( D ) RNA was isolated from irgm1 + / + and irgm1 - / - BMDMs and subjected to qRT - PCR with indicated viral restriction factor

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

(D) RNA was isolated from irgm1+/+ and irgm1-/- BMDMs and subjected to qRT-PCR with indicated viral restriction factor genes


( H - J ) RNA isolated from control and IRGM knockdown HT - 29 cells and subjected to qRT - PCR with indicated genes of ( H ) Immunoproteasome complex ( I ) TAP complex ( J ) human leukocyte antigen ( HLA ) system .

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

(H-J) RNA isolated from control and IRGM knockdown HT-29 cells and subjected to qRT-PCR with indicated genes of (H) Immunoproteasome complex (I) TAP complex (J) human leukocyte antigen (HLA) system.


( L , M ) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP - 1 cells treated with DQ - OVA ( green ) ( 10 µg / ml , 30 min ) . Scale Bar , 10 µm . ( M ) Graph depicts percentage of control and IRGM knockdown THP - 1 cells with DQ - OVA puncta '

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

(L, M) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP-1 cells treated with DQ-OVA (green) (10 µg/ml, 30 min). Scale Bar, 10 µm. (M) Graph depicts percentage of control and IRGM knockdown THP-1 cells with DQ-OVA puncta's


( P ) Representative confocal images of H - 2Kb - SIINFEKL ( red ) on the surface of Irgm1 + / + and irgm1 - / - mouse BMDMs treated with OVA ( 2 mg / ml , 3 h ) , Scale bar 5 µm or 8 µm ( as indicated ) .

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

(P) Representative confocal images of H-2Kb-SIINFEKL (red) on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h), Scale bar 5 µm or 8 µm (as indicated).


( C ) Representative images of the plaque assay in Vero cells performed from the culture supernatant of CHIKV ( MOI 1 , 24 h ) infected HT - 29 control and IRGM knockdown cells .

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(C) Representative images of the plaque assay in Vero cells performed from the culture supernatant of CHIKV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( F ) Representative images of plaque assay in Vero cells performed from the culture supernatant of HSV - 1 ( MOI 1 , 24 h ) infected HT - 29 control and IRGM knockdown cells .

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(F) Representative images of plaque assay in Vero cells performed from the culture supernatant of HSV-1 (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( I ) Representative images of plaque assay in Vero cells performed from the culture supernatant of JEV ( MOI 1 , 24 h ) infected HT - 29 control and IRGM knockdown cells .

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(I) Representative images of plaque assay in Vero cells performed from the culture supernatant of JEV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( L ) Representative images of plaque assay in Vero cells were performed from the culture supernatant of WNV ( MOI 0 . 3 , 16 h ) infected HuH7 control and IRGM knockdown cells .

(L) Representative images of plaque assay in Vero cells were performed from the culture supernatant of WNV (MOI 0.3, 16 h) infected HuH7 control and IRGM knockdown cells.


( O ) Representative images of plaque assay in Vero cells were performed from the culture supernatant of ZIKV ( MOI 5 , 48 h ) infected Huh7 control and IRGM knockdown cells .

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(O) Representative images of plaque assay in Vero cells were performed from the culture supernatant of ZIKV (MOI 5, 48 h) infected Huh7 control and IRGM knockdown cells.


( S ) Representative images of plaque assay in Vero E6 cells were performed from the culture supernatant of SARS - CoV2 ( MOI 1 , 24 h ) infected THP - 1 control and IRGM knockdown cells .

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(S) Representative images of plaque assay in Vero E6 cells were performed from the culture supernatant of SARS-CoV2 (MOI 1, 24 h) infected THP-1 control and IRGM knockdown cells.


( D ) Kaplan - Meier survival graph depicts percentage survival of mock and CHIKV infected irgm1 + / + and irgm1 - / - neonates during the course of

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

(D) Kaplan-Meier survival graph depicts percentage survival of mock and CHIKV infected irgm1+/+ and irgm1-/- neonates during the course of infection


( H , I ) Total RNA isolated from muscles and brains of mock and CHIKV infected ( MOI 1x107 PFU / mouse ) ( 6 dpi ) irgm1 + / + and irgm1 - / - mice and was subjected to qRT PCR with MX2 The total RNA used for qRT PCR with the brain is four times more than muscles .

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

(H, I) Total RNA isolated from muscles and brains of mock and CHIKV infected (MOI 1x107 PFU/mouse) (6 dpi) irgm1+/+ and irgm1-/-mice and was subjected to qRT PCR with MX2 The total RNA used for qRT PCR with the brain is four times more than muscles.


( A - D ) Control and si - IRGM transfected THP - 1 IFN reporter cells were kept uninfected ( Mock ) or infected with ( A ) JEV ( MOI 5 ) or ( B ) CHIKV ( MOI 5 ) or ( C ) HSV - 1 ( MOI 2 . 5 ) or ( D ) VSV - eGFP ( MOI 1 ) and the supernatant collected 8 hpi were subjected to luciferase assay . The graphs depict fold change in interferon response .

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

(A-D) Control and si-IRGM transfected THP-1 IFN reporter cells were kept uninfected (Mock) or infected with (A) JEV (MOI 5) or (B) CHIKV (MOI 5) or (C) HSV-1 (MOI 2.5) or (D) VSV-eGFP (MOI 1) and the supernatant collected 8 hpi were subjected to luciferase assay. The graphs depict fold change in interferon response.


( L ) Western blot analysis with cell lysates of mock and CHIKV ( MOI 5 , 24 h ) infected control and IRGM knockdown HT - 29 cells and probed with the indicated antibodies . ( M ) Western blot analysis with cell lysates of mock and JEV ( MOI 5 , 24 h ) infected control and si - IRGM transfected HT - 29 cells and probed with the indicated antibodies .

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

(L) Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT-29 cells and probed with the indicated antibodies. (M) Western blot analysis with cell lysates of mock and JEV (MOI 5, 24 h) infected control and si-IRGM transfected HT-29 cells and probed with the indicated antibodies.


( Q ) Western blot analysis with cell lysates of mock and CHIKV ( MOI 1 , 24 h ) infected control and IRGM knockdown HT - 29 cells transfected with indicated siRNA and probed with the indicated antibodies .

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

(Q) Western blot analysis with cell lysates of mock and CHIKV (MOI 1, 24 h) infected control and IRGM knockdown HT-29 cells transfected with indicated siRNA and probed with the indicated antibodies.


( B , C ) Representative immunoblots ( B ) and quantification of Tau degradation ( C ) from 14 DIV primary neurons transduced with EGFP or shTSG101 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau and tubulin . shTSG101 - expressing neurons exhibit markedly decreased Tau degradation compared to EGFP - expressing controls ( n = 18 - 19 per condition , unpaired student ' s t - test , * p = 0 . 0108 )

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

(B, C) Representative immunoblots (B) and quantification of Tau degradation (C) from 14 DIV primary neurons transduced with EGFP or shTSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shTSG101-expressing neurons exhibit markedly decreased Tau degradation compared to EGFP-expressing controls (n=18-19 per condition, unpaired student's t-test, *p=0.0108)


( D , E ) Representative immunoblots ( D ) and quantification of Tau degradation ( E ) from 14 DIV neurons transduced with mCh or mCh - TSG101 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau and tubulin . Overexpression of TSG101 increases Tau degradation ( n = 17 / condition , unpaired student ' s t - test , * p = 0 . 0115 )

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

(D, E) Representative immunoblots (D) and quantification of Tau degradation (E) from 14 DIV neurons transduced with mCh or mCh-TSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. Overexpression of TSG101 increases Tau degradation (n=17/condition, unpaired student's t-test, *p=0.0115)


( F , G ) Representative immunoblots ( F ) and quantification of Tau degradation ( G ) from 14 DIV neurons transduced with mCh or shRab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau and tubulin . shRab35 - expressing neurons exhibit markedly decreased Tau degradation compared to mCh - expressing controls ( n = 23 - 26 per condition , unpaired student ' s t - test , * * * p = 0 . 0008 )

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O 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, G) Representative immunoblots (F) and quantification of Tau degradation (G) from 14 DIV neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shRab35-expressing neurons exhibit markedly decreased Tau degradation compared to mCh-expressing controls (n=23-26 per condition, unpaired student's t-test, ***p=0.0008)


