( f ) Nox2 - specific intracellular ROS production was measured using p47 - roGFP redox biosensor .

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(f) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor.


g ) Extracellular ROS production was assessed using Amplex - red dye .

g) Extracellular ROS production was assessed using Amplex-red dye.


( h ) Plasma calcium influx was measured by analysing Fura - 2 quench rate upon addition of extracellular Mn2 + .

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(h) Plasma membrane calcium influx was measured by analysing the Fura-2 fluorescence quench rate upon addition of extracellular Mn2+.


( i ) Intracellular RNS generation was measured using DAF - FM . Bars represent average ± s . e . m . from n = 15 fibres for each condition in ( a , b , f , g ,

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(i) Intracellular RNS generation was measured using DAF-FM. Bars represent average±s.e.m. from n=15 individual fibres for each condition in (a,b,f,g,i


, j ) . Markers of autophagy were analysed in isolated fibres ( incubated with or without PP2 ) from FDBs .

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,j). Markers of autophagy were analysed in isolated fibres (incubated with or without PP2) from FDBs.


( k ) Autophagosome formation was analysed using microscopy ( scale bar = 100 μm ) illustrated LC3 localization autophagosome formation . ( l ) Confocal microscopy detected p62 - LC3 localization in single fibres from FDBs ( scale bar = 140 50 μm for box areas ) . immunoblots were performed with isolated from FDBs probed with antibodies as indicated . Glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) was detected as a loading control . Representative are shown . Bars represent average ± s . e . m . from n = 3 independent biological experiments . Statistical differences between groups were determined using of variance ( ANOVA ) with Tukey ' s post hoc . * P0 . 05 * * P0 . 01 .

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(k) Autophagosome formation was analysed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. (l) Confocal microscopy detected p62-LC3 localization in single fibres from FDBs (scale bar=140 and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using analysis of variance (ANOVA) with Tukey's post hoc test. *P0.05 and **P0.01.


( a ) FDB lysates were analysed using immunoblotting with antibodies as indicated .

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(a) FDB lysates were analysed using immunoblotting with antibodies as indicated.


( b ) Confocal microscopy shows effect of Nox2 inhibition on p62 - LC3 localization in single FDB fibres . Representative of independent biological experiments ( n = 3 ) are shown ( scale bar = 100 35 μm for box areas ) .

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(b) Confocal microscopy shows the effect of Nox2 inhibition on p62-LC3 localization in single FDB fibres. Representative images of independent biological experiments (n=3) are shown (scale bar=100 and 35 μm for white box areas).


( c ) Autophagic flux was analysed by immunoblotting with anti - LC3B anti - p62 antibodies .

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(c) Autophagic flux was analysed by immunoblotting with anti-LC3B and anti-p62 antibodies.


( d ) Effect of gp91 ds PP2 on LC3 - LAMP1 colocalization was analysed using confocal microscopy . Representative from independent biological experiments ( n = 3 ) are shown ( scale bar = 140 40 μm for red box areas ) .

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(d) Effect of gp91 ds and PP2 on LC3-LAMP1 colocalization was analysed using confocal microscopy. Representative images from independent biological experiments (n=3) are shown (scale bar=140 and 40 μm for red box areas).


(e) Immunoblot analysis of LAMP1,


( g ) Immunoblot of Caspase3 activity . immunoblots were performed with isolated from FDBs probed with antibodies as indicated . GAPDH was detected as a loading control . Representative are shown . Bars represent average ± s . e . m . from n = 3 independent biological experiments . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc . * P0 . 05 * * P0 . 01 .

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(g) Immunoblot of Caspase3 activity. All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. GAPDH was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


( a ) Intracellular ROS production was assessed using DCFH - DA . ( b ) Plasma calcium influx was measured by analysing quench of Fura - 2 upon addition of extracellular Mn2 + . ( a , b ) Were conducted in enzymatically digested single FDBs from WT , mdx p47 − / − - mdx mice . Bars represent average ± s . e . m . from n = 15 fibres for each condition .

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(a) Intracellular ROS production was assessed using DCFH-DA. (b) Plasma membrane calcium influx was measured by analysing the quench of Fura-2 fluorescence upon addition of extracellular Mn2+. (a,b) Were conducted in enzymatically digested single FDBs from WT, mdx and p47−/−-mdx mice. Bars represent average±s.e.m. from n=15 individual fibres for each condition.


( c ) Immunoblot of Src with anti - P - Src or anti - Src antibodies .

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(c) Immunoblot analysis of Src protein with anti-P-Src or anti-Src antibodies.


( d ) Autophagy marker were analysed by immunoblotting with antibodies as indicated ( c ) ( d ) were conducted in enzymatically isolated fibres from FDBs of WT , mdx p47 − / − - mdx mice . Representative are shown . GAPDH was detected as a loading control . Bars represent average ± s . e . m . from n = 3 independent biological experiments .

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(d) Autophagy marker proteins were analysed by immunoblotting with antibodies as indicated (c) and (d) were conducted in enzymatically isolated fibres from FDBs of WT, mdx and p47−/−-mdx mice. Representative images are shown. GAPDH was detected as a loading control. Bars represent average±s.e.m. from n=3 independent biological experiments.


( e ) LC3 - LAMP1 colocalization in single FDBs from mdx p47 − / − - mdx mice was analysed using confocal microscopy . Representative from n = 3 independent biological experiments are shown ( scale bar = 100 40 μm for red box areas ) .

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(e) LC3-LAMP1 colocalization in single FDBs from mdx and p47−/−-mdx mice was analysed using confocal microscopy. Representative images from n=3 independent biological experiments are shown (scale bar=100 and 40 μm for red box areas).


f ) Immunofluorescence to detect lysosomes ( LAMP1 ) in TA muscle from WT , mdx p47 − / − - mdx mice . arrows indicate lysosome yellow arrows indicate nucleus .

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f) Immunofluorescence assay to detect lysosomes (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. White arrows indicate lysosome and yellow arrows indicate nucleus.


( g ) Immunohistochemistry to detect lysosome ( LAMP1 ) in TA muscle from WT , mdx p47 − / − - mdx mice . Black arrows indicate LAMP1 positive ( brown ) structures . Histogram plot quantifying number of immunopositive LAMP1 structures per fibre . Representative from n = 3 independent biological experiments are shown . Scale bar represents 100 μm for f 140 μm for g . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc . * P0 . 05 * * P0 . 01 .

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(g) Immunohistochemistry to detect lysosome (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. Black arrows indicate LAMP1 positive (brown) structures. Histogram plot quantifying the number of immunopositive LAMP1 structures per fibre. Representative images from n=3 independent biological experiments are shown. Scale bar represents 100 μm for f and 140 μm for g. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


( a ) Haematoxylin eosin of cross - sections from Dia showing central nuclei ( arrow head ) smaller fibres ( arrow ) .

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(a) Haematoxylin and eosin staining of cross-sections from Dia showing central nuclei (arrow head) and smaller fibres (arrow).


( b ) Immunoblot of macrophage content ( CD68 ) .

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(b) Immunoblot analysis of macrophage content (CD68).


( c ) Immunofluorescent bright field of Dia cross - sections showing fibre - type distribution . Type I ( red ) , IIA ( green ) , IIB / IIX ( x , unstained viewed from bright field overlay ) .

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(c) Immunofluorescent and bright field images of Dia cross-sections showing fibre-type distribution. Type I (red), IIA (green), IIB/IIX ( white x, unstained and viewed from bright field overlay).


( d ) Serum creatine kinase activity . ( e ) Force - frequency relationship in Dia muscle strips from WT ( black ) , mdx ( red ) p47 − / − - mdx ( blue ) mice . Scale bars represent 55 μm . For e , # P0 . 01 p47 − / − - mdx versus mdx . # # P0 . 01 p47 − / − - mdx versus WT mdx . Mdx was statistically different than WT at frequencies of stimulation . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc . * P0 . 05 * * P0 . 01 .

