C ) Purified centrosomes from KE37 were analyzed by SRM with stable isotope dilution . Histogram a priori represents relative copy numbers per centrosome ( Results presented are means ± . ) . To predict abundance in absolute numbers ( average number of copies / centrosome ) , data were normalized to 1340 γ - tubulin molecules , i . e . average number of γ - tubulin per centrosome as deduced by combining SRM EGFP measurements ( Figs 4 5 ) . Bars for Sas - 6 STIL are marked in red to indicate that corresponding values should be approximately doubled to correct for fact that some 50 % of purified centrosomes are derived from G1 phase that mostly lack these two . Note different scale for γ - tubulin on y - axis .

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C) Purified centrosomes from KE37 cells were analyzed by SRM with stable isotope dilution. Histogram a priori represents relative copy numbers per centrosome (Results presented are means ± SEM.). To predict protein abundance in absolute numbers (average number of copies/centrosome), data were normalized to 1340 γ-tubulin molecules, i.e. the average number of γ-tubulin per centrosome as deduced by combining SRM and EGFP fluorescence measurements (Figs 4 and 5). Bars for Sas-6 and STIL are marked in red to indicate that the corresponding values should be approximately doubled to correct for the fact that some 50% of purified centrosomes are derived from G1 phase cells that mostly lack these two proteins. Note the different scale for γ-tubulin on the y-axis.


Centrosome preparations from KE37 were analyzed by shotgun proteomics relative abundances determined using iBAQ shotgun proteomics method . iBAQ values were normalized with trendline as obtained from SRM measurements ( Fig EV1 , panel E ) γ - tubulin was normalized to 1340 copies / centrosome . A subset of centrosomal is shown as described in ( Azimzadeh et al , 2012 ) . Black bars represent centrosome subset red bars represent values obtained by SRM . Results presented are means ± .

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Centrosome preparations from KE37 cells were analyzed by shotgun proteomics and relative protein abundances determined using the iBAQ shotgun proteomics method. The iBAQ values were normalized with the trendline as obtained from the SRM measurements (Fig EV1, panel E) and γ-tubulin was normalized to 1340 copies/centrosome. A subset of centrosomal proteins is shown as described in (Azimzadeh et al, 2012). Black bars represent the centrosome subset and red bars represent the values obtained by SRM. Results presented are means ± SEM.


A ) of RPE - 1 expressing endogenously tagged γ - tubulin - EGFP . were synchronized in G2 , using RO - 3306 , to allow for a clear distinction of two centrosomes present at this cycle stage . Panels illustrate sum projections of stacks from an original picture ( left ) , a whole mask ( middle ) a centrosome mask ( right ) ; both masks are on thresholding . Bar : 10 μm .

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A) Fluorescence analysis of RPE-1 cells expressing endogenously tagged γ-tubulin-EGFP. Cells were synchronized in G2, using RO-3306, to allow for a clear distinction of the two centrosomes present at this cell cycle stage. Panels illustrate sum projections of the stacks from an original picture (left), a whole cell mask (middle) and a centrosome mask (right); both masks are based on intensity thresholding. Bar: 10 μm.


B ) EGFP - tagged viral like particles ( GFP - VLP2 / 6 ) are used as a reference for of γ - tubulin - EGFP . histogram shows distribution of total associated with single GFP - VLP2 / 6 ; 2 dashed lines delimit population used for assigning an average value to represent 120 GFP molecules associated with each VLP2 / 6 particle ( Charpilienne et al , 2001 ) . inset shows a sum projection of EGFP - VLPs image . Bar : 10 μm .

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B) EGFP-tagged viral like particles (GFP-VLP2/6) are used as a reference for quantification of γ-tubulin-EGFP. The histogram shows the distribution of the total fluorescence intensity associated with single GFP-VLP2/6; the 2 dashed lines delimit the population used for assigning an average fluorescence intensity value to represent the 120 GFP molecules associated with each VLP2/6 particle (Charpilienne et al, 2001). The inset shows a sum projection of EGFP-VLPs fluorescence image. Bar: 10 μm.


C ) Scatter plots show numbers of γ - tubulin molecules per centrosome determined by calibrations using either SRM measurements ( n = 46 ) or EGFP - VLP2 / 6 ( n = 40 ) . black lines represent mean values ; numbers ± are indicated on top of each scatter plot .

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C) Scatter plots show the numbers of γ-tubulin molecules per centrosome determined by calibrations using either SRM measurements (n=46 cells) or EGFP-VLP2/6 fluorescence (n=40 cells). The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot.


To determine cycle dependence of centrosomal abundance by , endogenously tagged RPE - 1 were synchronized as described under Materials Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 expressing ( endogenous ) γ - tubulin - EGFP ( A ) at indicted cycle stages . of representative are shown as maximum projections . Bars : 10μm . Bottom panels : quantifications were performed using EGFP - VLP2 / 6 method for calibration . Scatter plots represent number of γ - tubulin ( A ) molecules per centrosome at indicated cycle stages . black lines represent mean values ; numbers ± are indicated on top of each scatter plot . following numbers of were analyzed in early S phase : γ - tubulin : 45 ; in late S / G2 phase : γ - tubulin : 45 ; in late G2 phase : γ - tubulin : 48 ; in early mitosis : γ - tubulin : 31 . Note that were recorded using identical settings . P values from t - are indicated by following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) ns ( P > 0 . 05 ) .

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To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing (endogenous) γ-tubulin-EGFP (A) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of γ-tubulin (A) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: γ-tubulin: 45; in late S/G2 phase: γ-tubulin: 45; in late G2 phase: γ-tubulin: 48; in early mitosis: γ-tubulin: 31. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


To determine cycle dependence of centrosomal abundance by , endogenously tagged RPE - 1 were synchronized as described under Materials Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 expressing Sas - 6 ( B ) at indicted cycle stages . of representative are shown as maximum projections . Bars : 10μm . Bottom panels : quantifications were performed using EGFP - VLP2 / 6 method for calibration . Scatter plots represent number of molecules per centrosome at indicated cycle stages . black lines represent mean values ; numbers ± are indicated on top of each scatter plot . following numbers of were analyzed in early S phase : Sas - 6 : 43 ; in late S / G2 phase : Sas - 6 : 46 ; in late G2 phase : Sas - 6 : 48 . Note that were recorded using identical settings . P values from t - are indicated by following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) ns ( P > 0 . 05 ) .

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To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing Sas-6 (B) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: Sas-6: 43; in late S/G2 phase: Sas-6: 46; in late G2 phase: Sas-6: 48. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


To determine cycle dependence of centrosomal abundance by , endogenously tagged RPE - 1 were synchronized as described under Materials Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 expressing STIL ( C ) at indicted cycle stages . of representative are shown as maximum projections . Bars : 10μm . Bottom panels : quantifications were performed using EGFP - VLP2 / 6 method for calibration . Scatter plots represent number of STIL ( C ) molecules per centrosome at indicated cycle stages . black lines represent mean values ; numbers ± are indicated on top of each scatter plot . following numbers of were analyzed in early S phase : STIL : 79 ; in late S / G2 phase : STIL : 68 ; in late G2 phase : STIL : 68 . Note that were recorded using identical settings . P values from t - are indicated by following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) ns ( P > 0 . 05 ) .

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To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing STIL (C) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of STIL (C) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: STIL: 79; in late S/G2 phase: STIL: 68; in late G2 phase: STIL: 68. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


a , Correlative electron microscopy of HeLa / GFP - LC3 infected with SIN / mCherry . capsid . Top left , deconvolved image of red green channels ; middle , three - dimensional surface reconstruction of red green channels ; right , yellow ( red + green ) colocalization channel . Arrows denote yellow that correspond to ' 1 ' ' 2 ' in electron microscopy below . Bottom left , electron microscopy of identical ; middle right , high magnification of insets ' 1 ' ' 2 ' . Scale bars , left , 10 µm ; middle right , 200 nm .

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a, Correlative light and electron microscopy of HeLa/GFP-LC3 cell infected with SIN/mCherry.capsid virus. Top left, deconvolved image of red and green fluorescence channels; middle, three-dimensional surface reconstruction of red and green channels; right, yellow (red + green) colocalization channel. Arrows denote yellow puncta that correspond to '1' and '2' in electron microscopy images below. Bottom left, electron microscopy of identical cell; middle and right, high magnification images of insets '1' and '2'. Scale bars, left, 10 µm; middle and right, 200 nm.


a , Electron microscopy of wild - type ( WT ) Smurf1 - / - ( KO ) MEFs grown in normal media or EBSS ( starvation ) for 4 h . Arrowheads , representative autophagosomes ; arrows , representative autolysosomes . Scale bars , 500 nm .

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a, Electron microscopy analysis of wild-type (WT) and Smurf1-/- (KO) MEFs grown in normal media or EBSS (starvation) for 4 h. Arrowheads, representative autophagosomes; arrows, representative autolysosomes. Scale bars, 500 nm.


b , Western blot analyses of LC3 - I / II ( non - lipidated lipidated forms of MAP1LC3 , respectively ) p62 in MEFs of indicated genotype .

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b, Western blot analyses of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in MEFs of indicated genotype.


c , of colocalization of SIN / mCherry . capsid GFP - LC3 in indicated MEFs 16 h after SIN / mCherry . capsid / GFP - LC3 infection . Data shown represent mean ± s . e . m . number of colocalized red green per for 50 per well in triplicate samples . * P < 0 . 001 against wild - type , Students t - .

