Figure 7 ( Left ) Kaplan - Meier curve of the probability of survival for 155 GBM patients with low or high QKI RNA level , using median cut - off , based on the TCGA RNAseq dataset . ( Right ) Differential expression analysis related to either MALT1 or QKI expression highlighted a lysosomal lumen GO function . Venn diagram of overlapping lysosomal enriched protein encoding genes from this comparison showed 7 shared genes , together with 9 and 10 specific genes for MALT1 and QKI expression , respectively . ( Bottom ) Correlation between MALT1 and QKI expression was analyzed using The Cancer Genome Atlas ( TCGA , HG - U133A dataset ) on the GlioVis platform ( Bowman et al , 2007 ) . Pearson correlation factor = - 0 . 21 , p - value = 0 . 03 . GSCs # 1 and # 9 protein lysates ( input ) were processed for immunoprecipitation ( IP ) using control immunoglobulins ( Ig ) , anti - QKI or anti - BCL10 antibodies . Input and IP fractions were separated on SDS - PAGE and western - blots for MALT1 , QKI and BCL10 antibodies were performed as specified . Total protein lysates ( input ) from GSC # 9 treated with vehicle ( - , DMSO ) or MPZ ( + , 20 μM , 1 hour ) or with vehicle ( - , H2O ) or Z - VRPR - FMK ( + , 75 μM , 4 hours ) , were processed for control immunoglobulins ( Ig ) or anti - QKI antibodies immunoprecipitation ( IP ) . Western - blots were performed with indicated antibodies . Western - blots were performed with indicated antibodies . Confocal analysis of Lysotracker ( green ) or FLAG ( red ) in GSC # 9 overexpressing either empty vector ( mock ) or FLAG - QKI . Scale bars : 10 μm . Nuclei ( DAPI ) are shown in blue . Confocal analysis of LAMP2 ( green ) or FLAG ( red ) in GSC # 9 transfected with either empty vector ( mock ) or FLAG - QKI . Scale bars : 10 μm . Nuclei ( DAPI ) are shown in blue . Quantification of LAMP2 staining pixel intensity on GSC # 9 transfected with mock and FLAG - QKI . Data are presented as the mean + s . e . m . on 3 independent experiments . Each dot represents one cell . n > 15 . Western - blot analysis of QKI , LAMP2 and LC3B in GSC # 9 overexpressing either empty vector ( mock ) or FLAG - QKI . GAPDH served as a loading control . Confocal analysis of mTOR ( green ) or FLAG ( red ) in GSC # 9 transfected with either empty vector ( mock ) or Flag - QKI . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm . GSC # 1 were transfected with either empty vector ( mock ) or FLAG - QKI . Total protein lysates were processed for western - blots against p - S6 and FLAG . Total S6 served as a loading control . Fraction of surviving cells over time in GSCs # 1 and # 9 , transduced with empty vector ( mock ) or FLAG - QKI bi - cistronic GFP plasmids . Data are plotted as the percentage of GFP - positive cells at the day of the analysis ( Dx ) , normalized to the starting point ( Day 4 post - infection , D4 ) . Data are representative of n = 3 . GSC # 9 transfected with non - silencing RNA duplexes ( sic ) or QKI targeting siRNA duplexes ( siQKI ) were treated for 16 hours with vehicle ( DMSO ) or MPZ ( 10 μM ) . Total protein lysates were processed for western - blots against LAMP2 , CTSD , QKI , and LC3B expression , as indicated . GAPDH served as a loading control . Confocal analysis of mTOR ( green ) and LAMP2 ( red ) in GSC # 9 transfected with sic or siQKI , and treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm . Quantification of mTOR colocalization score with LAMP2 is shown . The Coloc2 plug - in from ImageJ was used to measure Mander ' s tM1 correlation factor in LAMP2 - positive ROI , using Costes threshold regression . Data are presented as the mean + s . e . m . on 3 independent experiments . Each dot represents one cell . n > 10 . GSC # 9 transfected with non - silencing RNA duplexes ( sic ) or QKI targeting siRNA duplexes ( siQKI ) were treated for 1 hour with vehicle ( DMSO ) or MPZ ( 20 μM ) . Total protein lysates were processed for western - blots against QKI and p - S6 . TUBULIN and Total S6 served as loading controls . FACS analysis of EdU staining was performed on GSC # 9 cells transfected with non - silencing RNA duplexes ( sic , pink ) , QKI targeting siRNA duplexes ( siQKI , light purple ) , MALT1 targeting siRNA duplexes ( siMALT1 , blue ) or double transfected with siQKI and siMALT1 ( purple ) . FACS analysis of propidium iodide ( PI ) staining in GSC # 9 transfected with non - silencing RNA duplexes ( sic ) , QKI targeting siRNA duplexes ( siQKI ) , MALT1 targeting siRNA duplexes ( siMALT1 ) or double transfected with siQKI and siMALT1 and analyzed 72 hours later . Percentage of PI - positive cells normalized to vehicle treated controls are presented as the mean + s . e . m . on 3 independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(Left) Kaplan-Meier curve of the probability of survival for 155 GBM patients with low or high QKI RNA level, using median cut-off, based on the TCGA RNAseq dataset. (Right) Differential expression analysis related to either MALT1 or QKI expression highlighted a lysosomal lumen GO function. Venn diagram of overlapping lysosomal enriched protein encoding genes from this comparison showed 7 shared genes, together with 9 and 10 specific genes for MALT1 and QKI expression, respectively. (Bottom) Correlation between MALT1 and QKI expression was analyzed using The Cancer Genome Atlas (TCGA, HG-U133A dataset) on the GlioVis platform (Bowman et al, 2007). Pearson correlation factor=-0.21, p-value=0.03.GSCs #1 and #9 protein lysates (input) were processed for immunoprecipitation (IP) using control immunoglobulins (Ig), anti-QKI or anti-BCL10 antibodies. Input and IP fractions were separated on SDS-PAGE and western-blots for MALT1, QKI and BCL10 antibodies were performed as specified.Total protein lysates (input) from GSC#9 treated with vehicle (-, DMSO) or MPZ (+, 20 μM, 1 hour) or with vehicle (-, H2O) or Z-VRPR-FMK (+, 75 μM, 4 hours), were processed for control immunoglobulins (Ig) or anti-QKI antibodies immunoprecipitation (IP). Western-blots were performed with indicated antibodies. Western-blots were performed with indicated antibodies.Confocal analysis of Lysotracker (green) or FLAG (red) in GSC#9 overexpressing either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue.Confocal analysis of LAMP2 (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue. Quantification of LAMP2 staining pixel intensity on GSC#9 transfected with mock and FLAG-QKI. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>15.Western-blot analysis of QKI, LAMP2 and LC3B in GSC#9 overexpressing either empty vector (mock) or FLAG-QKI. GAPDH served as a loading control.Confocal analysis of mTOR (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or Flag-QKI. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.GSC#1 were transfected with either empty vector (mock) or FLAG-QKI. Total protein lysates were processed for western-blots against p-S6 and FLAG. Total S6 served as a loading control.Fraction of surviving cells over time in GSCs #1 and #9, transduced with empty vector (mock) or FLAG-QKI bi-cistronic GFP plasmids. Data are plotted as the percentage of GFP-positive cells at the day of the analysis (Dx), normalized to the starting point (Day 4 post-infection, D4). Data are representative of n=3.GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 16 hours with vehicle (DMSO) or MPZ (10 μM). Total protein lysates were processed for western-blots against LAMP2, CTSD, QKI, and LC3B expression, as indicated. GAPDH served as a loading control.Confocal analysis of mTOR (green) and LAMP2 (red) in GSC#9 transfected with sic or siQKI, and treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. Quantification of mTOR colocalization score with LAMP2 is shown. The Coloc2 plug-in from ImageJ was used to measure Mander's tM1 correlation factor in LAMP2-positive ROI, using Costes threshold regression. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>10.GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 1 hour with vehicle (DMSO) or MPZ (20 μM). Total protein lysates were processed for western-blots against QKI and p-S6. TUBULIN and Total S6 served as loading controls.FACS analysis of EdU staining was performed on GSC#9 cells transfected with non-silencing RNA duplexes (sic, pink), QKI targeting siRNA duplexes (siQKI, light purple), MALT1 targeting siRNA duplexes (siMALT1, blue) or double transfected with siQKI and siMALT1 (purple).FACS analysis of propidium iodide (PI) staining in GSC#9 transfected with non-silencing RNA duplexes (sic), QKI targeting siRNA duplexes (siQKI), MALT1 targeting siRNA duplexes (siMALT1) or double transfected with siQKI and siMALT1 and analyzed 72 hours later. Percentage of PI-positive cells normalized to vehicle treated controls are presented as the mean + s.e.m. on 3 independent experiments.