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(d) Serum creatine kinase activity. (e) Force-frequency relationship in Dia muscle strips from WT (black), mdx (red) and p47−/−-mdx (blue) mice. Scale bars represent 55 μm. For e, #P0.01 p47−/−-mdx versus mdx.##P0.01 p47−/−-mdx versus WT and mdx. Mdx was statistically different than WT at all frequencies of stimulation. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


( B ) Representative immunohistochemical with anti - pan keratin antibodies of a pair of lesions formed by parallel injections of versus Black HKC strains together with of pan - keratin positive areas ( mm2 ) of lesions ( dots ) formed by 6 HKCs strains of two ancestries . n ( tumors per ancestry ) = 8 . * p < 0 . 05 . Horizontal : median . Two - unpaired t - . Scale bar : 500μm .

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(B) Representative immunohistochemical analysis with anti-pan keratin antibodies of a pair of tumor lesions formed by parallel injections of White versus Black HKC strains together with quantification of the pan-keratin positive areas (mm2) of individual lesions (dots) formed by 6 HKCs strains of the two ancestries. n(tumors per ancestry) = 8. *p<0.05. Horizontal line: median. Two-tailed unpaired t-test. Scale bar: 500μm.


Double immunofluorescence of tumors formed by six HKC strains , as in previous panels , with antibodies against Ki67 ( C ) together with anti - pan - keratin antibodies for identification . Shown are representative of results of lesions ( dots ) . Digitally acquired were used to determine % of Ki67 positive ( C ) in pan - keratin positive areas , examining 3 - 5 fields in each case . For Ki67 , K10 K1 : n ( tumors per ancestry ) = 24 , 22 6 , respectively . * * * * p < 0 . 0001 ; * * p < 0 . 005 , horizontal : median , two - unpaired t . Scale bars : 100μm .

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Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against Ki67 (C) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of Ki67 positive cells (C) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm.


Double immunofluorescence of tumors formed by six HKC strains , as in previous panels , with antibodies against keratin 10 1 ( K10 , K1 ) ( D ) together with anti - pan - keratin antibodies for identification . Shown are representative of results of lesions ( dots ) . Digitally acquired were used to determine % of K1 / K10 positivity ( D ) in pan - keratin positive areas , examining 3 - 5 fields in each case . For Ki67 , K10 K1 : n ( tumors per ancestry ) = 24 , 22 6 , respectively . * * * * p < 0 . 0001 ; * * p < 0 . 005 , horizontal : median , two - unpaired t . Scale bars : 100μm .

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Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against keratin 10 and 1 (K10, K1) (D) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of K1/K10 positivity (D) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm.


( B ) Kaplan Meier plots of long - term overall survival of H / NSCC from TCGA dataset divided on basis of high versus low HSD17B7 expression relative to its median expression value among ( darkgreen lightgreen lines ) ( red orange lines ) , showing statistically significant association of elevated HSD17B7 expression with poorer survival . Statistical significance was calculated by log - rank between groups . High _ : n = 57patients . Low _ : n = 79 . High _ : n = 203 . Low _ : n = 181 .

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(B) Kaplan Meier plots of long-term overall survival of H/NSCC patients from the TCGA dataset divided on the basis of high versus low HSD17B7 expression levels relative to its median expression value among males (darkgreen and lightgreen lines) and females (red and orange lines), showing statistically significant association of elevated HSD17B7 expression with poorer patient survival. Statistical significance was calculated by the log-rank test between the groups. High_female: n=57patients. Low_female: n= 79 patients. High_male: n= 203 patients. Low_male: n= 181 patients.


( G ) Immunofluorescence ( IF ) of HSD17B7 expression in Black HKCs . Shown are representative as well as IF signal at level of ( dots ) for indicated strains . 19 - 46 were counted in each case . n ( HKC strains per ancestry ) = 5 , * * * p < 0 . 0005 , horizontal : median , 2 - unpaired t - . Unit of Y - axis : pixels . Scale bar : 20 μm .

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(G) Immunofluorescence (IF) analysis of HSD17B7 protein expression in White and Black HKCs. Shown are representative images as well as IF signal quantification at the level of individual cells (dots) for the indicated strains. 19-46 cells were counted in each case. n(HKC strains per ancestry) = 5, ***p<0.0005, horizontal line: median, 2-tailed unpaired t-test. Unit of Y-axis: pixels. Scale bar: 20 μm.


( D - F ) Intradermal tumorigenicity assays with HKCs ( 269W ) stably infected with an HSD17B7 overexpressing lentivirus ( HSD17B7 - oe ) versus control ( Ctrl ) . Experimental conditions were same as in Fig 2 . Mice were sacrificed 15 days after injection . Shown are representative of density assessed by immunohistochemical with anti - pan keratin antibodies ( D , ) as well as % of Ki67 positive ( E ) K1 positivity ( F ) of pan - keratin positive areas . For ( D ) , n ( tumors per condition ) = 9 ; for ( E F ) n ( 3 fields per , 5 tumors per condition ) = 15 . * p < 0 . 05 , * * * * p < 0 . 0001 . Horizontal : median . 2 - tail unpaired t - . Scale bar : 500μm ( D ) , 100μm ( F ) .

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(D-F) Intradermal tumorigenicity assays with HKCs (269W) stably infected with an HSD17B7 overexpressing lentivirus (HSD17B7-oe) versus control (Ctrl). Experimental conditions were the same as in Fig 2. Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density assessed by immunohistochemical staining with anti-pan keratin antibodies (D,) as well as % of Ki67 positive cells (E) and K1 positivity (F) of pan-keratin positive areas. For (D), n(tumors per condition) = 9; for (E and F) n(3 fields per tumor, 5 tumors per condition) = 15. *p<0.05, ****p<0.0001. Horizontal line: median. 2-tail unpaired t-test. Scale bar: 500μm (D), 100μm (F).


( G , H ) Intradermal tumorigenicity assays with SCC13 overexpressing HSD17B7 ( HSD17B7 - oe ) versus control ( Ctrl ) . Mice were sacrificed 15 days after injection . Shown are representative of density , assessed by immunohistochemical with anti - pan keratin antibodies ( G ) as well as % of Ki67 positive in pan - keratin positive areas ( H ) . For ( G ) , n ( tumors per condition ) = 4 ; for ( H ) , n ( 3 - 5 fields per , 4 tumors per condition ) 15 , 19 . * p < 0 . 05 ; Horizontal : median . One - paired t - . Scale bar in ( G , H ) : 100μm .

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(G, H) Intradermal tumorigenicity assays with SCC13 cells overexpressing HSD17B7 (HSD17B7-oe) versus control (Ctrl). Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density, assessed by immunohistochemical staining with anti-pan keratin antibodies (G) as well as % of Ki67 positive cells in pan-keratin positive areas (H). For (G), n(tumors per condition) = 4; for (H), n(3-5 fields per tumor, 4 tumors per condition) 15, 19. *p<0.05; Horizontal line: median. One-tailed paired t-test. Scale bar in (G, H): 100μm.


( D ) Colony forming assays of indicated SCC lines plus / minus HSD17B7 silencing as in previous panels . plated in triplicate dishes 5 days after lentiviral vector infection selection were tested for clonogenic capability as in ( A ) . Data are represented as number of colonies + / - SD . n ( dishes per condition ) = 3 * p < 0 . 05 , * * p < 0 . 01 ; p * * * * < 0 . 0001 , 2 - unpaired t - .

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(D) Colony forming assays of the indicated SCC cell lines plus/minus HSD17B7 silencing as in the previous panels. Cells plated in triplicate dishes 5 days after lentiviral vector infection and selection were tested for clonogenic capability as in (A). Data are represented as number of colonies +/- SD. n(dishes per condition)= 3 *p<0.05, **p<0.01; p****<0.0001, 2-tailed unpaired t-test.