( H , I ) Representative immunoblots ( H ) and quantification of Tau degradation ( I ) from 14 DIV neurons transduced with mCh or mCh - Rab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau or tubulin . Rab35 overexpression increases Tau degradation ( n = 18 per condition , unpaired student ' s t - test , * p = 0 . 0126 )

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

(H, I) Representative immunoblots (H) and quantification of Tau degradation (I) from 14 DIV neurons transduced with mCh or mCh-Rab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau or tubulin. Rab35 overexpression increases Tau degradation (n=18 per condition, unpaired student's t-test, *p=0.0126)


( J ) Flow cytometry distribution of N2a cells cotransfected with either HA vector ( black ) or HA - Rab35 ( green ) and medium fluoresence timer - tagged wild - type Tau ( FT - Tau ) ; blue fluorescence ( y axis ) indicates " younger " Tau protein while red fluorescence ( x axis ) indicates " older "

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

(J) Flow cytometry distribution of N2a cells cotransfected with either HA vector (black) or HA-Rab35 (green) and medium fluoresence timer-tagged wild-type Tau (FT-Tau); blue fluorescence (y axis) indicates "younger" Tau protein while red fluorescence (x axis) indicates "older" Tau


( K ) The ratio of cells expressing red to blue ( older : younger ) FT - Tau is reduced in cells overexpressing Rab35 , indicating faster Tau turnover ( n = 9 / condition , unpaired student ' s t - test , * * * * p < 0 . 0001 )

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(K) The ratio of cells expressing red to blue (older:younger) FT-Tau is reduced in cells overexpressing Rab35, indicating faster Tau turnover (n=9/condition, unpaired student's t-test , ****p<0.0001)


( A , B ) Representative immunoblots ( A ) and quantification of Tau degradation ( B ) from 14 DIV primary neurons transduced with mCh or shRab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for pSer396 / 404 - Tau ( PHF1 ) , pSer262 - Tau , or pSer202 - Tau ( CP13 ) and tubulin . shRab35 - expressing neurons exhibit markedly decreased pSer262 - and p396 / 404 - Tau degradation compared to mCh - expressing controls , while pSer202 - Tau degradation is unaffected ( n = 4 per condition for pSer396 / 404 - Tau and pSer202 - Tau , n = 3 for pSer262 - Tau , unpaired student ' s t - test , * * p = 0 . 0027 , * p = 0 . 0120 )

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

(A, B) Representative immunoblots (A) and quantification of Tau degradation (B) from 14 DIV primary neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for pSer396/404-Tau (PHF1), pSer262-Tau, or pSer202-Tau (CP13) and tubulin. shRab35-expressing neurons exhibit markedly decreased pSer262- and p396/404-Tau degradation compared to mCh-expressing controls, while pSer202-Tau degradation is unaffected (n=4 per condition for pSer396/404-Tau and pSer202-Tau, n=3 for pSer262-Tau, unpaired student's t-test, **p=0.0027, *p=0.0120)


( C ) Images of PLA signal ( green ) for Hrs / Tau interaction in N2a cells co - transfected with FLAG - Hrs and either mCh or mCh - Rab35 ( red ) , and probed with antibodies against FLAG and total ( DA9 ) or phospho - Tau species ( pSer396 / 404 - Tau , pSer262 - Tau , or pSer202 - Tau ) ; scale bar : 5 μm . ( D ) Rab35 overexpression significantly increases PLA puncta for total , pSer396 / 404 - Tau , pSer262 - Tau but not pSer202 - Tau ( n = 121 - 132 cells for total Tau , 178 - 195 cells for pSer396 / 404 - Tau , 71 - 98 cells for pSer262 - Tau , 25 - 29 cells for pSer202 - Tau ; Mann - Whitney test , * * * * p < 0 . 0001 )

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

(C) Images of PLA signal (green) for Hrs/Tau interaction in N2a cells co-transfected with FLAG-Hrs and either mCh or mCh-Rab35 (red), and probed with antibodies against FLAG and total (DA9) or phospho-Tau species (pSer396/404-Tau, pSer262-Tau, or pSer202-Tau); scale bar: 5 μm. (D) Rab35 overexpression significantly increases PLA puncta for total, pSer396/404-Tau, pSer262-Tau but not pSer202-Tau (n=121-132 cells for total Tau, 178-195 cells for pSer396/404-Tau, 71-98 cells for pSer262-Tau, 25-29 cells for pSer202-Tau; Mann-Whitney test, ****p<0.0001)


( A , B ) Representative immunoblots ( A ) and quantification of Hrs , TSG101 , CHMP2b , and Rab35 protein levels ( B ) from 14 DIV neurons treated with either DMSO ( CON ) or glucocorticoids ( GC ) . GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs , TSG101 , or CHMP2b ( n = 9 - 10 / condition , unpaired student ' s t - test , * p = 0 . 0221 )

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

(A, B) Representative immunoblots (A) and quantification of Hrs, TSG101, CHMP2b, and Rab35 protein levels (B) from 14 DIV neurons treated with either DMSO (CON) or glucocorticoids (GC). GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs, TSG101, or CHMP2b (n=9-10/condition, unpaired student's t-test, *p=0.0221)


( C ) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells ( for neurons , n = 9 / condition , unpaired student ' s t - test , * * p = 0 . 0036 ; for N2a cells , n = 6 / condition , unpaired student ' s t - test , * p = 0 . 0138 )

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(C) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells (for neurons, n=9/condition, unpaired student's t-test, **p=0.0036; for N2a cells, n=6/condition, unpaired student's t-test, *p=0.0138)


( E , F ) Representative immunoblots ( E ) and quantification of levels of different Rab proteins ( F ) in hippocampus of GC - treated and CON animals . Protein levels of Rab35 , but not other Rabs , are decreased in GC - treated animals compared to CON ones ( n = 5 animals / condition , unpaired student ' s t - test , * p = 0 . 0257 )

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(E, F) Representative immunoblots (E) and quantification of levels of different Rab proteins (F) in hippocampus of GC-treated and CON animals. Protein levels of Rab35, but not other Rabs, are decreased in GC-treated animals compared to CON ones (n=5 animals/condition, unpaired student's t-test, *p=0.0257)


( G , H ) Immunofluorescence staining of Rab35 ( green ) and DAPI ( blue ) ( G ) showing that Rab35 fluorescence intensity is reduced in hippocampal area ( CA1 ) of GC - treated aniamls ( H ) ( n = 15 slices / condition ; unpaired student ' s t - test , * * * p = 0 . 0003 ) . Data information : All numeric data represent mean ± SEM

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

(G, H) Immunofluorescence staining of Rab35 (green) and DAPI (blue) (G) showing that Rab35 fluorescence intensity is reduced in hippocampal area (CA1) of GC-treated aniamls (H) (n=15 slices/condition; unpaired student's t-test, ***p=0.0003). Data information: All numeric data represent mean ± SEM


( A , B ) Representative immunoblots ( A ) and quantification of Tau degradation ( B ) in 14 DIV neurons expressing mCh - Rab35 or mCh , treated for 24 h with cycloheximide ( CHX ) or DMSO ( CON ) under GC conditions . GC significantly decreases Tau degradation , whereas Rab35 overexpression blocks this effect ( n = 12 - 14 / condition ; 1 - way ANOVA , Dunnet post - hoc analysis , * pmCh vs mCh GC = 0 , 0319 , * pmCh vs Rab35 GC = 0 , 047 )

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

(A, B) Representative immunoblots (A) and quantification of Tau degradation (B) in 14 DIV neurons expressing mCh-Rab35 or mCh, treated for 24 h with cycloheximide (CHX) or DMSO (CON) under GC conditions. GC significantly decreases Tau degradation, whereas Rab35 overexpression blocks this effect (n=12-14/condition; 1-way ANOVA, Dunnet post-hoc analysis, *pmCh vs mCh GC=0,0319, *pmCh vs Rab35 GC=0,047)


( D , E ) Representative immunoblots ( D of Tau levels ( E ) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP , but not Rab35 ( n = 5 - 6 animals / group , 2 replicates , 2 - way ANOVA , GC x Rab35 interaction F1 , 39 = 10 , 51 p = 0 . 002 ; Sidak posthoc analysis * p = 0 . 0168 )

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

(D, E) Representative immunoblots (D of Tau levels (E) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP, but not Rab35 (n=5-6 animals/group, 2 replicates, 2-way ANOVA, GC x Rab35 interaction F1, 39=10,51 p=0.002; Sidak posthoc analysis *p=0.0168)


