( a ) Subcellular localization of ceramide , generated by wild - type CerS1 induction ( + tet ) , was visualized in mitochondria by colocalization of ceramides and mitochondria with anti - ceramide and anti - Tom20 , respectively , using confocal microscopy .

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

(a) Subcellular localization of ceramide, generated by wild-type CerS1 induction (+ tet), was visualized in mitochondria by colocalization of ceramides and mitochondria with anti-ceramide and anti-Tom20, respectively, using confocal microscopy.


( b , c ) Mitochondrial ( Mito ) targeting of LC3B - II and C18 - ceramide , generated by induction of wild - type CerS1 ( b ) , and that of the products of the catalytically inactive mutant CerS1 ( + tet ) ( c ) was visualized by colocalization of ceramide ( Cer ; green ) , CFP - LC3B ( blue ) and MTR using anti - ceramide and confocal microscopy . Noninduced cells ( − tet ) were used as controls . Scale bars , 10 μm .

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

(b,c) Mitochondrial (Mito) targeting of LC3B-II and C18-ceramide, generated by induction of wild-type CerS1 (b), and that of the products of the catalytically inactive mutant CerS1 (+ tet) (c) was visualized by colocalization of ceramide (Cer; green), CFP-LC3B (blue) and MTR using anti-ceramide and confocal microscopy. Noninduced cells (− tet) were used as controls. Scale bars, 10 μm.


( a ) Binding of Flag - tagged wild - type LC3B and LC3BI35A , LC3BF52A or LC3BG120A with endogenous C18 - ceramide ( C18 - Cer ) , generated in response to wild - type CerS1 induction ( left ) or exogenous C18 - Pyr - Cer ( right ) , was measured using LC / MS / MS after pulldown using anti - Flag - conjugated beads . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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

(a) Binding of Flag-tagged wild-type LC3B and LC3BI35A, LC3BF52A or LC3BG120A with endogenous C18-ceramide (C18-Cer), generated in response to wild-type CerS1 induction (left) or exogenous C18-Pyr-Cer (right), was measured using LC/MS/MS after pulldown using anti-Flag-conjugated beads. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( b ) Effects of CerS1 and C18 - ceramide induction on the lipidation of Flag - tagged wild - type , LC3BI35A , LC3BF52A and LC3BG120A proteins were examined by western blotting . Noninduced cells ( − tet ) were used as controls . β - actin was used as a loading control . Full blots can be found in Supplementary Figure 13 .

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

(b) Effects of CerS1 and C18-ceramide induction on the lipidation of Flag-tagged wild-type, LC3BI35A, LC3BF52A and LC3BG120A proteins were examined by western blotting. Noninduced cells (− tet) were used as controls. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.


( a , b ) Effects of G120A and F52A mutations , which perturbed C18 - ceramide binding of LC3B , on targeting of mitochondria by LC3B - containing autophagosomes ( visualized by confocal microscopy using anti - Flag and anti - Tom20 ) ( a ) or mitochondrial function ( measurement of OCR ) ( b ) . V , vector .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effects of G120A and F52A mutations, which perturbed C18-ceramide binding of LC3B, on targeting of mitochondria by LC3B-containing autophagosomes (visualized by confocal microscopy using anti-Flag and anti-Tom20) (a) or mitochondrial function (measurement of OCR) (b). V, vector.


( c ) Effects of siRNA - mediated knockdown of p62 , NIX and Drp1 on OCR in the absence or presence of CerS1 and C18 - ceramide induction were measured using the SeaHorse . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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

(c) Effects of siRNA-mediated knockdown of p62, NIX and Drp1 on OCR in the absence or presence of CerS1 and C18-ceramide induction were measured using the SeaHorse. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( d ) Effects of siRNA - mediated knockdown of Drp1 ( lower left and right ) on the localization of ceramide within mitochondrial membranes were visualized using the colocalization of anti - ceramide ( red ) and anti - Tom20 ( green ) under confocal microscopy , in the absence ( upper and lower left ) or presence ( upper and lower right ) of CerS1 and C18 - ceramide induction ( − / + tet ) compared to Scr siRNA - transfected controls ( upper left and right ) . Scale bars , 10 μm in a and d . Enlarged images are shown in boxed areas in d .

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

(d) Effects of siRNA-mediated knockdown of Drp1 (lower left and right) on the localization of ceramide within mitochondrial membranes were visualized using the colocalization of anti-ceramide (red) and anti-Tom20 (green) under confocal microscopy, in the absence (upper and lower left) or presence (upper and lower right) of CerS1 and C18-ceramide induction (−/+ tet) compared to Scr siRNA-transfected controls (upper left and right). Scale bars, 10 μm in a and d. Enlarged images are shown in boxed areas in d.


( a , b ) Roles of shRNA - mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function ( a ) or UM - SCC - 22A xenograft - derived tumor growth ( b ) in the absence or presence of wild - type CerS1 and C18 - ceramide induction ( − / + tet ) were determined using the SeaHorse or measurement of tumor volumes in the flanks of SCID mice ( n = 6 per group ) , respectively . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Roles of shRNA-mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function (a) or UM-SCC-22A xenograft-derived tumor growth (b) in the absence or presence of wild-type CerS1 and C18-ceramide induction (−/+ tet) were determined using the SeaHorse or measurement of tumor volumes in the flanks of SCID mice (n = 6 per group), respectively. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


A ) Visualisation of the pathology load at 9M of age of TAUwt , TAUtg and APPtg mice at 9M of age . Immunofluorescent staining for X34 ( a fluorescent derivative of Congo Red ) , Iba1 ( microglia ) and TO - PRO - 3 ( nuclei ) has been pseudocolored . Scale bar = 100μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Visualisation of the pathology load at 9M of age of TAUwt, TAUtg and APPtg mice at 9M of age. Immunofluorescent staining for X34 (a fluorescent derivative of Congo Red), Iba1 (microglia) and TO-PRO-3 (nuclei) has been pseudocolored. Scale bar = 100μm.


Log2 fold change ( LFC ) in TAUtg ( x - axis ) and APPtg mice ( y - axis ) after differential expression analysis . Upregulated genes are on the right part ( TAUtg mice ) or upper part ( APPtg mice ) of the graph ; downregulated genes are on the left part ( TAUtg ) or lower part ( APPtg ) of the graph . Colored dots represent significantly differentially expressed genes ( Benjamini - Yuketieli - adjusted p - values ( Padj ) < 0 . 05 ) for APPtg ( green dots ) , TAUtg ( yellow dots ) or for both ( red dots ) . Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up - to most down - regulated on a combined score of LFC and padj ( i . e . signed log10 ( p - value ) , where the sign is determined by the LFC ) . A ) Genes differentially expressed as a function of age , i . e . 4M vs 10M , independently of genotype . Thus , genes with a positive LFC are more highly expressed in 10M mice over 4M mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis. Upregulated genes are on the right part (TAUtg mice) or upper part (APPtg mice) of the graph; downregulated genes are on the left part (TAUtg) or lower part (APPtg) of the graph. Colored dots represent significantly differentially expressed genes (Benjamini-Yuketieli-adjusted p-values (Padj) <0.05) for APPtg (green dots), TAUtg (yellow dots) or for both (red dots). Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up- to most down-regulated on a combined score of LFC and padj (i.e. signed log10(p-value), where the sign is determined by the LFC). A) Genes differentially expressed as a function of age, i.e. 4M vs 10M, independently of genotype. Thus, genes with a positive LFC are more highly expressed in 10M mice over 4M mice.


Log2 fold change ( LFC ) in TAUtg ( x - axis ) and APPtg mice ( y - axis ) after differential expression analysis . Upregulated genes are on the right part ( TAUtg mice ) or upper part ( APPtg mice ) of the graph ; downregulated genes are on the left part ( TAUtg ) or lower part ( APPtg ) of the graph . Colored dots represent significantly differentially expressed genes ( Benjamini - Yuketieli - adjusted p - values ( Padj ) < 0 . 05 ) for APPtg ( green dots ) , TAUtg ( yellow dots ) or for both ( red dots ) . Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up - to most down - regulated on a combined score of LFC and padj ( i . e . signed log10 ( p - value ) , where the sign is determined by the LFC ) . B ) Genes differentially expressed due to genotype , i . e . WT vs TG , independently of age . Thus , genes with a positive LFC are more highly expressed in TG mice than in WT mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis. Upregulated genes are on the right part (TAUtg mice) or upper part (APPtg mice) of the graph; downregulated genes are on the left part (TAUtg) or lower part (APPtg) of the graph. Colored dots represent significantly differentially expressed genes (Benjamini-Yuketieli-adjusted p-values (Padj) <0.05) for APPtg (green dots), TAUtg (yellow dots) or for both (red dots). Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up- to most down-regulated on a combined score of LFC and padj (i.e. signed log10(p-value), where the sign is determined by the LFC). B) Genes differentially expressed due to genotype, i.e. WT vs TG, independently of age. Thus, genes with a positive LFC are more highly expressed in TG mice than in WT mice.