Figure 1B . Chromatin accessibility and p300 binding at differential ATAC - seq peaks visualized using K - means clustering .

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Figure 1B. Chromatin accessibility and p300 binding at differential ATAC-seq peaks visualized using K-means clustering.


Figure 2B . Boxplots showing differential gene expression ( AC versus DMZ ) and associated ATAC - seq signals ( Two biological replicates ) . The central band within the boxplot represents the median and the whiskers show 1 . 5 times the range between the first and third quartile ( IQR ) . F . Genomic tracks showing aggregated accessibility of single - cell ATAC - seq clusters at the t ( tbxt ) , tfap2a , gsc , ctcf , lhx1 and sox11 loci .

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Figure 2B. Boxplots showing differential gene expression (AC versus DMZ) and associated ATAC-seq signals (Two biological replicates). The central band within the boxplot represents the median and the whiskers show 1.5 times the range between the first and third quartile (IQR).F. Genomic tracks showing aggregated accessibility of single-cell ATAC-seq clusters at the t (tbxt), tfap2a, gsc, ctcf, lhx1 and sox11 loci.


Figure 4C . Feature maps , showing the expression of selected genes in single cells ( cf . Appendix Fig . S2 ) . Color scale represents the gene expression value ( log1p - transformed counts per 10 , 000 unique reads ) for each cell for a given gene , from low ( grey ) to high ( orange ) .

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Figure 4C. Feature maps, showing the expression of selected genes in single cells (cf. Appendix Fig. S2). Color scale represents the gene expression value (log1p-transformed counts per 10,000 unique reads) for each cell for a given gene, from low (grey) to high (orange).


Figure 6A . Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1 , Foxb1 - Eomes , Eomes , Irx3 , Irx3 - Otx2 , Otx2 and Lhx8 . C . Fold enrichment of genes with zygotically defined ( ZyD ) H3K4me3 at their promoter in AC with transcription factor overexpression . Asterisk indicates hypergeometric p - value < = 0 . 01 .

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Figure 6A. Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1, Foxb1-Eomes, Eomes, Irx3, Irx3-Otx2, Otx2 and Lhx8.C. Fold enrichment of genes with zygotically defined (ZyD) H3K4me3 at their promoter in AC with transcription factor overexpression. Asterisk indicates hypergeometric p-value <= 0.01.


Figure 1B ) Expression of the 21U sensor ( left ) and DAPI staining ( right ) of gonad arms in the indicated genetic backgrounds . Gonads are outlined by a dashed line . The mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in the intensity of the signal . Number of animals analysed and with indicated phenotype are given in the panel . Animals not showing the activation of the 21U sensor ( + ) were still silenced , and animals that did not show the silenced 21U sensor ( RNAe ) state were expressing weakly , comparable to the 21U sensor ( + ) . Scale bar : 25 μm . C ) Tc1 reversion assay in different genetic backgrounds . All the strains tested carried the unc - 22 : : Tc1 ( st136 ) allele . Tc1 excision can result in restoration of unc - 22 function , which can be scored visually . Negative control = unc - 22 : : Tc1 ( st136 ) in a wild type background ; positive control = wago - 1 / - 2 / - 3 . Two independent experiments per strain are represented . D ) Crossing scheme to address the re - initiation of silencing of the 21U sensor . A mut - 7 mutant male expressing the 21U sensor is crossed with either a wild type ( left ) , prg - 1 ( middle ) or pid - 2 ( right ) mutant hermaphrodite . Their F1 offspring was scored for expression of the 21U sensor by microscopy . Three states of expression were scored , and represented in a pie chart . The three expression states are exemplified by representative images at the bottom : OFF ( left ) , FAINT ( right ) or ON ( middle ) . DIC images are shown above the fluorescence panels . Gonads are outlined by a dashed line . Scale bar : 25 µm . E ) Crossing scheme to address if maternal 21U RNAs are sufficient to re - initiate the silencing of the 21U sensor . A pid - 1 mutant male expressing the 21U sensor is crossed with a hermaphrodite , heterozygous for the same mutation . All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite , but in 50 % of the F1 , which is pid - 1 homozygous mutant , no zygotic PID - 1 is present , hence no zygotic 21U RNAs can be made . The silencing or expression of the 21U sensor in the pid - 1 homozygous mutant F1 has been scored by microscopy , and depicted in a pie chart . At the bottom , a representative image of an animal carrying a silenced 21U sensor ( lower : mCherry signal ; upper : DAPI staining ) in pid - 1 mutant offspring . Gonads are outlined by a dashed line . Scale bar : 25 μm . F ) Crossing scheme to test the role of PID - 2 in re - initiating the silencing of the 21U sensor , mediated by maternally provided 21U RNAs only . The expression of the 21U sensor in the F1 has been scored by microscopy , and depicted in a pie chart . White arrow heads indicate the many arrayed oocytes , typical of a feminized germline . DIC and fluorescence image of a representative animal are shown at the bottom . Gonads are outlined by a dashed line . Scale bar : 25 μm .