( F ) Golgi - based 3D neuronal reconstruction of hippocampal pyramidal neurons ( CA1 area )

(F) Golgi-based 3D neuronal reconstruction of hippocampal pyramidal neurons (CA1 area)


( G ) GC treatment reduces the length of apical dendrites in animals expressing EGFP , but not EGFP - Rab35 ( n = 6 - 7 animals / group ; 6 - 8 neurons / animal , 2 - way ANOVA , GC x Rab35 interaction F1 , 155 = 3 . 969 p = 0 . 0481 , overall GC effect F1 , 155 = 8 . 998 p = 0 . 0031 , Sidak posthoc analysis * * p = 0 . 0021 )

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

(G) GC treatment reduces the length of apical dendrites in animals expressing EGFP, but not EGFP-Rab35 (n=6-7 animals/group; 6-8 neurons/animal, 2-way ANOVA , GC x Rab35 interaction F1, 155=3.969 p=0.0481, overall GC effect F1, 155=8.998 p=0.0031, Sidak posthoc analysis **p=0.0021)


( H ) GC treatment reduces mature spine density in animals expressing EGFP , but not EGFP - Rab35 ( 2 - way ANOVA , GC x Rab35 interaction F1 , 499 = 12 . 33 p = 0 . 0005 , overall GC effect F1 , 499 = 4 . 373 p = 0 . 0370 , Sidak posthoc analysis * * * p = 0 . 0003 ; n = 6 - 7 animals / group ; 6 - 8 neurons per animal )

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(H) GC treatment reduces mature spine density in animals expressing EGFP, but not EGFP-Rab35 (2-way ANOVA, GC x Rab35 interaction F1, 499=12.33 p=0.0005, overall GC effect F1, 499=4.373 p=0.0370, Sidak posthoc analysis *** p=0.0003; n=6-7 animals/group; 6-8 neurons per animal)


( I , J ) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP - expressing animals ; however , this effect is not seen in EGFP - Rab35 - expressing animals ( 3 - way ANOVA , GC x Rab35 interaction F1 , 4212 = 14 , 926 p < 0 . 0001 , Simple effect analysis , Sidak test for multiple comparisons * p120 = 0 . 012 , * * p140 = 0 . 001 , * * p220 = 0 . 002 , * * * p240 < 0 . 001 , * * * p260 < 0 . 001 , * * p280 = 0 . 001 , * * p300 = 0 . 001 , * p320 = 0 . 021 , * p340 = 0 . 047 , n = 6 - 7 animals / group , 6 - 8 neurons per animal )

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(I, J) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP-expressing animals; however, this effect is not seen in EGFP-Rab35-expressing animals (3-way ANOVA, GC x Rab35 interaction F1, 4212= 14,926 p<0.0001, Simple effect analysis, Sidak test for multiple comparisons *p120=0.012, **p140=0.001, **p220=0.002, ***p240<0.001, ***p260<0.001, **p280=0.001, **p300=0.001, *p320=0.021, *p340=0.047, n=6-7 animals/group, 6-8 neurons per animal)


( A ) Volcano plot representation of PARL interaction partners . Mitochondria isolated from PARL - / - FITR293Tcells expressing PARL - FLAG were subjected to co - immunoprecipitation ( IP ) . Co - purifying proteins were separated by SDS - PAGE and identified by quantitative MS ( n = 3 ) ( Dataset EV1 ) .

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(A) Volcano plot representation of PARL interaction partners. Mitochondria isolated from PARL-/- FITR293Tcells expressing PARL-FLAG were subjected to co-immunoprecipitation (IP). Co-purifying proteins were separated by SDS-PAGE and identified by quantitative MS (n=3)(Dataset EV1).


( B ) Heat map of Log2 transformed LFQ intensities for three independent experiments . All significant ( FDR < 0 . 05 ) proteins showing a positive ratio between PARLIP and control in ( A ) are shown . Grey color shows missing quantitative information . Clustering was performed using the complete method with Euclidean distance .

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(B) Heat map of Log2 transformed LFQ intensities for three independent experiments. All significant (FDR < 0.05) proteins showing a positive ratio between PARLIP and control in (A) are shown. Grey color shows missing quantitative information. Clustering was performed using the complete method with Euclidean distance.


( C ) Complexome analysis of MEFmitochondria ( n = 3 ) . Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0 . 05 ( A ) and identified by mass spectrometry after BN - PAGE analysis are shown after hierarchical clustering .

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(C) Complexome analysis of MEFmitochondria (n=3). Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0.05 (A) and identified by mass spectrometry after BN-PAGE analysis are shown after hierarchical clustering.


( D ) BN - PAGE analysis of mitochondria lacking SLP2 , YME1L or PARL . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a protein concentration of 2 . 5 mg / ml . Solubilized proteins were separated by a 3 - 13 % blue native gradient gel and analyzed using SLP2 - , YME1L - and PARL - specific antibodies .

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(D) BN-PAGE analysis of mitochondria lacking SLP2, YME1L or PARL. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-13% blue native gradient gel and analyzed using SLP2-, YME1L- and PARL-specific antibodies.


( E ) High resolution complexome analysis of SPY complex members SLP2 , YME1L and PARL using MEFmitochondria ( n = 3 ) . Heat maps and migration profiles are shown after separation using a 3 - 9 % BN - PAGE . SLP2 , YME1L and PARL migrate in a high molecular weight complex ( 2 MDa ) , whereas SLP2 additionally is present in complexes of 1 . 6 MDa ) .

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(E) High resolution complexome analysis of SPY complex members SLP2, YME1L and PARL using MEFmitochondria (n=3). Heat maps and migration profiles are shown after separation using a 3-9% BN-PAGE. SLP2, YME1L and PARL migrate in a high molecular weight complex (2 MDa), whereas SLP2 additionally is present in complexes of 1.6 MDa).


( A ) Co - immunoprecipitation of endogenous SLP2 , PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2 - or PARL - specific antibodies . IgG was used as a negative control . In , input ( 10 % ) .

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(A) Co-immunoprecipitation of endogenous SLP2, PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2- or PARL-specific antibodies. IgG was used as a negative control. In, input (10%).


( B , C ) Co - immunoprecipitation of endogenous SLP2 and YME1L with PARL - specific antibodies in mitochondria isolated from wild type MEFs ( WT ) and MEFs lacking YME1L , SLP2 or PARL . In , input ( 10 % ) .

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(B, C) Co-immunoprecipitation of endogenous SLP2 and YME1L with PARL-specific antibodies in mitochondria isolated from wild type MEFs (WT) and MEFs lacking YME1L, SLP2 or PARL. In, input (10%).


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( D ) Assembly of SLP2 , PARL and YME1L into a high molecular weight complex . Mitochondria isolated from FITR293T cells ( Con ) and cells inducibly expressing SLP2 - FLAG ( FLAG ) were solubilized in digitonin and subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of the precipitate were analyzed by BN - PAGE and immunoblotting using SLP2 - , PARL - , YME1L - and PHB2 - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) .

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(D) Assembly of SLP2, PARL and YME1L into a high molecular weight complex. Mitochondria isolated from FITR293T cells (Con) and cells inducibly expressing SLP2-FLAG (FLAG) were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, PARL-, YME1L- and PHB2-specific antibodies. In, input (8%); E, eluate (100%).


( E ) High resolution BN - PAGE analysis of mitochondria lacking PARL , YME1L or SLP2 . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a protein concentration of 2 . 5 mg / ml . Solubilized proteins were separated by a 3 - 9 % gradient gel containing 0 - 10 % glycerol and analyzed using SLP2 - specific antibodies .

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(E) High resolution BN-PAGE analysis of mitochondria lacking PARL, YME1L or SLP2. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-9% gradient gel containing 0-10% glycerol and analyzed using SLP2-specific antibodies.


( F , G ) Submitochondrial localization of SLP2 . ( F ) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated . Fractions were analyzed by SDS - PAGE and immunoblotting . MFN2 served as OM marker , TIMM23 , SMAC / DIABLO , and YME1L as IMS markers , and AFG3L1 and AFG3L2 as matrix marker proteins .

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(F, G) Submitochondrial localization of SLP2. (F) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated. Fractions were analyzed by SDS-PAGE and immunoblotting. MFN2 served as OM marker, TIMM23, SMAC/DIABLO, and YME1L as IMS markers, and AFG3L1 and AFG3L2 as matrix marker proteins.