Log2 fold change ( LFC ) in TAUtg ( x - axis ) and APPtg mice ( y - axis ) after differential expression analysis . Upregulated genes are on the right part ( TAUtg mice ) or upper part ( APPtg mice ) of the graph ; downregulated genes are on the left part ( TAUtg ) or lower part ( APPtg ) of the graph . Colored dots represent significantly differentially expressed genes ( Benjamini - Yuketieli - adjusted p - values ( Padj ) < 0 . 05 ) for APPtg ( green dots ) , TAUtg ( yellow dots ) or for both ( red dots ) . Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up - to most down - regulated on a combined score of LFC and padj ( i . e . signed log10 ( p - value ) , where the sign is determined by the LFC ) . C ) Genes differentially expressed in the age * genotype interaction comparison , i . e . comparing the TG - 10M mice to all other experimental groups ( see Fig . 1C ) . Thus , genes with a positive LFC are more highly expressed in the TG - 10M mice compared to all other experimental groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis. Upregulated genes are on the right part (TAUtg mice) or upper part (APPtg mice) of the graph; downregulated genes are on the left part (TAUtg) or lower part (APPtg) of the graph. Colored dots represent significantly differentially expressed genes (Benjamini-Yuketieli-adjusted p-values (Padj) <0.05) for APPtg (green dots), TAUtg (yellow dots) or for both (red dots). Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up- to most down-regulated on a combined score of LFC and padj (i.e. signed log10(p-value), where the sign is determined by the LFC). C) Genes differentially expressed in the age*genotype interaction comparison, i.e. comparing the TG-10M mice to all other experimental groups (see Fig.1C). Thus, genes with a positive LFC are more highly expressed in the TG-10M mice compared to all other experimental groups.


Log2 fold change ( LFC ) in TAUtg ( x - axis ) and APPtg mice ( y - axis ) after differential expression analysis . Upregulated genes are on the right part ( TAUtg mice ) or upper part ( APPtg mice ) of the graph ; downregulated genes are on the left part ( TAUtg ) or lower part ( APPtg ) of the graph . Colored dots represent significantly differentially expressed genes ( Benjamini - Yuketieli - adjusted p - values ( Padj ) < 0 . 05 ) for APPtg ( green dots ) , TAUtg ( yellow dots ) or for both ( red dots ) . Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up - to most down - regulated on a combined score of LFC and padj ( i . e . signed log10 ( p - value ) , where the sign is determined by the LFC ) . D ) Depicts the 314 Marioni - based GWAS genes at p < 0 . 001 onto the LFC / LFC plot of the age * genotype comparison ( panel C ) . Green dots are significantly changed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis. Upregulated genes are on the right part (TAUtg mice) or upper part (APPtg mice) of the graph; downregulated genes are on the left part (TAUtg) or lower part (APPtg) of the graph. Colored dots represent significantly differentially expressed genes (Benjamini-Yuketieli-adjusted p-values (Padj) <0.05) for APPtg (green dots), TAUtg (yellow dots) or for both (red dots). Spearman correlation assesses the correlation between APPtg and TAUtg mice when ranking genes that are significantly differentially expressed in either APPtg or TAUtg mice from most up- to most down-regulated on a combined score of LFC and padj (i.e. signed log10(p-value), where the sign is determined by the LFC). D) Depicts the 314 Marioni-based GWAS genes at p<0.001 onto the LFC/LFC plot of the age*genotype comparison (panel C). Green dots are significantly changed.


B ) Log2 fold change ( LFC ) in TAUtg ( x - axis ) and APPtg mice ( y - axis ) after differential expression analysis , assessing the effects of age * genotype interaction . Color code for dots / numbers : grey = genes in hippocampus ( n = 15824 ) ; black = genes in APPtg - Blue ( n = 4236 ) ; green = genes significantly differentially expressed in APPtg mice ( n = 493 ) ; yellow = significantly differentially expressed genes in TAUtg mice ( n = 9 ) ; red = significantly differentially expressed genes in both ( n = 9 ) .

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B) Log2 fold change (LFC) in TAUtg (x-axis) and APPtg mice (y-axis) after differential expression analysis, assessing the effects of age*genotype interaction. Color code for dots/numbers: grey = genes in hippocampus (n=15824); black = genes in APPtg-Blue (n=4236); green = genes significantly differentially expressed in APPtg mice (n=493); yellow = significantly differentially expressed genes in TAUtg mice (n=9); red = significantly differentially expressed genes in both (n=9).


C ) Z - score distribution per experimental group for all genes within the APPtg - Blue module . Boxplots : center line , median ; box limits , 25th - 75th quartiles ; whiskers , 1 . 5x interquartile range . Empirical p - values are Bonferroni adjusted ( * * * pbonf < 0 . 001 ) and indicate significant shift in z - score

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C) Z-score distribution per experimental group for all genes within the APPtg-Blue module. Boxplots: center line, median; box limits, 25th-75th quartiles; whiskers, 1.5x interquartile range. Empirical p-values are Bonferroni adjusted (***pbonf<0.001) and indicate significant shift in z-score distribution


A ) UMAP clustering of sorted Cd11b + / Cd45 + myeloid cells from mouse hippocampus . ARM : activated response microglia ; CAM : CNS - associated macrophages ; CPM : cycling and proliferating microglia ; HM . 1 : homeostatic microglia cluster 1 ; HM . 2 : homeostatic microglia cluster 2 ; IRM : interferon - response microglia ; MHC . high : high MHC - expressing microglia ; Mnc : monocytes ; TRM : transitioning microglia .

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A) UMAP clustering of sorted Cd11b+/Cd45+ myeloid cells from mouse hippocampus. ARM: activated response microglia; CAM: CNS-associated macrophages; CPM: cycling and proliferating microglia; HM.1: homeostatic microglia cluster 1; HM.2: homeostatic microglia cluster 2; IRM: interferon-response microglia; MHC.high: high MHC-expressing microglia; Mnc: monocytes; TRM: transitioning microglia.


B ) Distribution of cells across the different clusters per experimental group ( n = 2 mice pooled for all experimental groups , except for TAUtg - 11M and TAUwt - 11M with n = 3 mice pooled ) .

O O O O O O O O O O O O O O O O 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) Distribution of cells across the different clusters per experimental group (n=2 mice pooled for all experimental groups, except for TAUtg-11M and TAUwt-11M with n=3 mice pooled).


C ) Distribution of cells per experimental group over the different clusters , expressed in percentages as stated on top of each bar . E . g . of all cells within APPtg - 11M , 56 . 7 % of cells are ARM , while 12 . 5 % are HM . 1 cells .

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C) Distribution of cells per experimental group over the different clusters, expressed in percentages as stated on top of each bar. E.g. of all cells within APPtg-11M, 56.7% of cells are ARM, while 12.5% are HM.1 cells.


E ) Differential expression of ARM vs HM . 1 microglia for APPtg and TAUtg mice separately . Genes with a positive log ( fold change ) ( LFC ) are higher expressed in ARMs compared to HM . 1 microglia . As can be seen which genes are more highly expressed in ARMs vs HM . 1 cells are highly similar among APPtg and TAUtg mice ( Spearman correlation r = 0 . 91 , p = 2 . 2e - 16 ) . LFC > | 0 . 2 | with padj < 0 . 05 are considered significant .

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E) Differential expression of ARM vs HM.1 microglia for APPtg and TAUtg mice separately. Genes with a positive log(fold change) (LFC) are higher expressed in ARMs compared to HM.1 microglia. As can be seen which genes are more highly expressed in ARMs vs HM.1 cells are highly similar among APPtg and TAUtg mice (Spearman correlation r=0.91, p=2.2e-16). LFC > |0.2| with padj<0.05 are considered significant.