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Figure 1B) Expression of the 21U sensor (left) and DAPI staining (right) of gonad arms in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. Number of animals analysed and with indicated phenotype are given in the panel. Animals not showing the activation of the 21U sensor(+) were still silenced, and animals that did not show the silenced 21U sensor(RNAe) state were expressing weakly, comparable to the 21U sensor(+). Scale bar: 25 μm.C) Tc1 reversion assay in different genetic backgrounds. All the strains tested carried the unc-22::Tc1(st136) allele. Tc1 excision can result in restoration of unc-22 function, which can be scored visually. Negative control = unc-22::Tc1(st136) in a wild type background; positive control = wago-1/-2/-3. Two independent experiments per strain are represented.D) Crossing scheme to address the re-initiation of silencing of the 21U sensor. A mut-7 mutant male expressing the 21U sensor is crossed with either a wild type (left), prg-1 (middle) or pid-2 (right) mutant hermaphrodite. Their F1 offspring was scored for expression of the 21U sensor by microscopy. Three states of expression were scored, and represented in a pie chart. The three expression states are exemplified by representative images at the bottom: OFF (left), FAINT (right) or ON (middle). DIC images are shown above the fluorescence panels. Gonads are outlined by a dashed line. Scale bar: 25 µm.E) Crossing scheme to address if maternal 21U RNAs are sufficient to re-initiate the silencing of the 21U sensor. A pid-1 mutant male expressing the 21U sensor is crossed with a hermaphrodite, heterozygous for the same mutation. All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite, but in 50% of the F1, which is pid-1 homozygous mutant, no zygotic PID-1 is present, hence no zygotic 21U RNAs can be made. The silencing or expression of the 21U sensor in the pid-1 homozygous mutant F1 has been scored by microscopy, and depicted in a pie chart. At the bottom, a representative image of an animal carrying a silenced 21U sensor (lower: mCherry signal; upper: DAPI staining) in pid-1 mutant offspring. Gonads are outlined by a dashed line. Scale bar: 25 μm.F) Crossing scheme to test the role of PID-2 in re-initiating the silencing of the 21U sensor, mediated by maternally provided 21U RNAs only. The expression of the 21U sensor in the F1 has been scored by microscopy, and depicted in a pie chart. White arrow heads indicate the many arrayed oocytes, typical of a feminized germline. DIC and fluorescence image of a representative animal are shown at the bottom. Gonads are outlined by a dashed line. Scale bar: 25 μm.


Figure 2The 22G RNAs mapping to the 21U sensor transgene were identified from small RNA sequencing data , and read density was plotted over the transgene , which is schematically depicted at the bottom . The aggregated results of three replicates is shown for each indicated genotype in the different panels . The shading , in grey , represents the standard deviation of the read density from the three replicates . ( + ) means that the sensor was detectably expressed . ( RNAe ) means that the sensor was crossed into the respective mutant background in an RNAe state ( i . e . its silencing was PRG - 1 - independent ) , and its expression remained undetectable by microscopy .

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Figure 2The 22G RNAs mapping to the 21U sensor transgene were identified from small RNA sequencing data, and read density was plotted over the transgene, which is schematically depicted at the bottom. The aggregated results of three replicates is shown for each indicated genotype in the different panels. The shading, in grey, represents the standard deviation of the read density from the three replicates. (+) means that the sensor was detectably expressed. (RNAe) means that the sensor was crossed into the respective mutant background in an RNAe state (i.e. its silencing was PRG-1-independent), and its expression remained undetectable by microscopy.


Figure 3A ) MA - plot of log2 fold changes ( Y - axis ) versus the mean of normalized counts of 22G RNAs ( X - axis ) for pid - 2 ( xf23 ) mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . B ) Heatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in pid - 2 ( xf23 ) mutants . Significance was tested with Fisher ' s exact test and p - values adjusted with the Benjamini - Hochberg Procedure . Colour scheme represents the odds ratio of overlaps representing the strength of association . N . S . : not significant . C - D ) Cumulative 22G coverage along the gene body of Mutator and CSR - 1 targets . Values represent 22G coverage normalized to the total coverage of each gene , for wild type ( N2 ) and pid - 2 ( xf23 ) mutants . The lines represent the average of biological replicates , whereas the shading represents the standard deviation of biological replicates . a . u . : arbitrary units .

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Figure 3A) MA-plot of log2 fold changes (Y-axis) versus the mean of normalized counts of 22G RNAs (X-axis) for pid-2(xf23) mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.B) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-2(xf23) mutants. Significance was tested with Fisher's exact test and p-values adjusted with the Benjamini-Hochberg Procedure. Colour scheme represents the odds ratio of overlaps representing the strength of association. N.S.: not significant.C-D) Cumulative 22G coverage along the gene body of Mutator and CSR-1 targets. Values represent 22G coverage normalized to the total coverage of each gene, for wild type (N2) and pid-2(xf23) mutants. The lines represent the average of biological replicates, whereas the shading represents the standard deviation of biological replicates. a.u.: arbitrary units.


Figure 4A ) Volcano plot representing the enrichment of proteins interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 protein , followed by quantitative label - free mass spectrometry . As control , eGFP IPs were performed on protein extracts from wild - type , non - transgenic animals . For each IP - MS experiment , quadruplicates were measured and analysed . The dashed line reflects a significance threshold of p - value < 0 . 05 at two - fold enrichment . Volcano plot representing the enrichment of proteins interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 protein , followed by quantitative label - free mass spectrometry . In this experiment , the endogenous PID - 2 protein was immunoprecipitated , and pid - 2 ( xf23 ) mutant protein extracts were used as control . Volcano plot , representing the enrichment of proteins interacting with PID - 4 , by immunoprecipitating endogenously tagged PID - 4 protein . IPs from protein extracts from wild - type , non - tagged animals were used as control . Volcano plot , representing the enrichment of proteins interacting with PID - 5 , by immunoprecipitating endogenously tagged PID - 5 protein . IPs from protein extracts from wild - type , non - tagged animals were used as control .