( F , G ) Submitochondrial localization of SLP2 . ( G ) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet ( P ) and supernatant ( S ) fraction by centrifugation . Fractions were analyzed by SDS - PAGE and immunoblotting .

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(F, G) Submitochondrial localization of SLP2. (G) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fraction by centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting.


( A ) Steady state levels of proteolytic substrates of YME1L . Whole cell extracts of Yme1l - / - , Parl - / - , and Slp2 - / - MEFs were analyzed by SDS - PAGE and immunoblotting using the indicated antibodies .

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(A) Steady state levels of proteolytic substrates of YME1L. Whole cell extracts of Yme1l-/-, Parl-/-, and Slp2-/- MEFs were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies.


( B ) PGAM5 processing depends on proteolytically active PARL . PARL and catalytic inactive PARLS277A harboring C - terminal FLAG - epitopes were expressed under the control of a tetracycline ( tet ) - inducible promoter in wild type ( WT ) and PARL - / - FITR293T cells as indicated . Processing of L - PGAM5 to S - PGAM5 was monitored by immunoblotting .

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(B) PGAM5 processing depends on proteolytically active PARL. PARL and catalytic inactive PARLS277A harboring C-terminal FLAG-epitopes were expressed under the control of a tetracycline (tet)-inducible promoter in wild type (WT) and PARL-/- FITR293T cells as indicated. Processing of L-PGAM5 to S-PGAM5 was monitored by immunoblotting.


( C ) Accelerated processing of L - PGAM5 in Slp2 - / - cells . Processing of L - PGAM5 was analyzed by immunoblotting in wild type ( WT ) , Yme1l - / - , Parl - / - and Slp2 - / - MEFs after inhibition of cytosolic protein synthesis by cycloheximide ( CHX ) . A quantification of L - PGAM5 levels at different time points is shown in the lower panel . Two - way ANOVA analysis ( n = 3 ; * * * * , p < 0 . 0001 ) .

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(C) Accelerated processing of L-PGAM5 in Slp2-/- cells. Processing of L-PGAM5 was analyzed by immunoblotting in wild type (WT), Yme1l-/-, Parl-/- and Slp2-/-MEFs after inhibition of cytosolic protein synthesis by cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown in the lower panel. Two-way ANOVA analysis (n=3; ****, p<0.0001).


( D ) The accelerated processing of L - PGAM5 in Slp2 - / - cells is mediated by PARL . PARL was depleted from Slp2 - / - cells by RNAi prior to CHX treatment . A quantification of L - PGAM5 levels at different time points is shown . Two - way ANOVA analysis ( n = 3 ; * * , p < 0 . 01 . * * * * , p < 0 . 0001 ) .

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(D) The accelerated processing of L-PGAM5 in Slp2-/- cells is mediated by PARL. PARL was depleted from Slp2-/- cells by RNAi prior to CHX treatment. A quantification of L-PGAM5 levels at different time points is shown. Two-way ANOVA analysis (n=3; **, p<0.01. ****, p<0.0001).


( E ) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL . Mitochondria isolated from FITR293T cells ( WT ) or PARL - / - FITR293T cells expressing PARL - FLAG ( PARL - WT ) or PARLS277A - FLAG ( PARL - S277A ) were solubilized in digitonin were analyzed by BN - PAGE and immunoblotting using FLAG - and PGAM5 - specific antibodies .

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(E) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin were analyzed by BN-PAGE and immunoblotting using FLAG- and PGAM5-specific antibodies.


( A ) PARL dependent cleavage of PINK1 - HA is reduced in Slp2 - / - and Yme1l - / - MEFs . Whole cell extracts of Slp2 - / - , Parl - / - and Yme1l - / - MEFs expressing PINK1 - HA were analysed by SDS - PAGE and immunoblotting using the indicated antibodies . Cells were treated with 20 µM MG132 or 20 µM CCCP for 4 h . ( B ) Quantification of the protein ratio ( Log2 ) PINK1 - HA 52 kDa / 63 kDa ( n = 3 ; * , p < 0 . 05 ; One - way ANOVA ) . n . s . , not significant . Error bars indicate SEM .

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(A) PARL dependent cleavage of PINK1-HA is reduced in Slp2-/- and Yme1l-/- MEFs. Whole cell extracts of Slp2-/-, Parl-/ -and Yme1l-/- MEFs expressing PINK1-HA were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Cells were treated with 20 µM MG132 or 20 µM CCCP for 4 h.(B) Quantification of the protein ratio (Log2) PINK1-HA 52 kDa/63 kDa (n=3; *, p<0.05; One-way ANOVA). n.s., not significant. Error bars indicate SEM.


( C ) PINK1 - HA associates with the SPY complex harboring proteolytically inactive PARL . Mitochondria isolated from FITR293T cells ( WT ) or PARL - / - FITR293T cells expressing PARL - FLAG ( PARL - WT ) or PARLS277A - FLAG ( PARL - S277A ) were solubilized in digitonin and analysed by BN - PAGE and immunoblotting using FLAG - , HA - and SLP2 - specific antibodies .

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(C) PINK1-HA associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using FLAG-, HA- and SLP2-specific antibodies.


( D ) PINK1 associates with SLP2 . Mitochondria isolated from FITR293T cells ( WT ) or PARL - / - FITR293T cells , depleted of SLP2 by siRNA as indicated , were solubilized in digitonin and analysed by BN - PAGE and immunoblotting using SLP2 - and PINK1 - specific antibodies .

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(D) PINK1 associates with SLP2. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells, depleted of SLP2 by siRNA as indicated, were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using SLP2- and PINK1-specific antibodies.


( A ) Processing of L - PGAM5 to S - PGAM5 in depolarized mitochondria of wild type ( WT ) , Slp2 - / - , Yme11 - / - , and Parl - / - MEFs . PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide ( CHX ) . A quantification of L - PGAM5 levels at different time points is shown . L - PGAM5 levels at t = 0 was set to 100 % . Two - way ANOVA analysis ( n = 3 ; * , p < 0 . 05 , * * , p < 0 . 01 . * * * * , p < 0 . 0001 ) . Arrowheads denote intermediate PGAM5 cleavage products .

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(A) Processing of L-PGAM5 to S-PGAM5 in depolarized mitochondria of wild type (WT), Slp2-/-, Yme11-/-, and Parl-/- MEFs. PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown. L-PGAM5 levels at t=0 was set to 100%. Two-way ANOVA analysis (n=3; *, p<0.05, **, p<0.01. ****, p<0.0001). Arrowheads denote intermediate PGAM5 cleavage products.


( B ) OMA1 - mediated processing of PGAM5 in depolarized Parl - / - mitochondria . Processing of L - PGAM5 to S - PGAM5 in Parl - / mitochondria was assessed as in ( A ) in the presence or absence of CCCP ( 20 µM ) , which were depleted of OMA1 by siRNA as indicated . The arrowhead denotes an intermediate PGAM5 cleavage product . SCR , scrambled .

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(B) OMA1-mediated processing of PGAM5 in depolarized Parl-/-mitochondria. Processing of L-PGAM5 to S-PGAM5 in Parl-/ mitochondria was assessed as in (A) in the presence or absence of CCCP (20 µM), which were depleted of OMA1 by siRNA as indicated. The arrowhead denotes an intermediate PGAM5 cleavage product. SCR, scrambled.


( C ) Impaired PGAM5 processing in depolarized Oma1 - / - mitochondria lacking PARL . Processing of L - PGAM5 to S - PGAM5 was examined as in ( A ) after 2 h in wild type ( WT ) and Oma1 - / - mitochondria in the presence or absence of CCCP ( 20 µM , 2 h ) , which were depleted of PARL by siRNA as indicated . * unspecific cross - reaction .

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(C) Impaired PGAM5 processing in depolarized Oma1-/- mitochondria lacking PARL. Processing of L-PGAM5 to S-PGAM5 was examined as in (A) after 2 h in wild type (WT) and Oma1-/- mitochondria in the presence or absence of CCCP (20 µM, 2 h), which were depleted of PARL by siRNA as indicated. * unspecific cross-reaction.


( D ) OMA1 mediates accelerated processing of L - OPA1 in Slp2 - / - MEFs . Processing of OPA1 and PGAM5 was monitored in Slp2 - / - , Oma1 - / - , and Slp2 - / - Oma1 - / - cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide ( CHX ) .

