( G , H ) Western blot ( G ) quantitative ( H ) of dystrophin expression in - wide from mdx mice treated with PMO at 12 . 5 mg / kg / week for 3 mixed with MOTS - c ( 500 μg ) intravenously ( n = 3 ; * p < 0 . 05 ) . 0 . 5 µg , 1 µg , 2 . 5 µg , 5 µg 10 µg total from C57BL / 6 50 µg of muscle samples from untreated treated mdx mice were loaded . α - actinin was used as loading control . Fold change refers to PMO - M relative to PMO alone .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O

(G, H) Western blot (G) and quantitative analysis (H) of dystrophin expression in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) intravenously (n=3; *p<0.05). 0.5 µg, 1 µg, 2.5 µg, 5 µg and 10 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. Fold change refers to PMO-M relative to PMO alone.


Immunohistochemistry of dystrophin - positive fibres in - wide from mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( scale bar : 100 μm ) ( * p < 0 . 05 , * * p < 0 . 001 ; two - t ) .

B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunohistochemistry of dystrophin-positive fibres in body-wide muscles from mdx mice treated with repeated doses of PMO-M (n=4) or PMO alone (n=3) (scale bar: 100 μm) (*p<0.05, **p<0.001; two-tailed t test).


quantitative of dystrophin - positive fibres in - wide from mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( scale bar : 100 μm ) ( * p < 0 . 05 , * * p < 0 . 001 ; two - t ) .

O O B-GENEPROD O O B-SUBCELLULAR O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

quantitative analysis of dystrophin-positive fibres in body-wide muscles from mdx mice treated with repeated doses of PMO-M (n=4) or PMO alone (n=3) (scale bar: 100 μm) (*p<0.05, **p<0.001; two-tailed t test).


( D ) Western blot for dystrophin expression in - wide from mdx mice treated with PMO - M or PMO alone . 0 . 5 µg , 2 . 5 µg , 5 µg , 15 µg total from C57BL / 6 50 µg of muscle samples from untreated treated mdx mice were loaded .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O 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-ORGANISM O O O

(D) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO alone. 0.5 µg, 2.5 µg, 5 µg, and 15 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded.


( E ) Quantitative of dystrophin expression in - wide from mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( * p < 0 . 05 ; two - t ) .

O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(E) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05; two-tailed t test).


( F ) Muscle function was assessed to determine physical improvement with grip strength for mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) , untreated mdx controls ( n = 3 ) C57BL / 6 ( n = 3 ) ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Muscle function was assessed to determine the physical improvement with grip strength test for mdx mice treated with PMO-M (n=4) or PMO alone (n=3), untreated mdx controls (n=3) and C57BL/6 (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).


( G ) Measurement of muscle endurance with running wheel for wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls ) . Muscle functional were conducted two after last injection .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(G) Measurement of muscle endurance with the running wheel test for wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). Muscle functional tests were conducted two weeks after last injection.


( A ) Measurement of serum creatine kinase ( CK ) in wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls ) .

O O O O O B-TISSUE B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O 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-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O 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) Measurement of serum creatine kinase (CK) levels in wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).


( B ) Re - localization of DAPC components in treated mdx mice to assess dystrophin function recovery of normal myoarchitecture ( scale bar : 100 μm ) . arrowheads point to identical myofibers . C57 means wild - type C57BL / 6 ; mdx represents untreated mdx controls .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

(B) Re-localization of DAPC components in treated mdx mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar: 100 μm). The arrowheads point to identical myofibers. C57 means wild-type C57BL/6; mdx represents untreated mdx controls.


( C ) Measurement of - changes of wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) .

O O O O O B-EXP_ASSAY O O O O O 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-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O

(C) Measurement of body-weight changes of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3).


( D ) Quantitative of centrally nucleated fibres ( CNFs ) in triceps quadriceps of wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO ( n = 3 ) ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls ) .

O O O O O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O O O O 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-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantitative analysis of centrally nucleated fibres (CNFs) in triceps and quadriceps of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman- Keuls test).


( E ) Measurement of cross - sectional ( CSA ) of muscle fibres from mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO ( n = 3 ) ( * p < 0 . 05 , two - t ) .

O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(E) Measurement of cross-sectional area (CSA) of muscle fibres from mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, two-tailed t test).


( A Measurement of serum indices including liver enzymes ( AST , ALT GGT ) from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) to reflect liver kidney functions ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls ) .

O O O O B-TISSUE O O B-TISSUE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O 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-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O

(A Measurement of serum indices including liver enzymes (AST, ALT and GGT) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).


B ) Measurement of kidney markers ( CREA UA ) from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) to reflect liver kidney functions ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls ) .

O O O O B-TISSUE O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O 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-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O

B) Measurement of kidney markers (CREA and UA) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).


( C ) Morphological examination of liver kidney from wild - type C57BL / 6 ( C57 ) , untreated mdx controls ( mdx ) treated mdx mice ( scale bar : 100 μm ) .

O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O

(C) Morphological examination of liver and kidney from wild-type C57BL/6 (C57), untreated mdx controls (mdx) and treated mdx mice (scale bar: 100 μm).


( D Immunohistochemistry of macrophages , T monocytes in quadriceps diaphragm from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) mdx mice treated with PMO - M ( n = 4 ) or PMO ( n = 3 ) ( scale bar : 100 μm ) . arrowheads point to CD68 + macrophages , CD3 + T or CD11b + monocytes ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls ) .

O O B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O O O O 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-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D Immunohistochemistry of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).


E ) of macrophages , T monocytes in quadriceps diaphragm from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) mdx mice treated with PMO - M ( n = 4 ) or PMO ( n = 3 ) ( scale bar : 100 μm ) . arrowheads point to CD68 + macrophages , CD3 + T or CD11b + monocytes ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls ) .

O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O O O O 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-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) quantification of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).


A . Endogenous localization of VAPA or VAPB Syt in hippocampal neurons ( DIV16 ) expressing GFP - Sec61β . Zooms represent ( 1 ) an axonal structure with presynaptic boutons ( arrowheads ) , ( 2 ) a dendritic structure . Scale bars : 10 µm ( full ) 5 µm ( zoom ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O

A. Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons (DIV16) expressing GFP-Sec61β. Zooms represent (1) an axonal structure with presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 µm (full size) and 5 µm (zoom).


C . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP pSuper empty vector or VAPA / B shRNAs . Yellow grey arrowheads mark presynaptic boutons with without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full ) 2 µm ( zoom ) .

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

C. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).


D . Quantifications of of internalized endogenous Syt ( B , C ) at single presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 288 - 541 boutons . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U .

O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N=2, n=288-541 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


E . Western blot of endogenous SCRN1 expression in indicated adult rat neuronal non - neuronal tissues . Cereb . , cerebellum . Hippoc . , hippocampus . Spin . , spinal .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O B-ORGANISM B-CELL_TYPE O O B-CELL_TYPE O O B-TISSUE O O B-TISSUE O B-TISSUE O O B-TISSUE O B-TISSUE O O B-TISSUE O

E. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal.