Fig 1 : Urinary triclosan levels are elevated in TC mothers following 6 months of exposure . Summaries are provided as the interquartile range with median of triclosan molecules in a picogram / microliter ( pg / L ) . Urinary triclosan measurements are available for 38 mothers ( 17 TC , 21 nTC ) and 33 infants ( 15 TC , 18 nTC ) . The p - value for the mothers in Figure 1 ( represented by * * * ) is p = 5 . 07e - 5 ( Mann - Whitney U test ) .

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

Fig 1: Urinary triclosan levels are elevated in TC mothers following 6 months of exposure. Summaries are provided as the interquartile range with median of triclosan molecules in a picogram/microliter (pg/L). Urinary triclosan measurements are available for 38 mothers (17 TC, 21 nTC) and 33 infants (15 TC, 18 nTC). The p-value for the mothers in Figure 1 (represented by ***) is p=5.07e-5 (Mann-Whitney U test).


( A ) PCoA of Bray - Curtis dissimilarity for all ( n = 221 ) samples shows that gut communities cluster by mothers and infants .

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(A) PCoA of Bray-Curtis dissimilarity for all (n=221) samples shows that gut communities cluster by mothers and infants.


( B ) PCoA separated by time and treatment .

(B) PCoA separated by time and treatment.


Fig 3 : TC randomization does not decrease gut microbial diversity in infants or mothers . Shannon diversity measures are plotted as the interquartile range with median for each TC exposure class and time point grouping for infants and mothers .

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Fig 3: TC randomization does not decrease gut microbial diversity in infants or mothers. Shannon diversity measures are plotted as the interquartile range with median for each TC exposure class and time point grouping for infants and mothers.


( A ) Differentially abundant taxa between nTC and TC households . Values left of the grey line indicate an enrichment in nTC household and values to the right indicate an enrichment in TC households . ( A ) Analyses are separated by mothers and infants for all samples across the 3 time points ( FDR adjusted p - value < 0 . 01 ) .

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(A) Differentially abundant taxa between nTC and TC households. Values left of the grey line indicate an enrichment in nTC household and values to the right indicate an enrichment in TC households. (A) Analyses are separated by mothers and infants for all samples across the 3 time points (FDR adjusted p-value < 0.01).


Differentially abundant taxa are displayed for ( B ) infantsat per visit ( FDR adjusted p - value < 0 . 05 ) .

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Differentially abundant taxa are displayed for (B) infantsat per visit (FDR adjusted p-value < 0.05).


Differentially abundant taxa are displayed for ( C ) mothers at per visit ( FDR adjusted p - value < 0 . 05 ) .

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Differentially abundant taxa are displayed for(C) mothers at per visit (FDR adjusted p-value < 0.05).


A , Quantification of Nox4 mRNA levels ( A ) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum - replete cells ( 15 % serum ) . n = 4 cell preparations / group .

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A, Quantification of Nox4 mRNA levels (A) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum-replete cells (15% serum). n=4 cell preparations/group.


B Quantification of Nox4 protein levels ( B ) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum - replete cells ( 15 % serum ) . n = 4 cell preparations / group . A representative immunoblot for Nox4 and tubulin as a loading control is shown in panel B .

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B Quantification of Nox4 protein levels (B) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum-replete cells (15% serum). n=4 cell preparations/group. A representative immunoblot for Nox4 and tubulin as a loading control is shown in panel B.


C Cell death after 48 h serum starvation in cardiomyocytes in which Nox4 was depleted by an adenoviral shRNA ( Ad . shNox4 ) as compared to cells infected with a control vector ( Ad . Ctl ) . n = 6 / group . The representative immunoblot shows reduction in Nox4 protein levels after shRNA - mediated knockdown .

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C Cell death after 48 h serum starvation in cardiomyocytes in which Nox4 was depleted by an adenoviral shRNA (Ad.shNox4) as compared to cells infected with a control vector (Ad.Ctl). n=6/group. The representative immunoblot shows reduction in Nox4 protein levels after shRNA-mediated knockdown.


D Lactate dehydrogenase ( LDH ) activity in cell supernatants . Staurosporine ( Stauro , 1 μmol / L ) was used to induce cell death by apoptosis . n = 4 cell preparations / group .

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D Lactate dehydrogenase (LDH) activity in cell supernatants. Staurosporine (Stauro, 1 μmol/L) was used to induce cell death by apoptosis. n=4 cell preparations/group.


E Levels of high mobility group box1 protein ( HMGB1 ) in cell supernatants . n = 3 / group . A representative immunoblot is shown at the top .

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E Levels of high mobility group box1 protein (HMGB1) in cell supernatants. n=3/group. A representative immunoblot is shown at the top.


F Protein levels of cytochrome c in the cytoplasmic fractions of cardiomyocytes . n = 4 / group . A representative immunoblot is shown at the top . The cellular membrane fraction ( Mem ) containing mitochondria was used as a positive control .

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F Protein levels of cytochrome c in the cytoplasmic fractions of cardiomyocytes. n=4/group. A representative immunoblot is shown at the top. The cellular membrane fraction (Mem) containing mitochondria was used as a positive control.


G Percentage of Nox4 - depleted and control cells with depolarized mitochondria , quantified by flow cytometry . n = 3 cell preparations / group ( 100 , 000 cells per experiment ) .

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G Percentage of Nox4-depleted and control cells with depolarized mitochondria, quantified by flow cytometry. n=3 cell preparations/group (100,000 cells per experiment).


H Effect of cyclosporin A ( CsA , 1 μmol / L ) on the percentage of cells with depolarized mitochondria . n = 6 - 8 / group .

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H Effect of cyclosporin A (CsA, 1 μmol/L) on the percentage of cells with depolarized mitochondria. n=6-8/group.


I Effect of cyclosporin A ( CsA , 1 μmol / L ) on cell death in serum - starved cardiomyocytes . n = 4 / group .

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I Effect of cyclosporin A (CsA, 1 μmol/L) on cell death in serum-starved cardiomyocytes. n=4/group.


J Cell death in serum starved Nox4 knockout MEFs ( Nox4KO ) and wild - type MEFs ( WT ) . Cat = PEG - catalase ( 500 U / mL ) . Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant , Nox4P437H ( Mut ) . n = 6 - 12 / group .

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J Cell death in serum starved Nox4 knockout MEFs (Nox4KO) and wild-type MEFs (WT). Cat = PEG-catalase (500 U/mL). Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant, Nox4P437H (Mut). n=6-12/group.


K HMGB1 levels in the supernatants of serum - starved MEFs in similar experiments to those in ( J ) . n = 4 / group . A representative immunoblot is shown to the top .

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K HMGB1 levels in the supernatants of serum-starved MEFs in similar experiments to those in (J). n=4/group. A representative immunoblot is shown to the top.


L Percentage of MEFs with depolarized mitochondria . n = 6 / group .

L Percentage of MEFs with depolarized mitochondria. n=6/group.


A Basal mitochondrial calcium levels assessed in serum - starved rat cardiomyocytes using a mitochondrial - targeted cameleon CFP / YFP FRET probe . Nox4 was depleted with silencing RNAs ( siRNAs ) or cells were transfected with a control scrambled siRNA ( siScr ) . Representative photomicrographs are shown at the top . The spectrum color scale represents the ratio of emitted fluorescence ( YFP / CFP ) . The mean changes in Nox4 - depleted cells as compared to scrambled control ( dotted line ) at the bottom . The representative immunoblot shows depletion of Nox4 protein levels . Scale bars : 10 µm . n = 3 / group ( with > 50 cells per individual experiment ) .

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A Basal mitochondrial calcium levels assessed in serum-starved rat cardiomyocytes using a mitochondrial-targeted cameleon CFP/YFP FRET probe. Nox4 was depleted with silencing RNAs (siRNAs) or cells were transfected with a control scrambled siRNA (siScr). Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). The mean changes in Nox4-depleted cells as compared to scrambled control (dotted line) at the bottom. The representative immunoblot shows depletion of Nox4 protein levels. Scale bars: 10 µm. n=3/group (with >50 cells per individual experiment).


B Basal mitochondrial calcium levels in WT and Nox4KO MEFs after serum starvation . Representative photomicrographs are shown at the top . The spectrum color scale represents the ratio of emitted fluorescence ( YFP / CFP ) . Mean data at the bottom . Scale bars : 10 µm . n = 3 / group ( with > 100 cells per individual experiment ) .

