTN1 at human telomeres . ChIP was preformed using anti - FLAG antibodies Southern blotting was performed using a human telomeric probe . of 3 independent ChIP experiments * p ≤ 0 . 05 * * p ≤ 0 . 01 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

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B) hSSU72 is required for efficient loading of hSTN1 at human telomeres. ChIP was preformed using anti-FLAG antibodies and Southern blotting was performed using a human telomeric probe. Quantification of 3 independent ChIP experiments *p ≤0.05 **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


A ) Volcano plot of proteome . Plotted is negative log10 of p - value ( y - axis ) versus log2 LFQ of SPPL2c overexpressing control ( x - axis ) of a given identified . A permutation FDR correction for multiple hypotheses was applied ( p = 0 . 05 ; s0 = 0 . 1 ; dashed ) . Open grey circles indicate not - significantly altered in SPPL2c expressing , red circles significantly altered after FDR correction . Filled red circles indicate type IV filled blue circles with red blue names type II altered in SPPL2c expressing , n = 6 biological repl . per .

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

A) Volcano plot of proteome analysis. Plotted is the negative log10 of the p-value (y-axis) versus the log2 LFQ ratio of SPPL2c overexpressing and control cells (x-axis) of a given identified protein. A permutation based FDR correction for multiple hypotheses was applied (p = 0.05; s0 = 0.1; dashed line). Open grey circles indicate proteins not-significantly altered in SPPL2c expressing cells, red circles significantly altered proteins after FDR correction. Filled red circles indicate type IV membrane proteins and filled blue circles with red line and blue names type II proteins altered in SPPL2c expressing cells, n=6 biological repl. per cell line.


C ) Western Blot of selected type IV SPPL2c candidate substrates . Endogenous of VAPA , VAPB , VAMP8 , Syntaxin 8 , 18 ( Stx8 & Stx18 ) Membrin ( encoded by gene Golgi SNAP Receptor Member 2 , GOSR2 ) were analyzed in HEK293 ectopically expressing wild type SPPL2c ( SPPL2c WT ) or a catalytically inactive SPPL2c ( SPPL2c D / A ) . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as controls . VAPA , VAPB , VAMP8 , Stx8 Membrin were decreased in SPPL2c expressing but not in control or SPPL2c D / A expressing . For Stx18 a cleavage product was detected in SPPL2c WT expressing . Expression of SPPL2c are depicted Calnexin serves as loading control .

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

C) Western Blot of selected type IV SPPL2c candidate substrates. Endogenous protein levels of VAPA, VAPB, VAMP8, Syntaxin 8, 18 (Stx8 & Stx18) and Membrin (encoded by the gene Golgi SNAP Receptor Complex Member 2, GOSR2) were analyzed in HEK293 cells ectopically expressing wild type SPPL2c (SPPL2c WT) or a catalytically inactive SPPL2c (SPPL2c D/A). Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. VAPA, VAPB, VAMP8, Stx8 and Membrin protein levels were decreased in SPPL2c expressing cells but not in control or SPPL2c D/A expressing cells. For Stx18 a cleavage product was detected in SPPL2c WT expressing cells. Expression levels of SPPL2c are depicted and Calnexin serves as loading control.


A ) SPPL2c overexpression causes a decrease on SPPL3 . Endogenous of SPPL3 were assessed in ectopically expressing wild type SPPL2c ( SPPL2c WT ) or catalytically inactive SPPL2c ( SPPL2c D / A ) by Western Blot . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as controls . Note that SPPL3 expression is reduced in expressing SPPL2c wt . Data information : Expression of ectopically expressed SPPL proteases are shown Calnexin serves as loading

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

A) SPPL2c overexpression causes a decrease on SPPL3 levels. Endogenous levels of SPPL3 were assessed in cells ectopically expressing wild type SPPL2c (SPPL2c WT) or catalytically inactive SPPL2c (SPPL2c D/A) by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. Note that SPPL3 expression is reduced in cells expressing SPPL2c wt. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading control


B ) SPPL2c impairs secretion maturation of glycan - modifying enzymes . of endogenous EXTL3 , B4GALT1 , OGFOD3 GnT - V were monitored in lysates of treated with control siRNA ( siCtr ) or with siRNA targeting SPPL3 ( siSPPL3 ) in ectopically expressing either SPPL2c or SPPL3 by Western Blot . In addition , secreted forms ( sEXTL3 , sB4GALT1 , sOGFOD3 , sGnTV , ) were analyzed in corresponding conditioned media using Western Blot . Note that SPPL2c reduces secretion of glycosyltransferases similar to SPPL3 knock down , but additionally induces a decrease of mature glycosyltransferases in lysate similar to ectopic expression of SPPL3 . Data information : Expression of ectopically expressed SPPL proteases are shown Calnexin serves as loading