F . Pull down of HEK293T co - expressing Myc - VAPA with BioGFP or BioGFP - SCRN1 . G . Pull down of HEK293T co - expressing Myc - VAPB with BioGFP or BioGFP - SCRN1 . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Pull down assay of HEK293T cells co-expressing Myc-VAPA with BioGFP or BioGFP-SCRN1. G. Pull down assay of HEK293T cells co-expressing Myc-VAPB with BioGFP or BioGFP-SCRN1.Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


H . Scaled representation of SCRN1 - associated identified with pull - down followed by mass spectrometry of purified BioGFP or BioGFP - SCRN1 from HEK293T lysates with adult rat brain extracts . candidates showed > 10 enrichment of PSM compared to control .

O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O

H. Scaled representation of SCRN1-associated proteins identified with pull-down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP-SCRN1 from HEK293T cell lysates with adult rat brain extracts. All candidates showed >10 enrichment of PSM compared to control.


I . Endogenous localization of SCRN1 Syt in cortical neurons ( DIV18 ) expressing GFP . Zoom represents an axon structure with presynaptic boutons ( arrowheads ) . Scale bars : 10 µm ( full ) 5 µm ( zoom ) .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O

I. Endogenous localization of SCRN1 and Syt in cortical neurons (DIV18) expressing GFP. Zoom represents an axon structure with presynaptic boutons (arrowheads). Scale bars: 10 µm (full size) and 5 µm (zoom).


J . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 . Yellow grey arrowheads mark presynaptic boutons with without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full ) 2 µm ( zoom ) .

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

J. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).


K . Quantifications of of internalized endogenous Syt ( B , C ) at single presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP pSuper empty vector , SCRN1 shRNA alone or SCRN1 shRNA with GFP - SCRN1 . N = 2 , n = 201 - 300 boutons . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U .

O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA alone or SCRN1 shRNA with GFP-SCRN1. N=2, n=201-300 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


A . Localization of GFP - SCRN1 TagRFP - ER in hippocampal neurons ( DIV16 - 18 ) . Scale bars : 10 µm ( bottom panel , full ) 5 µm ( top panel ; bottom panel , zoom ) .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O 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. Localization of GFP-SCRN1 and TagRFP-ER in hippocampal neurons (DIV16-18). Scale bars: 10 µm (bottom panel, full size) and 5 µm (top panel; bottom panel, zoom).


B . Localization of GFP - SCRN1 ssRFP - KDEL in COS7 . Scale bars : 10 µm ( full ) 5 µm ( zoom ) .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O

B. Localization of GFP-SCRN1 and ssRFP-KDEL in COS7 cells. Scale bars: 10 µm (full size) and 5 µm (zoom).


C . Hippocampal neurons ( DIV16 ) co - expressing GFP - SCRN1 with HA - VAPA or HA - VAPB . Scale bars : 10 µm ( full ) 5 µm ( zoom ) .

O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

C. Hippocampal neurons (DIV16) co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).


D . COS7 co - expressing GFP - SCRN1 with HA - VAPA or HA - VAPB . Scale bars : 10 µm ( full ) 5 µm ( zoom ) .

O O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

D. COS7 cells co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).


F . COS7 co - expressing HA - VAPB with GFP - SCRN2 or GFP - SCRN3 . Scale bars : 10 µm ( full ) 5 µm ( zoom ) .

F. COS7 cells co-expressing HA-VAPB with GFP-SCRN2 or GFP-SCRN3. Scale bars: 10 µm (full size) and 5 µm (zoom).


B . COS7 co - expressing GFP - SCRN1 with HA - VAPB - K87D / M89D . Scale bars : 10 µm ( full ) 5 µm ( zoom ) .

O O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O

B. COS7 cells co-expressing GFP-SCRN1 with HA-VAPB-K87D/M89D. Scale bars: 10 µm (full size) and 5 µm (zoom).


C . COS7 co - expressing HA - VAPB with GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A . Scale bars : 10 µm ( full ) 5 µm ( zoom ) .

O O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

C. COS7 cells co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).


D . Hippocampal neurons ( DIV16 ) co - expressing HA - VAPB with GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A . Scale bars : 10 µm ( full ) 5 µm ( zoom ) .

O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

D. Hippocampal neurons (DIV16) co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).


E . Quantifications of SCRN1 recruitment to VAPB - positive structures in COS7 ( % ) . Left graph : co - expression of GFP - SCRN1 with HA - VAPB or HA - VAPB - K87D / M89D ( N = 2 - 3 , n = 44 - 46 ) . Right graph : co - expression of HA - VAPB with GFP or GFP - SCRN1 , GFP - SCRN1 - N , GFP - SCRN1 - C , GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A ( N = 2 - 3 , n = 41 - 64 ) . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Chi - square with post - hoc including Bonferroni correction .

O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Quantifications of SCRN1 recruitment to VAPB-positive structures in COS7 cells (%). Left graph: co-expression of GFP-SCRN1 with HA-VAPB or HA-VAPB-K87D/M89D ( N=2-3, n= 44-46). Right graph: co-expression of HA-VAPB with GFP or GFP-SCRN1, GFP-SCRN1-N, GFP-SCRN1-C, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A (N=2-3, n=41-64). Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Chi-square test with post-hoc analysis including Bonferroni correction.


F . Pull down of HEK293T co - expressing Myc - VAPB with GFP , GFP - SCRN1 , GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O

F. Pull down of HEK293T cells co-expressing Myc-VAPB with GFP, GFP-SCRN1, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A.


B . Live hippocampal neurons ( DIV17 - 18 ) co - expressing TagRFP - ER with GFP , GFP - SCRN1 , GFP - SCRN1 - F402A , SCRN1 shRNA or VAPA / B shRNAs . Scale bars : 5 µm ( full ) 2 µm ( zoom ) .

O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O

B. Live hippocampal neurons (DIV17-18) co-expressing TagRFP-ER with GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Scale bars: 5 µm (full size) and 2 µm (zoom).


C . Time - lapse of TagRFP - ER dynamics in hippocampal neurons ( DIV16 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A , SCRN1 shRNA or VAPA / B shRNAs . Intact ER structures ( arrowheads ) , intact dynamic ER structures ( arrowheads ) impaired an non - dynamic ER structures ( arrows ) are indicated . Scale bars : 5 µm ( full ) 2 µm ( zoom ) .

O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

C. Time-lapse of TagRFP-ER dynamics in hippocampal neurons (DIV16-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Intact ER structures (dark arrowheads), intact and dynamic ER structures (light arrowheads) and impaired an non-dynamic ER structures (arrows) are indicated. Scale bars: 5 µm (full size) and 2 µm (zoom).