( C , D ) SCC13 stably infected with an HSD17B7 - expressing lentivirus ( HSD - oe ) versus empty vector control ( Ctrl ) ( C ) or two HSD17B7 - silencing lentivirus ( shHSD2 , shHSD4 ) versus vector control ( shCtrl ) ( D ) were analyzed as in previous panels . Shown are values for 3 parallel cultures per condition together with mean + / - SD . * * p < 0 . 005 ; * * * p < 0 . 0005 ; * * * * p < 0 . 0001 . 2 - unpaired t - . Additional metabolic measurement experiments of SCC plus / minus HSD17B7 overexpression silencing , in context of additional manipulations are shown in Fig 9 .

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(C, D) SCC13 cells stably infected with an HSD17B7-expressing lentivirus (HSD-oe) versus empty vector control (Ctrl) (C) or two HSD17B7-silencing lentivirus (shHSD2, shHSD4) versus vector control (shCtrl) (D) were analyzed as in the previous panels. Shown are individual values for 3 parallel cultures per condition together with mean +/- SD. **p<0.005; ***p<0.0005; ****p<0.0001. 2-tailed unpaired t-test. Additional metabolic measurement experiments of SCC cells plus/minus HSD17B7 overexpression and silencing, in the context of additional manipulations are shown in Fig 9.


( C ) Purified mitochondrial preparation from SCC13 infected with lentiviruses expressing wild type ( HSD - wt ) Y193G K197G mutated ( HSD - mut ) forms versus empty vector control ( Ctrl ) as in ( B ) were assayed for of Electron Transport Chain activity , ATP mtROS production as in Fig 8A . Data are displayed as average values of triplicate measurements together with mean + / - SD . n ( dishes per condition ) = 3 . * * p < 0 . 005 ; * * * p < 0 . 0005 . 2 - unpaired t - . experimental values are provided in Dataset EV3 .

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

(C) Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild type (HSD-wt) and Y193G and K197G mutated (HSD-mut) forms versus empty vector control (Ctrl) as in (B) were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. **p<0.005; ***p<0.0005. 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.


( D ) SCC13 were infected with two HSD17B7 silencing lentiviruses ( shHSD2 , shHSD4 ) versus vector control ( shCtrl ) followed , 3 days after infection antibiotic selection , by treatment with Zymosterol ( 2 . 5uM ) or ethanol vehicle alone for 48hours . Purified mitochondrial preparation were assayed for of Electron Transport Chain activity , ATP mtROS production as in Fig 8A . Data are displayed as average values of triplicate measurements together with mean + / - SD . n ( dishes per condition ) = 3 . * * * * p < 0 . 0001 , 2 - unpaired t - . experimental values are provided in Dataset EV3 .

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

(D) SCC13 cells were infected with two HSD17B7 silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl) followed, 3 days after infection and antibiotic selection, by treatment with Zymosterol (2.5uM) or ethanol vehicle alone for 48hours. Purified mitochondrial preparation were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. ****p<0.0001, 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.


A , B methylation level of NTN1 ( A ) DAPK1 ( B ) 5 ' regions ( Illumina ' s HumanMethylation450K Array ( HM450 ) from Cancer Genome Atlas breast cohort ) in paired breast tissues ( normal : green circles , : red circles ) , n = 92 , Wilcoxon matched - pairs signed rank , P = 0 . 004 P = 0 . 0008 .

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

A, B DNA methylation level of NTN1 (A) and DAPK1 (B) 5' regions (Illumina's HumanMethylation450K Array (HM450) from The Cancer Genome Atlas breast cohort) in paired breast tissues (normal: green circles, tumor: red circles), n = 92, Wilcoxon matched-pairs signed rank test, P = 0.004 and P = 0.0008.


C , D NTN1 ( C ) DAPK1 ( D ) gene expression in paired breast tissues ( normal : green bars , : red bars , RNAseq from TCGA breast cohort , n = 112 114 , respectively .

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

C, D NTN1 (C) and DAPK1 (D) gene expression in paired breast tissues (normal: green bars, tumor: red bars, RNAseq from TCGA breast cohort, n = 112 and 114, respectively.


E , F Correlation between NTN1 ( E ) DAPK1 ( F ) gene expression methylation in breast cancer cohort ( TCGA , n = 807 ) . Pearson correlation , P = 6 . 7 . 10 - 5 , r = - 0 . 14 for NTN1 ( A ) P < 10 - 16 , r = - 0 . 32 for DAPK1 ( B ) , respectively . G , H / Normal methylation of NTN1 ( G ) DAPK1 ( H ) in humanbreasttumors ( data extracted from TCGA cohort , paired samples ) according to gene expression ( downregulated FC ≤ 0 . 5 , down , n = 33 , or upregulated FC ≥ 1 . 3 , up , n = 16 ) . P = 3 . 10 - 2 P = 3 . 10 - 4 two - sided Mann Whitney , for NTN1 DAPK1 , respectively .

O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-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 O O B-GENEPROD 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 B-GENEPROD O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O

E, F Correlation between NTN1 (E) and DAPK1 (F) gene expression and DNA methylation in the breast cancer cohort (TCGA, n = 807). Pearson correlation, P = 6.7.10-5, r = -0.14 for NTN1 (A) and P<10-16, r = -0.32 for DAPK1 (B), respectively.G, H Tumor/Normal DNA methylation ratio of NTN1 (G) and DAPK1 (H) in humanbreasttumors (data extracted from TCGA cohort, paired samples) according to gene expression (downregulated FC ≤ 0.5, down, n = 33, or upregulated FC ≥ 1.3, up, n = 16). P = 3.10-2 and P = 3.10-4 two-sided Mann Whitney test, for NTN1 and DAPK1, respectively.


A Methyl - Cap - seq read density profiles of 5 ' end of DAPK1 , UNC5B , NTN1in MDA - MB - 231 ( blue ) HMLER ( green ) . Red boxes represent CpG islands ( CGis ) ; gray boxes regions analyzed by bisulfite - PCR - sequencing ; gray boxes regions analyzed by parallel sequencing of amplicons from bisulfite modified ; black boxes exons UTR . Chromosome coordinates of each gene are given ( black lines ) .

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

A Methyl-Cap-seq read density profiles of the 5' end of DAPK1, UNC5B, and NTN1in MDA-MB-231 (blue) and HMLER (green) cells. Red boxes represent the CpG islands (CGis); light gray boxes the regions analyzed by bisulfite-PCR-sequencing; dark gray boxes the regions analyzed by parallel sequencing of amplicons from bisulfite modified DNA; and black boxes the exons and UTR. Chromosome coordinates of each gene are given (black lines).


B , C Gene expression was measured by Q - RT - PCR after 72 h for MDA - MB - 231 ( B ) HMLER ( C ) treated daily with DAC ( 10 µM ) . PBGD expression level was used as an internal control . Data are expressed as mean ± s . e . m . of at least 3 independent experiments . * * * * P < 0 . 0001 , two - unpaired Student ' s t - .

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

B, C Gene expression was measured by Q-RT-PCR after 72 h for MDA-MB-231 (B) and HMLER cells (C) treated daily with DAC (10 µM). PBGD expression level was used as an internal control. Data are expressed as mean ± s.e.m. of at least 3 independent experiments. **** P<0.0001, two-tailed unpaired Student's t-test.


D , E Measurement of hypomethylation of DAPK1 ( D ) NTN1 ( E ) promoters after decitabine treatment of MDA - MB - 231 HMLER . Over 1960 sequences were analyzed per group in 2 independent experiments . * * * * P < 0 . 0001 , two - way ANOVA post - hoc Tukey - .

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D, E Measurement of DNA hypomethylation of the DAPK1 (D) and NTN1 (E) promoters after decitabine treatment of MDA-MB-231 and HMLER cells. Over 1960 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.


A , B were treated with DAC ( 10 μM , 72 h ) , / or net1 - mAb ( 10 μg / ml , 48 h ) / or recombinant netrin - 1 ( 5 μg / ml , 48 h ) . TUNEL assays from 3 independent experiments . * * * P < 0 . 0001 one - way ANOVA .