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Figure 4A) Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. As control, eGFP IPs were performed on protein extracts from wild-type, non-transgenic animals. For each IP-MS experiment, quadruplicates were measured and analysed. The dashed line reflects a significance threshold of p-value < 0.05 at two-fold enrichment.Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. In this experiment, the endogenous PID-2 protein was immunoprecipitated, and pid-2(xf23) mutant protein extracts were used as control.Volcano plot , representing the enrichment of proteins interacting with PID-4, by immunoprecipitating endogenously tagged PID-4 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.Volcano plot, representing the enrichment of proteins interacting with PID-5, by immunoprecipitating endogenously tagged PID-5 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.


Figure 5A ) Expression of the 21U sensor in the indicated genetic backgrounds . Gonads are outlined by a dashed line . The mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in the intensity of the signal . The panels on the right are DIC images of the respective animals . Scale bar : 25 μm . B ) 22G RNA coverage of the 21U sensor ( RNAe ) in the indicated genetic backgrounds . C ) MA - plot of log2 fold changes ( on the Y - axis ) versus the mean of normalized counts of 22G RNAs ( on the X - axis ) for pid - 4 mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . D ) Heatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in pid - 4 mutants . Colour scheme represents the odds ratio of overlaps representing the strength of association . MA - plot of log2 fold changes ( on the Y - axis ) versus the mean of normalized counts of 22G RNAs ( on the X - axis ) , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . E ) for pid - 5 mutantHeatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in F ) pid - 5 mutantMA - plot of log2 fold changes ( on the Y - axis ) versus the mean of normalized counts of 22G RNAs ( on the X - axis ) compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . G ) for pid - 4 ; pid - 5 double mutantHeatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in H ) pid - 4 ; pid - 5 double mutantI , J ) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC . Six L2 - L3 larvae for each of the indicated backgrounds were hand - picked to a fresh plate every four or six days , counting as two or three generations , respectively , until no larvae were present on the plate to be picked . * : two plates of N2 were contaminated and were excluded from the assay .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Expression of the 21U sensor in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. The panels on the right are DIC images of the respective animals. Scale bar: 25 μm.B) 22G RNA coverage of the 21U sensor(RNAe) in the indicated genetic backgrounds.C) MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) for pid-4 mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.D) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-4 mutants. Colour scheme represents the odds ratio of overlaps representing the strength of association.MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) , compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. E) for pid-5 mutantHeatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in F) pid-5 mutantMA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. G) for pid-4;pid-5 double mutantHeatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in H) pid-4;pid-5 double mutantI, J) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC. Six L2-L3 larvae for each of the indicated backgrounds were hand-picked to a fresh plate every four or six days, counting as two or three generations, respectively, until no larvae were present on the plate to be picked. *: two plates of N2 were contaminated and were excluded from the assay.


Figure 6Expression of PID - 2 : : eGFP ( xfSi145 ) ( A ) in perinuclear granules in the germline . PGL - 1 : : mTagRFP - T ( A ) mark P granules . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm . Expression of , PID - 4 : : mTagRFP - T ( B ) in perinuclear granules in the germline . DEPS - 1 : : GFP ( B mark P granules . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm . Expression of PID - 5 : : mTagRFP - T ( C ) in perinuclear granules in the germline . DEPS - 1 : : GFP mark P granules . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm . Expression of PID - 4 : : mTagRFP - T ( D ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm . Expression of PID - 5 : : mTagRFP - T ( E ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm . F ) Box - plots representing the distance ( µm ) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot . Between 4 - 10 different gonads were analyzed for each condition . Expression of PID - 2 : : eGFP ( xfSi145 ) together with PID - 4 : : mTagRFP - T ( G ) The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar = 25 µExpression of PID - 2 : : eGFP ( xfSi145 ) together with PID - 5 : : mTagRFP - T ( H ) . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar = 25 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Expression of PID-2::eGFP(xfSi145) (A) in perinuclear granules in the germline. PGL-1::mTagRFP-T (A) mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.Expression of , PID-4::mTagRFP-T (B) in perinuclear granules in the germline. DEPS-1::GFP (B mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.Expression of PID-5::mTagRFP-T (C) in perinuclear granules in the germline. DEPS-1::GFP mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.Expression of PID-4::mTagRFP-T (D) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.Expression of PID-5::mTagRFP-T (E) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.F) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition.Expression of PID-2::eGFP (xfSi145) together with PID-4::mTagRFP-T (G) The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µExpression of PID-2::eGFP (xfSi145) together with PID-5::mTagRFP-T (H). The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µm.


Figure 7A - E ) Expression of 3xFLAG : : GFP : : ZNFX - 1 and PGL - 1 : : mTagRFP - T in a wild type ( A ) , pid - 2 ( B ) , pid - 4 ; pid - 5 ( C ) , pid - 4 ( D ) , and pid - 5 ( E ) mutant backgrounds . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown . Arrow heads indicate individual condensates . Scale bar : 25 µm . F ) Quantification of the ratio Z / P granules in wild type , pid - 2 , pid - 4 / - 5 , pid - 4 and pid - 5 mutant backgrounds . The number of granules is indicated in brackets , next to the genotype . Expression of PID - 4 : : mTagRFP - T ( G ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker , in a pid - 2 mutant background . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Scale bar : 25 µm . Expression of PID - 4 : : mTagRFP - T H ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker , in a pid - 2 mutant background . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Scale bar : 25 µm . I ) Box - plots representing the distance ( µm ) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot . Between 4 - 10 different gonads were analyzed for each condition . The median is represented by a line . The interquartile range ( IQR ) , 25th to 75th percentile , is represented by the upper and lower lines , respectively , and whiskers represent the first quartile ( down to - 1 . 5 * IQR ) or the third quartile ( up to + 1 . 5 * IQR ) . P - values were calculated using a t - test ( two - tailed ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-E) Expression of 3xFLAG::GFP::ZNFX-1 and PGL-1::mTagRFP-T in a wild type (A), pid-2 (B), pid-4;pid-5 (C), pid-4 (D), and pid-5 (E) mutant backgrounds. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown. Arrow heads indicate individual condensates. Scale bar: 25 µm. F) Quantification of the ratio Z / P granules in wild type, pid-2, pid-4/-5, pid-4 and pid-5 mutant backgrounds. The number of granules is indicated in brackets, next to the genotype. Expression of PID-4::mTagRFP-T (G) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.Expression of PID-4::mTagRFP-T H) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.I) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition. The median is represented by a line. The interquartile range (IQR), 25th to 75th percentile, is represented by the upper and lower lines, respectively, and whiskers represent the first quartile (down to -1.5*IQR) or the third quartile (up to +1.5*IQR). P-values were calculated using a t-test (two-tailed).