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c, Quantification of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection. Data shown represent mean ± s.e.m. number of colocalized red and green puncta per cell for 50 cells per well in triplicate samples. *P<0.001 against wild-type, Students t-test.


d , Representative confocal of colocalization of SIN / mCherry . capsid GFP - LC3 in indicated MEFs 16 h after SIN / mCherry . capsid / GFP - LC3 infection used for in c . Arrows , colocalized red green . Scale bar , 15 & amp ; amp ; mgr ; m .

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d, Representative confocal micrographs of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection images used for quantification in c. Arrows, colocalized red and green puncta. Scale bar, 15 &amp;mgr;m.


e , Representative electron microscopy of indicated MEFs infected with HSV - 1 ( 17termA ) . arrowheads , partially degraded viral nucleocapsids inside autolysosomes ; black arrowheads , intact viral nucleocapsids inside viral vesicles . Scale bar , 0 . 5 µm . For a - e , similar results were obtained in 3 - 5 independent experiments .

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e, Representative electron microscopy images of indicated MEFs infected with HSV-1 (strain 17termA). White arrowheads, partially degraded viral nucleocapsids inside autolysosomes; black arrowheads, intact viral nucleocapsids inside viral vesicles. Scale bar, 0.5 µm. For a-e, similar results were obtained in 3-5 independent experiments.


a , Representative mitochondrial in Smurf1 + / + ( wild - type ) Smurf1 − / − ( KO ) MEFs transfected with indicated construct treated with DMSO or 10 µM CCCP for 24 h . b , of of total with a diffuse accumulation of abnormal fragmented mitochondria lack of mitochondrial clearance . Results shown represent combined data from 3 - 5 experiments per condition with triplicate wells ( of at least 100 per well ) analysed for each condition per experiment . Shown are mean ± s . e . m . for average values from each experiment . Similar results were observed in each independent experiment . * P 0 . 001 , Students t - . c , Measurement of mitochondrial fractional ( of total ) in MEFs treated as in a . Results shown represent mean ± s . e . m . for 50 per condition .

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a, Representative mitochondrial morphology in Smurf1+/+ (wild-type) and Smurf1−/− (KO) MEFs transfected with indicated construct and treated with DMSO or 10 µM CCCP for 24 h. b, Quantification of percentage of total cells with a diffuse accumulation of abnormal fragmented mitochondria and lack of mitochondrial clearance. Results shown represent combined data from 3-5 experiments per condition with triplicate wells (of at least 100 cells per well) analysed for each condition per experiment. Shown are mean ± s.e.m. for average values from each experiment. Similar results were observed in each independent experiment. *P  0.001, Students t-test. c, Measurement of mitochondrial fractional area (percentage of total cellular area) in MEFs treated as in a. Results shown represent mean ± s.e.m. for 50 cells per condition.


d , Representative confocal of KO MEFs transfected with YFP - SMURF1 wild - type or YFP - SMURF1Dgr ; C2 ( Dgr ; C2 ) treated for 4 h with DMSO or CCCP .

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d, Representative confocal micrographs of KO MEFs transfected with YFP-SMURF1 wild-type or YFP-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with DMSO or CCCP.


e , Representative confocal of KO MEFs transfected with GFP - LC3 wild - type mCherry - SMURF1 ( WT ) or mCherry - SMURF1Dgr ; C2 ( Dgr ; C2 ) treated for 4 h with CCCP . Inset , upper right , formation of completed autophagosome around a damaged mitochondrion associated with wild - type SMURF1 ; insets , lower right , incomplete autophagosomes or absence of LC3 signal around mitochondria associated with SMURF1Dgr ; C2 . See also Supplementary Figs 10 11 for enlarged .

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e, Representative confocal micrographs of KO MEFs transfected with GFP-LC3 and wild-type mCherry-SMURF1 (WT) or mCherry-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with CCCP. Inset, upper right, formation of completed autophagosome around a damaged mitochondrion associated with wild-type SMURF1; insets, lower right, incomplete autophagosomes or absence of LC3 signal around mitochondria associated with SMURF1Dgr;C2. See also Supplementary Figs 10 and 11 for enlarged images.


f , Representative electron microscopy of indicated tissues from 10 - month - oldSmurf1 + / + ( WT ) or Smurf1 - / - ( KO ) mice . Arrows , representative abnormal mitochondria . Scale bars , 1 µm . Similar abnormalities were observed throughout entire electron microscopy tissue section for each mouse in tissue samples from three different mice for each genotype .

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f, Representative electron microscopy images of indicated tissues from 10-month-oldSmurf1+/+ (WT) or Smurf1-/- (KO) mice. Arrows, representative abnormal mitochondria. Scale bars, 1 µm. Similar abnormalities were observed throughout entire electron microscopy tissue section for each mouse and in tissue samples from three different mice for each genotype.


B Latency to enter compartment for Gadd45a - WT ( bars , n = 11 ) Gadd45a - KO ( blue bars , n = 11 ) . A significant reduction was found in Gadd45a - KO at 24 hours 7 days after training . Values shown are mean ± ; 2 - Way ANOVA Bonferroni post - hoc : * = p < 0 . 05 .

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B Latency to enter dark compartment for Gadd45a-WT (white bars, n=11) and Gadd45a-KO (blue bars, n=11). A significant reduction was found in the Gadd45a-KO at 24 hours and 7 days after the training. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05.


C In situ hybridization to detect Gadd45a mRNA in adult wild - type hippocampus ( n = 8 ; antisense , left panel ; sense , middle panel ) in Gadd45a - KO sections ( n = 6 ; right panel ) . Scale bars : 500 µm ( black bar ) .

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C In situ hybridization to detect Gadd45a mRNA in adult wild-type hippocampus (n=8 animals; antisense, left panel; sense, middle panel) and in Gadd45a-KO sections (n=6 animals; right panel). Scale bars: 500 µm (black bar).


D Experience - dependent changes in mRNA were evaluated under control conditions ( home cage ) exposed to PA ( context , context + shock groups ; n = 6 for groups ) , respectively . Hippocampal Gadd45a mRNA remained unchanged , while Gadd45b was induced ( context ; t10 = 2 . 427 , context + shock t10 = 2 . 319 ) . Concomitantly , mRNA of activity - regulated cytoskeleton ( Arc ) , as a proxy of neuronal activation , were increased in mice exposed to PA context ( t10 = 3 . 406 ) context + shock ( t10 = 3 . 141 ) . Values shown are mean ± ; 1 - Way ANOVA unpaired t - : * = p < 0 . 05 , * * * = p < 0 . 001 .

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D Experience-dependent changes in mRNA levels were evaluated under control conditions (home cage) and exposed to the PA (context, and context + shock groups; n=6 for all groups), respectively. Hippocampal Gadd45a mRNA levels remained unchanged, while Gadd45b was induced (context; t10=2.427, context + shock t10=2.319). Concomitantly, mRNA levels of activity-regulated cytoskeleton protein (Arc), as a proxy of neuronal activation, were increased in mice exposed to the PA context (t10=3.406) and context + shock (t10=3.141). Values shown are mean ± SEM; 1-Way ANOVA and unpaired t-test: * = p<0.05, *** = p<0.001.


Gadd45a mRNA level in hippocampus of Gadd45a - overexpressing mice ( red , n = 7 ) was significantly increased ( t10 = 4 . 189 ) as compared to Gadd45a - WT ( n = 5 ) . Values shown are mean ± ; unpaired t - : * * = p < 0 . 01 .

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Gadd45a mRNA level in the hippocampus of Gadd45a-overexpressing mice (red, n=7) was significantly increased (t10=4.189) as compared to Gadd45a-WT (n=5). Values shown are mean ± SEM; unpaired t-test: ** = p<0.01.


C Immunohistochemistry for hemagglutinin ( HA , fused to Gadd45α ) in Gadd45a - WT ( Nex - Cre ( - / - ) , n = 5 ) Gadd45a - overexpressing hippocampi ( Nex - Cre ( + / - ) , n = 5 ) 8 after AAV injection . Note that Gadd45α - HA signal in Gadd45a - overexpressing mice is predominantly found in stratum pyramidale ( sp ) but also in stratum oriens stratum radiatum ( so sr , respectively ) . Scale bar : 250 µm .

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C Immunohistochemistry for hemagglutinin (HA, fused to Gadd45α) in Gadd45a-WT (Nex-Cre(-/-), n=5) and Gadd45a-overexpressing hippocampi (Nex-Cre(+/-), n=5) 8 weeks after AAV injection. Note that the Gadd45α-HA signal in Gadd45a-overexpressing mice is predominantly found in the stratum pyramidale (sp) but also in the stratum oriens and stratum radiatum (so and sr, respectively). Scale bar: 250 µm.


D distribution of Gadd45α was analyzed by HA - western blot on fractions of Gadd45a - WT Gadd45a - overexpressing mice ( n = 2 for both groups ) . HA was found predominantly in fraction of Gadd45a - overexpressing hippocampi , with a smaller but recognizable expression also in cytoplasm .

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D Cellular distribution of Gadd45α was analyzed by HA-western blot on nuclear and cytoplasmic protein fractions of Gadd45a-WT and Gadd45a-overexpressing mice (n=2 for both groups). HA was found predominantly in the nuclear fraction of Gadd45a-overexpressing hippocampi, with a smaller but recognizable expression also in the cytoplasm.