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A, B Cells were treated with DAC (10 μM, 72 h), and/or net1-mAb (10 μg/ml, 48 h) and/or recombinant netrin-1 (5 μg/ml, 48 h). TUNEL assays from 3 independent experiments. *** P<0.0001 one-way ANOVA.


C mortality was assessed using toxilight . Data represent mean ± s . e . m . from 3 independent experiments . * * * * P < 0 . 0001 , one - way ANOVA .

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C Cellular mortality was assessed using the toxilight assay. Data represent mean ± s.e.m. from 3 independent experiments. **** P<0.0001, one-way ANOVA.


D Loss of methylation of NTN1 DAPK1 promoters in decitabine treated tumors , compared with PBS treated tumors . > 1700 sequences were analyzed per group in 2 independent experiments . * * * * P < 0 . 0001 , two - way ANOVA post - hoc Tukey - .

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D Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated tumors, compared with PBS treated tumors. >1700 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.


E , F After one week of treatment , tumors from xenografted mice were fixed in formalin , embedded in paraffin , sliced into 4 μm sections . ( E ) of DAPK1 , UNC5B , netrin - 1 were measured in 4 independent tumors per treatment group by immunohistochemistry , expressed as a of total surface normalized against control PBS - group . Data represented as means ± s . e . m . * * * * P < 0 . 0001 , two - way ANOVA post - hoc Tukey - . ( F ) Representative sections corresponding to MDA - MB - 231xenografts , scale bars = 50 μm .

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

E, F After one week of treatment, tumors from xenografted mice were fixed in formalin, embedded in paraffin, and sliced into 4 μm sections. (E) Levels of DAPK1, UNC5B, and netrin-1 were measured in 4 independent tumors per treatment group by immunohistochemistry staining, and expressed as a percentage of total tumor surface normalized against the control PBS-group. Data represented as means ± s.e.m. **** P<0.0001, two-way ANOVA and post-hoc Tukey-test. (F) Representative tumor sections corresponding to MDA-MB-231xenografts, scale bars = 50 μm.


G , H When tumors reached 100 mm3 , mice were injected subcutaneously with decitabine ( 0 . 4 mg / kg ) or PBS / or intraperitoneally with net1 - mAb ( 10 mg / kg ) . ( G ) volumes were measured twice a week . statistical significance of differences obtained between control PBS - group treated ( DAC + net1 - mAb ) group was determined by two - way ANOVA post - hoc Tukey - . * * * P < 0 . 0001 . ( H ) weights were measured 3 days after end of experiment . statistical significance of differences obtained between groups was determined by one - way ANOVA , * * * P < 0 . 0001 . Error bars = s . e . m .

O O O O B-TISSUE O O O O B-ORGANISM O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE 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

G, H When tumors reached 100 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). (G) Tumor volumes were measured twice a week. The statistical significance of the differences obtained between the control PBS-group and treated (DAC + net1-mAb) group was determined by two-way ANOVA and post-hoc Tukey-test. *** P<0.0001. (H) Tumor weights were measured 3 days after the end of the experiment. The statistical significance of the differences obtained between the groups was determined by one-way ANOVA, *** P<0.0001. Error bars = s.e.m.


A Loss of methylation of NTN1 DAPK1 promoters in decitabine treated PDX tumors , compared with control PBS - group . of mean methylation of 11 DAPK1 - CpGs was 94 % ( 550 amplicons analyzed ) , while that of 7 NTN1 - CpGs was 64 % ( 213 amplicons analyzed ) . Two - way ANOVA post - hoc Tukey - .

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A Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated PDX tumors, compared with the control PBS-group. The percentage of mean DNA methylation of the 11 DAPK1-CpGs was 94% (550 amplicons analyzed), while that of the 7 NTN1-CpGs was 64% (213 amplicons analyzed). Two-way ANOVA and post-hoc Tukey-test.


B Expression of DAPK1 , UNC5B , NTN1 was measured by Q - RT - PCR in PDX tumors after 7 days of in vivo DAC treatment ( 0 . 4 mg / kg ) . level of PBGD expression was used as an internal control . Data are expressed as mean ± s . e . m . for at least 3 grafts per group . * * * * P < 0 . 0001 , , two - way ANOVA post - hoc Tukey - .

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

B Expression of DAPK1, UNC5B, and NTN1 was measured by Q-RT-PCR in PDX tumors after 7 days of in vivo DAC treatment (0.4 mg/kg). The level of PBGD expression was used as an internal control. Data are expressed as mean ± s.e.m. for at least 3 grafts per group. **** P<0.0001,, two-way ANOVA and post-hoc Tukey-test.


C of DAPK1 , UNC5B , netrin - 1 were measured in at least 3 independent paraffin embedded xenografts per treatment group by immunohistochemistry . Data are expressed as of total surface normalized against median surface of PBS - group . * * * * P < 0 . 0001 , one - way ANOVA . Error bars = s . e . m .

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

C Levels of DAPK1, UNC5B, and netrin-1 were measured in at least 3 independent paraffin embedded xenografts per treatment group by immunohistochemistry. Data are expressed as the percentage of the total tumor surface normalized against the median tumor surface of the PBS-group. **** P<0.0001, one-way ANOVA. Error bars = s.e.m.


D caspase - 3 fragmentation ( TUNEL ) median number of per mm2 were measured in treated xenografts . Data represent mean ± s . e . m . from at least 3 tumors per group . * * * * P < 0 . 0001 , one - way ANOVA . E Representative sections of xenograft PDX analyzed in ( D ) . Scale bars = 50 μm . Arrows indicate positive .

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

D Cleaved caspase-3 and DNA fragmentation (TUNEL) median number of cells per mm2 were measured in treated xenografts. Data represent mean ± s.e.m. from at least 3 tumors per group. **** P<0.0001, one-way ANOVA.E Representative sections of the xenograft PDX analyzed in (D). Scale bars = 50 μm. Arrows indicate positive staining.


F combination of net1 - mAb DAC reduces humanbreasttumor growth in immuno - compromised mice . After anesthesia , mice were engrafted in interscapular with a ≈ 60 mm3 - derived . When tumors reached 120 - 150 mm3 , mice were injected subcutaneously with decitabine ( 0 . 4 mg / kg ) or PBS / or intraperitoneally with net1 - mAb ( 10 mg / kg ) or with a humanIgG1 control isotype antibody ( Ctrl IgG1 , 10 mg / kg ) from day 1 to day 21 . volumes were measured twice a week ; n = 7 mice per group . statistical significance of differences obtained between DAC + Ctrl IgG1 - group DAC + net1 - mAb - group was determined by two - way ANOVA2 a post - hoc Tukey - , * * * * P < 0 . 0001 . Error bars = s . e . m .

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

F The combination of net1-mAb and DAC reduces humanbreasttumor growth in immuno-compromised mice. After anesthesia, mice were engrafted in the interscapular area with a ≈ 60 mm3 patient-derived tumor. When the tumors reached 120-150 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg) or with a humanIgG1 control isotype antibody (Ctrl IgG1, 10 mg/kg) from day 1 to day 21. Tumor volumes were measured twice a week; n = 7 mice per group. The statistical significance of the differences obtained between the DAC + Ctrl IgG1-group and the DAC + net1-mAb-group was determined by two-way ANOVA2 and a post-hoc Tukey-test, **** P<0.0001. Error bars = s.e.m.


A , D Stably transfected MDA - MB - 231 bearing a control ( A ) , DAPK1 ( B ) , UNC5B ( C ) , or NTN1 shRNA ( D ) were injected into mammary fat pad of immuno - compromised mice . When tumors reached 100 - 120 mm3 , mice were injected subcutaneously with DAC ( 0 . 4 mg / kg ) or PBS / or intraperitoneally with net1 - mAb ( 10 mg / kg ) . volumes were measured twice a week , n = 8 mice per group . statistical significance of differences obtained between DAC - group DAC + net1 - mAb - group for shControl , DAC - group DAC + net1 - mAb - group for shDAPK1 shUNC5B , PBS - group DAC - group for shNTN1 , respectively , were determined by two - way ANOVA a post - hoc Tukey - . * * * * P < 0 . 0001 , , ns = not significant . Error bars = s . e . m .