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(D) OMA1 mediates accelerated processing of L-OPA1 in Slp2-/- MEFs. Processing of OPA1 and PGAM5 was monitored in Slp2-/-, Oma1-/-, and Slp2-/-Oma1-/- cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide (CHX).


( E ) OMA1 interacts with SLP2 . OMA1 - / - FITR293T cells ectopically expressing OMA1 - myc were transfected with pcDNA5 ( Control ) or pcDNA5 - SLP2 - FLAG ( FLAG ) . Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of the precipitate were analyzed by BN - PAGE and immunoblotting using SLP2 - , YME1L - , PARL - , and myc - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) . Samples were analyzed by SDS - PAGE in parallel ( lower panel ) . ( F ) Quantification of immunoprecipitation efficiencies in ( E ) .

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(E) OMA1 interacts with SLP2. OMA1-/-FITR293T cells ectopically expressing OMA1-myc were transfected with pcDNA5 (Control) or pcDNA5-SLP2-FLAG (FLAG). Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, YME1L-, PARL-, and myc-specific antibodies. In, input (8%); E, eluate (100%). Samples were analyzed by SDS-PAGE in parallel (lower panel).(F) Quantification of immunoprecipitation efficiencies in (E).


( a ) Western blot analysis showing the replacement of endogenous Beclin 1 with Beclin 1 - EGFP in Becn1 - / - ; Becn1 - EGFP / + mice , as detected by an anti - Beclin 1 antibody .

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(a) Western blot analysis showing the replacement of endogenous Beclin 1 with Beclin 1-EGFP in Becn1-/-;Becn1-EGFP/+ mice, as detected by an anti-Beclin 1 antibody.


( b ) Coomassie - stained SDS - PAGE showing the Beclin 1 - interacting proteins immuno - isolated from brain and liver of the ' rescued ' mice ( lanes 2 and 4 ) and of control Becn1 + / - littermates ( lanes 1 and 3 ) , using an anti - GFP antibody . Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15 / p150 ( band 1 ) , Vps34 / class III PI ( 3 ) K ( band 3 ) , UVRAG ( band 4 ) , Beclin 1 - EGFP ( band 5 ) , Atg14L ( band 6 , asterisk , gi | 27369860 ) and Rubicon ( band 2 , asterisk , gi | 45708948 ) . UVRAG levels varied with different affinity - purification conditions , suggesting an unstable association of UVRAG with the complex .

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(b) Coomassie-stained SDS-PAGE showing the Beclin 1-interacting proteins immuno-isolated from brain and liver of the 'rescued' mice (lanes 2 and 4) and of control Becn1+/- littermates (lanes 1 and 3), using an anti-GFP antibody. Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15/p150 (band 1), Vps34/class III PI(3)K (band 3), UVRAG (band 4), Beclin 1-EGFP (band 5), Atg14L (band 6, asterisk, gi|27369860) and Rubicon (band 2, asterisk, gi|45708948). UVRAG levels varied with different affinity-purification conditions, suggesting an unstable association of UVRAG with the complex.


( d ) Western blot analysis of Atg14L , Rubicon , Vps34 and Beclin 1 in gel filtration fractions from wild - type mouse liver extract showed co - elution of these proteins in fractions 38 - 45 . Atg14L was also eluted in later fractions 51 - 56 . The fractions for the peak elution of thyroglobulin ( 670K ) and γ - globulin ( 158K ) are indicated by arrows . Control siRNA - transfected NIH 3T3 cell lysate was loaded as a positive control ( labelled as 1 ) for the migration position of the Atg14L protein on SDS - PAGE ; Atg14L siRNA - transfected NIH 3T3 cell lysate was loaded as a negative control ( labelled as 2 ) .

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(d) Western blot analysis of Atg14L, Rubicon, Vps34 and Beclin 1 in gel filtration fractions from wild-type mouse liver extract showed co-elution of these proteins in fractions 38-45. Atg14L was also eluted in later fractions 51-56. The fractions for the peak elution of thyroglobulin (670K) and γ-globulin (158K) are indicated by arrows. Control siRNA-transfected NIH 3T3 cell lysate was loaded as a positive control (labelled as 1) for the migration position of the Atg14L protein on SDS-PAGE; Atg14L siRNA-transfected NIH 3T3 cell lysate was loaded as a negative control (labelled as 2).


( e , f ) Co - immunoprecipitation confirmed protein - protein interaction between Atg14L and Rubicon . HEK 293 cells were co - transfected with Atg14L - EGFP and Flag - Rubicon ( e ) or Rubicon - EGFP and Flag - Atg14L ( f ) . Cell lysates were used for immunoprecipitation with an anti - GFP antibody and the resulting immunoprecipitates were blotted with an anti - Flag antibody . Our results show immunoprecipitation of Rubicon by Atg14L ( e ) and vice versa ( f ) . WCL , whole cell lysate ; IP , immunoprecipitated . See Supplementary Information , Fig . S6 for full scans of blots in a , d , e and f .

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(e, f) Co-immunoprecipitation confirmed protein-protein interaction between Atg14L and Rubicon. HEK 293 cells were co-transfected with Atg14L-EGFP and Flag-Rubicon (e) or Rubicon-EGFP and Flag-Atg14L (f). Cell lysates were used for immunoprecipitation with an anti-GFP antibody and the resulting immunoprecipitates were blotted with an anti-Flag antibody. Our results show immunoprecipitation of Rubicon by Atg14L (e) and vice versa (f). WCL, whole cell lysate; IP, immunoprecipitated. See Supplementary Information, Fig. S6 for full scans of blots in a, d, e and f.


( a ) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62 / SQSTM1 and LC3 II levels under normal and nutrient - starvation conditions in NIH 3T3 cells .

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(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under normal and nutrient-starvation conditions in NIH 3T3 cells.


( b ) Compared with control siRNA , Atg14L siRNA decreased long - lived protein degradation in NIH 3T3 cells under normal ( * P = 0 . 007 ) and starvation ( * P = 5 × 10 − 6 ) conditions ( one - tailed Student ' s t - test with equal variances , n = 4 ) . This difference was diminished when the starved cells were treated with 3 - methyladenine ( 3MA , 10 mM ) , a PI ( 3 ) K inhibitor .

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(b) Compared with control siRNA, Atg14L siRNA decreased long-lived protein degradation in NIH 3T3 cells under normal (*P = 0.007) and starvation (*P = 5 × 10−6) conditions (one-tailed Student's t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA, 10 mM), a PI(3)K inhibitor.


( c ) Vps34 kinase assay . HEK 293T cells were co - transfected with Myc - Vps34 - Vps15 and Flag - Atg14L or Flag vector , either in the absence or in the presence of Beclin 1 - EGFP . Myc - Vps34 - Vps15 was immunoprecipitated by anti - Myc antibody for the in vitro kinase assay . The resulting radioactive PI ( 3 ) P was separated by thin - layer chromatography ( TLC ) , quantified and normalized against the amount of immunoprecipitated Myc - tagged Vps34 as measured by western blot ( upper panel ) . The quantified results ( lower panel ) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2 . 5 - fold , but only when Beclin 1 was also overexpressed ( * P = 0 . 04 , one - tailed Student ' s t - test with unequal variances , n = 5 ) .

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(c) Vps34 kinase assay. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Atg14L or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-Myc antibody for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by thin-layer chromatography (TLC), quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34 as measured by western blot (upper panel). The quantified results (lower panel) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2.5-fold, but only when Beclin 1 was also overexpressed (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 5).


( d ) Colocalization of co - expressed Atg14L - EGFP ( green ) and Beclin 1 - AsRed ( red ) in punctate structures in transiently transfected HeLa cells . Scale bar , 10 μm .

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(d) Colocalization of co-expressed Atg14L-EGFP (green) and Beclin 1-AsRed (red) in punctate structures in transiently transfected HeLa cells. Scale bar, 10 μm.


( e ) Electron microscopy images show large structures ( asterisks ) that are often enwrapped with double membranes in the HEK 293T cells co - transfected with Atg14L - EGFP and Beclin 1 - AsRed : concentric membrane ' rings ' ( panel 1 ) ; two large structures ( 3 - 5 μm in diameter , panel 2 ) containing material with high electron density ( inset , enwrapping double membranes ) ; numerous autophagosomes ( arrows , panel 3 ) in the cytoplasm ; immuno - electron microscopy image of a Atg14L - Beclin 1 structure ( labelled with anti - GFP antibody and developed by DAB , panel 4 ) enwrapped with concentric membrane ' rings ' . M , mitochondria ; N , nucleus . Scale bar , 500 nm .