E Latency to enter compartment for Gadd45a - WT ( bars , n = 10 ) Gadd45a - overexpressing mice ( red bars , n = 12 ) in PA . A significant increase was found 24 hours 7 days after PA training in Gadd45a - overexpresion group . Values shown are mean ± ; 2 - Way ANOVA Bonferroni Post - hoc : * = p < 0 . 05 .

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E Latency to enter the dark compartment for Gadd45a-WT (white bars, n=10) and Gadd45a-overexpressing mice (red bars, n=12) in the PA test. A significant increase was found 24 hours and 7 days after the PA training in the Gadd45a-overexpresion group. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: * = p<0.05.


B LTP protocol was applied to 16 slices of Gadd45a - WT mice ( dots , n = 5 ) 11 slices of Gadd45a - KO mice ( blue dots , n = 5 ) , resulting in a significantly ( p < 0 . 05 , Student t - ) lowered induction of LTP in Gadd45a - KO mice as compared with Gadd45a - WT . Values shown are mean ± . C Gadd45a - overexpressing mice ( red dots , n = 10 slices from 6 ) presented a slightly increased LTP as compared with Gadd45a - WT ( dots , n = 8 slices from 6 ) , which became more pronounced during late phase of experiment . Values shown are mean ± .

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B LTP protocol was applied to 16 slices of Gadd45a-WT mice (white dots, n=5 animals) and 11 slices of Gadd45a-KO mice (blue dots, n=5 animals), resulting in a significantly (p<0.05, Student t-test) lowered induction of LTP in Gadd45a-KO mice as compared with Gadd45a-WT. Values shown are mean ± SEM. C Gadd45a-overexpressing mice (red dots, n=10 slices from 6 animals) presented a slightly increased LTP as compared with Gadd45a-WT (white dots, n=8 slices from 6 animals), which became more pronounced during the late phase of the experiment. Values shown are mean ± SEM.


A MA plot of DESeq differential expression with 1 % false discovery rate ( FDR ) hits in red ( 5 / 402 ; with > 2 - fold change 1 / 65 , up - / down - regulated respectively ) .

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A MA plot of DESeq differential expression analysis with the 1% false discovery rate (FDR) hits in red (5/402; and with >2-fold change 1/65, up-/down-regulated respectively).


D UCSC browser view of reads - per - million - normalised - seq coverage for Grin2a in Gadd45a - WT ( black ) Gadd45a - KO ( blue ) representative samples . Gene structure of Grin2a ( colored in purple ) , direction of transcription ( red arrow ) . Upper pannel covers entire transcript while lower panel is an enlargement of exons 4 - 8 .

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D UCSC browser view of reads-per-million-normalised RNA-seq coverage for Grin2a in Gadd45a-WT (black) and Gadd45a-KO (blue) representative samples. Gene structure of Grin2a (colored in purple), and the direction of transcription (red arrow). Upper pannel covers the entire transcript while lower panel is an enlargement of exons 4-8.


E Detailed view of extended 3 ´ UTR of Grin2a showing a significant 5 ´ < 3 ´ gradient in - seq coverage . Note that normalised read coverage is similar between genotypes at actual 3 ´ UTR end but differs significantly upstream including annotated exons . For subsequent experiments , position of designed primers covering proximal distal parts of 3 ´ UTR are indicated below .

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E Detailed view of the extended 3´UTR of Grin2a showing a significant 5´<3´ gradient in RNA-seq coverage. Note that the normalised read coverage is similar between genotypes at the actual 3´UTR end but differs significantly upstream including the annotated exons. For subsequent experiments, position of the designed primers covering proximal and distal parts of the 3´UTR are indicated below.


F , G Validation of Gadd45α - regulated of Grin2a mRNA by qPCR with primers covering exons 3 - 4 . Grin2a mRNA were analyzed in hippocampus of mice under control conditions ( bars ; Gadd45a - WT = 7 Gadd45a - KO = 7 ) 1 hour after PA ( Gadd45a - WT = 5 , black bars ; Gadd45a - KO = 6 , blue bars ) . Random primed cDNA ( F ) , were very similar in experimental groups . Oligo ( dT ) - primed cDNA ( G ) revealed a significant increase of Grin2a 1h after PA , which was only observed in Gadd45a - WT mice . Values shown are mean ± ; 2 - Way ANOVA Bonferroni post - hoc : * * = p < 0 . 01 , * * * = p < 0 . 001 . H , I mRNA of proximal vs . distal parts of 3 ´ UTR analyzed in same set of oligo ( dT ) - primed cDNA . ( H ) Significant increase in Grin2a mRNA was found for proximal part only in Gadd45a - WT . ( I ) Distal parts of 3 ´ UTR remained unchanged ( n = 6 for groups ) . Values shown are mean ± ; 2 - Way ANOVA Bonferroni post - hoc : * = p < 0 . 05 .

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F, G Validation of Gadd45α-regulated levels of Grin2a mRNA by qPCR with primers covering exons 3-4. Grin2a mRNA levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT=7 and Gadd45a-KO=7) and 1 hour after PA (Gadd45a-WT=5, black bars; Gadd45a-KO=6, blue bars). Random primed cDNA levels (F), were very similar in all the experimental groups. Oligo(dT)-primed cDNA (G) revealed a significant increase of Grin2a 1h after PA, which was only observed in Gadd45a-WT mice. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01, *** = p<0.001. H, I mRNA levels of proximal vs. distal parts of the 3´UTR analyzed in the same set of oligo(dT)-primed cDNA. (H) Significant increase in Grin2a mRNA levels was found for the proximal part only in Gadd45a-WT. (I) Distal parts of the 3´UTR remained unchanged (n=6 for all groups). Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05.


A , B NR2A were analyzed in hippocampus of mice under control conditions ( bars ; Gadd45a - WT , n = 13 Gadd45a - KO , n = 14 ) 1 hour after PA ( black bars ; Gadd45a - WT = 14 , Gadd45a - KO = 14 ) . NR2A signal ( 160 kDa ) was normalized to loading control ( tubulin ( 55 kDa ) for total fraction , synaptotagmin ( 68 kDa ) for synaptosomal fraction ) calculated as of control . In Gadd45a - WT samples ( A ) , NR2A were unchanged in total fraction during memory formation , while in synaptosomal fraction , a significant increase was detected ( t20 = 3 . 199 ) . In Gadd45a - KO hippocampi ( B ) , of NR2A in both fractions remained unaltered during memory consolidation . Values shown are mean ± ; 2 - Way ANOVA Bonferroni post - hoc : * * = p < 0 . 01 .

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A, B NR2A protein levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT, n=13 and Gadd45a-KO, n=14) and 1 hour after PA (black bars; Gadd45a-WT=14, Gadd45a-KO=14). NR2A signal (160 kDa) was normalized to the loading control (tubulin (55 kDa) for the total fraction, and synaptotagmin (68 kDa) for the synaptosomal fraction) and calculated as percentage of control. In Gadd45a-WT samples (A), NR2A protein levels were unchanged in the total fraction during memory formation, while in the synaptosomal fraction, a significant increase was detected (t20=3.199). In Gadd45a-KO hippocampi (B), levels of NR2A protein in both fractions remained unaltered during memory consolidation. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01.


C Gadd45α pulldown experiments were carried out in wild - type hippocampal lysates ( n = 12 for groups ) . of input recovery was unchanged between GFP - FLAG ( green bars ) Gadd45α - FLAG ( black bars ) for genes that are not regulated by Gadd45α , such as succinate dehydrogenase flavoprotein subunit A ( Sdha , no 3 ´ UTR extension ) , mitogen - activated kinase 6 ( Map2K6 , containing 3 ´ UTR extension of 10 kb ) . On contrary , Gadd45α - FLAG bound fraction revealed a significant increase in Grin2a ( t22 = 4 . 992 ) Grm5 mRNA ( t22 = 4 . 939 ) as compared to GFP - FLAG fraction . Values shown are mean ± ; 2 - Way ANOVA Bonferroni Post - hoc : * * * = p < 0 . 001 .

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C Gadd45α pulldown experiments were carried out in wild-type hippocampal lysates (n=12 for all groups). Percentage of input recovery was unchanged between GFP-FLAG (green bars) and Gadd45α-FLAG (black bars) for genes that are not regulated by Gadd45α, such as succinate dehydrogenase complex flavoprotein subunit A (Sdha, no 3´UTR extension), and mitogen-activated protein kinase 6 (Map2K6, containing 3´UTR extension of 10 kb). On the contrary, Gadd45α-FLAG bound fraction revealed a significant increase in Grin2a (t22=4.992) and Grm5 mRNA (t22=4.939) as compared to GFP-FLAG fraction. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: *** = p<0.001.


B Hippocampal Gadd45a mRNA were significantly upregulated in both Gadd45a - overexpression ( q24 = 4 . 094 ) Gadd45aK45E - overexpression groups ( q24 = 4 . 726 ) ( n = 9 for groups ) . Values shown are mean ± ; Tukey ´ s multiple comparison : * = p < 0 . 05 , * * = p < 0 . 01 .

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B Hippocampal Gadd45a mRNA levels were significantly upregulated in both Gadd45a-overexpression (q24 = 4.094) and Gadd45aK45E-overexpression groups (q24 = 4.726) (n=9 for all groups). Values shown are mean ± SEM; Tukey´s multiple comparison test: * = p<0.05, ** = p<0.01.