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B Basal mitochondrial calcium levels in WT and Nox4KO MEFs after serum starvation. Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). Mean data at the bottom. Scale bars: 10 µm. n=3/group (with >100 cells per individual experiment).


Changes in mitochondrial calcium levels in serum - starved WT and Nox4KO MEFs after the addition of histamine ( 100 μmol / L , C ) n = 3 / group ( with > 30 cells per individual experiment ) .

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Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, C) n=3/group (with >30 cells per individual experiment).


D Changes in mitochondrial calcium levels in serum - starved WT and Nox4KO MEFs after the addition of ATP ( 100 μmol / L , D ) . n = 3 / group ( with > 30 cells per individual experiment ) .

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D Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, D). n=3/group (with >30 cells per individual experiment).


E , Changes in ER calcium levels measured with an ER - targeted cameleon probe in serum - starved WT and Nox4KO MEFs after the addition of histamine ( 100 μmol / L , E ) n = 3 / group ( with > 30 cells per individual experiment ) .

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E, Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, E) n=3/group (with >30 cells per individual experiment).


F Changes in ER calcium levels measured with an ER - targeted cameleon probe in serum - starved WT and Nox4KO MEFs after the addition of ATP ( 100 μmol / L , F ) . n = 3 / group ( with > 30 cells per individual experiment ) .

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F Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, F). n=3/group (with >30 cells per individual experiment).


G Peak increase in mitochondrial calcium levels response in response to histamine ( 100 μmol / L ) in WT MEFs with or without treatment with PEG - catalase ( Cat ) , and in Nox4KO MEFs with or without transfection with active Nox4 or a catalytically inactive Nox4P437H mutant ( Mut ) . n = 3 / group ( with > 30 cells per individual experiment ) .

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G Peak increase in mitochondrial calcium levels response in response to histamine (100 μmol/L) in WT MEFs with or without treatment with PEG-catalase (Cat), and in Nox4KO MEFs with or without transfection with active Nox4 or a catalytically inactive Nox4P437H mutant (Mut). n=3/group (with >30 cells per individual experiment).


H Representative time - course of histamine - induced changes in mitochondrial calcium for the experiments shown in ( I ) .

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H Representative time-course of histamine-induced changes in mitochondrial calcium for the experiments shown in (I).


A Immunoblotting of subcellular fractions from serum - starved rat cardiomyoblasts ( H9c2 cells ) . Input : homogenate before fractionation ; Cyto : cytosol ; ER : endoplasmic reticulum ; Crude M : crude mitochondrial fraction , also containing MAM ; Pure Mito : mitochondria ; MAM : mitochondria associated - ER membranes . Cytochrome c ( Cyt c ) was used as a mitochondrial marker , VDAC as an outer mitochondrial membrane marker , calnexin as an ER marker , Sigma1R and FACL4 as MAM markers , and tubulin as a cytosolic marker . B Immunoblotting of subcellular fractions from rat heart . C Immunoblotting of subcellular fractions from rat kidney .

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A Immunoblotting of subcellular fractions from serum-starved rat cardiomyoblasts (H9c2 cells). Input: homogenate before fractionation; Cyto: cytosol; ER: endoplasmic reticulum; Crude M: crude mitochondrial fraction, also containing MAM; Pure Mito: mitochondria; MAM: mitochondria associated-ER membranes. Cytochrome c (Cyt c) was used as a mitochondrial marker, VDAC as an outer mitochondrial membrane marker, calnexin as an ER marker, Sigma1R and FACL4 as MAM markers, and tubulin as a cytosolic marker. B Immunoblotting of subcellular fractions from rat heart. C Immunoblotting of subcellular fractions from rat kidney.


D Electron micrographs showing subcellular localization of Nox4 in wild - type ( WT ) and Nox4KO mouse hearts , and human induced pluripotent stem cell - derived cardiomyocytes ( hiPSC - CM ) . hiPSC - CM were depleted of Nox4 using a lentiviral shRNA ( Nox4KD ) or infected with a control lentivirus . FACL4 was used as a MAM marker . Arrow heads show peri - mitochondrial localization of Nox4 and FACL4 , in the proximity of ER cisternae and ribosomes . M = mitochondria , ER = endoplasmic reticulum , T = T - tubule . Scale bars : 100 nm .

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D Electron micrographs showing subcellular localization of Nox4 in wild-type (WT) and Nox4KO mouse hearts, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). hiPSC-CM were depleted of Nox4 using a lentiviral shRNA (Nox4KD) or infected with a control lentivirus. FACL4 was used as a MAM marker. Arrow heads show peri-mitochondrial localization of Nox4 and FACL4, in the proximity of ER cisternae and ribosomes. M=mitochondria, ER=endoplasmic reticulum, T=T-tubule. Scale bars: 100 nm.


A Simplified photomicrographs of proximity ligation studies in WT and Nox4KO MEFs , showing cell borders , nuclei ( blue ) and yellow dots corresponding to co - localization of proteins . Quantification of the number of dots / cell in each condition is shown to the right . Proximity was tested for the following protein couples : FACL4 / Nox4 , InsP3R / Nox4 , and FACL4 / InsP3R . Scale bars : 10 µm . n = 3 / group ( with > 30 cells / individual experiment ) .

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A Simplified photomicrographs of proximity ligation studies in WT and Nox4KO MEFs, showing cell borders, nuclei (blue) and yellow dots corresponding to co-localization of proteins. Quantification of the number of dots/cell in each condition is shown to the right. Proximity was tested for the following protein couples: FACL4/Nox4, InsP3R/Nox4, and FACL4/InsP3R. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).


B Simplified photomicrographs of proximity ligation staining in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control ( siScr ) , with quantification as in ( A ) . Proximity was tested for the FACL4 / Nox4 protein couple . Scale bars : 10 µm . n = 3 / group ( with > 30 cells / individual experiment ) .

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B Simplified photomicrographs of proximity ligation staining in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control (siScr), with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).


C Simplified photomicrographs of proximity ligation staining in hiPSC - CM ( human cardiomyocytes ) transduced with Nox4 shRNA or scrambled control , with quantification as in ( A ) . Proximity was tested for the FACL4 / Nox4 protein couple . Scale bars : 10 µm . n = 3 / group ( with > 30 cells / individual experiment ) .

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C Simplified photomicrographs of proximity ligation staining in hiPSC-CM (human cardiomyocytes) transduced with Nox4 shRNA or scrambled control, with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).


A Immunoblotting of subcellular fractions from serum - starved cardiomyoblasts for InsP3R , Akt and PP2a , as in Fig 3A .

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A Immunoblotting of subcellular fractions from serum-starved cardiomyoblasts for InsP3R, Akt and PP2a, as in Fig 3A.


B Fractionation of crude mitochondrial fractions ( containing mitochondria and MAM ) from WT MEFs on a sucrose gradient ( 5 - 60 % ) and immunoblotting for relevant proteins . Fraction size : 200 µL . Representative of 3 independent experiments .

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B Fractionation of crude mitochondrial fractions (containing mitochondria and MAM) from WT MEFs on a sucrose gradient (5-60%) and immunoblotting for relevant proteins. Fraction size: 200 µL. Representative of 3 independent experiments.


C Immunoblotting for phosphorylated and total Akt or InsP3R protein in crude mitochondrial fractions from serum - starved WT and Nox4KO MEFs . FACL4 was used as a loading control for the MAM . InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt - substrate motif RXRXX ( pS / T ) ( p - InsP3R ) . Representative immunoblots are shown at the top and mean data at the bottom . Cat = PEG - catalase . Nox4KO MEFs were transfected either with active Nox4 or a Nox4P437H mutant ( Mut ) . n = 3 / group .

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C Immunoblotting for phosphorylated and total Akt or InsP3R protein in crude mitochondrial fractions from serum-starved WT and Nox4KO MEFs. FACL4 was used as a loading control for the MAM. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Representative immunoblots are shown at the top and mean data at the bottom. Cat = PEG-catalase. Nox4KO MEFs were transfected either with active Nox4 or a Nox4P437H mutant (Mut). n=3/group.