E . ER nanostructures visualized with GFP - Sec61β in axons of hippocampal neurons ( DIV18 ) immunostained for α - Tubulin co - expressed with pSuper empty vector , SCRN1 shRNA or VAPA / B shRNAs , subjected to expansion microscopy . Scale bars : 2 µm ( full ) 500 nm ( zoom ) .

O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

E. ER nanostructures visualized with GFP-Sec61β in axons of hippocampal neurons (DIV18) immunostained for α-Tubulin and co-expressed with pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs, and subjected to expansion microscopy. Scale bars: 2 µm (full size) and 500 nm (zoom).


F . FRAP experiment of TagRFP - ER in hippocampal neurons ( DIV17 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A . Scale bar : 2 µm .

O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O

F. FRAP experiment of TagRFP-ER in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. Scale bar: 2 µm.


G . Average normalized TagRFP - ER recovery after photo - bleaching in hippocampal neurons ( DIV17 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A . N = 2 , n = 9 neurons . H . Normalized TagRFP - ER recovery after photo - bleaching at T = 60 sec in hippocampal neurons ( DIV17 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A . N = 2 , n = 9 neurons . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U .

O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. H. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


I . Average normalized TagRFP - ER recovery after photo - bleaching in hippocampal neurons ( DIV18 ) co - expressing pSuper empty vector , SCRN1 shRNA , VAPA / B shRNAs . N = 4 , n = 6 - 12 neurons . J . Normalized TagRFP - ER recovery after photo - bleaching at T = 60 sec in hippocampal neurons ( DIV18 ) co - expressing pSuper empty vector , SCRN1 shRNA , VAPA / B shRNAs . N = 4 , n = 6 - 12 neurons . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U .

O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. J. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


A . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 - F402A . Yellow grey arrowheads mark presynaptic boutons with without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full ) 2 µm ( zoom ) . B . Quantifications of of internalized endogenous Syt at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 - F402A . N = 2 , n = 201 - 300 boutons . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U .

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O 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. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1-F402A. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom). B. Quantifications of fluorescence intensity of internalized endogenous Syt at individual presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1-F402A. N=2, n=201-300 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


C . Representative time - lapse of GCaMP6f upon electric field stimulation ( 50 APs , 20 Hz ) in axons of hippocampal neurons ( DIV21 ) co - expressed with RIM1a - mCherry to visualize presynaptic sites ( arrowheads ) . Scale bar : 5 µm .

O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

C. Representative time-lapse of cytosolic GCaMP6f upon electric field stimulation (50 APs, 20 Hz) in axons of hippocampal neurons (DIV21) co-expressed with RIM1a-mCherry to visualize presynaptic sites (arrowheads). Scale bar: 5 µm.


D . Time - lapses of GCaMP6f upon electric field stimulation ( 50 APs , 20 Hz ) in axons of hippocampal neurons ( DIV21 ) co - expressing pSuper control , SCRN1 shRNA or VAPA / B shRNAs . Arrowheads mark presynaptic boutons . Scale bar : 5 µm .

O O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

D. Time-lapses of cytosolic GCaMP6f upon electric field stimulation (50 APs, 20 Hz) in axons of hippocampal neurons (DIV21) co-expressing pSuper control, SCRN1 shRNA or VAPA/B shRNAs. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.


E . Average normalized response of GCaMP6f at presynaptic boutons upon stimulation ( 50 APs , 20 Hz ) in hippocampal neurons ( DIV21 ) co - expressing pSuper empty vector , SCRN1 shRNA or VAPA / B shRNAs . N = 5 - 6 , n = 15 - 27 . F . Average normalized peak response of GCaMP6f at presynaptic boutons upon stimulation ( 50 APs , 20 Hz ) in hippocampal neurons ( DIV21 ) co - expressing pSuper empty vector , SCRN1 shRNA or VAPA / B shRNAs . N = 5 - 6 , n = 15 - 27 . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U .

O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Average normalized response of GCaMP6f fluorescent intensity at presynaptic boutons upon stimulation (50 APs, 20 Hz) in hippocampal neurons (DIV21) co-expressing pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs. N=5-6, n=15-27. F. Average normalized peak response of GCaMP6f at presynaptic boutons upon stimulation (50 APs, 20 Hz) in hippocampal neurons (DIV21) co-expressing pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs. N=5-6, n=15-27. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


G . Representative time - lapse of GCaMP6f before ( F0 ) after ( Fmax ) ionomycin treatment at axons of hippocampal neurons ( DIV18 ) co - expressed with mRFP pSuper empty vector or VAPA / B shRNAs . Arrowheads mark presynaptic boutons . Scale bar : 5 µm .

O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

G. Representative time-lapse of cytosolic GCaMP6f before (F0) and after (Fmax) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressed with mRFP and pSuper empty vector or VAPA/B shRNAs. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.


H . Basal GCaMP6f ( F ) normalized to maximum GCaMP6f ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing mRFP with pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 47 - 50 . I . Average basal GCaMP6f ( F0 ) normalized to max GCaMP6f ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing mRFP with pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 47 - 50 . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U .

O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Basal GCaMP6f fluorescence (F) normalized to the maximum GCaMP6f fluorescence (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N=2, n=47-50. I. Average basal GCaMP6f fluorescence (F0) normalized to the max GCaMP6f fluorescence intensity (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N=2, n=47-50. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


J . Representative time - lapse of R - GECO1 before ( F0 ) after ( Fmax ) ionomycin treatment at axons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . Arrowheads mark presynaptic boutons . Scale bar : 5 µm .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

J. Representative time-lapse of cytosolic R-GECO1 before (F0) and after (Fmax) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.


K . Basal R - GECO1 ( F ) normalized to maximum R - GECO1 ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . N = 3 , n = 70 - 89 . L . Average basal R - GECO1 ( F0 ) normalized to maximum R - GECO1 ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . N = 3 , n = 70 - 89 . Data information : Data represent Mean ± ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Basal R-GECO1 fluorescence (F) normalized to the maximum R-GECO1 fluorescence (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. L. Average basal R-GECO1 fluorescence (F0) normalized to the maximum R-GECO1 fluorescence intensity (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


B . Flow cytometry for Bu - 1a expression in wild type with ( GAA ) n tracts of different knocked into BU - 1A locus ( in blue ) . DT40 are heterozygous carry one BU - 1A one BU - 1B allele . experiments introducing repeats into BU - 1A are carried out in in which + 3 . 5 G4 has been deleted from both A B alleles , to avoid transvection between allele Black outline : positive control ( wild type ) ; red outline : negative control ( carrying a genetic disruption of BU - 1 )

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O

B. Flow cytometry for Bu-1a expression in wild type cells with (GAA)n tracts of different length knocked into the BU-1A locus (in blue). DT40 cells are heterozygous and carry one BU-1A and one BU-1B allele. All experiments introducing repeats into BU-1A are carried out in cells in which the +3.5 G4 has been deleted from both A and B alleles, to avoid transvection between the allele Black outline: positive control (wild type cells); red outline: negative control (cells carrying a genetic disruption of BU-1)