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(e) Electron microscopy images show large structures (asterisks) that are often enwrapped with double membranes in the HEK 293T cells co-transfected with Atg14L-EGFP and Beclin 1-AsRed: concentric membrane 'rings' (panel 1); two large structures (3-5 μm in diameter, panel 2) containing material with high electron density (inset, enwrapping double membranes); numerous autophagosomes (arrows, panel 3) in the cytoplasm; immuno-electron microscopy image of a Atg14L-Beclin 1 structure (labelled with anti-GFP antibody and developed by DAB, panel 4) enwrapped with concentric membrane 'rings'. M, mitochondria; N, nucleus. Scale bar, 500 nm.


( f , g ) EGFP - Atg12 ( f ) or EGFP - Atg5 ( g ) ( green ) was colocalized with the large structures ( arrows ) that were labelled by Atg14L - AsRed ( red ) and Beclin 1 - Myc ( blue ) in transfected HeLa cells . Some of these structures seemed to be ' ring ' - shaped ( yellow arrows and inset ) . Scale bar , 10 μm . See Supplementary Information , Fig . S6 for full scans of blots in a and c .

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(f, g) EGFP-Atg12 (f) or EGFP-Atg5 (g) (green) was colocalized with the large structures (arrows) that were labelled by Atg14L-AsRed (red) and Beclin 1-Myc (blue) in transfected HeLa cells. Some of these structures seemed to be 'ring'-shaped (yellow arrows and inset). Scale bar, 10 μm. See Supplementary Information, Fig. S6 for full scans of blots in a and c.


( a ) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient - starvation conditions .

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(a) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient-starvation conditions.


( b ) Overexpression of Rubicon resulted in increased levels of p62 under both normal and nutrient - starved conditions in HEK 293 cells either stably expressing ( upper rows ) or transiently transfected with ( lower rows ) Rubicon - EGFP . The control cells were either stably expressing or transiently transfected with the EGFP - N3 vector .

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(b) Overexpression of Rubicon resulted in increased levels of p62 under both normal and nutrient-starved conditions in HEK 293 cells either stably expressing (upper rows) or transiently transfected with (lower rows) Rubicon-EGFP. The control cells were either stably expressing or transiently transfected with the EGFP-N3 vector.


( c ) Vps34 kinase activity . HEK 293T cells were co - transfected with Myc - Vps34 - Vps15 and Flag - Rubicon or Flag vector , either in the absence or in the presence of Beclin 1 - EGFP . Myc - Vps34 - Vps15 was immunoprecipitated by an anti - Myc antibody and used for the in vitro kinase assay . The resulting radioactive PI ( 3 ) P was separated by TLC , quantified and normalized against the amount of immunoprecipitated Myc - tagged Vps34 , as measured by western blotting ( upper panel ) . The quantified results ( lower panel ) show that overexpressing Rubicon significantly downregulated the Vps34 kinase activity to 0 . 58 - fold , but only without Beclin 1 overexpression ( * P = 0 . 04 , one - tailed Student ' s t - test with unequal variances , n = 4 ) .

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(c) Vps34 kinase activity. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Rubicon or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by an anti-Myc antibody and used for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by TLC, quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34, as measured by western blotting (upper panel). The quantified results (lower panel) show that overexpressing Rubicon significantly downregulated the Vps34 kinase activity to 0.58-fold, but only without Beclin 1 overexpression (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 4).


( d ) Effect of overexpressing Flag - Rubicon on autophagosome acidification , as monitored by mCherry - GFP - LC3 fluorescence . HeLa cells were transiently co - transfected with mCherry - GFP - LC3 and Flag - Rubicon ( or control Flag vector ) . Cells co - expressing mCherry - GFP - LC3 and control Flag vector contained many red - only puncta along with yellow ( indicating the presence of both red and green ) puncta , suggesting the presence of both autolysosomes and nascent autophagosomes ( upper panel ) . In contrast , cells co - expressing mCherry - GFP - LC3 and Flag - Rubicon contained primarily yellow or white puncta , suggesting the presence of only nascent autophagosomes ( lower panel , white arrows ) . Notably , some cells , which were co - transfected with mCherry - GFP - LC3 and Flag - Rubicon but expressed high levels of mCherry - GFP - LC3 and undetectable levels of FLAG - Rubicon , contained many red - only puncta ( lower panel , yellow arrows ) . ( e ) Quantification of the results in d show that overexpressing Flag - Rubicon markedly reduced the percentage of red - only puncta ( mCherry - LC3 ) from 39 % in the control Flag vector - transfected cells to 2 % in the Flag - Rubicon - transfected cells ( * P = 2 × 10 − 26 , one - tailed Student ' s t - test with unequal variances , n = 30 ) , indicating that overexpression of Rubicon blocks autophagosome acidification or maturation . See Supplementary Information , Fig . S6 for full scans of blots in a - c .

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(d) Effect of overexpressing Flag-Rubicon on autophagosome acidification, as monitored by mCherry-GFP-LC3 fluorescence. HeLa cells were transiently co-transfected with mCherry-GFP-LC3 and Flag-Rubicon (or control Flag vector). Cells co-expressing mCherry-GFP-LC3 and control Flag vector contained many red-only puncta along with yellow (indicating the presence of both red and green) puncta, suggesting the presence of both autolysosomes and nascent autophagosomes (upper panel). In contrast, cells co-expressing mCherry-GFP-LC3 and Flag-Rubicon contained primarily yellow or white puncta, suggesting the presence of only nascent autophagosomes (lower panel, white arrows). Notably, some cells, which were co-transfected with mCherry-GFP-LC3 and Flag-Rubicon but expressed high levels of mCherry-GFP-LC3 and undetectable levels of FLAG-Rubicon, contained many red-only puncta (lower panel, yellow arrows). (e) Quantification of the results in d show that overexpressing Flag-Rubicon markedly reduced the percentage of red-only puncta (mCherry-LC3) from 39% in the control Flag vector-transfected cells to 2% in the Flag-Rubicon-transfected cells (*P = 2 × 10−26, one-tailed Student's t-test with unequal variances, n = 30), indicating that overexpression of Rubicon blocks autophagosome acidification or maturation. See Supplementary Information, Fig. S6 for full scans of blots in a-c.


( a ) Colocalization of Rubicon - EGFP - associated structures with the late endosome / lysosome marker Lamp1 ( arrows ) in HeLa cells transfected with Rubicon - EGFP . Note that some of the Rubicon - EGFP - associated structures show a ' ring ' shape ( yellow arrows ) . Scale bar , 10 μm .

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(a) Colocalization of Rubicon-EGFP-associated structures with the late endosome/lysosome marker Lamp1 (arrows) in HeLa cells transfected with Rubicon-EGFP. Note that some of the Rubicon-EGFP-associated structures show a 'ring' shape (yellow arrows). Scale bar, 10 μm.


( b ) Partial colocalization of Rubicon - EGFP - associated structures with the MVB marker LBPA ( arrows ) in HeLa cells transfected with Rubicon - EGFP . Scale bar , 10 μm .

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(b) Partial colocalization of Rubicon-EGFP-associated structures with the MVB marker LBPA (arrows) in HeLa cells transfected with Rubicon-EGFP. Scale bar, 10 μm.


( c ) Representative ultrastructural images show aberrant expansion of late endosomal / lysosomal structures in HEK 293T cells overexpressing Rubicon - EGFP . These abnormal organelles are large in size , with high ( orange arrows ) or low ( black arrows ) electron density . Some enclose small vesicles ( purple arrows ) and some resemble the MVB ( blue arrows ) . Scale bars , 500 nm .

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(c) Representative ultrastructural images show aberrant expansion of late endosomal/lysosomal structures in HEK 293T cells overexpressing Rubicon-EGFP. These abnormal organelles are large in size, with high (orange arrows) or low (black arrows) electron density. Some enclose small vesicles (purple arrows) and some resemble the MVB (blue arrows). Scale bars, 500 nm.


( d ) Representative ultrastructural images show late endosome / lysosome - like structures that are labelled with anti - GFP gold particles ( panels 3 , 4 ) in HEK 293T cells transiently transfected with Rubicon - EGFP . These structures are enwrapped by double membranes ( panel 4 inset ) and co - labelled by anti - GFP ( developed by DAB ) and anti - Lamp1 ( gold enhanced ) ( panels 5 - 7 ) antibodies . Note that mitochondria are mostly negative for Rubicon - EGFP ( panel 4 ) . The negative controls are without antibody ( panels 1 - 2 ) . M , mitochondria . Scale bars , 200 nm .