D ROS levels indexed in MEFs using H2O2 specific HyPer probes targeted to the MAM ( FACL4 HyPer ) or the cytosol ( Cyto HyPer ) . Imaging was performed after serum starvation for 12h . Representative photomicrographs of the fluorescence ratio are shown to the right and mean data for changes in fluorescence ratio presented to the left . The signal obtained using corresponding ROS - insensitive SypHer probes was used to correct for any pH - induced changes in fluorescence ; the change in fluorescence ratio between HyPer and corresponding SypHer probe ( ΔR ) for each condition is reported . n = 5 independent cell preparations / group , with at least 20 cells imaged / preparation . Scale bars : 10 µm .

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D ROS levels indexed in MEFs using H2O2 specific HyPer probes targeted to the MAM (FACL4 HyPer) or the cytosol (Cyto HyPer). Imaging was performed after serum starvation for 12h. Representative photomicrographs of the fluorescence ratio are shown to the right and mean data for changes in fluorescence ratio presented to the left. The signal obtained using corresponding ROS-insensitive SypHer probes was used to correct for any pH-induced changes in fluorescence; the change in fluorescence ratio between HyPer and corresponding SypHer probe (ΔR) for each condition is reported. n=5 independent cell preparations/group, with at least 20 cells imaged/preparation. Scale bars: 10 µm.


B Basal mitochondrial calcium levels in serum - starved WT and Nox4KO MEFs in the presence or absence of Akti ( 1 µmol / L ) normalized versus WT MEF control . n = 6 / group with > 20 cells imaged per experiment .

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B Basal mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs in the presence or absence of Akti (1 µmol/L) normalized versus WT MEF control. n=6/group with >20 cells imaged per experiment.


C Effect of Akti ( 1 µmol / L ) or XeC ( 1 µmol / L ) on histamine - induced changes in mitochondrial calcium levels in MEFs . The time course of changes in calcium levels is shown on the left and the mean data for peak mitochondrial calcium levels on the right . n = 3 / group with > 40 cells imaged per experiment .

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C Effect of Akti (1 µmol/L) or XeC (1 µmol/L) on histamine-induced changes in mitochondrial calcium levels in MEFs. The time course of changes in calcium levels is shown on the left and the mean data for peak mitochondrial calcium levels on the right. n=3/group with >40 cells imaged per experiment.


D Quantification of cell death in serum - starved WT and Nox4KO MEFs in the absence or presence of Akti or XeC . n = 6 / group .

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D Quantification of cell death in serum-starved WT and Nox4KO MEFs in the absence or presence of Akti or XeC. n=6/group.


E Cardiac troponin I ( cTnI ) levels as a marker of cardiomyocyte necrosis in the perfusate of isolated WT and Nox4KO hearts subjected to ischemia - reperfusion ( I / R ) . XeC was added at a final concentration of 2 µmol / L prior to ischemia . n = 6 - 7 / group .

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E Cardiac troponin I (cTnI) levels as a marker of cardiomyocyte necrosis in the perfusate of isolated WT and Nox4KO hearts subjected to ischemia-reperfusion (I/R). XeC was added at a final concentration of 2 µmol/L prior to ischemia. n=6-7/group.


F Immunoblotting for the phosphorylation levels of Akt and InsP3R in crude mitochondrial fractions from WT and Nox4KO hearts after I / R . InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt - substrate motif RXRXX ( pS / T ) ( p - InsP3R ) . Mean data shown to the right . n = 3 / group .

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F Immunoblotting for the phosphorylation levels of Akt and InsP3R in crude mitochondrial fractions from WT and Nox4KO hearts after I/R. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Mean data shown to the right. n=3/group.


G Cardiac left ventricular contractile function in isolated Langendorff - perfused WT and Nox4KO hearts at baseline ( Basal ) and then after I / R . Hearts were treated with XeC ( 1 μmol / L ) or vehicle control for 20 min prior to ischemia . RPP , heart rate x left ventricular pressure product ; DEVP , left ventricular developed pressure . n = 6 - 7 / group .

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G Cardiac left ventricular contractile function in isolated Langendorff-perfused WT and Nox4KO hearts at baseline (Basal) and then after I/R. Hearts were treated with XeC (1 μmol/L) or vehicle control for 20 min prior to ischemia. RPP, heart rate x left ventricular pressure product; DEVP, left ventricular developed pressure. n=6-7/group.


( C ) Immunoblot of WT , KO and A537T prostates probed with specific antibodies to detect ELAC2 and normalise its levels to Hsp60 that was used as a loading control ( n = 3 ) . * * * p = 1 . 09909E - 07 for KO and * * * p = 3 . 55091E - 05 for A537T , Student ' s two - way t - test , values are means ± SD of biological replicates .

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(C) Immunoblot of WT, KO and A537T prostates probed with specific antibodies to detect ELAC2 and normalise its levels to Hsp60 that was used as a loading control (n=3). ***p= 1.09909E-07 for KO and ***p= 3.55091E-05 for A537T, Student's two-way t-test, values are means ± SD of biological replicates.


( E ) Survival data for ELAC2 WT ( n = 30 ) , KO ( n = 30 ) and A537T ( n = 30 ) mice .

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(E) Survival data for ELAC2 WT (n=30), KO (n=30) and A537T (n=30) mice.


( F ) ELAC2 WT - TRAMP , KO - TRAMP and A537T - TRAMP prostates from 10 - and 30 - week - old mice . Bladder and seminal vesicles were removed in 10 - week - old mice . Images show the typical differences between WT , KO and A537T prostates of at least 10 different mice per genotype .

(F) ELAC2 WT-TRAMP, KO-TRAMP and A537T-TRAMP prostates from 10- and 30-week-old mice. Bladder and seminal vesicles were removed in 10-week-old mice. Images show the typical differences between WT, KO and A537T prostates of at least 10 different mice per genotype.


( G ) Survival data for ELAC2 WT - TRAMP ( n = 30 ) , KO - TRAMP ( n = 30 ) and A537T - TRAMP ( n = 30 ) mice ( biological replicates ) . Gehan - Breslow - Wilcoxon test showed significant difference ( * p = 0 . 045 ) between the WT - TRAMP and KO - TRAMP or A537T - TRAMP survival curves .

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(G) Survival data for ELAC2 WT-TRAMP (n=30), KO-TRAMP (n=30) and A537T-TRAMP (n=30) mice (biological replicates). Gehan-Breslow-Wilcoxon test showed significant difference (*p=0.045) between the WT-TRAMP and KO-TRAMP or A537T-TRAMP survival curves.


( A ) Representative images of anterior , dorsal , ventral and lateral lobe sections from 30 - week - old ELAC2 WT ( n = 7 ) , KO ( n = 7 ) and A537T ( n = 5 ) mice . 5 µm sections of each lobe were cut and stained with haematoxylin and eosin .

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(A) Representative images of anterior, dorsal, ventral and lateral lobe sections from 30-week-old ELAC2 WT (n=7), KO (n=7) and A537T (n=5) mice. 5 µm sections of each lobe were cut and stained with haematoxylin and eosin.


( B ) Histological changes were scored according additional categories added to the scoring system ( n = 6 - 8 biological replicates ) . Values are means ± SD of biological replicates , * * * * p = 0 . 001 compared with WT by two - tailed Student ' s t - test .

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(B) Histological changes were scored according additional categories added to the scoring system (n=6-8 biological replicates). Values are means ± SD of biological replicates, ****p =0.001 compared with WT by two-tailed Student's t-test.


( A ) Representative images of anterior , dorsal , ventral and lateral lobe sections from 30 - week - old WT - TRAMP ( n = 5 ) , KO - TRAMP ( n = 5 ) and A537T - TRAMP mice ( n = 5 ) . 5 µm sections of each lobe were cut and stained with haematoxylin and eosin . ( B ) Histological changes scored according to Suttie et al . with additional categories ( n = 6 - 7 biological replicates ) , values are means ± SD of biological replicates . Anterior lobe * p = 0 . 013 , Dorsal lobe * * p = 0 . 003 , * p = 0 . 025 , Ventral lobe * p = 0 . 031 and Lateral lobe * * p = 0 . 0038 , * p = 0 . 032 , compared with WT - TRAMP by two - tailed Student ' s t - testing .

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(A) Representative images of anterior, dorsal, ventral and lateral lobe sections from 30-week-old WT-TRAMP (n=5), KO-TRAMP (n=5) and A537T-TRAMP mice (n=5). 5 µm sections of each lobe were cut and stained with haematoxylin and eosin. (B) Histological changes scored according to Suttie et al. with additional categories (n=6-7 biological replicates), values are means ± SD of biological replicates. Anterior lobe * p =0.013, Dorsal lobe ** p = 0.003, * p = 0.025, Ventral lobe * p =0.031 and Lateral lobe ** p = 0.0038, * p = 0.032, compared with WT-TRAMP by two-tailed Student's t-testing.