C Immunohistochemistry for hemagglutinin ( HA , fused to Gadd45α ) in Gadd45a - WT ( left ) , Gadd45a - overexpression ( middle ) Gadd45aK45E - overexpression ( right ) samples 8 after AAV injection ( n = 3 for groups ) . Upper row depicts entire hippocampus , while center lower rows show magnifications of CA3 in which HA signal ( red ) is not restricted to nuclei ( blue ) . Scale bar : 250 µm .

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C Immunohistochemistry for hemagglutinin (HA, fused to Gadd45α) in Gadd45a-WT (left), Gadd45a-overexpression (middle) and Gadd45aK45E-overexpression (right) samples 8 weeks after AAV injection (n=3 for all groups). Upper row depicts the entire hippocampus, while center and lower rows show magnifications of the CA3 area in which the HA signal (red) is not restricted to the nuclei (blue and white). Scale bar: 250 µm.


D Gadd45α - overexpression was confirmed at level by HA - western blot on hippocampal samples from Gadd45a - WT , Gadd45a - overexpression Gadd45aK45E - overexpression mice ( n = 3 for groups ) . Hippocampus scale bars : 250 µm ; CA3 scale bars : 50µm .

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D Gadd45α-overexpression was confirmed at the protein level by HA-western blot on hippocampal samples from Gadd45a-WT, Gadd45a-overexpression and Gadd45aK45E-overexpression mice (n=3 for all groups). Hippocampus scale bars: 250 µm; CA3 scale bars: 50µm.


E Latency to enter compartment for Gadd45a - WT ( bars , n = 12 ) , Gadd45a - overexpression ( red bars , n = 11 ) Gadd45aK45E - overexpression mice ( grey bars , n = 11 ) in PA . Note that , unlike Gadd45a - overexpression mice , those expressing mutated form of Gadd45α ( no - binding capacity ) did not show an increase in memory retention . Values shown are mean ± ; 2 - Way ANOVA Bonferroni Post - hoc : * = p < 0 . 05 , * * * = p < 0 . 001 .

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E Latency to enter the dark compartment for Gadd45a-WT (white bars, n=12), Gadd45a-overexpression (red bars, n=11) and Gadd45aK45E-overexpression mice (grey bars, n=11) in the PA test. Note that, unlike Gadd45a-overexpression mice, those expressing the mutated form of Gadd45α (no RNA-binding capacity) did not show an increase in memory retention. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: * = p<0.05, *** = p<0.001.


A . Mutations in kinase motif abolished yeast toxicity of MavQ . S . cerevisiae was transformed with plasmids expressing Flag - tagged wild - type MavQ or indicated MavQ mutants under galactose - inducible promoter . Yeast were spotted on synthetic medium supplemented with glucose or galactose for 3 days before image acquisition .

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A. Mutations in the kinase motif abolished the yeast toxicity of MavQ. S. cerevisiae was transformed with plasmids expressing Flag-tagged wild-type MavQ or the indicated MavQ mutants under the galactose-inducible promoter. Yeast cells were spotted on synthetic medium supplemented with glucose or galactose for 3 days before image acquisition.


B . expression of MavQ in yeast was detected by Flag antibody . PGK ( 3 - phosphoglyceric phosphokinase ) was used as a loading control .

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B. The expression of MavQ in the yeast was detected by Flag antibody. The PGK (3-phosphoglyceric phosphokinase) was used as a loading control.


A . Biochemical assays for kinase activity of MavQ . Purified MavQ was incubated with or without specific PI substrates , conversion of ATP to ADP was measured by ADP - Glo . RLU , relative luminescence units .

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A. Biochemical assays for the kinase activity of MavQ. Purified MavQ was incubated with or without specific PI substrates, the conversion of ATP to ADP was measured by the ADP-Glo assay. RLU, relative luminescence units.


B . H149 , N152 D160 in MavQ are critical for its kinase activity . Purified wild - type mutant MavQ were reacted with PtdIns ATP . kinase activity was determined by ADP - Glo .

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B. H149, N152 and D160 in MavQ are critical for its kinase activity. Purified wild-type and mutant MavQ were reacted with PtdIns and ATP. The kinase activity was determined by the ADP-Glo assay.


C . PI product produced by MavQ determined by TLC assays . di - C8 - Bodipy - FL - PtdIns was used to react with MavQ ( lane 4 ) samples were further incubated with 3 - phosphatase MTM which hydrolyzes both PtdIns3P PtdIns ( 3 , 5 ) P2 ( lane 5 ) or with PI4K LepB _ NTD which phosphorylates PtdIns3P into PtdIns ( 3 , 4 ) P2 ( lane 6 ) . A schematic illustration of enzymatic reactions is shown in lower panel .

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C. The PI product produced by MavQ determined by TLC assays. di-C8-Bodipy-FL-PtdIns was used to react with MavQ (lane 4) and samples were further incubated with the 3-phosphatase MTM which hydrolyzes both PtdIns3P and PtdIns(3,5)P2 (lane 5) or with the PI4K LepB_NTD which phosphorylates PtdIns3P into PtdIns (3,4)P2 (lane 6). A schematic illustration of the enzymatic reactions is shown in the lower panel.


D . Mutations in H149 , N152 D160 abolish ability of MavQ to convert PtdIns into PtdIns3P .

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D. Mutations in H149, N152 and D160 abolish the ability of MavQ to convert PtdIns into PtdIns3P.


A . Representative of HeLa expressing PtdIns3P probe GFP - 2xFYVEHrs MavQ with or without wortmannin treatment . Scale bars , 5 μm .

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A. Representative fluorescence images of HeLa cells expressing the PtdIns3P probe GFP-2xFYVEHrs and MavQ with or without wortmannin treatment. Scale bars, 5 μm.


B . of HeLa transfected with indicated plasmids in which GFP - 2xFYVEHrs localizes to vesicle - like structures . At least 100 ( n ≥ 100 ) were counted for each sample . Wort , wortmannin .

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B. Percentage of HeLa cells transfected with the indicated plasmids in which GFP-2xFYVEHrs localizes to vesicle-like structures. At least 100 cells (n≥100) were counted for each sample. Wort, wortmannin.


C . Induction of ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin . Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room prior to addition of PtdIns ATP . wortamannin - sensitive PI3K p110α was used as a control . ADP - Glo was used to measure kinase activity .

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C. Induction of the ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin. Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room temperature prior to the addition of PtdIns and ATP. The wortamannin-sensitive PI3K p110α was used as a control. The ADP-Glo assay was used to measure the kinase activity.


D . Wortmannin does not affect production of PtdIns3P by MavQ . Purified MavQ was pretreated with 100 μM of wortmannin for 30 min at room . di - C8 - Bodipy - FL - PtdIns ATP were then added to reaction mixtures . TLC was employed to detect generation of PtdIns3P by MavQ . wortamannin - sensitive PI3K p110α was included as a control .

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D. Wortmannin does not affect the production of PtdIns3P by MavQ. Purified MavQ was pretreated with 100 μM of wortmannin for 30 min at room temperature. di-C8-Bodipy-FL-PtdIns and ATP were then added to the reaction mixtures. The TLC assay was employed to detect the generation of PtdIns3P by MavQ. The wortamannin-sensitive PI3K p110α was included as a control.


A . Representative immunofluorescence of association of MavQ with LCV . BMDM infected with wild - type or Dot / Icm - deficient ( dotA - ) L . pneumophila were sequentially stained with antibodies against L . pneumophila MavQ . were taken under a microscope . Insets represent 3x magnification of regions defined by dash lines . Scale bar , 5 μm .

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A. Representative immunofluorescence images of the association of MavQ with the LCV. BMDM cells infected with wild-type or Dot/Icm-deficient (dotA-) L. pneumophila were sequentially stained with antibodies against L. pneumophila and MavQ. Images were taken under a fluorescence microscope. Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.


B . rates of of LCVs positively stained for MavQ 2 h post - infection . At least 100 LCVs ( n ≥ 100 ) were scored for each sample .

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B. The rates of of LCVs positively stained for MavQ 2 h post-infection. At least 100 LCVs (n≥100) were scored for each sample.


C . Dynamics of MavQ association with LCV . BMDMs were infected with wild - type L . pneumophila at an MOI of 2 for indicated experimental durations . At least 100 phagosomes ( n ≥ 100 ) were counted for each sample to obtain of MavQ - positive LCVs .

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C. Dynamics of MavQ association with LCV. BMDMs were infected with the wild-type L. pneumophila strain at an MOI of 2 for the indicated experimental durations. At least 100 phagosomes (n≥100) were counted for each sample to obtain the percentage of MavQ-positive LCVs.


D . Intracellular growth of L . pneumophila in macrophages . BMDMs were infected with indicated L . pneumophila strains at an MOI of 0 . 05 . At indicated time points , CFUs were determined by plating saponin - solubilized lysates of infected on CYE plates .

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D. Intracellular growth of L. pneumophila in macrophages. BMDMs were infected with indicated L. pneumophila strains at an MOI of 0.05. At the indicated time points, CFUs were determined by plating the saponin-solubilized lysates of infected cells on CYE plates.


E . Expression of mavQ in L . pneumophila strains used for infection . Total of cultured to post - exponential phase separated by SDS - PAGE were probed with MavQ - specific antibodies . ICDH was detected as a loading control .