Figure 1 ( A ) ATX serum protein levels were measured in blood serum samples from IBD patients and healthy controls using ELISA : active UC ( n = 26 ) , active CD ( n = 34 ) , control ( Con ) ( n = 26 ) . ( B ) Age - and sex - matched Atx ∆ ΜΕ / ∆ ΜΕ ; Il10 - / - mice ( n = 9 ) and Atx + / + ; Il10 - / - littermates ( n = 9 ) were examined for the development of spontaneous colitis . Body weight changes were monitored every other day , starting at the age of 37 days and ending at the age of 6 months . Results are means ± SD . Data were compared by Two - way ANOVA ( with treatment and times ) , followed by the multiple - comparison Bonferroni t test to assess differences between groups . ( C ) Gross appearance of the full - length mouse colon . Each line on a ruler represents a millimeter ( mm ) . ( D and E ) H & E stained section of the mouse colon . Insets are enlarged in the lower panel . Colonic mucosa thickness was measured using ZEISS Axio Imager Z1 microscope ( n = 14 / group ) ( E ) . ( F ) Histological parameters were quantified with H & E - colon sections ( n = 10 / group ) ( D ) and shown as mean ± SEM . ( G ) Survival of Atx ∆ ΜΕ / ∆ ΜΕ ; Il10 - / - ( n = 17 ) and Atx + / + ; Il10 - / - littermates ( n = 18 ) was analyzed by the Kaplan - Meier method ( Log - rank P = 0 . 0046 ) . ( H ) Presented are H & E - stained sections of the small intestine in a ' Swiss - Roll ' form . Inset areas are enlarged in the right panel .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) ATX serum protein levels were measured in blood serum samples from IBD patients and healthy controls using ELISA: active UC (n=26), active CD (n=34), control (Con) (n=26).(B) Age- and sex-matched Atx∆ΜΕ/∆ΜΕ;Il10-/- mice (n=9) and Atx+/+;Il10-/- littermates (n=9) were examined for the development of spontaneous colitis. Body weight changes were monitored every other day, starting at the age of 37 days and ending at the age of 6 months. Results are means ± SD. Data were compared by Two-way ANOVA (with treatment and times), followed by the multiple-comparison Bonferroni t test to assess differences between groups.(C) Gross appearance of the full-length mouse colon. Each line on a ruler represents a millimeter (mm).(D and E) H&E stained section of the mouse colon. Insets are enlarged in the lower panel. Colonic mucosa thickness was measured using ZEISS Axio Imager Z1 microscope (n=14/group) (E).(F) Histological parameters were quantified with H&E-colon sections (n=10/group) (D) and shown as mean ± SEM.(G) Survival of Atx∆ΜΕ/∆ΜΕ;Il10-/- (n=17) and Atx+/+;Il10-/- littermates (n=18) was analyzed by the Kaplan-Meier method (Log-rank P=0.0046).(H) Presented are H&E-stained sections of the small intestine in a 'Swiss-Roll' form. Inset areas are enlarged in the right panel.


Figure 2 ( A ) Total cell lysates of peritoneal macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were subjected to immunoblotting analysis to confirm Atx deletion . ( B ) Peritoneal macrophages from the mice were subjected to the ESI - MS / MS method to quantify endogenous LPA . Individual molecular species of LPA and total LPA ( inset graph ) were compared . The data are shown as mean ± SEM ( n = 3 / group ) * P < 0 . 05 ( one - tailed unpaired t - test ) . ( C and D ) Thioglycolate - elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages ( C ) and DCs ( D ) . Scale bar indicates 1 μm . Lipid rafts from peritoneal macrophages ( E , F ) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100 , 000g for 17 h . Protein was purified and concentrated with the 3K cut - off filter , followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Caveolin or Flotillin - 1 . Lipid rafts from mouse macrophage Raw264 . 7 cells treated with Atx inhibitor ( Atx - i ) PF8380 ( 50 μM , 30 min ) or vehicle ( Veh . , DMSO 0 . 1 % ) ( G ) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100 , 000g for 17 h . Protein was purified and concentrated with the 3K cut - off filter , followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Flotillin - 1 . ( H ) Peritoneal macrophages were stained with Alexa Fluor 594 - cholera toxin subunit B ( Red ) . The plasma membrane was examined with FV10i confocal scanning microscopy . Inset areas were magnified at the right panel . Each scale bar indicates 10 μm .

O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Total cell lysates of peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were subjected to immunoblotting analysis to confirm Atx deletion.(B) Peritoneal macrophages from the mice were subjected to the ESI-MS/MS method to quantify endogenous LPA. Individual molecular species of LPA and total LPA (inset graph) were compared. The data are shown as mean ± SEM (n=3/group) *P < 0.05 (one-tailed unpaired t-test).(C and D) Thioglycolate-elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages (C) and DCs (D). Scale bar indicates 1 μm.Lipid rafts from peritoneal macrophages (E, F) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Caveolin or Flotillin-1.Lipid rafts from mouse macrophage Raw264.7 cells treated with Atx inhibitor (Atx-i) PF8380 (50 μM, 30 min) or vehicle (Veh., DMSO 0.1%) (G) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Flotillin-1.(H) Peritoneal macrophages were stained with Alexa Fluor 594-cholera toxin subunit B (Red). The plasma membrane was examined with FV10i confocal scanning microscopy. Inset areas were magnified at the right panel. Each scale bar indicates 10 μm.


Figure 3Peritoneal macrophages ( A ) were stimulated with LPS ( 20 ng / mL , 20 min ) Cell lysates were subjected to co - immunoprecipitation ( IP ) with CD14 antibody , followed by immunoblotting ( IB ) with TLR4 antibody . Raw264 . 7 cells ( B ) were stimulated with LPS ( 20 ng / mL , 20 min ) in the presence / absence of ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) . Cell lysates were subjected to co - immunoprecipitation ( IP ) with CD14 antibody , followed by immunoblotting ( IB ) with TLR4 antibody . The physical interaction of TLR4 - CD14 was examined by FRET analysis . HEK293 cells were transfected with TLR4 - EYFP ( yellow ) and CD14 - ECFP ( blue ) ( C ) followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . Cells were washed with PBS and fixed , then visualized with filter sets for CFP and YFP . FRET images were visualized with the FRET filter set , expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins ( arbitrary linear units of fluorescence intensity ) . Presented are the representative images from at least three independent experiments . Scale bar indicates 5 μm . The physical interaction of TLR4 - TLR4 was examined by FRET analysis . HEK293 cells were transfected with TLR4 - EYFP and TLR4 - ECFP ( D ) , followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . Cells were washed with PBS and fixed , then visualized with filter sets for CFP and YFP . FRET images were visualized with the FRET filter set , expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins ( arbitrary linear units of fluorescence intensity ) . Presented are the representative images from at least three independent experiments . Scale bar indicates 5 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Peritoneal macrophages (A) were stimulated with LPS (20 ng/mL, 20 min) Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.Raw264.7 cells (B) were stimulated with LPS (20 ng/mL, 20 min) in the presence/absence of ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%). Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.The physical interaction of TLR4-CD14 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP (yellow) and CD14-ECFP (blue) (C) followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.The physical interaction of TLR4-TLR4 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP and TLR4-ECFP (D), followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.