C , D . Bu - 1a fluctuation of wildtype primpol in which endogenous + 3 . 5 G4 has been deleted ( ΔG4 ) or with ( GAA ) 10 ( GAA ) 20 orientated such that it is replicated as leading ( C ) or lagging ( D ) strand template for a fork entering from 3 ' end of locus as shown in panel ( A ) . At least two independent fluctuation analyses were performed . Circles represent of Bu - 1a loss variants in at least 24 clones from these experiments , with mean ± SD reported . * * * * p < 0 . 0001 , * p ≤ 0 . 05 , ns = not significant ; one - way ANOVA .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Bu-1a fluctuation analysis of wildtype and primpol cells in which the endogenous +3.5 G4 has been deleted (ΔG4) or with (GAA)10 and (GAA)20 sequence orientated such that it is replicated as the leading (C) or lagging (D) strand template for a fork entering from the 3' end of the locus as shown in panel (A). At least two independent fluctuation analyses were performed. Circles represent the percentage of Bu-1a loss variants in at least 24 individual clones from these experiments, with mean ± SD reported. ****p < 0.0001, * p ≤ 0.05, ns = not significant; one-way ANOVA.


Human PrimPol , or mutants , tagged with YFP were expressed in primpol harbouring ( GAA ) 10 in BU - 1A locus . Bu - 1a YFP double positive were sorted expanded for 2 , then analysed for Bu - 1a expression variants . For each complementation , at least two independently derived clones were subjected to fluctuation . As previously observe expression of hPrimPol [ AxA ] hPrimPol [ ZfKO ] is deleterious unstable . expressing these mutations remaining YFP - positive at end of expansion period will have been through fewer divisions other lines in this . Pooled results from at least three independent fluctuation analyses are represented with mean ± SD indicated with red bar whiskers . Statistical significance : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p ≤ 0 . 05 , ns = not significant ; Kruskal - Wallis

B-ORGANISM B-GENEPROD O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human PrimPol, or mutants, tagged with YFP were expressed in primpol cells harbouring (GAA)10 sequence in the BU-1A locus. Bu-1a and YFP double positive cells were sorted and expanded for 2 weeks, and then analysed for Bu-1a expression variants. For each complementation, at least two independently derived clones were subjected to fluctuation analysis. As previously observe expression of hPrimPol[AxA] and hPrimPol[ZfKO] is deleterious and unstable. Cells expressing these mutations and remaining YFP-positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis. Pooled results from at least three independent fluctuation analyses are represented with mean ±SD indicated with red bar and whiskers. Statistical significance: **** p < 0.0001, *** p<0.001, * p ≤ 0.05, ns = not significant; Kruskal-Wallis test


A . DRIP - qPCR reveals accumulation of R - loops across BU - 1 locus in primpol . DRIP signal was calculated as enrichment over RNase H - treated samples was normalised to - 0 . 5 kb amplicon . mean SD for three biological replicates is presented . An unpaired t - was used to compare differences between matched amplicons in primpol BU - 1A ( GAA ) 10 other lines indicated . * * * * p ≤ 0 . 0001 , ns = not

O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

A. DRIP-qPCR analysis reveals accumulation of R-loops across the BU-1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H-treated samples and was normalised to -0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t-test was used to compare differences between matched amplicons in primpol BU-1A(GAA)10 and the other cell lines indicated. **** p ≤ 0.0001, ns = not significant


B . : hybrids in primpol BU - 1A ( GAA ) 10 : GgRNase H primpol BU - 1A ( GAA ) 10 : hPrimPol . An unpaired t - on three biological replicates was used to compare differences to primpol BU - 1A ( GAA ) 10 for each matched amplicon . bar represent mean whiskers SD . * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , ns = not

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O 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. DNA:RNA hybrids in primpol BU-1A(GAA)10:GgRNase H and primpol BU-1A(GAA)10:hPrimPol. An unpaired t-test on three biological replicates was used to compare differences to primpol BU-1A(GAA)10 for each matched amplicon. The bar represent the mean and whiskers the SD. *** p ≤ 0.001, ** p ≤ 0.01, ns = not significant


C . Overexpression of chicken RNase H1 prevents ( GAA ) 10 induced BU - 1A epigenetic instability in primpol . Fluctuation was performed on three primpol BU - 1A ( GAA ) 10 clones . One - way ANOVA was used to calculate significance of differences in BU - 1 instability between primpol BU - 1A ΔG4 other lines . * * * * p ≤ 0 . 0001 , ns = not

O O O O B-ORGANISM B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O

C. Overexpression of chicken RNase H1 prevents (GAA)10 induced BU-1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU-1A (GAA)10 clones. One-way ANOVA was used to calculate the significance of differences in BU-1 instability between primpol BU-1A ΔG4 and other cell lines. **** p ≤ 0.0001, ns = not significant


D . Diagram of RNase H1 hybrid binding domain ( HBD ) - mCherry fusion flow cytometry expression profiles of construct in four

O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

D. Diagram of the RNase H1 hybrid binding domain (HBD) - mCherry fusion and flow cytometry expression profiles of the construct in four clones


E . R - loop stabilisation induces epigenetic instability of BU - 1 . Bu - 1a fluctuation of wild type expressing HBD - mCherry . scatter plots pool results from at least two different clones with matched HBD - expression . Mean ± SD reported . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , ns = not significant ; one - way ANOVA .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. R-loop stabilisation induces epigenetic instability of BU-1. Bu-1a fluctuation analysis of wild type cells expressing HBD-mCherry. The scatter plots pool results from at least two different clones with matched HBD-expression. Mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant; one-way ANOVA.


A . Expression of geminin - tagged chicken RNase H1 - YFP . Phases of cycle were determined by content in live by Hoechst 33342 ( X - axis ) . RNase H1 - YFP with or without geminin degron is detected on Y - axis . RNase H1 - YFP - geminin degron is degraded in G1 . In contrast , RNase H1 - YFP remain stable irrespective of phase of cycle . 2n 4n indicate chromosome number before after

O O B-EXP_ASSAY O B-GENEPROD O O B-ORGANISM B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O

A . Expression of geminin-tagged chicken RNase H1-YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 (X-axis). RNase H1-YFP with or without the geminin degron protein is detected on the Y-axis. The RNase H1-YFP-geminin degron is degraded in G1. In contrast, RNase H1-YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication


B . Bu - 1a fluctuation of two independently derived primpol BU - 1A ( GAA ) 10 : Gg RNase H1 - YFP - geminin degron clones . Since expression of RNase H1 - YFP - geminin degron construct is not stable ( unlike RNase H1 - YFP construct without degron ) , Bu - 1a expression was assessed separately in YFP + ve YFP - ve within each clone . Statistical differences calculated Kruskal - Wallis . For panels , at least 36 clones were analysed ; mean ± SD reported . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , ns = not

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-ORGANISM B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bu-1a fluctuation analysis of two independently derived primpol BU-1A(GAA)10: Gg RNase H1-YFP-geminin degron clones. Since the expression of the RNase H1-YFP-geminin degron construct is not stable (unlike the RNase H1-YFP construct without the degron), Bu-1a expression was assessed separately in the YFP +ve and YFP -ve cells within each clone. Statistical differences calculated the Kruskal-Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant


C . DRIP - qPCR for R - loops around engineered + 3 . 5 ( GAA ) 10 repeat in BU - 1 in different phases of cycle . location of qPCR amplicons is indicated in map at top of panel . BU - 1 DRIP signal was normalised to - 0 . 5 kb amplicon in G1 - arrested ( t = 0 h ) Black : wild type ; red : primpol . Error bars = SD . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 .