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

B) SPPL2c impairs secretion and maturation of glycan-modifying enzymes. Protein levels of endogenous EXTL3, B4GALT1, OGFOD3 and GnT-V were monitored in lysates of cells treated with control siRNA (siCtr) or with siRNA targeting SPPL3 (siSPPL3) and in cells ectopically expressing either SPPL2c or SPPL3 by Western Blot. In addition, the secreted forms (sEXTL3, sB4GALT1, sOGFOD3, sGnTV,) were analyzed in the corresponding conditioned media using Western Blot. Note that SPPL2c reduces secretion of the glycosyltransferases similar to SPPL3 knock down, but additionally induces a decrease of the mature glycosyltransferases in the cell lysate similar to ectopic expression of SPPL3. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading control


C ) Processing of ectopically expressed GnT - V with an N - terminal Flag - C - terminal V5 - tag ( Flag - GnTV - V5 ) was analyzed in lysates conditioned media ( sup . ) of either SPPL2c wt or SPPL2c D / A expressing by Western Blot . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as controls . In addition , maturation of glycoproteins Nicastrin ( NCT ) , Lamp2 SPPL2a was monitored by detection of respective mature ( mat ) immature ( im ) version . Note that secretion of GnT - V is impaired in SPPL2c wt expressing immature glycoproteins accumulate . Data information : Expression of ectopically expressed SPPL proteases are shown Calnexin serves as loading

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

C) Processing of ectopically expressed GnT-V with an N-terminal Flag- and C-terminal V5-tag (Flag-GnTV-V5) was analyzed in lysates and conditioned media (sup.) of either SPPL2c wt or SPPL2c D/A expressing cells by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. In addition, maturation of the glycoproteins Nicastrin (NCT), Lamp2 and SPPL2a was monitored by detection of the respective mature (mat) and immature (im) version. Note that secretion of GnT-V is impaired in SPPL2c wt expressing cells and immature glycoproteins accumulate. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading control


D ) SPPL2c induces mislocalization of GnT - V . Ectopically expressed GnT - V was visualized by immunofluorescence using anti - V5 antibody either in control ( Ctr ) or in co - expressing either SPPL2c wt or SPPL2c D / A . ER was stained using anti - BiP antibody Golgi with anti - TGN46 antibody . While in control GnT - V mainly localizes to Golgi , its localization shifts to more ER - like structures in SPPL2c expressing . Scale bar 5 µm .

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

D) SPPL2c induces mislocalization of GnT-V. Ectopically expressed GnT-V was visualized by immunofluorescence staining using the anti-V5 antibody either in control cells (Ctr) or in cells co-expressing either SPPL2c wt or SPPL2c D/A. The ER was stained using the anti-BiP antibody and the Golgi with the anti-TGN46 antibody. While in control cells GnT-V mainly localizes to the Golgi, its localization shifts to more ER-like structures in SPPL2c expressing cells. Scale bar 5 µm.


of validated SPPL2c substrates VAPA ( A ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

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

Protein levels of the validated SPPL2c substrates VAPA (A) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates VAPB ( B ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

Protein levels of the validated SPPL2c substrates VAPB (B) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates VAMP8 ( C ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

Protein levels of the validated SPPL2c substrates VAMP8 (C) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates Syntaxin 8 ( Stx8 ) ( D ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

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

Protein levels of the validated SPPL2c substrates Syntaxin 8 (Stx8) (D) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates Syntaxin 18 ( Stx18 ) ( E ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

Protein levels of the validated SPPL2c substrates Syntaxin 18 (Stx18) (E) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates Membrin ( F ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

Protein levels of the validated SPPL2c substrates Membrin (F) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


B ) Processing of endogenous Syntaxin 5 ( Stx5 ) Syntaxin 6 ( Stx6 ) by SPPL2c wt by Western Blot . Non - transfected ( - ) , stably transfected but non - induced SPPL2c wt ( n / i ) induced stably expressing SPPL2c D / A served as controls . Results are derived from one experiment , thus expression ( SPPL2c ) loading ( Calnexin ) control are same . Note , that Syntaxin 5 is by SPPL2c while Syntaxin 6 is not .