Figure 4 ( A and B ) Peritoneal macrophages from Atx + / + ( A ) and Atx ∆ ΜΕ / ∆ ΜΕ ( B ) mice were stimulated with LPS ( 20 ng / mL , 20 min ) , followed by fixation in 4 % paraformaldehyde for 15 min at room temperature and staining with TLR4 - anti rabbit FITC ( Green ) , early endosomal marker EEA1 - anti mouse Dylight405 ( Blue ) , and Alexa Fluor 594 - CTXB ( Red ) . TLR4 internalization was examined with confocal microscopy . Presented are the representative from 3 independent experiments in which more than 95 % of the cells exhibited similar results . Scale bar , 10 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A and B) Peritoneal macrophages from Atx+/+ (A) and Atx∆ΜΕ/∆ΜΕ (B) mice were stimulated with LPS (20 ng/mL, 20 min), followed by fixation in 4% paraformaldehyde for 15 min at room temperature and staining with TLR4-anti rabbit FITC (Green), early endosomal marker EEA1-anti mouse Dylight405 (Blue), and Alexa Fluor 594-CTXB (Red). TLR4 internalization was examined with confocal microscopy. Presented are the representative from 3 independent experiments in which more than 95% of the cells exhibited similar results. Scale bar, 10 μm.


Figure 5The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated with LPS ( 20 ng / mL , 20 min ) The cell lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) analysis with the antibodies indicated . Presented is the representative from three independent experiments . Raw264 . 7 cells were stimulated by LPS with the ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) The cell lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) analysis with the antibodies indicated . Presented is the representative from three independent experiments . The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated with LPS ( 20 ng / mL , 20 min ) The cell lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) analysis with the antibodies indicated . Presented is the representative from three independent experiments . Raw264 . 7 cells were stimulated by LPS with the ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) The cell lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) analysis with the antibodies indicated . Presented is the representative from three independent experiments . The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated with LPS ( 20 ng / mL , 20 min ) The cell lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) analysis with the antibodies indicated . Presented is the representative from three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.Raw264.7 cells were stimulated by LPS with the ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.Raw264.7 cells were stimulated by LPS with the ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.


Figure 6The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated by LPS [ 20 ng / mL , 20 min : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 , were used for a loading control . Presented are the representative image from at least three independent experiments . The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated by LPS [ 20 ng / mL , 20 min The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . regular Akt were used for a loading control . Presented are the representative image from at least three independent experiments . Atx - heterozygous ( Atx - Het ) mice and wild type littermates ( C ) were stimulated by LPS indicated time point The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . RasGAP levels were used for a loading control . Presented are the representative image from at least three independent experiments . ( D ) Raw264 . 7 cells were stimulated by LPS with the ATX inhibitor . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . , β - Actin were used for a loading control . Presented are the representative image from at least three independent experiments . The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with TLR2 ligand Peptidoglycan ( PGN , 30 ng / mL ) ( E ) for the indicated time points . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments . The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with Pam3CSK4 ( 1 μg / mL ) ( F ) for the indicated time points . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments . The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with IL - 1β ( 100 ng / mL ) for the indicated time points . : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments . The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with TNFα ( 25 ng / mL ) ( H ) for the indicated time points . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2, were used for a loading control. Presented are the representative image from at least three independent experiments.The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. regular Akt were used for a loading control. Presented are the representative image from at least three independent experiments.Atx-heterozygous (Atx-Het) mice and wild type littermates (C) were stimulated by LPS indicated time point The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. RasGAP levels were used for a loading control. Presented are the representative image from at least three independent experiments.(D) Raw264.7 cells were stimulated by LPS with the ATX inhibitor. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. , β-Actin were used for a loading control. Presented are the representative image from at least three independent experiments.The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TLR2 ligand Peptidoglycan (PGN, 30 ng/mL) (E) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with Pam3CSK4 (1 μg/mL) (F) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with IL-1β (100 ng/mL) for the indicated time points. : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TNFα (25 ng/mL) (H) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


Figure 7Peritoneal macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates ( A ) were stimulated with LPS ( 50 ng / mL , 8 h ) The culture medium was collected for ELISA to measure the level of secreted cytokines . All assays were performed in triplicate , and data are shown as mean ± SEM . Raw264 . 7 cells ( B ) were stimulated with LPS ( 50 ng / mL , 8 h ) in the presence / absence of the ATX inhibitor ( 50 μM , 30 min ) . The culture medium was collected for ELISA to measure the level of secreted cytokines . All assays were performed in triplicate , and data are shown as mean ± SEM . Peritoneal macrophages were stimulated with LPS or vehicle ( endotoxin free water ) , after which intracellular cytokine production was measured through FACS analysis . With the scatter dot plot , the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry . The overlay plot represents the cytometry data . Peritoneal macrophages were stimulated with LPS or vehicle ( endotoxin free water ) , after which intracellular cytokine production was measured through FACS analysis . With the scatter dot plot , the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry . The overlay plot represents the cytometry data . ( F ) To induce sepsis via LPS injection ( Shirey et al . , 2013 ; Voss et al . , 2016 ) , age ( 9 - 10 weeks ) - and sex - matched Atx ∆ ΜΕ / ∆ ΜΕ mice ( n = 9 ) and Atx + / + littermates ( n = 21 ) were injected with LPS ( i . p . 25 mg / kg ) . Survival was monitored for up to 16 h , and analyzed by the Kaplan - Meier method ( Log - rank P = 0 . 0004 ) . ( G ) Blood samples were collected from Atx ∆ ΜΕ / ∆ ΜΕ ( n = 7 ) and Atx + / + ( n = 6 ) mice 10 h after LPS injection , after which the serum TNFα protein level was measured . The data are shown as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates (A) were stimulated with LPS (50 ng/mL, 8 h) The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.Raw264.7 cells (B) were stimulated with LPS (50 ng/mL, 8 h) in the presence/absence of the ATX inhibitor (50 μM, 30 min). The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.Peritoneal macrophages were stimulated with LPS or vehicle (endotoxin free water), after which intracellular cytokine production was measured through FACS analysis. With the scatter dot plot, the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry. The overlay plot represents the cytometry data.Peritoneal macrophages were stimulated with LPS or vehicle (endotoxin free water), after which intracellular cytokine production was measured through FACS analysis. With the scatter dot plot, the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry. The overlay plot represents the cytometry data.(F) To induce sepsis via LPS injection (Shirey et al., 2013; Voss et al., 2016), age (9-10 weeks)- and sex-matched Atx∆ΜΕ/∆ΜΕ mice (n = 9) and Atx+/+ littermates (n = 21) were injected with LPS (i.p. 25 mg/kg). Survival was monitored for up to 16 h, and analyzed by the Kaplan-Meier method (Log-rank P=0.0004).(G) Blood samples were collected from Atx∆ΜΕ/∆ΜΕ (n = 7) and Atx+/+ (n = 6) mice 10 h after LPS injection, after which the serum TNFα protein level was measured. The data are shown as mean ± SEM.