( C ) Representative histological images of complete prostate sections from 30 - week - old WT - TRAMP , KO - TRAMP and A537T - TRAMP showing the prominent tumours ( black arrows ) in the mutant strains compared to the WT - TRAMP mice . Images show the typical differences between WT - TRAMP , KO - TRAMP and A537T - TRAMP prostates of at least 10 different mice per genotype .

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(C) Representative histological images of complete prostate sections from 30-week-old WT-TRAMP, KO-TRAMP and A537T-TRAMP showing the prominent tumours (black arrows) in the mutant strains compared to the WT-TRAMP mice. Images show the typical differences between WT-TRAMP, KO-TRAMP and A537T-TRAMP prostates of at least 10 different mice per genotype.


Specific protein changes that were in common between the KO and A537T lines at 30 weeks are shown in volcano plots ( B ) increased proteins are shown in red and reduced proteins are shown in blue .

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Specific protein changes that were in common between the KO and A537T lines at 30 weeks are shown in volcano plots (B) increased proteins are shown in red and reduced proteins are shown in blue.


( E - F ) Dot plots showing expression change of mRNA / miRNA pairs in the 30 - week mutant lines compared to their respective controls . mRNA log2 fold change compared to controls is on the y - axis while the colour and x - axis denote the log2 fold change observed in the miRNA compared to controls .

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(E-F) Dot plots showing expression change of mRNA/miRNA pairs in the 30-week mutant lines compared to their respective controls. mRNA log2 fold change compared to controls is on the y-axis while the colour and x-axis denote the log2 fold change observed in the miRNA compared to controls.


WT ELAC2 - EGFP or A537T ELAC2 - EGFP were expressed in LNCaP cells and the associated proteins that immunoprecipitated with the EGFP antibody were detected by mass spectrometry . EGFP - only protein was used as a control . Volcano plots are showing the enriched proteins with proteins and ( C ) shows the proteins that have reduced or increased association with A537T ELAC2 . Volcano plots comparing Log2 fold change against the - log10 p - value and significant ( p < 0 . 05 ) enriched proteins are shown in red and proteins with reduced association are shown in blue for three biologically independent experiments .

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WT ELAC2-EGFP or A537T ELAC2-EGFP were expressed in LNCaP cells and the associated proteins that immunoprecipitated with the EGFP antibody were detected by mass spectrometry. EGFP-only protein was used as a control. Volcano plots are showing the enriched proteins with proteins and (C) shows the proteins that have reduced or increased association with A537T ELAC2. Volcano plots comparing Log2 fold change against the -log10 p-value and significant (p < 0.05) enriched proteins are shown in red and proteins with reduced association are shown in blue for three biologically independent experiments.


( B ) Immunoblots from cytosolic ( cyt ) and nuclear ( nuc ) fractionations of RPE - 1 cells in early - G1 showing the presence of endogenous ACTN4 in nuclear fractions . Proteins were detected with antibodies against ( α ) ACTN4 , α - Tubulin or Histone 3 as

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots from cytosolic (cyt) and nuclear (nuc) fractionations of RPE-1 cells in early-G1 showing the presence of endogenous ACTN4 in nuclear fractions. Proteins were detected with antibodies against (α) ACTN4, α-Tubulin or Histone 3 as indicated


( C ) Upper and middle panels : immunoblots showing precipitation of ACTN4 and β - actin from RPE - 1 nuclear lysates in early - G1 in the presence or absence of biotin - phalloidin , as indicated . Lower panel : Immunoblot of cytosolic ( cyt ) and nuclear ( nuc ) fractions . Proteins were detected with antibodies against ( α ) ACTN4 , β - Actin , α - Tubulin or Histone 3 as indicated .

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(C) Upper and middle panels: immunoblots showing precipitation of ACTN4 and β-actin from RPE-1 nuclear lysates in early-G1 in the presence or absence of biotin-phalloidin, as indicated. Lower panel: Immunoblot of cytosolic (cyt) and nuclear (nuc) fractions. Proteins were detected with antibodies against (α) ACTN4, β-Actin, α-Tubulin or Histone 3 as indicated.


( D ) Immmunofluorescence images of endogenous ACTN4 in non - dividing and post - mitotic NIH3T3 and RPE - 1 cells in as indicated . Shown are maximum intensity projections . Cells were fixed in 4 % PFA and stained for ACTN4 and nuclei ( DAPI ) as indicated . Scale bars 10 μm .

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(D) Immmunofluorescence images of endogenous ACTN4 in non-dividing and post-mitotic NIH3T3 and RPE-1 cells in as indicated. Shown are maximum intensity projections. Cells were fixed in 4% PFA and stained for ACTN4 and nuclei (DAPI) as indicated. Scale bars 10 μm.


( A ) Cells in early G1 stably expressing nAC - mCherry and doxycycline - inducible ACTN4 - SNAP were analyzed by time - lapse microscopy . ACTN4 - SNAP was labeled by SNAP - Cell 647SiR dye ( green ) . Different arrows mark dynamic ACTN4 clusters . The white square 1 is shown as a timeseries on the right . The white square 2 is shown as a magnification below . The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B . Scale bar overview ( nAC ) represents 10 µm . Scale bar time series and magnification 5 µm .

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(A) Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). Different arrows mark dynamic ACTN4 clusters. The white square 1 is shown as a timeseries on the right. The white square 2 is shown as a magnification below. The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. Scale bar overview (nAC) represents 10 µm. Scale bar time series and magnification 5 µm.


Cells in early G1 stably expressing nAC - mCherry and doxycycline - inducible ACTN4 - SNAP were analyzed by time - lapse microscopy . ACTN4 - SNAP was labeled by SNAP - Cell 647SiR dye ( green ) . The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B . B ) Linescan of an actin filament with associated ACTN4 from square 3 in A .

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Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. B) Linescan of an actin filament with associated ACTN4 from square 3 in A.


( C ) Automated tracking ( autoregressive motion ) of nuclear actin and ACTN4 in nuclei by Imaris software . Cells were treated like in A . Tracks are visualized by red ( actin ) or green ( ACTN4 ) lines in a representative image ( see corresponding Movie EV2 ) . Scale bar is 2 µm . The track length , track displacement ( distance between starting - and end point ) , average speed and maximum speed were quantified from 3 independent experiments . The black line shows the median and grey dashed lines show the quartiles . Quantifications and images show similar motion characteristics for actin and ACTN4 .

(C) Automated tracking (autoregressive motion) of nuclear actin and ACTN4 in nuclei by Imaris software. Cells were treated like in A. Tracks are visualized by red (actin) or green (ACTN4) lines in a representative image (see corresponding Movie EV2). Scale bar is 2 µm. The track length, track displacement (distance between starting- and end point), average speed and maximum speed were quantified from 3 independent experiments. The black line shows the median and grey dashed lines show the quartiles. Quantifications and images show similar motion characteristics for actin and ACTN4.


( A ) NIH3T3 cells expressing nAC - mCherry were imaged for 120 min . Image frames represent maximum projections of 30 Z planes . Lower panel is a magnification of the indicated area ( orange box ) ; scale bars 10 µm .

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(A) NIH3T3 cells expressing nAC-mCherry were imaged for 120 min. Image frames represent maximum projections of 30 Z planes. Lower panel is a magnification of the indicated area (orange box); scale bars 10 µm.


( B ) NIH3T3 cells stably expressing H2B - mCherry were transfected with siCtrl or siACTN1 + siACTN4 . After mitotic exit z - stacks ( 30 planes ) were acquired every 5 min . Nuclei were 3D - reconstructed and volume was measured by Imaris . Data are mean ± SEM from 3 biological replicates with 22 - 27 ( siCtrl ) and 32 - 42 ( siACTN1 + 4 ) analyzed nuclei ; * * * p < 0 . 001 by Wilcoxon test and * * * * p < 0 . 0001 by t - test at 90 min .

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(B) NIH3T3 cells stably expressing H2B-mCherry were transfected with siCtrl or siACTN1 + siACTN4. After mitotic exit z-stacks (30 planes) were acquired every 5 min. Nuclei were 3D-reconstructed and volume was measured by Imaris. Data are mean ± SEM from 3 biological replicates with 22-27 (siCtrl) and 32-42 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by t-test at 90 min.