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

B) Processing of endogenous Syntaxin 5 (Stx5) and Syntaxin 6 (Stx6) by SPPL2c wt by Western Blot. Non-transfected cells (-), stably transfected but non-induced SPPL2c wt cells (n/i) and induced cells stably expressing SPPL2c D/A served as controls. Results are derived from one experiment, thus expression (SPPL2c) and loading (Calnexin) control are the same. Note, that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not.


C ) Syntaxin 5 were down regulated processing maturation of ectopically expressed GnT - V , as well maturation of endogenous glycoproteins Nicastrin ( NCT ) SPPL2a was analyzed in Western Blot . Calnexin served as loading control ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody . While GnT - V Nicastrin maturation were similarly affected by Syntaxin 5 knock down SPPL2c overexpression , SPPL2a maturation was hardly impaired in Syntaxin 5 knock down .

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

C) Syntaxin 5 protein levels were down regulated and processing and maturation of ectopically expressed GnT-V, as well maturation of the endogenous glycoproteins Nicastrin (NCT) and SPPL2a was analyzed in Western Blot. Calnexin served as loading control and ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody. While GnT-V and Nicastrin maturation were similarly affected by Syntaxin 5 knock down and SPPL2c overexpression, SPPL2a maturation was hardly impaired in Syntaxin 5 knock down cells.


D ) Syntaxin 5 localization is impaired by overexpression of SPPL2c . Endogenous Syntaxin 5 was visualized in control without SPPL2c expression ( Ctr ) in overexpressing SPPL2c wt or SPPL2c D / A using immunofluorescence . BiP was used as a marker of ER TGN46 as Golgi marker . arrow indicating with disturbed Golgi Syntaxin 5 . Scale bar 5 µm .

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

D) Syntaxin 5 localization is impaired by overexpression of SPPL2c. Endogenous Syntaxin 5 was visualized in control cells without SPPL2c expression (Ctr) and in cells overexpressing SPPL2c wt or SPPL2c D/A using immunofluorescence. BiP was used as a marker protein of the ER and TGN46 as Golgi marker. White arrow indicating cells with disturbed Golgi and Syntaxin 5 staining. Scale bar 5 µm.


E ) Syntaxin 5 knock down induces mislocalization of GnT - V similar to SPPL2c overexpression ectopically expressing GnT - V were treated either with non - specific siRNA ( siCtr ) or with siRNA targeting Syntaxin 5 ( siStx5 ) . GnT - V was visualized in immunofluorescence using anti - V5 antibody targeting its C - terminal tag either BIP was co - stained as ER marker or TGN46 as Golgi marker . Scale bar 5 µm .

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

E) Syntaxin 5 knock down induces mislocalization of GnT-V similar to SPPL2c overexpression Cells ectopically expressing GnT-V were treated either with non-specific siRNA (siCtr) or with siRNA targeting Syntaxin 5 (siStx5). GnT-V was visualized in immunofluorescence using anti-V5 antibody targeting its C-terminal tag and either BIP was co-stained as ER marker protein or TGN46 as Golgi marker protein. Scale bar 5 µm.


After seeding were cultured for 72h expression of catalytically active ( wt ) or non - active ( D / A ) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated . Non - transfected ( Ctr ) or non - induced SPPL2c ( n / i ) served as controls . ER was stained with anti - BiP antibody , cis / medial - Golgi with anti - Giantin , trans - Golgi Network with anti - TGN46 antibody Golgi subcompartments with CAB45 - specific antibody in immunofluorescence . Note that only upon expression of catalytically active SPPL2c morphologies of ER cis - Golgi change compared to controls . Scale bar 5 µm .

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

After seeding cells were cultured for 72h and expression of catalytically active (wt) or non-active (D/A) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated. Non-transfected cells (Ctr) or non-induced SPPL2c cells (n/i) served as controls. The ER was stained with the anti-BiP antibody, the cis/medial-Golgi with the anti-Giantin, the trans-Golgi Network with the anti-TGN46 antibody and all Golgi subcompartments with the CAB45-specific antibody in immunofluorescence. Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis-Golgi change compared to controls. Scale bar 5 µm.


A ) Western Blot of SPPL2c in preparations from HEK293 inducibly overexpressing SPPL2c , human testis mouse testis tissue . For human mouse tissue , 5 μg of each total testis preparation is loaded . fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 is diluted 1 : 50 ( 0 . 1 μg total ) .

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

A) Western Blot analysis of SPPL2c in membrane preparations from HEK293 inducibly overexpressing SPPL2c, human testis and mouse testis tissue. For human and mouse tissue, 5 μg protein of each total testis preparation is loaded. The membrane fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 cells is diluted 1:50 (0.1 μg total protein).