O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O 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-GENEPROD O O O O O O O O O O O O O O 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. DRIP-qPCR for R-loops around the engineered +3.5 (GAA)10 repeat in BU-1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU-1 DRIP signal was normalised to -0.5 kb amplicon in G1-arrested cells (t = 0 h) Black: wild type; red: primpol. Error bars = SD. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05


E . Validation of of nascent : hybrid formation in BU - 1 locus . Enrichment of 4SU labelled moiety of : hybrids was calculated relative to input in three independent asynchronous wild type ( black ) or primpol ( red ) , with or without exogenous RNase H treatment . Error bars = SD . * * p ≤ 0 . 01 , * p ≤ 0 . 05 , ns = not significant ; unpaired t -

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Validation of analysis of nascent DNA:RNA hybrid formation in BU-1 locus. Enrichment of 4SU labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** p ≤ 0.01, * p ≤ 0.05, ns = not significant; unpaired t-test


G . Newly synthesised R - loops in BU - 1 during S phase in wild type ( black ) primpol ( red ) . Error bars represent 1SD of three biological repeats of experiment . * * * p ≤ 0 . 001 , * p ≤ 0 . 05 ; unpaired t -

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Newly synthesised R-loops in BU-1 during S phase in wild type (black) and primpol (red). Error bars represent 1SD of three biological repeats of the experiment. *** p ≤ 0.001, * p ≤ 0.05; unpaired t-test


A . Representative normalised DRIP - seq data in two genes COL22A1 , spanning over 200 kb , MYC . locations of H - G4 motifs are shown below gene map . Wild type in blue ; primpol in red . corresponding RNAse H - treated samples are dashed . See Methods for further details of graphic generation . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative normalised DRIP-seq data in two genes COL22A1, spanning over 200 kb, and MYC. The locations of H-DNA and G4 motifs are shown below the gene map. Wild type in blue; primpol in red. The corresponding RNAse H-treated samples are dashed. See Methods for further details of graphic generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


C . DRIP peak heights in wild type primpol DT40 normalised to Drosophila S2 spike - in . n ( wild type ) = 41445 ; n ( primpol ) = 48648 . D . Correlation of normalised DRIP peak heights in overlapping peaks between wild type primpol . Blue = 1 : 1 correlation ; red = linear regression through data . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O B-EXP_ASSAY O O O O O B-GENEPROD B-CELL_LINE O O B-ORGANISM B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike-in. n (wild type) = 41445; n (primpol) = 48648. D. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


E . Correlation between H - forming sequences genes ( bar ) , genes with DRIP peaks in wild type ( blue ) primpol ( red ) . F . Normalised DRIP peak heights in genes identified as associating with H - . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). F. Normalised DRIP peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


G . Correlation between G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) genes ( bar ) , genes with DRIP peaks in wild type ( blue ) primpol ( red ) . H . Normalised DRIP peak heights in genes identified as associating with G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). H. Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


A . Representative normalised DIP - seq data in SKI locus . Wild type in blue ; primpol in red . locations of H - G4 motifs are shown below gene map . corresponding RNAse H - treated samples are dashed . Since so little material was recovered following RNase H treatment , samples were pooled prior to library generation . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative normalised RNA DIP-seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H-DNA and G4 motifs are shown below the gene map. The corresponding RNAse H-treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


C . DIP - seq peak heights in wild type primpol BOBSC normalised to a DT40 spike - in . n ( wild type ) = 32740 ; n ( primpol ) = 33721 . D . Correlation of normalised DIP - seq peak heights in overlapping peaks between wild type primpol . Blue = 1 : 1 correlation ; red = linear regression through data . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RNA DIP-seq peak heights in wild type and primpol BOBSC cells normalised to a DT40 spike-in. n (wild type) = 32740; n (primpol) = 33721. D. Correlation of normalised RNA DIP-seq peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


E . Correlation between H - forming sequences genes ( bar ) , genes with DRIP peaks in wild type ( blue ) primpol ( red ) . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


F . Correlation between G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) genes ( bar ) , genes with DRIP peaks in wild type ( blue ) primpol ( red ) . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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F. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


G . Normalised DIP - seq peak heights in genes identified as associating with H - . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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G. Normalised RNA DIP-seq peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


H . Normalised DIP - seq peak heights in genes identified as associating with G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) . Data information : P values calculated with Mann - Whitney U . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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H. Normalised RNA DIP-seq peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


A Immunohistochemistry was used to assess STAMP2 expression in normal malignant human prostate specimens . Tissue microarrays ( TMAs ) with normal prostate ( n = 27 ) early - stage ( Gleason grade < 7 ) ( n = 35 ) or late - stage prostatetumors ( Gleason grade ≥ 7 ) ( n = 149 ) were subjected to immunohistochemistry as described in Materials Methods . Representative are shown . B of cytosolicSTAMP2 in TMAs is shown in ( A ) . One - way of variance ( ANOVA ) with a post hoc , * P < 0 . 0001 . Error bars indicate . C , D details of ( C ) ( D ) STAMP2 in TMAs described in ( A ) .

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A Immunohistochemistry was used to assess STAMP2 expression in normal and malignant human prostate specimens. Tissue microarrays (TMAs) with normal prostate (n = 27) and early-stage (Gleason grade < 7) (n = 35) or late-stage prostatetumors (Gleason grade ≥ 7) (n = 149) were subjected to immunohistochemistry as described in Materials and Methods. Representative images are shown.B Quantification of cytosolicSTAMP2 staining in TMAs is shown in (A). One-way analysis of variance (ANOVA) with a post hoc test, *P < 0.0001. Error bars indicate SEM.C, D The details of cytosolic (C) and membrane (D) STAMP2 staining in the TMAs described in (A).


A LNCaP were transfected with either control siRNA or two independent STAMP2 siRNAs , fractions of were prepared followed by Western blotting . STAMP1 is used as a loading control .