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E. Expression of mavQ in L. pneumophila strains used for infection. Total proteins of bacteria cultured to the post-exponential phase separated by SDS-PAGE were probed with MavQ-specific antibodies. ICDH was detected as a loading control.


A . Biochemical synthesis of PtdIns4P from PtdIns by sequential reactions catalyzed by MavQ , LepB _ NTD SidF . MavQ was reacted with di - C8 - Bodipy - FL - PtdIns , resulting in production of PtdIns3P ( lane 6 ) . PtdIns3P - specific PI4K LepB - NTD was then added into reaction , yielding PtdIns ( 3 , 4 ) P2 ( lane 7 ) . product of sample receiving LepB _ NTD was further incubated with 3 - phosphatase SidF to yield PtdIns4P ( lane 8 ) . lipid products were detected visualized by TLC . A schematic illustration of enzymatic reactions is shown in lower panel .

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A. Biochemical synthesis of PtdIns4P from PtdIns by sequential reactions catalyzed by MavQ, LepB_NTD and SidF. MavQ was reacted with di-C8-Bodipy-FL-PtdIns, resulting in the production of PtdIns3P (lane 6). The PtdIns3P-specific PI4K LepB-NTD was then added into the reaction, yielding PtdIns(3,4)P2 (lane 7). The product of the sample receiving LepB_NTD was further incubated with the 3-phosphatase SidF to yield PtdIns4P (lane 8). The lipid products were detected and visualized by TLC. A schematic illustration of the enzymatic reactions is shown in the lower panel.


B . Representative of SidC anchoring on LCVs . BMDMs infected with indicated L . pneumophila strains at an MOI of 2 for 2 h were sequentially stained with antibodies against SidC . were acquired using a microscope . Insets represent 3x magnification of regions defined by dash lines . Scale bar , 5 μm .

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B. Representative images of SidC anchoring on LCVs. BMDMs infected with the indicated L. pneumophila strains at an MOI of 2 for 2 h were sequentially stained with antibodies against the bacteria and SidC. Images were acquired using a fluorescence microscope. Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.


C . Quantitation of SidC - positive LCVs . At least 100 phagosomes ( n ≥ 100 ) were scored for each sample .

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C. Quantitation of SidC-positive LCVs. At least 100 phagosomes (n≥100) were scored for each sample.


D . Expression translocation of SidC . lysates of L . pneumophila strains used for infection were probed for expression , ICDH was detected as a loading control ( top panel ) . SidC translocation was detected by probing saponin - soluble fractions of infected U937 with SidC - specific antibodies . Tubulin was probed as a loading control ( bottom panel ) .

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D. Expression and translocation of SidC. The lysates of L. pneumophila strains used for infection were probed for protein expression, ICDH was detected as a loading control (top panel). SidC translocation was detected by probing the saponin-soluble fractions of infected U937 cells with SidC-specific antibodies. Tubulin was probed as a loading control (bottom panel).


A . Direct interactions between SidP MavQ in GST pull - down . GST beads coated with GST - SidP or GST were incubated with His6 - MavQ . After washing beads with GST binding buffer , resolved by SDS - PAGE were detected by Coomassie brilliant blue .

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A. Direct interactions between SidP and MavQ in the GST pull-down assay. GST beads coated with GST-SidP or GST were incubated with His6-MavQ. After washing the beads with GST binding buffer, the proteins resolved by SDS-PAGE were detected by Coomassie brilliant blue staining.


B , SidP does not impair kinase activity of MavQ in vitro . MavQ was preincubated with increasing amounts of SidP , kinase activity of MavQ was measured by ADP - Glo ( B )

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B, SidP does not impair the kinase activity of MavQ in vitro. MavQ was preincubated with increasing amounts of SidP, and the kinase activity of MavQ was measured by the ADP-Glo assay (B)


C . SidP does not impair kinase activity of MavQ in vitro . MavQ was preincubated with increasing amounts of SidP , kinase activity of MavQ was measured by TLC ( C ) .

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C. SidP does not impair the kinase activity of MavQ in vitro. MavQ was preincubated with increasing amounts of SidP, and the kinase activity of MavQ was measured by TLC assay (C).


D . Co - expression of SidP with MavQ in HeLa does not influence production of PtdIns3P caused by MavQ . association of probe GFP - 2xFYVEHrs with vesicle - like structures was used to indicate distribution of PtdIns3P in . At least 100 were scored in each sample ( n ≥ 100 ) ( left panel ) . Wort , wortmannin . Expression of MavQ SidP in samples were shown in right panel . EV represents empty vector .

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D. Co-expression of SidP with MavQ in HeLa cells does not influence the production of PtdIns3P caused by MavQ. The association of the fluorescence probe GFP-2xFYVEHrs with vesicle-like structures was used to indicate the distribution of PtdIns3P in cells. At least 100 cells were scored in each sample (n≥100) (left panel). Wort, wortmannin. Expression of MavQ and SidP in the samples were shown in the right panel. EV represents empty vector.


E . Deletion of sidP does not impact association of SidC with LCV . BMDMs infected with indicated L . pneumophila stains were stained association of SidC with phagosomes was similarly determined ( n ≥ 100 ) ( left panel ) . Representative of infected harboring phagosomes were also shown ( right panel ) . Insets represent 3x magnification of regions defined by dash lines . Scale bar , 5 μm .

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E. Deletion of sidP does not impact the association of SidC with the LCV. BMDMs infected with the indicated L. pneumophila stains were stained and the association of SidC with bacterial phagosomes was similarly determined (n≥100) (left panel). Representative images of infected cells harboring bacterial phagosomes were also shown (right panel). Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.


A WT MDFs were treated ± TSI individually or in combination for 3 hrs . Whole lysates ( WCL ) UBA - pull down ( UBA - PD ) fractions were analysed by Western blot probed with antibodies as indicated . Representative of three independent experiments . Samples of UBA - pull down in following figures were analysed in same way unless otherwise indicated .

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A WT MDFs were treated ± TSI individually or in combination for 3 hrs. Whole cell lysates (WCL) and UBA-pull down (UBA-PD) fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments. Samples of UBA-pull down in following figures were analysed in the same way unless otherwise indicated.


B WT , Ripk3 - / - , Mlkl - / - , Tnfr1 - / - Traf2 - / - MDFs were untreated ( UT ) or treated with TSI for 3 hrs . Nec - 1 GSK872 were added to inhibit RIPK1 RIPK3 kinase activities respectively .

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B WT, Ripk3-/-, Mlkl-/-, Tnfr1-/- and Traf2-/- MDFs were untreated (UT) or treated with TSI for 3 hrs. Nec-1 and GSK872 were added to inhibit RIPK1 and RIPK3 kinase activities respectively.


E RIPK3 - gyrase were inducibly expressed in Ripk3 - / - MDFs by doxycycline ( dox ) for 5 hrs , were then treated ± combination of TSI , or coumermycin ( coum ) for 3 hrs .

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E RIPK3-gyrase were inducibly expressed in Ripk3-/- MDFs by doxycycline (dox) for 5 hrs, and cells were then treated ± combination of TSI, or coumermycin (coum) for 3 hrs.


B UBA - pull down from WT MDFs treated with TSI for 3 hrs were subjected to DUBs shown in ( A ) . Beads eluates were analysed by Western blot probed with antibodies as indicated . Representative of three independent experiments .

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B UBA-pull down from WT MDFs treated with TSI for 3 hrs were subjected to the DUBs shown in (A). Beads eluates were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


C 1 . 4 mL cleared lysate from 4 x 106 TSI - treated WT MDFs was split into three parts of indicated volume , followed by UBA - pull down DUB incubation . Bead eluates were analysed by Western blot probed with indicated antibodies . Representative of three independent experiments .

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C 1.4 mL cleared cell lysate from 4 x 106 TSI-treated WT MDFs was split into three parts of the indicated volume, followed by UBA-pull down and DUB incubation. Bead eluates were analysed by Western blot and probed with the indicated antibodies. Representative of three independent experiments.


A WT MDFs were treated with TSI for indicated time . were fractionated into cytosol crude , followed by UBA - pull down . fractions were analysed by Western blot probed with antibodies as indicated . Representative of three independent experiments .

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A WT MDFs were treated with TSI for indicated time. Cells were fractionated into cytosol and crude membrane, followed by UBA-pull down. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


B WT USP21 - CaaX USP21C221R - CaaX ( CR ) were inducibly expressed in WT MDFs by doxycycline for 5 hrs . Ubiquitylated were enriched followed by TSI stimulation fractionation . fractions were analysed by Western blot probed with antibodies as indicated . Representative of three independent experiments .

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B WT USP21-CaaX and USP21C221R-CaaX (CR) were inducibly expressed in WT MDFs by doxycycline for 5 hrs. Ubiquitylated proteins were enriched followed by TSI stimulation and cellular fractionation. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


A WT R105AD106A mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline , at same time were untreated ( UT ) or treated with TSQ for 6 hrs . were fractionated into cytosol ( C ) crude ( M ) . Fractions were analysed by BN - or SDS - PAGE , Western blot probed with indicated antibodies . Representative of three independent experiments . B lysates from ( A ) were subjected to UBA - pull down analysed as described above .

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A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, at the same time cells were untreated (UT) or treated with TSQ for 6 hrs. Cells were fractionated into cytosol (C) and crude membrane (M). Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. B Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.