Figure 8Confocal laser scanning micrographs of the macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates . Cells were incubated with IgG - opsonized latex beads in the absence / presence of LPS ( 20 ng / mL , 2 h ) and stained with Phalloidin ( F - actin ) and DAPI . Internalized beads were examined under a higher ( A ) Confocal laser scanning micrographs of the macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates . Cells were incubated with IgG - opsonized latex beads in the absence / presence of LPS ( 20 ng / mL , 2 h ) and stained with Phalloidin ( F - actin ) and DAPI . Internalized beads were examined under a lower magnification ( B ) to quantify % phagocytosis by dividing latex bead - positive cell numbers by the total number of DAPI - positive cells ( n = 11 - 16 per group ) . The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM ( B ) . * * P < 0 . 01 ( Mann - Whitney U test ) . ( C ) LPS - stimulated macrophages were co - incubated with IgG - opsonized latex beads for 40 min . Phagocytic cups were visualized by F - actin staining with Phalloidin - iFluor 647 . The arrow indicates the phagocytic cup formed in the plasma membrane . Scale bar is 10 μm . After LPS ( 20 ng / mL , 4 h ) treatment , LPS - induced iNos , Arginase - 1 ( Arg - 1 ) , and Ym - 1 mRNA expression were evaluated by semi - quantitative PCR ( D ) . With LPS stimulation ( 20 ng / mL , 8 h ) , iNOS protein production was examined by immunoblotting analysis ( E ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a higher(A)Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a lower magnification (B) to quantify % phagocytosis by dividing latex bead-positive cell numbers by the total number of DAPI-positive cells (n=11 - 16 per group). The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM (B). **P < 0.01 (Mann-Whitney U test).(C) LPS-stimulated macrophages were co-incubated with IgG-opsonized latex beads for 40 min. Phagocytic cups were visualized by F-actin staining with Phalloidin-iFluor 647. The arrow indicates the phagocytic cup formed in the plasma membrane. Scale bar is 10 μm.After LPS (20 ng/mL, 4 h) treatment, LPS-induced iNos, Arginase-1 (Arg-1), and Ym-1 mRNA expression were evaluated by semi-quantitative PCR (D).With LPS stimulation (20 ng/mL, 8 h), iNOS protein production was examined by immunoblotting analysis (E).


Figure 1 ( A ) in vitro kinase assays were performed using purified recombinant kinases ( ULK1 and ULK2 ) and substrate ( ATG16L1 ) in the presence of radiolabelled ATP . ULK and ATG16L1 inputs were examined by western blot ( WB ) and substrate phosphorylation was analyzed by autoradiography ( AR ) . Data information : Unless otherwise indicated experiments were performed three times . ( B ) Full - length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay . ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography . Data information : Unless otherwise indicated experiments were performed three times . ( D ) Full - length or mutated HA - ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay . Inputs were analysed by WB and target phosphorylation by AR . Data information : Unless otherwise indicated experiments were performed three times . ( E ) HEK293A cells were transfected with wild - type or phospho - dead ATG16L1 in the presence of wild - type or kinase - dead ULK1 . Phosphorylation of ATG16L1 ( S278 or S287 ) and inputs were examined by WB . Data information : Unless otherwise indicated experiments were performed three times .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP. ULK and ATG16L1 inputs were examined by western blot (WB) and substrate phosphorylation was analyzed by autoradiography (AR). Data information: Unless otherwise indicated experiments were performed three times.(B) Full-length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay. ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography. Data information: Unless otherwise indicated experiments were performed three times.(D) Full-length or mutated HA-ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay. Inputs were analysed by WB and target phosphorylation by AR. Data information: Unless otherwise indicated experiments were performed three times.(E) HEK293A cells were transfected with wild-type or phospho-dead ATG16L1 in the presence of wild-type or kinase-dead ULK1. Phosphorylation of ATG16L1 (S278 or S287) and inputs were examined by WB. Data information: Unless otherwise indicated experiments were performed three times.


Figure 2 ( A ) Wild - type , ULK1 / 2 double knockout ( dKO ) , or IKKα KO mouse embryonic fibroblasts ( MEFs ) were incubated with either complete medium , amino acid - deficient DMEM , or HBSS for 1 hour . Samples were immunoblotted using the indicated antibodies . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test . ( B ) Wild - type , ULK1 / 2 dKO , or IKKα KO MEFs were infected with log phase Salmonella for 2 hours ; bacteria - containing media was then removed and cells were incubated with gentamycin ( 50 µg / mL ) - containing DMEM for 2 hours . Samples were immunoblotted using the indicated antibodies . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test . ( C ) Wild - type , ULK1 / 2 dKO , or IKKα KO MEFs cells were infected with Salmonella for 1 hours . Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B . Representative images are shown ( scale bars , 10 μm and 3 μm ) . Quantification was generated from 8 fields of view from a representative experiment . The experiments were repeated twice . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test . ( D ) Wild - type , ULK1 / 2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour . Xenophagy rates were examined through Colony Forming Unit ( CFU ) assays . Quantification of infection rates by immunofluorescence is demonstrated in the right panel . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

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Figure 2(A) Wild-type, ULK1/2 double knockout (dKO), or IKKα KO mouse embryonic fibroblasts (MEFs) were incubated with either complete medium, amino acid-deficient DMEM, or HBSS for 1 hour. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(B) Wild-type, ULK1/2 dKO, or IKKα KO MEFs were infected with log phase Salmonella for 2 hours; bacteria-containing media was then removed and cells were incubated with gentamycin (50 µg/mL)-containing DMEM for 2 hours. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(C) Wild-type, ULK1/2 dKO, or IKKα KO MEFs cells were infected with Salmonella for 1 hours. Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B. Representative images are shown (scale bars, 10 μm and 3 μm). Quantification was generated from 8 fields of view from a representative experiment. The experiments were repeated twice. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(D) Wild-type, ULK1/2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour. Xenophagy rates were examined through Colony Forming Unit (CFU) assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