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

A, D Stably transfected MDA-MB-231 cells bearing a control (A), DAPK1 (B), UNC5B (C), or NTN1 shRNA (D) were injected into the mammary fat pad of immuno-compromised mice. When tumors reached 100-120 mm3, mice were injected subcutaneously with DAC (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). Tumor volumes were measured twice a week, n = 8 mice per group. The statistical significance of the differences obtained between DAC-group and DAC + net1-mAb-group for shControl, DAC-group and DAC + net1-mAb-group for shDAPK1 and shUNC5B, and PBS-group and DAC-group for shNTN1, respectively, were determined by two-way ANOVA and a post-hoc Tukey-test. **** P<0.0001, , ns = not significant. Error bars = s.e.m.


A . Infection by a L . pneumophila overexpressing MvcA but not inactive mutant MvcAC83A interferes with UBE2N modification . Raw264 . 7 were infected with indicated strains at an MOI of 5 for 2 h . Saponin soluble resolved by SDS - PAGE were probed with a UBE2N - specific antibody ( top panel ) . delivery of MavC MvcA into infected was probed with antibodies specific to each of these two . Note that MavC but not MvcA is detectable in infected with wild type ( middle panels labeled as " translocation " ) . Tubulin was probed as a loading control . of MavC MvcA associated with were similarly probed in saponin insoluble fraction ( lower panels labeled as " expression " ) . metabolism enzyme isocitrate dehydrogenase ( ICDH ) was probed as a loading control . Note that endogenous MvcA was not detectable in grown in bacteriological medium ( middle panel of lower portion ) .

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

A. Infection by a L. pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification. Raw264.7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h. Saponin soluble proteins resolved by SDS-PAGE were probed with a UBE2N-specific antibody (top panel). The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins. Note that MavC but not MvcA is detectable in cells infected with wild type bacteria (middle panels labeled as "translocation"). Tubulin was probed as a loading control. Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction (lower panels labeled as "expression"). The bacterial metabolism enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control. Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium (the middle panel of the lower portion).


B . Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian . HEK293T were transfected with combinations of empty vector plasmid directing expression of MavC or MvcA . 16 h after transfection , modification of endogenous UBE2N was detected by immunoblotting ( upper level ) . canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis ( Ypt ) , which cannot modify UBE2N , was included as a control . Note that coexpression of MvcA led to accumulation of unmodified UBE2N ( 4th lane ) in samples that expressed MavC . expression of MavC MvcA Cif was detected using a Flag - specific antibody ( lower panel ) . Tubulin was probed as a loading control ( lower panel ) .

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B. Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells. HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA. 16 h after transfection, modification of endogenous UBE2N was detected by immunoblotting (upper level). The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis (Ypt), which cannot modify UBE2N, was included as a control. Note that coexpression of MvcA led to accumulation of unmodified UBE2N (4th lane) in samples that expressed MavC. The expression of MavC MvcA and Cif was detected using a Flag-specific antibody (lower panel). Tubulin was probed as a loading control (lower panel).


C . MvcA interferes with MavC - induced UBE2N ubiquitination . Reactions containing indicated combinations of were allowed to proceed for 30 min samples resolved by SDS - PAGE were detected by Coomassie Brilliant blue ( CBB ) . Note that MvcA ( inclusion of MvcA with MavC , 7th lane . Compare to MavC alone in 3rd lane ) but not MvcAC83A mutant ( 8th lane ) abolished MavC - induced UBE2N modification .

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C. MvcA interferes with MavC-induced UBE2N ubiquitination. Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by Coomassie Brilliant blue (CBB) staining. Note that MvcA (inclusion of MvcA with MavC, 7th lane. Compare to MavC alone in 3rd lane) but not the MvcAC83A mutant (8th lane) abolished MavC-induced UBE2N modification.


D . Dose - dependent modification of UBE2N by MavC MvcA . A series of reactions containing MavC MvcA at indicated molar ratios were established reactions were allowed to proceed for 30 min . Samples resolved by SDS - PAGE were detected by CBB .

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D. Dose-dependent modification of UBE2N by MavC and MvcA. A series of reactions containing MavC and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min. Samples resolved by SDS-PAGE were detected by CBB staining.


A . hydrolytic cleavage of UBE2N - Ub by MvcA into UBE2N ubiquitin ( Q40E variant ) . Reactions containing indicated were allowed to proceed for 30 min samples resolved by SDS - PAGE were detected by CBB ( left ) . Mass spectrometric of band corresponding to ubiquitin produced by MvcA from UBE2N - Ub ( right ) . ubiquitin product is Glu40 variant of ubiquitin .

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A. The hydrolytic cleavage of UBE2N-Ub by MvcA into UBE2N and ubiquitin (Q40E variant). Reactions containing the indicated proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by CBB staining (left). Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N-Ub (right). The ubiquitin product is the Glu40 variant of ubiquitin.


C . Dose - dependent cleavage of UBE2N - Ub by MvcA . A series of reactions containing UBE2N - Ub MvcA at indicated molar ratios were established reactions were allowed to proceed for 30 min before SDS - PAGE CBB . Note that activity was detectable in a reaction starting at molar of between UBE2N - Ub MvcA is 2048 : 1 .

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C. Dose-dependent cleavage of UBE2N-Ub by MvcA. A series of reactions containing UBE2N-Ub and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min before SDS-PAGE and CBB staining. Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N-Ub and MvcA is 2048:1.


D . Time - dependent cleavage of UBE2N - Ub by MvcA . UBE2N - Ub was incubated with MvcA at molar of 128 . Samples taken at indicated time points were resolved by SDS - PAGE detected by CBB .

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D. Time-dependent cleavage of UBE2N-Ub by MvcA. UBE2N-Ub was incubated with MvcA at the molar ratio of 128. Samples taken at the indicated time points were resolved by SDS-PAGE and detected by CBB staining.


E . UBE2N produced from UBE2N - Ub via MvcA catalyzed deubiquitination is active in catalyzing formation of polyubiquitin chains . UBE2N - Ub was preincubated with MvcA or its inactive mutant for 30 min . Then a cocktail containing E1 , UBE2V2 , 3xHA - ubiquitin , E3 enzyme TRAF6 ATP was added into reactions . Ubiquitination was allowed to proceed for 30 min . in samples resolved by SDS - PAGE were transferred onto nitrocellulose membranes blotted with HA specific antibody . Note robust formation of polyubiquitin chains in reaction receiving MvcA ( middle lane ) .

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E. UBE2N produced from UBE2N-Ub via MvcA catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains. UBE2N-Ub was preincubated with MvcA or its inactive mutant for 30 min. Then a cocktail containing E1, UBE2V2, 3xHA-ubiquitin, the E3 enzyme TRAF6 and ATP was added into reactions. Ubiquitination was allowed to proceed for 30 min. Proteins in samples resolved by SDS-PAGE were transferred onto nitrocellulose membranes and blotted with the HA specific antibody. Note the robust formation of polyubiquitin chains in the reaction receiving MvcA (middle lane).


H . Mutational of residues important for substrate recognition by MvcA predicted by structural . For deubiquitination reactions , indicated amounts of MvcA or its mutants were incubated with 2 . 0 μg UBE2N for 5 min in 25 μl reactions . For ubiquitin deamidase activity , indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions . of cleavage was analyzed by SDS - PAGE then densitometry . Each bar represents of cleavage or deamidation obtained from three independent experiments . Bars error bars : means standard error of mean ( ) .

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H. Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis. For deubiquitination reactions, the indicated amounts of MvcA or its mutants were incubated with 2.0 μg UBE2N for 5 min in 25 μl reactions. For ubiquitin deamidase activity, the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM).


I - J . roles of ubiquitin contact residues in activity of MvcA . I . indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N - Ub for 5 min in 25 μl reactions . of cleavage was analyzed by SDS - PAGE then densitometry . Each bar represents of cleavage obtained from three independent experiments . Bars error bars : means standard error of mean ( ) . J . activity of MvcA mutant harboring three mutations . 0 . 05 μg , 0 . 25 μg 1 . 25 μg of MvcA or mutant was incubated with 2 μg UBE2N - Ub in 25 μl reactions . After 30 min incubation , products resolved by SDS - PAGE were detected by CBB . Note that mutant did not detectable cleave UBE2N - Ub even in reaction received highest amount of .

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I-J. The roles of ubiquitin contact residues in the activity of MvcA. I. The indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N-Ub for 5 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM). J. The activity of the MvcA mutant harboring three mutations. 0.05 μg, 0.25 μg and 1.25 μg of MvcA or the mutant was incubated with 2 μg UBE2N-Ub in 25 μl reactions. After 30 min incubation, the products resolved by SDS-PAGE were detected by CBB staining. Note that the mutant did not detectable cleave UBE2N-Ub even in the reaction received the highest amount of protein.


A . Overexpression of MavC but not inactive mutant MavCC74A in ∆ mavC ∆ mvcA mutant background causes IκBα accumulation in infected . U937 infected with indicated strains at an MOI of 5 were probed for IκBα . Tubulin was probed as a loading control .

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A. Overexpression of MavC but not the inactive mutant MavCC74A in the ∆mavC∆mvcA mutant background causes IκBα accumulation in infected cells. U937 cells infected with the indicated bacterial strains at an MOI of 5 were probed for IκBα. Tubulin was probed as a loading control.


B . Prolonged UBE2N modification by MavC restricts vacuole maturation . HEK293 transfected to express FcγII receptor 4xFlag - MavC or C74A mutant were challenged at an MOI of 1 with opsonized L . pneumophila that expresses GFP for 12 h . vacuole was scored categorized by number of it contained . Data ( mean + / - SE ) shown were from three independent experiments , each with at least 300 vacuoles counted . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

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B. Prolonged UBE2N modification by MavC restricts bacterial vacuole maturation. HEK293 cells transfected to express the FcγII receptor and 4xFlag-MavC or the C74A mutant were challenged at an MOI of 1 with opsonized L. pneumophila that expresses GFP for 12 h. The vacuole was scored and categorized by the number of bacteria it contained. Data (mean +/- SE) shown were from three independent experiments, each with at least 300 vacuoles counted. *, p<0.05; **, p<0.01; n.s., not significant.


C . Overexpression of MavC but not MavCC74A affects intracellular growth of ∆ mavC ∆ mvcA mutant of L . pneumophila . Raw264 . 7 were infected with indicated strains intracellular were determined at indicated time points . Each was examined in triplicate values shown were mean + / - SE . Similar results were obtained in two independent experiments . * , p < 0 . 1 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ( t - by comparing p ( MavC ) with p ( MavCC74A ) ) .

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C. Overexpression of MavC but not MavCC74A affects intracellular growth of the ∆mavC∆mvcA mutant of L. pneumophila. Raw264.7 cells were infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points. Each strain was examined in triplicate and the values shown were mean +/- SE. Similar results were obtained in two independent experiments. *, p<0.1; **, p<0.01; ***, p<0.001(t-test by comparing p(MavC) with p(MavCC74A)).


D . MvcA is required for reversal of UBE2N modification during L . pneumophila infection . U937 infected with wild type or ∆ mvcA mutant were withdrawn at indicated time points . Samples resolved by SDS - PAGE were probed for UBE2N , translocated MavC MvcA . Tubulin was probed as a loading control . Note that in samples infected with ∆ mvcA , level of UBE2N - Ub decreased at slower rates .

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D. MvcA is required for reversal of UBE2N modification during L. pneumophila infection. U937 cells infected with wild type or the ∆mvcA mutant were withdrawn at the indicated time points. Samples resolved by SDS-PAGE were probed for UBE2N, translocated MavC and MvcA. Tubulin was probed as a loading control. Note that in samples infected with the ∆mvcA strain, the level of UBE2N-Ub decreased at slower rates.


E . MvcA becomes detectable in intracellular 5 h after uptake . U937 macrophages infected with wild type L . pneumophila at an MOI of 10 were lysed at indicated time recovered were probed by immunoblotting for MavC MvcA , respectively . isocitrate dehydrogenase ( ICDH ) was probed sequentially as a loading control .

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E. MvcA becomes detectable in intracellular bacteria 5 h after uptake. U937 macrophages infected with wild type L. pneumophila at an MOI of 10 were lysed at the indicated time and recovered bacteria were probed by immunoblotting for MavC and MvcA, respectively. The bacterial isocitrate dehydrogenase (ICDH) was probed sequentially as a loading control.


F . MvcA restores translocation of p65 inhibited by MavC during L . pneumophila infection . Raw264 . 7 were infected with indicated L . pneumophila strains for 2 h at an MOI of 1 . Fixed were immunostained with antibodies specific for p65 bacterium , respectively . Host nuclei were labeled with Hoechst . Representative of in each sample category were shown ( upper panels ) ; localization of p65 was determined by scoring at least 300 infected ( lower panel ) . Results shown were from three independent experiments done in triplicate values shown were mean + / - SE ; t - .

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F. MvcA restores the nuclear translocation of p65 inhibited by MavC during L. pneumophila infection. Raw264.7 cells were infected with the indicated L. pneumophila strains for 2 h at an MOI of 1. Fixed cells were immunostained with antibodies specific for p65 and the bacterium, respectively. Host nuclei were labeled with Hoechst. Representative images of cells in each sample category were shown (upper panels); nuclear localization of p65 was determined by scoring at least 300 infected cells (lower panel). Results shown were from three independent experiments done in triplicate and the values shown were mean +/- SE; t-test.


G . Overexpression of MvcA but not its inactive mutant MvcAC83A promotes IκBα degradation during L . pneumophila infection . U937 were infected with indicated strains at an MOI of 5 . Lysates of infected for indicated time were probed for IκBα . Tubulin was probed as a loading control . IκBα for infected with strains ∆ mvA ( pMvcA ) ∆ mvA ( pMvcAC83A ) at indicated time points were evaluated by measuring band from three independent experiments ( lower panel ) , values shown were mean + / - SE ; t - .

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

G. Overexpression of MvcA but not its inactive mutant MvcAC83A promotes IκBα degradation during L. pneumophila infection. U937 cells were infected with the indicated bacterial strains at an MOI of 5. Lysates of cells infected for the indicated time were probed for IκBα. Tubulin was probed as a loading control. IκBα levels for cells infected with the strains ∆mvA(pMvcA) and ∆mvA(pMvcAC83A) at the indicated time points were evaluated by measuring the band intensity from three independent experiments (lower panel), the values shown were mean +/- SE; t-test.


Live imaging of histone - GFP ( green ) ParB - mCherry ( magenta ) expressed in larval salivary nuclei using 1151 - Gal4 . H3 . 3 - GFP are increased at E ( spl ) - C in presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ ( A ) . ParB - mCherry binds to its cognate int inserted within E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( ) = 5 µm . Quantifications of relative of histone - GFP ParB - mCherry across E ( spl ) - C in Notch - OFF ( upper ) Notch - ON ( lower ) conditions . Mean + / - ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 nglands = 5 , 5 , 5 , 5 , 5 , 4 where each represents a biological replicate ( from top to bottom ) .

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

Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. H3.3-GFP levels are increased at the E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Live imaging of histone - GFP ( green ) ParB - mCherry ( magenta ) expressed in larval salivary nuclei using 1151 - Gal4 . E ( spl ) - C in presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ but there is little change in H3 - GFP between Notch - OFF Notch - ON nuclei ParB - mCherry binds to its cognate int inserted within E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( ) = 5 µm . Quantifications of relative of histone - GFP ParB - mCherry across E ( spl ) - C in Notch - OFF ( upper ) Notch - ON ( lower ) conditions . Mean + / - ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 nglands = 5 , 5 , 5 , 5 , 5 , 4 where each represents a biological replicate ( from top to bottom ) .

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

Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ but there is little change in H3-GFP between Notch-OFF and Notch-ON nuclei ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Live imaging of histone - GFP ( green ) ParB - mCherry ( magenta ) expressed in larval salivary nuclei using 1151 - Gal4 . E ( spl ) - C in presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ ( A ) . with H3 . 3core - GFP ParB - mCherry binds to its cognate int inserted within E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( ) = 5 µm . Quantifications of relative of histone - GFP ParB - mCherry across E ( spl ) - C in Notch - OFF ( upper ) Notch - ON ( lower ) conditions . Mean + / - ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 nglands = 5 , 5 , 5 , 5 , 5 , 4 where each represents a biological replicate ( from top to bottom ) .

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

Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). with H3.3core-GFP ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Effects from depleting remodelers histone chaperones , as indicated ( wide range shown in A , on recruitment of Su ( H ) - GFP in Notch - ON nuclei ( expressing NΔECD ) of larval salivary . Different OSA RNAi stocks used in C are denoted by ( 1 ) ( 2 ) . In conditions except Moira RNAi ( A ) nuclei exhibit a bright accumulation of Su ( H ) - GFP at a single locus when imaged live . Scale bars ( yellow ) = 5 µm . of Notch - ON nuclei retaining a single clear band of Su ( H ) - GFP when indicated RNAi is co - expressed with NΔECD . For each genotype , 5 nuclei from each of 10 were scored ( 50 nuclei total ) . * * * * A significant fraction of nuclei lost band when components of BRM were perturbed ; p < 0 . 0001 , two - Fisher ' s exact calculated using raw ( non - ) scoring data .

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Effects from depleting chromatin remodelers and histone chaperones, as indicated (wide range shown in A, on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. Different OSA RNAi stocks used in C are denoted by (1) and (2). In all conditions except Moira RNAi (A) nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.


Effects from depleting remodelers histone chaperones , BRM components shown in C ) , on recruitment of Su ( H ) - GFP in Notch - ON nuclei ( expressing NΔECD ) of larval salivary . w RNAi is a control BrmK804R is expression of dominant - negative Brm . Different OSA RNAi stocks used in C are denoted by ( 1 ) ( 2 ) . , Snr1 RNAi BrmK804R expression ( C ) , nuclei exhibit a bright accumulation of Su ( H ) - GFP at a single locus when imaged live . Scale bars ( yellow ) = 5 µm . of Notch - ON nuclei retaining a single clear band of Su ( H ) - GFP when indicated RNAi is co - expressed with NΔECD . For each genotype , 5 nuclei from each of 10 were scored ( 50 nuclei total ) . * * * * A significant fraction of nuclei lost band when components of BRM were perturbed ; p < 0 . 0001 , two - Fisher ' s exact calculated using raw ( non - ) scoring data .

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Effects from depleting chromatin remodelers and histone chaperones , BRM complex components shown in C), on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. w RNAi is a control and BrmK804R is expression of dominant-negative Brm. Different OSA RNAi stocks used in C are denoted by (1) and (2). , Snr1 RNAi and BrmK804R expression (C), nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.


accessibility in Notch - OFF ( B ) Notch - ON ( NΔECD expression , C ) salivary nuclei measured by ATAC - qPCR ; fold enrichment at indicated regions compared to a " closed ctrl " region . Expression of dominant - negative Brm , BrmK804R , led to reduced accessibility of E ( spl ) mβ - HLH E ( spl ) m3 - HLH enhancer regions in Notch - OFF conditions , to a more widespread reduction in accessibility in Notch - ON conditions . " Eip78C enh " corresponds to ecdysone receptor - binding region of Eip78C enhancer , which is highly accessible but not Notch - responsive ; " Rab11 tr " " Mst87F tr " represent highly lowly - expressed control genes respectively . Mean + / - ; n = 3 ; * p < 0 . 05 with two - Welch ' s t - comparing LacZ BrmK804R samples .

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Chromatin accessibility in Notch-OFF (B) and Notch-ON (NΔECD expression, C) salivary gland nuclei measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Expression of dominant-negative Brm, BrmK804R, led to reduced accessibility of E(spl)mβ-HLH and E(spl)m3-HLH enhancer regions in Notch-OFF conditions, and to a more widespread reduction in accessibility in Notch-ON conditions. "Eip78C enh" corresponds to the ecdysone receptor-binding region of the Eip78C enhancer, which is highly accessible but not Notch-responsive; "Rab11 tr" and "Mst87F tr" represent highly and lowly-expressed control genes respectively. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test comparing LacZ and BrmK804R samples.


accessibility in salivary nuclei depleted for Su ( H ) by RNAi , measured by ATAC - qPCR ; fold enrichment at indicated regions compared to a " closed ctrl " region . Accessibility is increased across most of E ( spl ) - C compared to controls expressing LacZ . Control primer regions are as in B C . Mean + / - ; n = 3 ; * p < 0 . 05 with two - Welch ' s t - compared to LacZ controls .

O O B-TISSUE B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD I-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 B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR 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 B-GENEPROD O O

Chromatin accessibility in salivary gland nuclei depleted for Su(H) by RNAi, measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Accessibility is increased across most of the E(spl)-C compared to controls expressing LacZ. Control primer regions are as in B and C. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test compared to LacZ controls.


Effect of brm Snr1 RNAi on brm Snr1 cDNA respectively , measured by reverse transcription - qPCR in Kc167 ; cDNA compared to GFP RNAi . knockdowns are highly effective , with only 1 - 2 % of brm Snr1 cDNA remaining detectable . Mean + / - ; n = 3 .

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Effect of brm and Snr1 RNAi on brm and Snr1 cDNA levels respectively, measured by reverse transcription-qPCR in Kc167 cells; percentage cDNA compared to GFP RNAi. The knockdowns are highly effective, with only 1-2% of brm and Snr1 cDNA remaining detectable. Mean +/- SEM; n = 3.


Knockdown of components of BRM reduces Su ( H ) recruitment both in Notch - OFF ( B ) Notch - ON ( C ) conditions . Fold enrichment of Su ( H ) occupancy at indicated positions detected by ChIP , relative to input , in Kc167 treated with brm , Snr1 or GFP RNAi as a control . Notch - ON conditions ( C ) were induced by 30 minutes of EGTA treatment . Mean + / - , n = 3 ( B ) ; Mean , n = 2 ( C ) ; * p < 0 . 05 with one - student ' s t - compared to GFP RNAi control .

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Knockdown of components of the BRM complex reduces Su(H) recruitment both in Notch-OFF (B) and Notch-ON (C) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with brm, Snr1 or GFP RNAi as a control. Notch-ON conditions (C) were induced by 30 minutes of EGTA treatment. Mean +/- SEM, n = 3 (B); Mean, n = 2 (C); * p<0.05 with one-tailed student's t-test compared to GFP RNAi control.


Effect of brm RNAi on E ( spl ) mβ - HLH ( mβ ) E ( spl ) m3 - HLH ( m3 ) induction by Notch activation ( EGTA treatment ) measured by reverse transcription - qPCR ; shown as fold difference to lacZ RNAi control . Mean + / - ; n = 3 .

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Effect of brm RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) induction by Notch activation (EGTA treatment) measured by reverse transcription-qPCR; shown as fold difference to lacZ RNAi control. Mean +/- SEM; n = 3.


Effect of Brm dominant - negative on expression of E ( spl ) mβ - HLH ( mβ ) E ( spl ) m3 - HLH ( m3 ) measured by reverse transcription - qPCR . Expression was analyzed in stable lines containing pMT - inducible BrmWT or BrmK804R in absence ( left , uninduced ) or presence of copper sulfate ( right , Cu2 + induced ) . response of E ( spl ) mβ - HLH E ( spl ) m3 - HLH to Notch activation ( " N - On " = EGTA treatment vs . " N - Off " = PBS control ) was reduced in BrmK804R - expressing compared to BrmWT - expressing , only when induced with copper ( right graph ) . Mean , n = 2 ( left ) ; Mean + / - , n = 3 ( right ) ; * p < 0 . 05 with one - student ' s t - comparing BrmWT BrmK804R .

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Effect of Brm dominant-negative on expression of E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) measured by reverse transcription-qPCR. Expression was analyzed in stable cell lines containing pMT-inducible BrmWT or BrmK804R in the absence (left, uninduced) or presence of copper sulfate (right, Cu2+ induced). The response of E(spl)mβ-HLH and E(spl)m3-HLH to Notch activation ("N-On" = EGTA treatment vs. "N-Off" = PBS control) was reduced in the BrmK804R-expressing cells compared to BrmWT-expressing cells, only when induced with copper (right graph). Mean, n = 2 (left); Mean +/- SEM, n = 3 (right); * p<0.05 with one-tailed student's t-test comparing BrmWT and BrmK804R.


Knockdown of actin - related subunit , BAP55 , reduces Su ( H ) recruitment in both Notch - OFF ( PBS treatment ) Notch - ON ( EGTA treatment ) conditions . Fold enrichment of Su ( H ) occupancy at indicated positions detected by ChIP , relative to input , in Kc167 treated with Bap55 or lacZ RNAi as a control . Mean + / - ; n = 3 ; * p < 0 . 05 with one - student ' s t - compared to lacZ RNAi control .

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Knockdown of actin-related subunit, BAP55, reduces Su(H) recruitment in both Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with Bap55 or lacZ RNAi as a control. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.


Effect of Bap55 RNAi on E ( spl ) mβ - HLH ( mβ ) E ( spl ) m3 - HLH ( m3 ) expression measured by reverse transcription - qPCR in Notch - OFF ( PBS treatment ) Notch - ON ( EGTA treatment ) conditions . Expression level of RpII215 is shown as a control gene . Mean + / - ; n = 3 ; * p < 0 . 05 with one - student ' s t - compared to lacZ RNAi control .

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Effect of Bap55 RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) expression levels measured by reverse transcription-qPCR in Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Expression level of RpII215 is shown as a control gene. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.


Nucleosome turnover measured by CATCH - IT - qPCR ; fold enrichment over input samples compared to Sec15 tr control region . Su ( H ) - bound enhancers show increased nucleosome turnover in response to Notch signaling . Notch signaling is activated in Kc167 by 6 hours of copper induction of pMT - NICD with copper excluded in control . Positions of E ( spl ) - C primers are shown in Fig 3A ; remaining primers are control non - Notch - responsive regions . Mean , n = 2 .

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

Nucleosome turnover measured by CATCH-IT-qPCR; fold enrichment over input samples compared to Sec15 tr control region. Su(H)-bound enhancers show increased nucleosome turnover in response to Notch signaling. Notch signaling is activated in Kc167 cells by 6 hours of copper induction of pMT-NICD with copper excluded in the control. Positions of E(spl)-C primers are shown in Fig 3A; the remaining primers are control non-Notch-responsive regions. Mean, n = 2.


brm RNAi reduces nucleosome turnover at Notch - responsive regions . CATCH - IT - qPCR results as in A after brm or lacZ RNAi as a control . Mean + / - ; n = 5 ; * p < 0 . 05 with one - student ' s t - comparing brm lacZ RNAi .

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brm RNAi reduces nucleosome turnover at Notch-responsive regions. CATCH-IT-qPCR results as in A after brm or lacZ RNAi as a control. Mean +/- SEM; n = 5; * p<0.05 with one-tailed student's t-test comparing brm and lacZ RNAi.


Brm is required for Notch - responsive nucleosome turnover . CATCH - IT - qPCR results after brm or lacZ RNAi as in B pMT - NICD expression as in A . Mean + / - ; n = 3 ; * p < 0 . 05 with two - student ' s t - compared to control ( lacZ RNAi Notch - ON bars are compared to lacZ RNAi Notch - OFF bars brm RNAi bars are compared to their respective lacZ RNAi control bars ) .

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Brm is required for Notch-responsive nucleosome turnover. CATCH-IT-qPCR results after brm or lacZ RNAi as in B and pMT-NICD expression as in A. Mean +/- SEM; n=3; * p<0.05 with two-tailed student's t-test compared to control (lacZ RNAi Notch-ON bars are compared to lacZ RNAi Notch-OFF bars and brm RNAi bars are compared to their respective lacZ RNAi control bars).


brm RNAi reduces incorporation of histone H3 . 3 . V5 ChIP - qPCR in Kc167 after lacZ or brm RNAi treatment in with H3 - V5 or H3 . 3 - V5 expression induced from pMT promoter by 3 hours of copper treatment , shown as fold enrichment over input samples . Mean + / - ; n = 3 . * p < 0 . 05 with two - Welch ' s t - compared to lacZ RNAi control .

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

brm RNAi reduces incorporation of histone H3.3. V5 ChIP-qPCR in Kc167 cells after lacZ or brm RNAi treatment in cells with H3-V5 or H3.3-V5 expression induced from the pMT promoter by 3 hours of copper treatment, shown as fold enrichment over input samples. Mean +/- SEM; n = 3. * p<0.05 with two-tailed Welch's t-test compared to lacZ RNAi control.


( B ) Epg5 wild - type copy of Epg5 are absent in Epg5 − / − mice . There is an intervening lane between Epg5 + / − Epg5 − / − lanes in Western blot .

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(B) Epg5 protein and the wild-type copy of Epg5 are absent in Epg5−/− mice. There is an intervening lane between Epg5+/− and Epg5−/− lanes in the Western blot.


( C ) curves of Epg5 + / − Epg5 − / − . Means ± of 12 mice are shown .

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(C) Body weight curves of Epg5+/− and Epg5−/− males. Means ± SEM of 12 mice are shown.


( D ) Time spent on a rotarod . horizontal lines indicate mean times .

(D) Time spent on a rotarod. The horizontal lines indicate mean times.


( E ) Epg5 − / − show gradually worsening limb - clasping reflexes .

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(E) Epg5−/− males show gradually worsening limb-clasping reflexes.


( F ) hindquarters of Epg5 − / − mice are completely paralyzed at 10 mo of .

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(F) The hindquarters of Epg5−/− mice are completely paralyzed at 10 mo of age.


( G ) Spinal deformation in an Epg5 − / − at 10 mo of .

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(G) Spinal deformation in an Epg5−/− male at 10 mo of age.


( H ) Epg5 − / − gradually develop limb - clasping phenotype .

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(H) Epg5−/− females gradually develop the limb-clasping phenotype.


( A ) Nissl of fifth layer of cortex in 10 - mo - old Epg5 + / − Epg5 − / − mice . ( B ) number of pyramidal in fifth layer per millimeters squared .

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(A) Nissl staining of the fifth layer of the cortex in 10-mo-old Epg5+/− and Epg5−/− mice. (B) The number of pyramidal cells in the fifth layer per millimeters squared.


( C D ) H & amp ; amp ; E of hippocampus shows reduced pyramidal numbers in Epg5 − / − mice . Means ± of three mice are shown in B D .

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(C and D) H&amp;E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5−/− mice. Means ± SEM of three mice are shown in B and D.


( E ) normally developed cerebellum in Epg5 − / − mice . ( F ) thickness of molecular layer in cerebellum in mutant control mice .

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(E) The normally developed cerebellum in Epg5−/− mice. (F) The thickness of the molecular layer in the cerebellum in mutant and control mice.


( G H ) Purkinje , stained by calbindin , are not reduced in number in Epg5 − / − mice . Means ± of five mice are shown in F H .

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(G and H) Purkinje cells, stained by calbindin, are not reduced in number in Epg5−/− mice. Means ± SEM of five mice are shown in F and H.