E HT29 were stimulated with TSI , ± NSA ( 500 nM ) , or left untreated ( UT ) for 16 hrs , followed by fractionation . Fractions were analysed by BN - or SDS - PAGE , Western blot probed with indicated antibodies . Representative of three independent experiments . F lysates from ( E ) were subjected to UBA - pulldown analysed as described above .

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E HT29 cells were stimulated with TSI, ± NSA (500 nM), or left untreated (UT) for 16 hrs, followed by cellular fractionation. Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. F Cell lysates from (E) were subjected to UBA-pulldown and analysed as described above.


A WT E109AE110A mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline , were untreated or treated with TSQ for 6 hrs ( please note that same WT control was used in Fig . EV3A ) . was measured by PI on flow cytometry . Data are plotted as mean ± of three independent experiments . B fractions from ( A ) were analysed by Western blot from BN - PAGE or SDS - PAGE using antibodies as indicated . same WT control was used in Fig . 4A . Representative of three independent experiments . C lysates from ( A ) were subjected to UBA - pull down analysed as described above .

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A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, cells were untreated or treated with TSQ for 6 hrs (please note that the same WT control was used in Fig. EV3A). Cell death was measured by PI staining based on flow cytometry. Data are plotted as mean ± SEM of three independent experiments. B Cellular fractions from (A) were analysed by Western blot from BN-PAGE or SDS-PAGE using antibodies as indicated. The same WT control was used in Fig. 4A. Representative of three independent experiments. C Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.


D N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline overnight , were treated with TSI for indicated time , followed by UBA - pull down . Representative of three independent experiments . E N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 16 hrs . were stimulated ± TSI for 3 hrs after withdrawal of doxycycline . Then TSI medium was removed replaced to medium containing inhibitors Bafilomycin A1 ( BAF ) , PS341 ( PS ) or left untreated ( UT ) . IDN - 6556 was added to conditions to block apoptosis . were collected 0 , 2 , 4 , 6 hrs after medium replacement , followed by UBA - pull down . Representative of three independent experiments .

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D N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline overnight, and cells were treated with TSI for indicated time, followed by UBA-pull down. Representative of three independent experiments. E N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 16 hrs. Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline. Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 (BAF), PS341 (PS) or left untreated (UT). IDN-6556 was added to all conditions to block apoptosis. Cells were collected 0, 2, 4, 6 hrs after medium replacement, followed by UBA-pull down. Representative of three independent experiments.


B WT 4KR mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 6 hrs were untreated ( UT ) or treated with TSI , followed by UBA - pull down . Representative of three independent experiments .

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B WT and 4KR mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 6 hrs and cells were untreated (UT) or treated with TSI, followed by UBA-pull down. Representative of three independent experiments.


D MLKL - / - HT29 stably transfected with constructs encoding human MLKL - USP21 MLKL - USP21C221R , were treated with doxycycline , NSA ( 1 μM ) , TSI or combinations thereof ( added simultaneously ) . Sytox Green positive were quantified in real time by live imaging . Data are plotted as mean ± of six independent experiments . ( A red dashed is shown to highlight delay in kinetics upon treatment with NSA ) . E Human MLKL - USP21 MLKL - USP21C221R were inducibly expressed in MLKL - / - HT29 by doxycycline ( 10 ng / mL ) for 16 hrs , TSI was added for indicated times but samples were collected 25 hrs post - induction , followed by UBA - pulldown . Representative of three independent experiments .

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D MLKL-/- HT29 cells stably transfected with constructs encoding human MLKL-USP21 and MLKL-USP21C221R, were treated with doxycycline, NSA (1 μM), TSI or combinations thereof (added simultaneously). Sytox Green positive cells were quantified in real time by live cell imaging. Data are plotted as mean ± SEM of six independent experiments. (A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA). E Human MLKL-USP21 and MLKL-USP21C221R were inducibly expressed in MLKL-/- HT29 cells by doxycycline (10 ng/mL) for 16 hrs, TSI was added for the indicated times but all samples were collected 25 hrs post-induction, followed by UBA-pulldown . Representative of three independent experiments.


( E ) Tadpoles at NF50 were amputated , fixed at indicated time then processed for whole mount in situ hybridisation using a probe specific for foxm1 . two last panels show transverse section in non - regenerating spinal cord ( nr ) regenerate at 3dpa . red circle highlights spinal cord asterisk notochord .

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(E) Tadpoles at NF50 were amputated, fixed at the indicated time and then processed for whole mount in situ hybridisation using a probe specific for foxm1. The two last panels show transverse section in the non-regenerating spinal cord (nr) and the regenerate at 3dpa. The red circle highlights the spinal cord and the asterisk the notochord.


( F ) Total was isolated from regenerating tails at indicated time points post amputation , reversed transcribed into cDNA analysed for foxm1 expression by qPCR , using ef1α as a reference gene . graph represents mean + / - SD of three independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons was used . * p = 0 .

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(F) Total RNA was isolated from regenerating tails at indicated time points post amputation, reversed transcribed into cDNA and analysed for foxm1 expression by qPCR, using ef1α as a reference gene. The graph represents the mean +/- SD of three independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was used. * p=0.0149


( A ) NF40 tadpoles with following genotypes foxm1 - / - ( mut ) , foxm1 + / - ( het ) foxm1 + / + ( wt ) were amputated left to regenerate for 7 days . show representative tails at 3 7 dpa . arrowheads indicate amputation site . ( B ) of rate of regeneration . of of regenerate to that has originally be amputated was compared for whole tail at 3 7 dpa . graph represents mean + / - SD of 5 independent experiments from 3 different clutches with at least 5 tadpoles in each experiment .

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(A) NF40 tadpoles with the following genotypes foxm1-/- (mut), foxm1+/- (het) and foxm1+/+ (wt) were amputated and left to regenerate for 7 days. The images show representative tails at 3 and 7 dpa. The white arrowheads indicate the amputation site. (B) Quantification of the rate of regeneration. The ratio of the length of the regenerate to the length that has originally be amputated was compared for the whole tail at 3 and 7 dpa. The graph represents the mean+/-SD of 5 independent experiments from 3 different clutches with at least 5 tadpoles in each experiment.


( D ) Representative of EdU ( red ) DAPI ( blue ) at 5 dpa . ( E ) graph represents mean + / - SD of EdU + over total number of in spinal cord of 5 to 12 tadpoles . Each data point represents a tadpole , with an average of 9 sections analysed per .

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(D) Representative images of EdU (red) and DAPI (blue) staining at 5 dpa. (E) The graph represents the mean +/- SD of EdU+ cells over the total number of cells in the spinal cord of 5 to 12 tadpoles. Each data point represents a tadpole, with an average of 9 sections analysed per animal.


( G ) Representative of EdU ( green ) , BrdU ( magenta ) , Sox3 ( ) DAPI ( blue ) at 3 dpa . ( H ) of in ( G ) . graph represents mean + / - SD of 6 tadpoles with an average of 13 sections per tadpole analysed .

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(G) Representative images of EdU (green), BrdU (magenta), Sox3 (white) and DAPI (blue) staining at 3 dpa. (H) Quantification of images in (G). The graph represents the mean +/- SD of 6 tadpoles with an average of 13 sections per tadpole analysed.


( C ) Representative sections labelled with an anti - PCNA antibody ( red ) DAPI ( blue ) at indicated day after amputation ( dpa ) . arrowheads point to in S phase in spinal cord yellow arrowhead at in G1 , G2 M phases . right panels correspond to inset indicated as a box in middle panels .

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(C) Representative sections labelled with an anti-PCNA antibody (red) and DAPI (blue) at the indicated day after amputation (dpa). The white arrowheads point to cells in S phase in the spinal cord and the yellow arrowhead at cells in G1, G2 and M phases. The right panels correspond to the inset indicated as a white box in the middle panels.


( A ) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa fixed at 5 dpa . After sectioning , samples were labelled with antibodies against Sox3 ( green ) , EdU ( magenta ) DAPI ( blue ) . arrowheads show Sox3 positive extensions .

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(A) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Sox3 (green), EdU (magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.


( B ) Sections of spinal cords at 5 dpa in stump ( non - regen . ) or in regenerate ( regen ) labelled with anti - Sox3 ( green ) , anti - Acetylated Tubulin ( AcTub , magenta ) DAPI ( blue ) . arrowheads show Sox3 positive extensions .

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(B) Sections of spinal cords at 5 dpa in the stump (non-regen.) or in the regenerate (regen) labelled with anti-Sox3 (green), anti-Acetylated Tubulin (AcTub, magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.


( E ) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa fixed at 5 dpa . After sectioning , samples were labelled with antibodies against Myt1 ( green ) , EdU ( magenta ) DAPI ( blue ) . ( F ) of shown in ( E ) . graph represents mean + / - SD from 3 tadpoles with an average of 12 sections each .

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(E) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Myt1 (green), EdU (magenta) and DAPI (blue). (F) Quantification of images shown in (E). The graph represents the mean +/- SD from 3 tadpoles with an average of 12 sections each.


( a ) microscopy of GFP - LC3 - transduced HeLa left unstimulated ( Unstim ) or stimulated for 4 h with C12 - iEDAP ( 2 . 5 μg / ml ) or for 2 h with rapamycin ( Rapa ; 50 μg / ml ) . Green , GFP - LC3 ; blue , DAPI ( - intercalating dye ) . Scale bar , 5 μm . ( b ) of dot - or ring - shaped GFP - LC3 signals ( representing autophagosomes ) in HeLa stimulated as described in a .

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(a) Fluorescence microscopy of GFP-LC3-transduced HeLa cells left unstimulated (Unstim) or stimulated for 4 h with C12-iEDAP (2.5 μg/ml) or for 2 h with rapamycin (Rapa; 50 μg/ml). Green, GFP-LC3; blue, DAPI (DNA-intercalating dye). Scale bar, 5 μm. (b) Quantification of dot- or ring-shaped GFP-LC3 signals (representing autophagosomes) in HeLa cells stimulated as described in a.


( c ) Confocal microscopy of wild - type Nod2 - deficient BMDMs transduced with GFP - LC3 ( green ) left unstimulated or stimulated for 4 h with LPS ( 1 μg / ml ) or MDP ( 100 μg / ml ) or for 2 h with rapamycin ( 50 μg / ml ) . Scale bar , 15 μm . ( d ) of dot - or ring - shaped GFP - LC3 ( green ) in BMDMs stimulated as described in c .

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(c) Confocal microscopy of wild-type and Nod2-deficient BMDMs transduced with GFP-LC3 (green) and left unstimulated or stimulated for 4 h with LPS (1 μg/ml) or MDP (100 μg/ml) or for 2 h with rapamycin (50 μg/ml). Scale bar, 15 μm. (d) Quantification of dot- or ring-shaped GFP-LC3 (green) in BMDMs stimulated as described in c.


( e ) Immunofluorescence microscopy of macrophages recovered from peritoneal cavities of thioglycolate - injected wild - type , Nod1 - Nod2 - deficient mice 4 h after intraperitoneal injection of MDP ( 300 μg / ml ) , FK565 ( 300 μg / ml ) or rapamycin ( 300 μg / ml ) ; after centrifugation by cytospin , were stained for endogenous LC3 ( red ) with DAPI ( blue ) . Scale bar , 10 μm . ( f ) of dot - or ring - shaped LC3 signals in macrophages recovered from peritoneal cavities of mice treated as described in c . Data are representative of four ( a - d ) or two ( e , f ) independent experiments with at least 100 per condition ( error bars ( b , d , f ) , s . d . ) .

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(e) Immunofluorescence microscopy of macrophages recovered from the peritoneal cavities of thioglycolate-injected wild-type, Nod1- and Nod2-deficient mice 4 h after intraperitoneal injection of MDP (300 μg/ml), FK565 (300 μg/ml) or rapamycin (300 μg/ml); after centrifugation by cytospin, cells were stained for endogenous LC3 (red) and with DAPI (blue). Scale bar, 10 μm. (f) Quantification of dot- or ring-shaped LC3 signals in macrophages recovered from the peritoneal cavities of mice treated as described in c. Data are representative of four (a-d) or two (e,f) independent experiments with at least 100 cells per condition (error bars (b,d,f), s.d.).


( a ) Confocal microscopy of wild - type Nod1 - deficient GFP - LC3 - expressing MEFs infected for 2 h with wild - type S . flexneri M90T - RFP . ( b ) of colocalization of GFP - LC3 signals with in a .

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(a) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3-expressing MEFs infected for 2 h with wild-type S. flexneri M90T-RFP. (b) Quantification of the colocalization of GFP-LC3 signals with bacteria in a.


( c ) Confocal microscopy of wild - type Nod1 - deficient GFP - LC3 - expressing MEFs infected for 2 h with S . flexneri ΔIcsB - RFP . ( d ) of colocalization of GFP - LC3 signals with in c .

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(c) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3-expressing MEFs infected for 2 h with S. flexneri ΔIcsB-RFP. (d) Quantification of the colocalization of GFP-LC3 signals with bacteria in c.


( e , f ) Plate of in wild - type or Nod1 - deficient MEFs infected for 0 . 5 h with S . flexneri ΔIcsB in presence or absence of autophagy blocker LY294002 ( LY ) or protease inhibitors ( PI ) , followed by 5 . 5 h of incubation in presence of gentamicin .

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(e,f) Plate assay of bacteria in wild-type or Nod1-deficient MEFs infected for 0.5 h with S. flexneri ΔIcsB in the presence or absence of the autophagy blocker LY294002 (LY) or protease inhibitors (PI), followed by 5.5 h of incubation in the presence of gentamicin.


( g - j ) Confocal microscopy ( g , i ) of colocalization of GFP - LC3 signals with M90T ΔIcsB ( h , j ) in wild - type RIP2 - deficient ( Ripk2 − / − ) GFP - LC3 - expressing MEFs infected for 2 h with wild - type S . flexneri ( M90T ; g , h ) or ΔIcsB S . flexneri ( i , j ) .

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(g-j) Confocal microscopy (g,i) and quantification of the colocalization of GFP-LC3 signals with M90T and ΔIcsB (h,j) in wild-type and RIP2-deficient (Ripk2−/−) GFP-LC3-expressing MEFs infected for 2 h with wild-type S. flexneri (M90T; g,h) or ΔIcsB S. flexneri (i,j).


( k ) Enzyme - linked immunosorbent of KC secretion by wild - type RIP2 - deficient MEFs infected for 0 . 5 h with M90T incubated for 5 . 5 h in presence of gentamicin .

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(k) Enzyme-linked immunosorbent assay of KC secretion by wild-type and RIP2-deficient MEFs infected for 0.5 h with M90T and incubated for 5.5 h in the presence of gentamicin.


( l , m ) Confocal microscopy ( l ) of colocalization of GFP - LC3 signals ( green ) with M90T ΔIcsB ( m ) of wild - type NEMO - deficient ( Ikbkg − / − ) GFP - LC3 - expressing MEFs infected for 0 . 5 h with wild - type S . flexneri ( M90T ) or S . flexneri ΔIcsB incubated for 1 . 5 h in presence of gentamicin . Red , RFP . Arrows indicate colocalization of GFP - LC3 signals with S . flexneri . Scale bars , 10 μm . * P 0 . 05 , * * P 0 . 01 ( t - ) . Data represent three independent experiments with at least 100 each ( error bars , s . e . m . ) .

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(l,m) Confocal microscopy (l) and quantification of the colocalization of GFP-LC3 signals (green) with M90T and ΔIcsB (m) of wild-type and NEMO-deficient (Ikbkg−/−) GFP-LC3-expressing MEFs infected for 0.5 h with wild-type S. flexneri (M90T) or S. flexneri ΔIcsB and incubated for 1.5 h in the presence of gentamicin. Red, RFP. Arrows indicate colocalization of GFP-LC3 signals with S. flexneri. Scale bars, 10 μm. *P 0.05, and **P 0.01 (t-test). Data represent three independent experiments with at least 100 cells each (error bars, s.e.m.).


( a ) Immunofluorescence of HeLa expressing hemagglutinin - tagged Nod1 ( HA - Nod1 ( red ) ; top ) or HA - Nod2 ( red ; bottom ) . Blue , DAPI .

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(a) Immunofluorescence of HeLa cells expressing hemagglutinin-tagged Nod1 (HA-Nod1 (red); top) or HA-Nod2 (red; bottom). Blue, DAPI.


( b ) Immunofluorescence of HeLa expressing Myc - tagged FL - ATG16L1 ( Myc - FL - ATG16L1 ) .

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(b) Immunofluorescence of HeLa cells expressing Myc-tagged FL-ATG16L1 (Myc-FL-ATG16L1).


( c , d ) Immunofluorescence of HeLa coexpressing HA - Nod1 ( red ; c ) or HA - Nod2 ( red ; d ) Myc - FL - ATG16L1 ( green ) . Blue , DAPI .

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(c,d) Immunofluorescence of HeLa cells coexpressing HA-Nod1 (red; c) or HA-Nod2 (red; d) and Myc-FL-ATG16L1 (green). Blue, DAPI.


( e ) Immunofluorescence of HeLa expressing a Flag - tagged truncated form of ATG16L1 ( Flag - ΔN85 - ATG16L1 ; green ) . Blue , DAPI .

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(e) Immunofluorescence of HeLa cells expressing a Flag-tagged truncated form of ATG16L1 (Flag-ΔN85-ATG16L1; green). Blue, DAPI.


( f ) Immunofluorescence of HeLa coexpressing HA - Nod2 ( red ) Flag - ΔN85 - ATG16L1 ( green ) . Blue , DAPI .

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(f) Immunofluorescence of HeLa cells coexpressing HA-Nod2 (red) and Flag-ΔN85-ATG16L1 (green). Blue, DAPI.


( g ) Immunofluorescence microscopy of Mode - K stained for endogenous Nod2 ( green ) ATG16L1 ( red ) with DAPI ( blue ) . For a - g , arrows indicate colocalization of ATG16 Nod2 ; outlined at left is enlarged at right ( a , b , e ) or in other ( c , d , f , ) ; scale bars , 8 μm .

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(g) Immunofluorescence microscopy of Mode-K cells stained for endogenous Nod2 (green) and ATG16L1 (red) and with DAPI (blue). For a-g, arrows indicate membrane colocalization of ATG16 and Nod2; area outlined at left is enlarged at right (a,b,e) or in other images (c,d,f,); scale bars, 8 μm.


( h , i ) Immunoprecipitation ( IP ) immunoblot ( IB ) of HEK293 transfected with Flag - ΔN85 - ATG16L1 ( h ) or Myc - FL - ATG16L1 ( i ) , together with HA - Nod1 , HA - Nod2 or HA - tagged enhanced GFP ( EGFP - HA ) . Ig , immunoglobulin ( loading control ) . Results are representative of one of at least three independent experiments ( a - g ) or one of three independent experiments ( h , i ) .

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(h,i) Immunoprecipitation (IP) and immunoblot (IB) analysis of HEK293 cells transfected with Flag-ΔN85-ATG16L1 (h) or Myc-FL-ATG16L1 (i), together with HA-Nod1, HA-Nod2 or HA-tagged enhanced GFP (EGFP-HA). Ig, immunoglobulin (loading control). Results are representative of one of at least three independent experiments (a-g) or one of three independent experiments (h,i).


Immunofluorescence microscopy of wild - type RIP2 - deficient MEFs expressing Flag - tagged ΔN85 - ATG16L1 HA - Nod1 ( a ) or HA - Nod2 ( b ) . Scale bars , 10 μm . outlined at far left is enlarged at right . Data are representative one of two independent experiments .

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Immunofluorescence microscopy of wild-type and RIP2-deficient MEFs expressing Flag-tagged ΔN85-ATG16L1 and HA-Nod1 (a) or HA-Nod2 (b). Scale bars, 10 μm. Area outlined at far left is enlarged at right. Data are representative one of two independent experiments.


( a ) microscopy of immortalized GFP - LC3 - transduced lymphoblasts from donors homozygous for normal ATG16L1 * 300T allele ( 300T ) or risk allele ATG16L1 * 300A ( 300A ) , left unstimulated or treated for 2 h with rapamycin ( 50 μg / ml ) or for 4 h with MDP ( 20 μg / ml ) . Green , GFP - LC3 . Scale bar , 5 μm . ( b ) Induction of GFP - LC3 signals by rapamycin , MDP or Gram - positive peptidoglycan ( PG + ; 20 μg / ml ) in treated as described in a , presented relative to GFP - LC3 signals in unstimulated control . * P 0 . 05 ( t - ) . Data represent three independent experiments ( error bars ( b ) , s . d . ) .

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(a) Fluorescence microscopy of immortalized GFP-LC3-transduced lymphoblasts from donors homozygous for the normal ATG16L1*300T allele (300T) or risk allele ATG16L1*300A (300A), left unstimulated or treated for 2 h with rapamycin (50 μg/ml) or for 4 h with MDP (20 μg/ml). Green, GFP-LC3. Scale bar, 5 μm. (b) Induction of GFP-LC3 signals by rapamycin, MDP or Gram-positive peptidoglycan (PG+; 20 μg/ml) in cells treated as described in a, presented relative to GFP-LC3 signals in unstimulated control cells. *P 0.05 (t-test). Data represent three independent experiments (error bars (b), s.d.).


( a ) Immunofluorescence microscopy of HeLa transfected with HA - Nod2 ( red ) Flag - ΔN85 - ATG16L1 ( green ) infected for 10 - 20 min with S . flexneri M90T . Blue , DAPI . Arrows indicate colocalization of Nod2 ATG16L1 around invading . Scale bars , 5 μm . Data are representative of one of three independent experiments .

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(a) Immunofluorescence microscopy of HeLa cells transfected with HA-Nod2 (red) and Flag-ΔN85-ATG16L1(green) and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.


( b ) Immunofluorescence microscopy of endogenous Nod2 ATG16L1 in Mode - K infected for 10 - 20 min with S . flexneri M90T . Blue , DAPI . Arrows indicate colocalization of Nod2 ATG16L1 around invading . Scale bars , 5 μm . Data are representative of one of three independent experiments .

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(b) Immunofluorescence microscopy of endogenous Nod2 and ATG16L1 in Mode-K cells and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.


( a , b ) Immunofluorescence microscopy of HeLa expressing Flag - ΔN85 - ATG16L1 ( green ; a ) or Myc - FL - ATG16L1 ( green ; b ) HA - tagged Nod2 L1007fsinsC ( HA - Nod2 , 1007fs ) . outlined at left is enlarged in other .

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(a,b) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (green; a) or Myc-FL-ATG16L1 (green; b) and HA-tagged Nod2 L1007fsinsC (HA-Nod2, 1007fs). Area outlined at left is enlarged in other images.


( c , d ) Immunofluorescence microscopy of HeLa expressing Flag - ΔN85 - ATG16L1 ( c ) or Myc - FL - ATG16L1 ( d ) together with HA - tagged Nod2fs ( red ) . Blue , DAPI . Scale bar , 8 μm .

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(c,d) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (c) or Myc-FL-ATG16L1 (d) together with HA-tagged Nod2fs (red). Blue, DAPI. Scale bar, 8 μm.


( e ) Immunofluorescence microscopy of wild - type Nod2fs - KI BMDMs transduced with GFP - LC3 ( green ) infected for 0 . 5 h with S . flexneri M90T - RFP ( red ) , followed by incubation for 1 h with gentamicin . Arrows indicate no colocalization of Nod2 with ΔN85 - ATG16L1 or FL - ATG16L1 . Scale bar , 15 μm . ( f ) of colocalization of GFP - LC3 signals with S . flexneri M90T - RFP in experiments as described in e . Data are representative of one of three independent experiments ( error bars ( f ) , s . d . ) .

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(e) Immunofluorescence microscopy of wild-type and Nod2fs-KI BMDMs transduced with GFP-LC3 (green) and infected for 0.5 h with S. flexneri M90T-RFP (red), followed by incubation for 1 h with gentamicin. Arrows indicate no colocalization of Nod2 with ΔN85-ATG16L1 or FL-ATG16L1. Scale bar, 15 μm. (f) Quantification of the colocalization of GFP-LC3 signals with S. flexneri M90T-RFP in experiments as described in e. Data are representative of one of three independent experiments (error bars (f), s.d.).


D ) heart , tibialis anterior ( TA ) triceps muscle were isolated analyzed to identify presence of duplication from exons 18 to 30 via RT - PCR using primers represented by arrows . Sanger sequencing confirmed correct splicing of exons 30 18 at duplication junction .

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D) The heart, tibialis anterior (TA) and triceps muscle were isolated and analyzed to identify the presence of the duplication from exons 18 to 30 via RT-PCR using primers represented by the arrows. Sanger sequencing confirmed correct splicing of exons 30 and 18 at the duplication junction.


A ) 8 - µm cross section of 15 - week - old WT Dup18 - 30 TA triceps were analyzed for dystrophin localization by immunofluorescence . Asterisk indicates clusters of revertant . Scale bars , 100 μm . B ) muscle architecture of same was investigated by H & E . A representative image is shown . Scale bars , 100 μm . Asterisk indicates areas with necrotic fibrosis . Arrows indicate with central nuclei .

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A) 8-µm cross section of 15-week-old WT and Dup18-30 TA and triceps were analyzed for dystrophin localization by immunofluorescence. Asterisk indicates clusters of revertant fibers. Scale bars, 100 μm. B) The muscle architecture of the same muscles was investigated by H&E staining. A representative image is shown. Scale bars, 100 μm. Asterisk indicates areas with necrotic fibers and fibrosis. Arrows indicate fibers with central nuclei.


G , H ) Mice were tested in an open - field chamber in which total travelled ( G ) vertical activity ( H ) were assessed . WT , n = 9 ; Dup18 - 30 n = 12 .

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G, H) Mice were tested in an open-field chamber in which total distance travelled (G) and vertical activity (H) were assessed. WT, n = 9; Dup18-30 n = 12.


F , G ) Western blotting detected restoration of dystrophin expression in ( F ) heart ( G ) TA in Dup18 - 30 mice . 25 % 50 % of WT compared to Dup18 - 30 mice have been loaded on gel . Calnexin was used as a loading control .

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F, G ) Western blotting detected restoration of dystrophin expression in (F) heart and (G) TA in the Dup18-30 mice. 25% and 50% of the WT proteins compared to Dup18-30 mice have been loaded on the gel. Calnexin was used as a loading control.


H ) Immunostaining showed restoration of dystrophin expression in heart TA . A representative sample is shown . Scale bars , 100 μm .

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H) Immunostaining showed restoration of dystrophin expression in the heart and TA. A representative sample is shown. Scale bars, 100 μm.


A ) Immunofluorescence for alpha - syntrophin beta - sarcoglycan in TA triceps of Dup18 - 30 mice . A representative image is shown . Scale bars , 100 μm .

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A) Immunofluorescence staining for alpha-syntrophin and beta-sarcoglycan in the TA and triceps muscles of the Dup18-30 mice. A representative image is shown. Scale bars, 100 μm.


B ) TA , triceps diaphragm muscle architecture was analyzed by H & E . Scale bars , 50 μm .

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B) TA, triceps and diaphragm muscle architecture was analyzed by H&E staining. Scale bars, 50 μm.


A ) Fibrosis was analyzed in diaphragm via Masson Trichrome . Scale bars , 100 μm .

A) Fibrosis was analyzed in the diaphragm via Masson Trichrome staining. Scale bars, 100 μm.


WT , Dup18 - 30 untreated Dup18 - 30 treated mice were tested 7 after treatment by measuring ( C ) forelimbs grip

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WT, Dup18-30 untreated and Dup18-30 treated mice were tested 7 weeks after treatment by measuring (C) forelimbs grip strength