Figure 3 ( A ) HEK293A cells were transfected with either flag - tagged WT ATG16L1 or T300A ATG16L1 . ULK1 was co - transfected in increasing amounts where indicated . Cleavage of ATG16L1 was analyzed by WB of whole cell lysates . Levels of ATG16L1 cleavage were quantified from 3 biological repeats ( right panel ) . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test . ( B ) HEK293A cells were transfected with either tagged wild - type , T300A , or S278 / T300A ATG16L1 in the presence or absence of ULK1 . Cleavage of ATG16L1 was analyzed by WB . Levels of ATG16L1 cleavage were measured from 3 biological repeats ( right panel ) . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test . ( C ) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan - caspase inhibitor Z - VAD - FMK ( 15 µM ) for 4 hours . Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test . ( D ) Wild - type or T300A - expressing HEK293A were treated with Salmonella in the presence or absence of ULK1 / 2 inhibitor ( 16 μM ) for the indicated time points . Expression of ATG16L1 was analysed by WB . The experiments were performed twice . ( E ) ATG16L1 knock - out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour . Xenophagy rates were examined through CFU assays . Quantification of infection rates by immunofluorescence is demonstrated in the right panel . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation and p values were determined by Student ' s T - Test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) HEK293A cells were transfected with either flag-tagged WT ATG16L1 or T300A ATG16L1. ULK1 was co-transfected in increasing amounts where indicated. Cleavage of ATG16L1 was analyzed by WB of whole cell lysates. Levels of ATG16L1 cleavage were quantified from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(B) HEK293A cells were transfected with either tagged wild-type, T300A, or S278/T300A ATG16L1 in the presence or absence of ULK1. Cleavage of ATG16L1 was analyzed by WB. Levels of ATG16L1 cleavage were measured from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(C) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan-caspase inhibitor Z-VAD-FMK (15 µM) for 4 hours. Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(D) Wild-type or T300A-expressing HEK293A were treated with Salmonella in the presence or absence of ULK1/2 inhibitor (16 μM) for the indicated time points. Expression of ATG16L1 was analysed by WB. The experiments were performed twice.(E) ATG16L1 knock-out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour. Xenophagy rates were examined through CFU assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


Figure 4 ( A ) Wild - type MEF cells were infected with Salmonella for 25 minutes . Phospho - ATG16L1 , total ATG16L1 , and LPS were stained and analysed by immunofluorescence . Representative immunofluorescent images are shown ( scale bars , 10 μm and 1 μm ) . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test . ( B ) Wild - type and ULK1 / 2 dKO were infected with Salmonella for 25 minutes . Immunofluorescence was performed using antibodies against LPS and ATG16L1 . Representative immunofluorescent images are shown on the left panel ( scale bars , 10 μm and 2 μm ) . Quantification of ATG16L1 - positive bacteria from 7 fields of view from a representative experiment is shown in the right panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test . ( C ) ATG16L1 knock - out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour . Bacteria were stained using anti - LPS antibodies to analyze localization in addition to ATG16L1 . Representative immunofluorescent images of ATG16L1 and LPS are shown ( scale bars , 5μm and 1 μm ) . Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test . ( D ) ATG16L1 knock - out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour . Bacteria were stained using anti - LPS antibodies to analyze localization in addition to the autophagy marker LC3B . Representative immunofluorescent images of LC3B and LPS are shown ( scale bars , 5μm and 1 μm ) . Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean and p values were determined by Student ' s T - Test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Wild-type MEF cells were infected with Salmonella for 25 minutes. Phospho-ATG16L1, total ATG16L1, and LPS were stained and analysed by immunofluorescence. Representative immunofluorescent images are shown (scale bars, 10 μm and 1 μm). Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.(B) Wild-type and ULK1/2 dKO were infected with Salmonella for 25 minutes. Immunofluorescence was performed using antibodies against LPS and ATG16L1. Representative immunofluorescent images are shown on the left panel (scale bars, 10 μm and 2 μm). Quantification of ATG16L1-positive bacteria from 7 fields of view from a representative experiment is shown in the right panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.(C) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to ATG16L1. Representative immunofluorescent images of ATG16L1 and LPS are shown (scale bars, 5μm and 1 μm). Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.(D) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to the autophagy marker LC3B. Representative immunofluorescent images of LC3B and LPS are shown (scale bars, 5μm and 1 μm). Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


Figure 1 ( D ) Number of putative enhancer regions per cell line . Bar depicts the mean . Diamonds represent individual numbers in each cell line and are color - coded according to cancer type ( E ) Heat maps of DNase I signal for enhancer regions ( rows ) . For intergenic enhancers ( left ) the signal is aligned at the upstream ( relative to + strand ) enhancer edge , shown from 4 kbp upstream to 5 kbp downstream , and ordered by increasing length of enhancer regions . For intragenic enhancers ( right ) the signal was aligned at the upstream and downstream enhancer edges and shown within ± 4 kbp . Exemplary data from LoVo colorectal cancer cells . ( F ) Heat maps as in ( E ) showing composite ChIP - seq peak coverage from 326 TFs

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Figure 1(D) Number of putative enhancer regions per cell line. Bar depicts the mean. Diamonds represent individual numbers in each cell line and are color-coded according to cancer type(E) Heat maps of DNase I signal for enhancer regions (rows). For intergenic enhancers (left) the signal is aligned at the upstream (relative to + strand) enhancer edge, shown from 4 kbp upstream to 5 kbp downstream, and ordered by increasing length of enhancer regions. For intragenic enhancers (right) the signal was aligned at the upstream and downstream enhancer edges and shown within ± 4 kbp. Exemplary data from LoVo colorectal cancer cells. (F) Heat maps as in (E) showing composite ChIP-seq peak coverage from 326 TFs


Figure 2 ( B ) Bidirectional promoters show higher mRNA synthesis . Boxplots showing TT - seq signal for mRNAs from unidirectional ( n = 39 , 376 ) and bidirectional ( n = 83 , 363 ) promoters . Bidirectional and unidirectional promoters were defined for each cell line separately and then plotted together . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Notches represent 95 % confidence intervals for the median values . Outliers not shown . P - value by two - sided Mann - Whitney U - test .

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Figure 2(B) Bidirectional promoters show higher mRNA synthesis. Boxplots showing TT-seq signal for mRNAs from unidirectional (n=39,376) and bidirectional (n=83,363) promoters. Bidirectional and unidirectional promoters were defined for each cell line separately and then plotted together. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.