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(d) Representative ultrastructural images show late endosome/lysosome-like structures that are labelled with anti-GFP gold particles (panels 3, 4) in HEK 293T cells transiently transfected with Rubicon-EGFP. These structures are enwrapped by double membranes (panel 4 inset) and co-labelled by anti-GFP (developed by DAB) and anti-Lamp1 (gold enhanced) (panels 5-7) antibodies. Note that mitochondria are mostly negative for Rubicon-EGFP (panel 4). The negative controls are without antibody (panels 1-2). M, mitochondria. Scale bars, 200 nm.


( b ) Colocalization of the PI ( 3 ) P - enriched lipid domain marker p40 ( phox ) - PX - EGFP ( green ) and Rubicon - AsRed ( red ) on large punctate structures ( arrows ) in the co - transfected HeLa cells ( upper panels ) . Treatment with the PI ( 3 ) K inhibitor wortmannin ( 75 nM ) for 1 h caused disappearance of the PI ( 3 ) P - enriched lipid domains , whereas the Rubicon - AsRed - positive structures were maintained ( lower panels ) . Scale bars , 10 μm .

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(b) Colocalization of the PI(3)P-enriched lipid domain marker p40 (phox)-PX-EGFP (green) and Rubicon-AsRed (red) on large punctate structures (arrows) in the co-transfected HeLa cells (upper panels). Treatment with the PI(3)K inhibitor wortmannin (75 nM) for 1 h caused disappearance of the PI(3)P-enriched lipid domains, whereas the Rubicon-AsRed-positive structures were maintained (lower panels). Scale bars, 10 μm.


( c ) Subcellular localization of transiently transfected Rubicon - EGFP , RubiconΔRUN - EGFP , RubiconΔC - EGFP or RubiconΔRUNΔC - EGFP in HeLa cells . In contrast to punctate Rubicon - EGFP and RubiconΔRUN - EGFP , RubiconΔC - EGFP and RubiconΔRUNΔC - EGFP were dispersed in the cytoplasm . ΔRUN , RUN domain deletion ; ΔC , cysteine - rich domain deletion . Scale bars , 10 μm .

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(c) Subcellular localization of transiently transfected Rubicon-EGFP, RubiconΔRUN-EGFP, RubiconΔC-EGFP or RubiconΔRUNΔC-EGFP in HeLa cells. In contrast to punctate Rubicon-EGFP and RubiconΔRUN-EGFP, RubiconΔC-EGFP and RubiconΔRUNΔC-EGFP were dispersed in the cytoplasm. ΔRUN, RUN domain deletion; ΔC, cysteine-rich domain deletion. Scale bars, 10 μm.


( d , e ) Absence of full - length Beclin 1 ( d ) or Beclin 1 - CE mutant ( e ) ( red ) on the Rubicon - EGFP - positive structures ( green ) in HEK 293 cells stably expressing Rubicon - EGFP . These cells were transiently transfected with either Beclin 1 - AsRed ( d ) or Flag - Beclin 1 - CE ( e ; that is , the Flag - tagged Beclin 1 mutant containing both CCD and ECD , which mediate the Beclin 1 - Rubicon interaction as shown in Supplementary Information , Fig . S3h ) . Scale bars , 10 μm .

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

(d, e) Absence of full-length Beclin 1 (d) or Beclin 1-CE mutant (e) (red) on the Rubicon-EGFP-positive structures (green) in HEK 293 cells stably expressing Rubicon-EGFP. These cells were transiently transfected with either Beclin 1-AsRed (d) or Flag-Beclin 1-CE (e; that is, the Flag-tagged Beclin 1 mutant containing both CCD and ECD, which mediate the Beclin 1-Rubicon interaction as shown in Supplementary Information, Fig. S3h). Scale bars, 10 μm.


( f ) The formation of the Rubicon - EGFP - positive structures was not affected by siRNA knockdown of Beclin 1 in HEK 293 cells stably expressing Rubicon - EGFP .

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

(f) The formation of the Rubicon-EGFP-positive structures was not affected by siRNA knockdown of Beclin 1 in HEK 293 cells stably expressing Rubicon-EGFP.


( a ) Beeswarm plot with Factor 1 values for each sample with colors corresponding to three clusters found by 3 - means clustering with low factor values ( LZ ) , intermediate factor values ( IZ ) and high factor values ( HZ )

O O O O O O O O O O O O O O O O O O O O O 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) Beeswarm plot with Factor 1 values for each sample with colors corresponding to three clusters found by 3-means clustering with low factor values (LZ), intermediate factor values (IZ) and high factor values (HZ)


( b ) Absolute loadings for the genes with the largest absolute weights in the mRNA data . Plus or minus symbols on the right indicate the sign of the loading . Genes highlighted in orange were previously described as prognostic markers in CLL and associated with IGHV status ( Maloum et al , 2009 ; Morabito et al , 2015 ; Plesingerova et al , 2017 ; Trojani et al , 2012 ; Vasconcelos et al , 2005 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Absolute loadings for the genes with the largest absolute weights in the mRNA data. Plus or minus symbols on the right indicate the sign of the loading. Genes highlighted in orange were previously described as prognostic markers in CLL and associated with IGHV status (Maloum et al, 2009; Morabito et al, 2015; Plesingerova et al, 2017; Trojani et al, 2012; Vasconcelos et al, 2005)


( c ) Heatmap of gene expression values for genes with the largest weights as in

(c) Heatmap of gene expression values for genes with the largest weights as in b


( d ) Absolute loadings of the drugs with the largest weights , annotated by target

(d) Absolute loadings of the drugs with the largest weights, annotated by target category


( e ) Drug response curves for two of the drugs , stratified by the clusters as in

(e) Drug response curves for two of the drugs, stratified by the clusters as in a


B . Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non - targeting siRNA ( siNT ) or silenced with the indicated oligos . 300 ng / mL MMC was added for 24 h . Each dot represents the number of PLA events in a single cell , from two to seven biological replicates ( siNT - MMC ( n = 6 ) , siNT + MMC ( n = 7 ) , siDPP9 - MMC ( n = 4 ) , siDPP9 + MMC ( n = 5 ) , siBRCA2 - MMC ( n = 2 ) , siBRCA2 + MMC ( n = 3 ) , siFLNA - MMC ( n = 5 ) , siFLNA + MMC ( n = 6 ) , NgtCntrl - MMC ( n = 3 ) , NgtCntrl + MMC ( n = 6 ) ) . The number of foci are shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * = p ≤ 0 . 05 , * * * * = p ≤ 0 . 0001 ) . C . Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells . Exposure of cells to MMC triggers more PLA events ( white ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _

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

B. Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non-targeting siRNA (siNT) or silenced with the indicated oligos. 300 ng / mL MMC was added for 24 h. Each dot represents the number of PLA events in a single cell, from two to seven biological replicates (siNT-MMC (n = 6), siNT+MMC (n = 7), siDPP9-MMC (n = 4), siDPP9+MMC (n = 5), siBRCA2-MMC (n = 2), siBRCA2+MMC (n = 3), siFLNA-MMC (n = 5), siFLNA+MMC (n = 6), NgtCntrl-MMC (n = 3), NgtCntrl+MMC (n = 6)). The number of foci are shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, **** = p ≤ 0.0001). C. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells. Exposure of cells to MMC triggers more PLA events (white). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071


D . Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S - Phase . Each dot represents the number of PLA events in a single cell , from three biological replicates . Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS ( 250 ng / mL for 30 min ) . Cells were sampled prior to NCS treatment , after NCS treatment and with 1 . 5 h and 3 h of recovery after NCS treatment . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * * = p ≤ 0 . 0001 ) . E . Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells . Exposure of cells to NCS triggers more PLA events ( white ) , which increase in time . Cells in S - Phase show a greater fold - increase in the number of PLA events in comparison to asynchronous cells . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Antibodies as in ( C ) .

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

D. Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S-Phase. Each dot represents the number of PLA events in a single cell, from three biological replicates. Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS (250 ng / mL for 30 min). Cells were sampled prior to NCS treatment, after NCS treatment and with 1.5 h and 3 h of recovery after NCS treatment. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001). E. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells. Exposure of cells to NCS triggers more PLA events (white), which increase in time. Cells in S-Phase show a greater fold-increase in the number of PLA events in comparison to asynchronous cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Antibodies as in (C).