( C ) RPE - 1 cells were analyzed as in B . Data are mean ± SEM from 5 biological replicates with 32 - 37 ( siCtrl ) and 31 - 39 ( siACTN1 + 4 ) analyzed nuclei ; * * * p < 0 . 001 by Wilcoxon test and * * * * p < 0 . 0001 by Mann Whitney test at 90 min .

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(C) RPE-1 cells were analyzed as in B. Data are mean ± SEM from 5 biological replicates with 32-37 (siCtrl) and 31-39 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by Mann Whitney test at 90 min.


( E ) NIH3T3 cells stably expressing H2B - mCherry and siRNA - resistant ACTN4 - NES were transfected with the indicated siRNAs and analyzed as described in B . Data are mean ± SEM from 5 biological replicates with 63 - 71 ( siCtrl ) , 37 - 41 ( siACTN1 + 4 ) , 52 - 58 ( siACTN1 ) and 52 - 55 ( siACTN4 ) analyzed nuclei ; * * * * p ( siACTN4 ) < 0 . 0001 ; * * * * p ( siACTN1 + 4 ) < 0 . 0001 by One Way ANOVA at 90 min .

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(E) NIH3T3 cells stably expressing H2B-mCherry and siRNA-resistant ACTN4-NES were transfected with the indicated siRNAs and analyzed as described in B. Data are mean ± SEM from 5 biological replicates with 63-71 (siCtrl), 37-41 (siACTN1+4), 52-58 (siACTN1) and 52-55 (siACTN4) analyzed nuclei; ****p(siACTN4) < 0.0001; ****p(siACTN1+4) < 0.0001 by One Way ANOVA at 90 min.


( F ) NIH3T3 cells stably expressing H2B - mCherry and the indicated ACTN4 mutants were transfected with the indicated siRNAs and analyzed as in B . Data are mean ± SEM from 3 biological replicates with 63 - 69 ( ACTN4 wt + siACTN4 ) , 63 - 71 ( ACTN4 - NES + siCtrl ) and 54 - 58 ( ACTN4 - NES + siACTN4 ) analyzed nuclei ; * * * * p ( ACTN4 - NES + siACTN4 ) < 0 . 0001 by One Way ANOVA at 90 min .

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(F) NIH3T3 cells stably expressing H2B-mCherry and the indicated ACTN4 mutants were transfected with the indicated siRNAs and analyzed as in B. Data are mean ± SEM from 3 biological replicates with 63-69 (ACTN4 wt + siACTN4), 63-71 (ACTN4-NES + siCtrl) and 54-58 (ACTN4-NES + siACTN4) analyzed nuclei; ****p(ACTN4-NES + siACTN4) < 0.0001 by One Way ANOVA at 90 min.


( G ) NIH3T3 cells expressing siRNA - resistant ACTN4 - NES were transfected with siCtrl or siACTN4 ; chromatin density was calculated from H2B - mCherry fluorescence intensities by total nuclear volumes ( intensity / μm3 ) and normalized to t = 0 ( mitotic exit ) ; Data are mean ± SEM from 5 biological replicates with 44 - 48 ( siCtrl ) and 44 - 47 ( siACTN4 ) analyzed nuclei ; * * * p < 0 . 001 by Wilcoxon test and * p < 0 . 05 by t - test at 90 min .

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(G) NIH3T3 cells expressing siRNA-resistant ACTN4-NES were transfected with siCtrl or siACTN4; chromatin density was calculated from H2B-mCherry fluorescence intensities by total nuclear volumes (intensity/μm3) and normalized to t=0 (mitotic exit); Data are mean ± SEM from 5 biological replicates with 44-48 (siCtrl) and 44-47 (siACTN4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and *p < 0.05 by t-test at 90 min.


( B ) Upper panel shows expression of ACTN4 constructs ( green , confocal image ) and phalloidin - AF647 staining in the same cells ( orange , dSTORM image ) scale bar 10 µm ; lower panel represents zoom of the indicated areas ( white boxes ) ; scale bars 5 µm .

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(B) Upper panel shows expression of ACTN4 constructs (green, confocal image) and phalloidin-AF647 staining in the same cells (orange, dSTORM image) scale bar 10 µm; lower panel represents zoom of the indicated areas (white boxes); scale bars 5 µm.


( C ) Phalloidin - AF647 localizations per post - mitotic nuclei ; data shown as mean ± 2 . 5 - 97 . 5 percentile : 52 ACTN4 - wt nuclei , 55 ACTN4ΔCH1 - NLS nuclei and 40 nuclei of non - dividing cells ( ACTN4 - wt ) from 5 biological replicates . * * * p ( ACTN4ΔCH1 - NLS post - mitotic ) < 0 . 001 , * * * * p ( ACTN4 - wt non - dividing ) < 0 . 0001 , * p ( ACTN4ΔCH1 - NLS post - mitotic vs . ACTN4 - wt non - dividing ) < 0 . 05 by One Way ANOVA .

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(C) Phalloidin-AF647 localizations per post-mitotic nuclei; data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei and 40 nuclei of non-dividing cells (ACTN4-wt) from 5 biological replicates. ***p(ACTN4ΔCH1-NLS post-mitotic) < 0.001, ****p(ACTN4-wt non-dividing) < 0.0001, *p (ACTN4ΔCH1-NLS post-mitotic vs. ACTN4-wt non-dividing) < 0.05 by One Way ANOVA.


( D ) Number of filaments in the post mitotic nuclei ; as in ( C ) ; data shown as mean ± 2 . 5 - 97 . 5 percentile : 52 ACTN4 - wt nuclei , 55 ACTN4ΔCH1 - NLS nuclei from 5 biological replicates , * * * p < 0 . 001 by t - test .

(D) Number of filaments in the post mitotic nuclei; as in (C); data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei from 5 biological replicates, ***p < 0.001 by t-test.


( E ) Actin filament widths for ACTN4 - wt and ACTN4ΔCH1 - NLS in early G1 nuclei ; ) ; data shown as mean ± 2 . 5 - 97 . 5 percentile with 96 filaments ( ACTN4 - wt ) and 36 filaments ( ACTN4ΔCH1 - NLS ) from 5 biological replicates , * * p < 0 . 01 by t - test .

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(E) Actin filament widths for ACTN4-wt and ACTN4ΔCH1-NLS in early G1 nuclei; ); data shown as mean ± 2.5-97.5 percentile with 96 filaments (ACTN4-wt) and 36 filaments (ACTN4ΔCH1-NLS) from 5 biological replicates, **p < 0.01 by t-test.


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Cell viability was assessed by CellTiter - Glo ( A ) after 16 hours . Data are means ± SEM of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cell viability was assessed by CellTiter-Glo (A) after 16 hours. Data are means ± SEM of three independent experiments. *P< 0.1, ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Cell viability was assessed by Crystal Violet ( B ) after 16 hours . Data are means ± SEM of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cell viability was assessed by Crystal Violet (B) after 16 hours. Data are means ± SEM of three independent experiments. *P< 0.1, ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Flow cytometric analysis of cells stained with TO - PRO - 3 nd Annexin V ( A5 ) . Histograms show means ± SEM of three independent experiments . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with TO-PRO-3 nd Annexin V (A5). Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Flow cytometric analysis of cells stained with Annexin V ( A5 ) . Histograms show means ± SEM of three independent experiments . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with Annexin V (A5). Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Flow cytometric analysis of cells stained with TO - PRO - 3 Histograms show means ± SEM of three independent experiments . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with TO-PRO-3 Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Cells were collected after 4 and 24 hours of treatment , and stained with TO - PRO - 3 Data are means ± SEM of three independent experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with TO-PRO-3 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Cells were collected after 4 hours of treatment , and stained with propidium iodide ( PI ) and A5 Data are means ± SEM of three independent experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 hours of treatment, and stained with propidium iodide (PI) and A5 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Cells were collected after 4 and 24 hours of treatment , and stained with propidium iodide ( PI ) Data are means ± SEM of three independent experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with propidium iodide (PI) Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . J Measurement of lactate deshydrogenase ( LDH ) released from necroptotic cells , as indicated ( means ± SEM , n = 4 biological replicates , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). J Measurement of lactate deshydrogenase (LDH) released from necroptotic cells, as indicated (means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA).