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A LNCaP cells were transfected with either control siRNA or two independent STAMP2 siRNAs, and membrane fractions of the cells were prepared followed by Western blotting analysis. STAMP1 is used as a loading control.


B LNCaP were cultured in RPMI 1640 medium containing 10 % CT - FBS treated with or without 1 nM R1881 for 24 h before being transfected with indicated siRNAs . were then cultured for indicated times , growth was measured by Counting Kit - 8 . n = 3 , * P < 0 . 0001 . Error bars indicate SD .

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B LNCaP cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h before being transfected with the indicated siRNAs. The cells were then cultured for the indicated times, and cell growth was measured by Cell Counting Kit-8. n = 3, *P < 0.0001. Error bars indicate SD.


C LNCaP were cultured transfected with indicated siRNAs as described in ( B ) . After transfection , were cultured for 10 days . colonies formed were stained photographed .

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C LNCaP cells were cultured and transfected with the indicated siRNAs as described in (B). After transfection, the cells were cultured for 10 days. The colonies formed were stained and photographed.


D LNCaP stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881 . were then collected , fractions were prepared followed by Western blot .

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D LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881. The cells were then collected, and membrane fractions were prepared followed by Western blot analysis.


E LNCaP stably expressing shRNA against STAMP2 or control shRNA were cultured for indicated times , numbers were determined using a hemocytometer . n = 3 , * P = 0 . 047 ; * * P = 0 . 0046 . Error bars indicate SD .

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E LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured for the indicated times, and the cell numbers were determined using a hemocytometer. n = 3, *P = 0.047; **P = 0.0046. Error bars indicate SD.


F LNCaP stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 as described in Materials Methods . plates were then stained photographed . G of data from ( F ) . n = 3 , * P = 0 . 022 . Error bars indicate SD .

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F LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were cultured in soft agar for 2 weeks as described in Materials and Methods. The plates were then stained and photographed.G Quantification of data from (F). n = 3, *P = 0.022. Error bars indicate SD.


H LNCaP stably expressing shRNA against STAMP2 or control shRNA were subcutaneously implanted into SCID mice . was measured after 8 . n = 9 , * P = 0 . 037 . Error bars indicate .

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H LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were subcutaneously implanted into male SCID mice. Tumor size was measured after 8 weeks. n = 9, *P = 0.037. Error bars indicate SEM.


I VCaP were transfected with indicated siRNA as described in ( B ) . After transfection , VCaP were cultured for 2 . colonies formed were stained photographed . J of data from ( I ) . n = 3 , * P < 0 . 0001 . Error bars indicate SD .

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I VCaP cells were transfected with the indicated siRNA as described in (B). After transfection, VCaP cells were cultured for 2 weeks. The colonies formed were stained and photographed.J Quantification of the data from (I). n = 3, *P < 0.0001. Error bars indicate SD.


A LNCaP were transfected with either control or STAMP2 - specific siRNA . Two days after transfection , were subjected to cycle as described in Materials Methods . Representative histograms showing PI - stained are shown . B proportion of in each stage of cycle from experiment in ( A ) is presented . Student ' s t - was performed to analyze statistical significance , n = 3 . * P = 0 . 0002 ; * * P = 0 . 0056 ; * * * P = 0 . 0099 . Error bars indicate SD .

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A LNCaP cells were transfected with either control or STAMP2-specific siRNA. Two days after transfection, cells were subjected to cell cycle analysis as described in Materials and Methods. Representative histograms showing PI-stained cells are shown.B The proportion of cells in each stage of the cell cycle from the experiment in (A) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.0002; **P = 0.0056; ***P = 0.0099. Error bars indicate SD.


C Two days after transfection with indicated siRNAs , LNCaP were harvested lysates were prepared subjected to Western blot with indicated antisera . Representative blots of three independent experiments are shown .

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C Two days after transfection with the indicated siRNAs, LNCaP cells were harvested and cell lysates were prepared and subjected to Western blot analysis with the indicated antisera. Representative blots of three independent experiments are shown.


D LNCaP were transfected with either control or STAMP2 - specific siRNA . Three days after transfection , were treated with either 50 ng / ml TRAIL or 20 μmol / l LY294002 ( LY ) for 24 h , or both agents for 6 h , then subjected to TUNEL / FACS . Representative histograms of TUNEL - stained are shown . FL1 - H refers to gating of for TUNEL with measurement . E extent of apoptosis from experiment in ( D ) is presented . Student ' s t - was performed to analyze statistical significance , n = 3 . * P = 0 . 027 ; * * P = 0 . 0058 ; * * * P = 0 . 0016 . Error bars indicate SD .

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D LNCaP cells were transfected with either control or STAMP2-specific siRNA. Three days after transfection, cells were treated with either 50 ng/ml TRAIL or 20 μmol/l LY294002 (LY) for 24 h, or both agents for 6 h, and then subjected to TUNEL/FACS analysis. Representative histograms of TUNEL-stained cells are shown. FL1-H refers to the gating of the cells for the TUNEL staining with fluorescence measurement.E The extent of apoptosis from the experiment in (D) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.027; **P = 0.0058; ***P = 0.0016. Error bars indicate SD.


F LNCaP were transfected with either control or two independent STAMP2 - specific siRNAs , ST2 - 1 ST2 - 2 . Three days after transfection , were treated in same way as in ( D ) . Whole - lysates were prepared subjected to Western blot with indicated antisera .

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F LNCaP cells were transfected with either control or two independent STAMP2-specific siRNAs, ST2-1 and ST2-2. Three days after transfection, cells were treated in the same way as in (D). Whole-cell lysates were prepared and subjected to Western blot analysis with the indicated antisera.


G LNCaP stably expressing either control or STAMP2 vector were treated in same way as in ( D ) . lysates were prepared subjected to Western blot with indicated antibodies .

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G LNCaP cells stably expressing either control or STAMP2 vector were treated in the same way as in (D). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies.


A STAMP2 expression was determined by of a neoadjuvant hormone therapy ( NHT ) TMA containing samples from hormone naïve ( untreated ) ( n = 28 ) , NHT 1 - 6 months ( n = 31 ) , NHT 7 - 12 months ( n = 41 ) , with PSA recurrence ( n = 21 ) as indicated . Representative are presented . B Quantitative presentation of data from ( A ) . Unt , untreated ; Recur , PSA recurrence . Kruskal - Wallis was used for significance . * P = 0 . 0499 . Error bars indicate .

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A STAMP2 expression was determined by IHC of a neoadjuvant hormone therapy (NHT) TMA containing samples from hormone naïve (untreated) (n = 28), NHT 1-6 months (n = 31), NHT 7-12 months (n = 41), and patients with PSA recurrence (n = 21) as indicated. Representative images are presented.B Quantitative presentation of the data from (A). Unt, untreated; Recur, PSA recurrence. The Kruskal-Wallis test was used for significance analysis. *P = 0.0499. Error bars indicate SEM.


C Boxplots of STAMP2 expression in normal prostate tissue , prostate cancer , metastatic CRPC were analyzed in two independent GEO datasets ( GSE6919 GSE35988 ) . values of samples are presented as dots . Thick horizontal lines represent median , with box representing upper lower quartile . whiskers represent 5th 95th percentiles . statistical significance , indicated in tables at bottom , was determined by Student ' s t - .

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C Boxplots of STAMP2 expression levels in normal prostate tissue, primary prostate cancer, and metastatic CRPC were analyzed in two independent GEO datasets (GSE6919 and GSE35988). The values of individual samples are presented as dots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles. The statistical significance, indicated in the tables at the bottom, was determined by Student's t-test.


D CWR22 xenografts were grown in nude mice , tumors were collected at different times after castration . mRNA was extracted from tumors used for qPCR of STAMP2 expression . results are presented as boxplots . Thick horizontal lines represent median , with box representing upper lower quartile . whiskers represent 5th 95th percentiles , outlier is presented as an open circle . statistical significance was determined by one - way ANOVA with a post hoc . n = 3 in group week 0 , 1 , 2 , 4 ; n = 5 in refractory group . * P = 0 . 011 ; * * P = 0 . 049 . Error bars indicate .

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D CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. mRNA was extracted from the tumors and used for qPCR analysis of STAMP2 expression. The results are presented as boxplots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles, and the outlier is presented as an open circle. The statistical significance was determined by one-way ANOVA with a post hoc test. n = 3 in group week 0, 1, 2, and 4; n = 5 in refractory group. *P = 0.011; **P = 0.049. Error bars indicate SEM.


A 22Rv1 were cultured in RPMI 1640 medium containing 10 % CT - FBS treated with or without 1 nM R1881 for 24 h were then transfected with indicated siRNAs . were then cultured for 10 days . colonies formed were stained photographed . B of data from ( A ) . Student ' s t - was performed to analyze statistical significance , n = 3 . * P = 0 . 0003 ; * * P < 0 . 0001 . Error bars indicate SD .

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A 22Rv1 cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h and were then transfected with the indicated siRNAs. The cells were then cultured for 10 days. The colonies formed were stained and photographed.B Quantification of data from (A). Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.0003; **P < 0.0001. Error bars indicate SD.


C 22Rv1 from Supplementary Fig S2 were subcutaneously implanted into both flanks of nude mice ( six mice per group ) . was measured at indicated time points . Student ' s t - was performed to analyze statistical significance , n = 10 . * P = 0 . 037 ; * * P = 0 . 039 . Error bars indicate . D Representative tumors from ( C ) right after resection at final time point are shown .

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C 22Rv1 cells from Supplementary Fig S2 were subcutaneously implanted into both flanks of male nude mice (six mice per group). Tumor size was measured at the indicated time points. Student's t-test was performed to analyze the statistical significance, n = 10. *P = 0.037; **P = 0.039. Error bars indicate SEM.D Representative tumors from (C) right after resection at the final time point are shown.


A LNCaP were transfected with either control or STAMP2 - specific siRNA in presence of 10 − 8 M R1881 . was isolated , qPCR was used to determine ATF4mRNA . Student ' s t - was used to analyze statistical significance , n = 3 . * P = 0 . 002 . Error bars indicate SD .

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A LNCaP cells were transfected with either control or STAMP2-specific siRNA in the presence of 10−8 M R1881. RNA was isolated, and qPCR was used to determine ATF4mRNA levels. Student's t-test was used to analyze the statistical significance, n = 3. *P = 0.002. Error bars indicate SD.


B lysates from ( A ) were subjected to Western blot .

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B Cell lysates from (A) were subjected to Western blot analysis.


C , D ATF4 expression in LNCaP ( C ) 22Rv1 ( D ) stably expressing control shRNA or shRNA against STAMP2 was analyzed by Western blot .

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C, D ATF4 expression in LNCaP (C) and 22Rv1 (D) cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by Western blot analysis.


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E ATF4 expression in xenografted tumors of 22Rv1 stably expressing control shRNA or shRNA against STAMP2 was analyzed by qPCR . Student ' s t - was used to analyze statistical significance , n = 4 . * P = 0 . 022 ; * * P = 0 . 015 ; * * * P = 0 . 044 . Error bars indicate .

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E ATF4 expression in xenografted tumors of 22Rv1 cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by qPCR. Student's t-test was used to analyze the statistical significance, n = 4. *P = 0.022; **P = 0.015; ***P = 0.044. Error bars indicate SEM.


F CWR22 xenografts were grown in nude mice , tumors were collected at different times after castration . association between ATF4 STAMP2 in samples was determined by Western blot .

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F CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. The association between ATF4 and STAMP2 in the tumor samples was determined by Western blot analysis.


G MSKCC Prostate Oncogenome cDNA microarray dataset was obtained from cBio Cancer Genomics Portal . was performed as described in Materials Methods . expression of STAMP2 ATF4 , as well as two ATF4 target genes ( ASNS SLC7A11 ) , are presented .

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G The MSKCC Prostate Oncogenome cDNA microarray dataset was obtained from the cBio Cancer Genomics Portal. Analysis was performed as described in Materials and Methods. The expression levels of STAMP2 and ATF4, as well as two ATF4 target genes (ASNS and SLC7A11), are presented.


A LNCaP were transfected with either control or ATF4 siRNA . Two days after transfection , were harvested whole - lysates were made used in Western blot .

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A LNCaP cells were transfected with either control or ATF4 siRNA. Two days after transfection, cells were harvested and whole-cell lysates were made and used in Western blot analysis.


B LNCaP were transfected with either control or ATF4 siRNA were cultured for 2 . colonies formed were stained photographed . C covered by colonies on each plate in ( B ) was quantified represented as of total of plate . * P = 0 . 0023 .

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B LNCaP cells were transfected with either control or ATF4 siRNA and were cultured for 2 weeks. The colonies formed were stained and photographed.C The area covered by the colonies on each plate in (B) was quantified and represented as percentage of the total area of the plate. *P = 0.0023.


D lysates of LNCaP stably expressing an empty vector ( Vector ) or a vector expressing ATF4 ( ATF4 ) were prepared subjected to Western blot with indicated antisera .

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D Cell lysates of LNCaP cells stably expressing an empty vector (Vector) or a vector expressing ATF4 (ATF4) were prepared and subjected to Western blot analysis with the indicated antisera.


E After transfection with indicated siRNAs , from ( D ) were cultured for 2 . colonies formed were stained photographed . F of colonies in ( E ) was quantitated using an image system represented as of total of plate . * P = 0 . 024 .

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E After transfection with the indicated siRNAs, cells from (D) were cultured for 2 weeks. The colonies formed were stained and photographed.F The area of the colonies in (E) was quantitated using an image analysis system and represented as percentage of the total area of the plate. *P = 0.024.


G LNCaP were transfected with either control ( Ctrl ) or ASNS siRNA . were then cultured for 2 . colonies formed were stained photographed . Knockdown of ASNS was confirmed by Western blot shown at top . H of data shown in ( G ) . * P = 0 . 0087 .

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G LNCaP cells were transfected with either control (Ctrl) or ASNS siRNA. The cells were then cultured for 2 weeks. The colonies formed were stained and photographed. Knockdown of ASNS was confirmed by Western blot analysis shown at the top.H Quantification of the data shown in (G). *P = 0.0087.


A 293T were transfected with plasmids expressing either an empty vector ( Vec ) or vectors expressing HA - tagged STEAP , STAMP1 ( ST1 ) , STAMP2 ( ST2 ) , or STAMP3 ( ST3 ) . Whole - extracts were prepared subjected to Western blot using anti - HA antibody or GAPDH as a loading control .

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A 293T cells were transfected with plasmids expressing either an empty vector (Vec) or vectors expressing HA-tagged STEAP, STAMP1 (ST1), STAMP2 (ST2), or STAMP3 (ST3). Whole-cell extracts were prepared and subjected to Western blot analysis using anti-HA antibody or GAPDH as a loading control.


B Relative ferrireductase activities were measured in expressing different constructs used in ( A ) as described in Materials Methods . * P < 0 . 0001 .

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B Relative ferrireductase activities were measured in the cells expressing the different constructs used in (A) as described in Materials and Methods. *P < 0.0001.


C 293T were transfected with different plasmids expressing either an empty vector ( Vec ) or vectors expressing His - tagged wild - type STAMP2 ( WT ) or three STAMP2 mutants ( dGSR , H304L , or H397L ) as indicated . Whole - extracts were prepared used in Western blot with anti - His antibody or GAPDH antibody as a loading control .

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C 293T cells were transfected with different plasmids expressing either an empty vector (Vec) or vectors expressing His-tagged wild-type STAMP2 (WT) or three STAMP2 mutants (dGSR, H304L, or H397L) as indicated. Whole-cell extracts were prepared and used in Western blot analysis with anti-His antibody or GAPDH antibody as a loading control.


D Relative ferrireductase activities were measured in from ( C ) as described in Materials Methods . * P = 0 . 0003 .

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D Relative ferrireductase activities were measured in the cells from (C) as described in Materials and Methods. *P = 0.0003.


E 293T with Dox - inducible STAMP2 expression or vector control was generated . Western blot confirmed STAMP2 expression in a Dox - inducible manner .

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E 293T cell line with Dox-inducible STAMP2 expression or vector control was generated. Western blot analysis confirmed STAMP2 expression in a Dox-inducible manner.


F from ( E ) were either left untreated or treated with increasing amounts of Dox , ferrireductase activity was determined . * P < 0 . 0001 .

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F The cells from (E) were either left untreated or treated with increasing amounts of Dox, and ferrireductase activity was determined. *P < 0.0001.


G with Dox - inducible STAMP2 expression from ( E ) were either left untreated or treated with Dox for 48 h . Prior to ferrireductase activity measurement , were treated with or without DPI as indicated for 1 h . * P < 0 . 0001 ; * * P = 0 . 0012 ; * * * P = 0 . 048 .

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G The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Prior to ferrireductase activity measurement, the cells were treated with or without DPI as indicated for 1 h. *P < 0.0001; **P = 0.0012; ***P = 0.048.


H from ( E ) were treated with 100 ng / ml Dox for 2 days . Oxidative stress was then measured by NBT , stained were photographed . There were equal numbers of on plates for − / + Dox as shown in Supplementary Fig S4 .

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H The cells from (E) were treated with 100 ng/ml Dox for 2 days. Oxidative stress was then measured by NBT staining, and the stained cells were photographed. There were equal numbers of cells on the plates for −/+ Dox as shown in Supplementary Fig S4.


I with Dox - inducible STAMP2 expression from ( E ) were either left untreated or treated with Dox for 48 h . Then , were treated with or without 10 μM DPI before being subjected to NBT . * P < 0 . 0001 .

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I The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Then, the cells were treated with or without 10 μM DPI before being subjected to NBT staining. *P < 0.0001.


A LNCaP stably expressing an empty vector ( LN - Vec ) or a vector expressing STAMP2 ( LN - ST2 ) were established by lentivirus delivery . lysates were prepared subjected to Western blot with indicated antisera .

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A LNCaP cells stably expressing an empty vector (LN-Vec) or a vector expressing STAMP2 (LN-ST2) were established by lentivirus delivery. Cell lysates were prepared and subjected to Western blot analysis with the indicated antisera.


B Relative ferrireductase activities were measured in described in ( A ) . * P = 0 .

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B Relative ferrireductase activities were measured in the cells described in (A). *P = 0.0003


C NBT was performed in LNCaP stably expressing either an empty vector ( LN - Vec ) or a vector expressing wild - type STAMP2 ( LN - ST2 / WT ) or a STAMP2 mutant ( LN - ST2 / dGSR ) as indicated . stained were then photographed quantified . * P = 0 . 004 .

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C NBT staining was performed in LNCaP cells stably expressing either an empty vector (LN-Vec) or a vector expressing wild-type STAMP2 (LN-ST2/WT) or a STAMP2 mutant (LN-ST2/dGSR) as indicated. The stained cells were then photographed and quantified. *P = 0.004.


D LNCaP were transfected with either scrambled siRNA ( siCtrl ) or siRNA against STAMP2 ( siST2 ) were cultured in presence of 10 nM R1881 or vehicle for 2 days . Intracellular ROS were then measured by CellROX reagent . * P = 0 . 002 . ns , not significant .

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D LNCaP cells were transfected with either scrambled siRNA (siCtrl) or siRNA against STAMP2 (siST2) and were cultured in the presence of 10 nM R1881 or vehicle for 2 days. Intracellular ROS levels were then measured by CellROX reagent staining. *P = 0.002. ns, not significant.


E LNCaP stably expressing an empty vector ( Vec ) , a vector expressing wild - type STAMP2 ( WT ) , or a STAMP2 mutant ( dGSR ) were cultured , NADPH / NADP + was determined as described in Materials Methods . * P = 0 . 015 .

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E LNCaP cells stably expressing an empty vector (Vec), a vector expressing wild-type STAMP2 (WT), or a STAMP2 mutant (dGSR) were cultured, and the NADPH/NADP+ ratio was determined as described in Materials and Methods. *P = 0.015.


F LNCaP were transfected with either control siRNA ( siCtrl ) or STAMP2 siRNA ( siST2 ) . were then cultured for 2 days harvested , NADPH / NADP + was determined as above . * P = 0 . 026 .

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F LNCaP cells were transfected with either control siRNA (siCtrl) or STAMP2 siRNA (siST2). Cells were then cultured for 2 days and harvested, and the NADPH/NADP+ ratio was determined as above. *P = 0.026.