F . Co - immunoprecipitation assays showing binding of BRCA2 and DPP9 - SWT . HEK293 DPP9 KO + DPP9WT cells , were treated with 1µg / mL Dox ( 24 h ) to induce the expression of DPP9 - FLAG . DNA damage was induced with 300 nM MMC treatment for 24h . Control cells do not express DPP9 ( - Dox ) . Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting ( anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - FLAG RRID : AB _ 262044 ) .

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

F. Co-immunoprecipitation assays showing binding of BRCA2 and DPP9-SWT. HEK293 DPP9 KO + DPP9WT cells, were treated with 1µg/mL Dox (24 h) to induce the expression of DPP9-FLAG. DNA damage was induced with 300 nM MMC treatment for 24h. Control cells do not express DPP9 (- Dox). Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting (anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-FLAG RRID:AB_262044).


B . Pull - down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N - terminal1 - 40 fragment immobilized on HA - beads . The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21 - 40HA for interaction with DPP9 . Representative data of three technical replicates is shown . Anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 .

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

B. Pull-down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N-terminal1-40 fragment immobilized on HA-beads. The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21-40HA for interaction with DPP9. Representative data of three technical replicates is shown. Anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.


C . Quantification of PLAs showing fewer MMC - induced DPP9 - BRCA2 PLA events in HeLa cells treated with 10µM 1G244 . Each dot represents the number of PLA events in a single cell , from three biological replicates . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 ) . D . Representative images of DPP9 - BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9 . Control cells were mock treated with DMSO . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _

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

C. Quantification of PLAs showing fewer MMC-induced DPP9-BRCA2 PLA events in HeLa cells treated with 10µM 1G244. Each dot represents the number of PLA events in a single cell, from three biological replicates. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (*** = p ≤ 0.001, **** = p ≤ 0.0001). D. Representative images of DPP9-BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9. Control cells were mock treated with DMSO. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071


F . Pull - down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N - terminus . While each protein can bind to the BRCA2 N - terminal peptide , in the presence of DPP9 , the PALB2 - BRCA2 binding is negatively affected . Representative data of three technical replicates is shown . Anti - PALB2 : RRID : AB _ 890607 , anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 .

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

F. Pull-down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N-terminus. While each protein can bind to the BRCA2 N-terminal peptide, in the presence of DPP9, the PALB2-BRCA2 binding is negatively affected. Representative data of three technical replicates is shown. Anti-PALB2: RRID:AB_890607, anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.


A - B . Representative Western Blots and accompanying graph from more than three biological replicates show that MMC ( 300 nM , 24 h ) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells , which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 ( 100µM ) , in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 ( siDPP9 ) . RAD51 stability is not altered in HeLa DPP9 KD cells . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown images originate from one representative cycloheximide ( CHX ) chase assay . The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test . ( * = p ≤ 0 . 03 ) . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - Vinculin : RRID : AB _ 477629 , anti - Tubulin : RRID : AB _ 628412 , anti - RAD51 : RRID : AB _ 1142428 .

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

A-B. Representative Western Blots and accompanying graph from more than three biological replicates show that MMC (300 nM, 24 h) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells, which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 (100µM), in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 (siDPP9). RAD51 stability is not altered in HeLa DPP9 KD cells. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test. (*= p ≤ 0.03). Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-Vinculin: RRID:AB_477629, anti-Tubulin: RRID:AB_628412, anti-RAD51: RRID:AB_1142428.


C - D . Representative Western Blots and graph summarizing results of more than three biological replicates . CHX chase assays show that the MMC - induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9 - SS729A cells overexpressing an inactive DPP9 - S mutant . Expression of DPP9 was induced ( + Dox , 1µg / ml ) simultaneously with MMC ( 300 nM ) , 24 h . Cells that were overexpressing the active variant ( DPP9 - SWT ) show similar levels of MMC - induced BRCA2 degradation . RAD51 stability is not altered by MMC and is similar in all cell lines . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown images originate from one representative cycloheximide ( CHX ) chase assay . The ratios of BRCA2 to Vinculin are defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test ( * = p ≤ 0 . 03 , * * = p ≤ 0 . 002 ) . Antibodies as described in ( A - B ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative Western Blots and graph summarizing results of more than three biological replicates. CHX chase assays show that the MMC-induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9-SS729A cells overexpressing an inactive DPP9-S mutant. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. Cells that were overexpressing the active variant (DPP9-SWT) show similar levels of MMC-induced BRCA2 degradation. RAD51 stability is not altered by MMC and is similar in all cell lines. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03, **= p ≤ 0.002). Antibodies as described in (A-B).


F - G . Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2 ∆ MP3 - 1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21 - 1000 . Cells were co - transfected with a GFP expressing plasmid , as transfection and loading control . Control cells were transfected with GFP only ( U . T ) . Image originates from one representative experiment . BRCA2 - FLAG signals are related to the transfection and loading control GFP . BRCA2 levels in relation to GFP were defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test ( * = p ≤ 0 . 03 ) . Anti - FLAG : RRID : AB _ 262044 , anti - GFP : RRID : AB _ 641123 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G. Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2∆MP3-1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21-1000. Cells were co-transfected with a GFP expressing plasmid, as transfection and loading control. Control cells were transfected with GFP only (U.T). Image originates from one representative experiment. BRCA2-FLAG signals are related to the transfection and loading control GFP. BRCA2 levels in relation to GFP were defined as 100 % at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03). Anti-FLAG: RRID:AB_262044, anti-GFP: RRID:AB_641123.


A . Representative immunofluorescence images of γH2AX signals ( white , RRID : AB _ 309864 ) in gki MEF DPP9S729A , showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin ( NCS ) , quantification in ( B ) . MEF DPP9S729A and control MEF cells were treated with 250 ng / mL NCS for 30 min and allowed to recover for the indicated time - points . γH2AX signals at time 0 , reflect 30 min of NCS and no recovery time . Nuclei are shown in blue ( DAPI ) . Scale bar 10 µm . B . Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in ( A ) . Signals from more than 1700 cells were quantified per condition per experiment . Mean ± SEM from six biological replicates , each in technical duplicates . Data were analysed by an unpaired two - way ANOVA with Sidak ' s multiple comparison test ( * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 ) .

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

A. Representative immunofluorescence images of γH2AX signals (white, RRID:AB_309864) in gki MEF DPP9S729A, showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin (NCS), quantification in (B). MEF DPP9S729A and control MEF cells were treated with 250 ng/mL NCS for 30 min and allowed to recover for the indicated time-points. γH2AX signals at time 0, reflect 30 min of NCS and no recovery time. Nuclei are shown in blue (DAPI). Scale bar 10 µm. B. Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in (A). Signals from more than 1700 cells were quantified per condition per experiment. Mean ± SEM from six biological replicates, each in technical duplicates. Data were analysed by an unpaired two-way ANOVA with Sidak's multiple comparison test (* = p ≤ 0.05, ** = p ≤ 0.01).


C . Representative PLA images of BRCA2 - PALB2 PLA experiments showing more PLA events ( white ) in HeLa cells silenced for DPP9 ( siDPP9 ) with respect to cells treated with non - targeting siRNA ( siNT ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - PALB2 : RRID : AB _ 890607 , anti - BRCA2 : RRID : AB _ 2259370 .

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

C. Representative PLA images of BRCA2-PALB2 PLA experiments showing more PLA events (white) in HeLa cells silenced for DPP9 (siDPP9) with respect to cells treated with non-targeting siRNA (siNT). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-PALB2: RRID:AB_890607, anti-BRCA2: RRID:AB_2259370.


D . Quantification of PLAs experiments showing more BRCA2 - PALB2 PLA events in cells silenced for DPP9 ( siDPP9 ) in comparison to non - targeting controls ( siNT ) . Each dot represents the number of PLA events in a single cell , from four biological replicates . The technical control samples ( NgtCntrl ) omitted the BRCA2 antibody . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) .

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

D. Quantification of PLAs experiments showing more BRCA2-PALB2 PLA events in cells silenced for DPP9 (siDPP9) in comparison to non-targeting controls (siNT). Each dot represents the number of PLA events in a single cell, from four biological replicates. The technical control samples (NgtCntrl) omitted the BRCA2 antibody. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).