HT - 29 cells were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . Cells pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . K Time lapsed microscopy analysis of A5 and TO - PRO - 3 staining upon induction of necroptosis . The presented data are representative of at least three independent experiments .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). K Time lapsed microscopy analysis of A5 and TO-PRO-3 staining upon induction of necroptosis. The presented data are representative of at least three independent experiments.


A HT - 29 cells were transfected with two individual siRNA for PANX1 , or scramble non - specific ( NS ) siRNA for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) together with 5 μM Birinapant ( S ) , prior stimulation with 10 ng . mL - 1 of TNFα ( T ) , as indicated . Western blotting for hallmarks of necroptosis , as indicated . The arrowhead indicates MLKL cleaved fragment . Molecular weight markers ( Mr ) are shown .

O O O O O O O O O O O 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 I-CONTROLLED_VAR I-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 O O O O O O O O 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 HT-29 cells were transfected with two individual siRNA for PANX1, or scramble non-specific (NS) siRNA for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) together with 5 μM Birinapant (S), prior stimulation with 10 ng.mL-1 of TNFα (T), as indicated. Western blotting for hallmarks of necroptosis, as indicated. The arrowhead indicates MLKL cleaved fragment. Molecular weight markers (Mr) are shown.


B Cells were exposed to TQS for 5 hours . Necrostatin - 1 ( Nec - 1s , 20 μM ) was also used . MLKL oligomers ( MLKLn ) were resolved by non - reducing SDS - PAGE after cross - linking .

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

B Cells were exposed to TQS for 5 hours. Necrostatin-1 (Nec-1s, 20 μM) was also used. MLKL oligomers (MLKLn) were resolved by non-reducing SDS-PAGE after cross-linking.


C The cell viability was assessed by CellTiter - Glo after 24 hours of treatment ( means ± SEM , n = 4 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

C The cell viability was assessed by CellTiter-Glo after 24 hours of treatment (means ± SEM, n=4 biological replicates).


Flow cytometric analysis of cells treated with TQS for 4 hours and stained with Annexin V ( D ) Data are means ± SEM of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometric analysis of cells treated with TQS for 4 hours and stained with Annexin V (D) Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA).


Flow cytometric analysis of cells treated with TQS for 4 hours and stained with TO - PRO - 3 ( E and F ) . Data are means ± SEM of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometric analysis of cells treated with TQS for 4 hours and stained with TO-PRO-3 (E and F). Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA).


HT - 29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1 . A control nonspecific sequence ( NC ) was also used . Cells were treated with the addition of 1 μM cycloheximide ( C ) to enhance cell death , and analyzed by Western blotting ( G ) .

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

HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death, and analyzed by Western blotting (G).


HT - 29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1 . A control nonspecific sequence ( NC ) was also used . Cells were treated with the addition of 1 μM cycloheximide ( C ) to enhance cell death Cell survival was assessed by CellTiter - Glo after 24 hours of treatment ( H ) . Histogram shows means ± SEM , n = 4 biological replicates , * * * * P < 0 . 0001 , ANOVA .

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

HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death Cell survival was assessed by CellTiter-Glo after 24 hours of treatment (H). Histogram shows means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA.


HT - 29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1 . A control nonspecific sequence ( NC ) was also used . Cells were treated as in ( A ) with the addition of 1 μM cycloheximide ( C ) to enhance cell death Annexin V staining was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM , n = 3 biological replicates , * P < 0 . 1 , * * * * P < 0 . 0001 , ANOVA .

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

HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated as in (A) with the addition of 1 μM cycloheximide (C) to enhance cell death Annexin V staining was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM, n=3 biological replicates, *P<0.1, ****P<0.0001, ANOVA.


HT - 29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1 . A control nonspecific sequence ( NC ) was also used . Cells were treated with the addition of 1 μM cycloheximide ( C ) to enhance cell death TO - PRO - 3 uptake was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM , n = 3 biological replicates , * P < 0 . 1 , * * * * P < 0 . 0001 , ANOVA .

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

HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death TO-PRO-3 uptake was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM, n=3 biological replicates, *P<0.1, ****P<0.0001, ANOVA.


PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP . Cell lysates were analyzed by Western blotting as indicated ( K ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP. Cell lysates were analyzed by Western blotting as indicated (K).


PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP . TO - PRO - 3 uptake was analyzed by flow cytometry Shown are means ± SEM , n = 3 biological replicates , * * * * P < 0 . 0001 ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP. TO-PRO-3 uptake was analyzed by flow cytometry Shown are means ± SEM, n=3 biological replicates, ****P<0.0001 ANOVA.


NS - and MLKL - silenced cells were pretreated with 100 μM carbenoxolone ( CBX ) , 2 mM Probenecid ( Probe ) or 30 μM Trovafloxacin ( Trova ) and stimulated Cell survival was evaluated by CellTiter - Glo after 24 hours ( N ) . Data are means ± SEM , n = 4 biological replicates , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA .

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

NS- and MLKL-silenced cells were pretreated with 100 μM carbenoxolone (CBX), 2 mM Probenecid (Probe) or 30 μM Trovafloxacin (Trova) and stimulated Cell survival was evaluated by CellTiter-Glo after 24 hours (N). Data are means ± SEM, n=4 biological replicates, ***P<0.001, ****P<0.0001, ANOVA.


NS - and MLKL - silenced cells were pretreated with 100 μM carbenoxolone ( CBX ) , 2 mM Probenecid ( Probe ) or 30 μM Trovafloxacin ( Trova ) and stimulated TO - PRO - 3 uptake was analyzed by flow cytometry ( O ) . Data are means ± SEM , n = 3 biological replicates , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA .

NS- and MLKL-silenced cells were pretreated with 100 μM carbenoxolone (CBX), 2 mM Probenecid (Probe) or 30 μM Trovafloxacin (Trova) and stimulated TO-PRO-3 uptake was analyzed by flow cytometry (O). Data are means ± SEM, n=3 biological replicates, ***P<0.001, ****P<0.0001, ANOVA.


P Cells were analyzed by Western blotting . The presented data are representative of at least three independent experiments .

P Cells were analyzed by Western blotting. The presented data are representative of at least three independent experiments.


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . NS - and MLKL - silenced cells were treated with 5 μM necrosulfonamide ( NSA ) . MLKL oligomers ( MLKLn ) were visualized by non - reducing SDS - PAGE after cross - linking proteins ( A ) . Data are means ± SEM of three independent experiments . * * * * P < 0 . 0001 ( ANOVA ) . Data information Arrowhead , MLKL cleaved fragment .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). NS- and MLKL-silenced cells were treated with 5 μM necrosulfonamide (NSA). MLKL oligomers (MLKLn) were visualized by non-reducing SDS-PAGE after cross-linking proteins (A). Data are means ± SEM of three independent experiments. ****P<0.0001 (ANOVA). Data information Arrowhead, MLKL cleaved fragment.


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . NS - and MLKL - silenced cells were treated with 5 μM necrosulfonamide ( NSA ) . MLKL oligomers ( MLKLn ) Cells were exposed to TQS for 5 hours . Molecular weight markers ( Mr ) are shown . TO - PRO - 3 uptake was analyzed by flow cytometry in cells treated with TQS for 4 hours ( B ) . Data are means ± SEM of three independent experiments . * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). NS- and MLKL-silenced cells were treated with 5 μM necrosulfonamide (NSA). MLKL oligomers (MLKLn) Cells were exposed to TQS for 5 hours. Molecular weight markers (Mr) are shown. TO-PRO-3 uptake was analyzed by flow cytometry in cells treated with TQS for 4 hours (B). Data are means ± SEM of three independent experiments. ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . C Cell lysates from NS - , and ITPK1 - silenced cells were analyzed by Western blotting as indicated . Data information : Arrowhead , MLKL cleaved fragment .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). C Cell lysates from NS-, and ITPK1-silenced cells were analyzed by Western blotting as indicated. Data information: Arrowhead, MLKL cleaved fragment.


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . D Western blotting analysis of MLKL

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). D Western blotting analysis of MLKL oligomerization


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . E Cell survival was evaluated by CellTiter - Glo after 24 hours of treatment ( means ± SEM , n = 4 biological replicates , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). E Cell survival was evaluated by CellTiter-Glo after 24 hours of treatment (means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA).