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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 animals ; antisense , left panel ; sense , middle panel ) and in Gadd45a - KO sections ( n = 6 animals ; 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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 ( 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 .

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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 analysis with the 1 % false discovery rate ( FDR ) hits in red ( 5 / 402 ; and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . The expression of MavQ in the yeast was detected by Flag antibody . The 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 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 .

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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 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 .

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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 . 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 .

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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 and D160 abolish the 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 fluorescence images of HeLa cells expressing the PtdIns3P probe GFP - 2xFYVEHrs and 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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . The 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 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 .

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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 the indicated time points , CFUs were determined by plating the saponin - solubilized lysates of infected cells 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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 )

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 - / - 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 .

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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 , and cells 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 the DUBs shown in ( A ) . Beads eluates were analysed by Western blot and 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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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

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 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 .

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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 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 .

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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 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 .

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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 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 .

(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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 . ) .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 ) .

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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 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 .

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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 ) 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 . ) .

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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 cells 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 cells 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 cells coexpressing HA - Nod1 ( red ; c ) or HA - Nod2 ( red ; d ) and 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 cells 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 cells coexpressing HA - Nod2 ( red ) and 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 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 .

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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 ) 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 ) .

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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 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 .

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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 ) 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 . ) .

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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 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 .

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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 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 .

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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 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 .

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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 cells 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 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 . ) .

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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 ) 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 .

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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 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 .

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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 distance travelled ( G ) and 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 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 .

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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 the heart and 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 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 .

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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 and diaphragm muscle architecture was analyzed by H & E staining . 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 the diaphragm via Masson Trichrome staining . Scale bars , 100 μm .

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


WT , Dup18 - 30 untreated and Dup18 - 30 treated mice were tested 7 weeks 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


A HEK293T were transfected with NF - kBluciferase reporter plasmid and FLAG - tagged full - length CARD14 ( FL ) or CARD14sh ( short ) at the indicated concentrations and analyzed 24 h later . ( A ) NF - kB reporter gene expression .

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A HEK293T were transfected with NF-kBluciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (A) NF-kB reporter gene expression.


HEK293T were transfected with NF - kB luciferase reporter plasmid and FLAG - tagged full - length CARD14 ( FL ) or CARD14sh ( short ) at the indicated concentrations and analyzed 24 h later . ( B ) IL - 8 secretion . Values are the mean of triplicates ± s . e . P - values are compared against empty vector treatment . Significance levels , ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 by student t test . Unless otherwise stated , p - values are p < 0 . 001 .

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HEK293T were transfected with NF-kB luciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (B) IL-8 secretion. Values are the mean of triplicates ± s.e. P-values are compared against empty vector treatment. Significance levels, ns P>0.05, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Unless otherwise stated, p-values are p<0.001.


HEK293T were transfected with NF - kB luciferase reporter plasmid and FLAG - tagged full - length CARD14 ( FL ) or CARD14sh ( short ) at the indicated concentrations and analyzed 24 h later . ( C ) Immunoblotting for c - Jun , phospho - c - Jun , JNK , phospho - JNK , p38 and phospho - p38 as indicated . Actin was used as a loading control . Data are representative of two independent experiments .

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HEK293T were transfected with NF-kB luciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (C) Immunoblotting for c-Jun, phospho-c-Jun, JNK, phospho-JNK, p38 and phospho-p38 as indicated. Actin was used as a loading control. Data are representative of two independent experiments.


( A ) MALT1 interacts with CARD14sh in a BCL10 - dependent manner . HEK293T cells were transfected with FLAG - CARD14sh , Myc - MALT1 and FLAG - BCL10 as indicated . 24 h later , MALT1 was immunoprecipitated ( IP ) from cell lysates with anti - MALT1 . CARD14sh and BCL10 co - immunoprecipitation with MALT1 was detected by immunoblotting with anti - FLAG . Immunoprecipitated MALT1 was detected by immunoblotting with anti - myc . The closed arrowhead shows the position of CARD14 , the double arrowhead shows the position of BCL10 . The asterisk indicates a non - specific band . Total expression levels of transfected proteins are shown by immunoblotting of a fraction of the cell lysates with the indicated antibodies ( bottom panel ) .

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(A) MALT1 interacts with CARD14sh in a BCL10-dependent manner. HEK293T cells were transfected with FLAG-CARD14sh, Myc-MALT1 and FLAG-BCL10 as indicated. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1. CARD14sh and BCL10 co-immunoprecipitation with MALT1 was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-myc. The closed arrowhead shows the position of CARD14, the double arrowhead shows the position of BCL10. The asterisk indicates a non-specific band. Total expression levels of transfected proteins are shown by immunoblotting of a fraction of the cell lysates with the indicated antibodies (bottom panel).


( B ) MALT1 co - expression potentiates CARD14sh - induced JNK and p38 MAP kinase activation . HEK293T cells were transfected with the indicated concentrations of FLAG - tagged CARD14sh , with or without MALT1 ( 30ng ) . Transfection with FLAG - TRAF6 and FLAG - CARD11 ( L232LI ) were used as a positive control . c - Jun accumulation , JNK and p38 phosphorylation were analyzed by immunoblotting with the antibodies indicated . Actin was used as a loading control .

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(B) MALT1 co-expression potentiates CARD14sh-induced JNK and p38 MAP kinase activation. HEK293T cells were transfected with the indicated concentrations of FLAG-tagged CARD14sh, with or without MALT1 (30ng). Transfection with FLAG-TRAF6 and FLAG-CARD11(L232LI) were used as a positive control. c-Jun accumulation, JNK and p38 phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control.


( C ) MALT1 co - expression potentiates NF - kB activation by pathogenic CARD14 mutants . HEK293T were transfected with NF - kB reporter plasmid and wild - type ( WT ) FLAG - CARD14 or the indicated psoriasis - associated CARD14 mutants ( 15ng ) , with or without MALT1 ( 15ng ) . Luciferase activity in cell lysates was measured after 24 h . Values are the mean of triplicates ± s . e . Significance levels , * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 by student t test . Expression of transfected proteins was verified by western blotting ( bottom panel ) .

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(C) MALT1 co-expression potentiates NF-kB activation by pathogenic CARD14 mutants. HEK293T were transfected with NF-kB reporter plasmid and wild-type (WT) FLAG-CARD14 or the indicated psoriasis-associated CARD14 mutants (15ng), with or without MALT1 (15ng). Luciferase activity in cell lysates was measured after 24 h. Values are the mean of triplicates ± s.e. Significance levels, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Expression of transfected proteins was verified by western blotting (bottom panel).


( D ) CARD14 ( E138A ) mutation enhances MALT1 binding . HEK293T cells were transfected with wild - type ( WT ) FLAG - CARD14sh or the indicated CARD14sh mutants and Myc - MALT1 . 24 h later , MALT1 was immunoprecipitated ( IP ) from cell lysates with anti - MALT1 and CARD14 co - immunoprecipitation was detected by immunoblotting with anti - FLAG . Immunoprecipitated MALT1 was detected by immunoblotting with anti - MALT1 . The asterisk indicates a non - specific band . Immunoprecipitation with a non - relevant antibody ( rIgG ) was used as a negative control ( first lane ) . Total expression levels of transfected proteins are shown by immunoblotting of total cell lysates ( bottom panel ) . Data are representative of two independent experiments .

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(D) CARD14(E138A) mutation enhances MALT1 binding. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14sh or the indicated CARD14sh mutants and Myc-MALT1. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1 and CARD14 co-immunoprecipitation was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-MALT1. The asterisk indicates a non-specific band. Immunoprecipitation with a non-relevant antibody (rIgG) was used as a negative control (first lane). Total expression levels of transfected proteins are shown by immunoblotting of total cell lysates (bottom panel). Data are representative of two independent experiments.


( A ) MALT1 expression is essential for CARD14sh - induced NF - kB activation . MALT1 - deficient HEK293T cells were transfected with NF - kB reporter plasmid and the indicated expression plasmids . Luciferase activity in cell lysates was measured 24 h later . CARD11 ( L232LI ) and TRAF6 transfection were used as MALT1 - dependent and MALT1 - independent controls , respectively . Values are the mean of triplicates ± s . e . Expression of the transfected plasmids was verified by immunoblotting ( lower panel ) .

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(A) MALT1 expression is essential for CARD14sh-induced NF-kB activation. MALT1-deficient HEK293T cells were transfected with NF-kB reporter plasmid and the indicated expression plasmids. Luciferase activity in cell lysates was measured 24 h later. CARD11(L232LI) and TRAF6 transfection were used as MALT1-dependent and MALT1-independent controls, respectively. Values are the mean of triplicates ± s.e. Expression of the transfected plasmids was verified by immunoblotting (lower panel).


( B ) MALT1 expression is essential for CARD14sh - induced JNK activation . MALT1 - deficient HEK293T cells were transfected with FLAG - CARD14sh ( 30ng ) either alone or together with either wild - type ( MALT1WT ) or catalytically inactive MALT1 ( MALT1C / A ) as indicated . c - Jun accumulation and JNK phosphorylation were analyzed by immunoblotting with the antibodies indicated . Actin was used as a loading control .

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(B) MALT1 expression is essential for CARD14sh-induced JNK activation. MALT1-deficient HEK293T cells were transfected with FLAG-CARD14sh (30ng) either alone or together with either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A) as indicated. c-Jun accumulation and JNK phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control.


( C ) MALT1 deficiency inhibits CARD14 - induced gene expression in HaCaT keratinocytes . HaCaT cells were transfected with scrambled ( scr ) or MALT1 - targeting siRNA prior to doxycycline - induced CARD14 expression as described in materials and methods . IL - 8 and MCP - 1 concentration in the cell supernatants was measured 8 h ( IL - 8 ) or 24 h ( MCP - 1 ) later by ELISA . Values are the mean of triplicates ± s . e . Doxycycline - induced CARD14 expression and MALT1 knock - down were verified by immunoblotting ( right panel ) . Data are representative of two independent experiments .

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(C) MALT1 deficiency inhibits CARD14-induced gene expression in HaCaT keratinocytes. HaCaT cells were transfected with scrambled (scr) or MALT1-targeting siRNA prior to doxycycline-induced CARD14 expression as described in materials and methods. IL-8 and MCP-1 concentration in the cell supernatants was measured 8 h (IL-8) or 24 h (MCP-1) later by ELISA. Values are the mean of triplicates ± s.e. Doxycycline-induced CARD14 expression and MALT1 knock-down were verified by immunoblotting (right panel). Data are representative of two independent experiments.