( C ) Immunofluorescence microscopy . Nbr1f / f hepatocytes were infected with LacZ or Cre adenovirus for 48 hrs and , when indicated , challenged with 10 μM As ( III ) for 12 hrs . The cells were then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * P < 0 . 01 , and * * * P < 0 . 001 as determined by Welch ' s t - test . ( D ) Role of NBR1 in the p62 - liquid droplets and the p62 - mediated Nrf2 activation . Oxidative stress up - regulates the level of NBR1 , which enhances the phosphorylation and liquid - droplet formation of p62 . The resulting p62 - droplets are resistant to autophagic degradation and serve as signaling nodes for the activation of Nrf2 . Since p62 is one of the targets of Nrf2 , increased level of p62 in cooperation with NBR1 promote the formation of liquid droplets and contributes to the persistent activation of Nrf2 against oxidative stress .

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(C) Immunofluorescence microscopy. Nbr1f/f hepatocytes were infected with LacZ or Cre adenovirus for 48 hrs and, when indicated, challenged with 10 μM As (III) for 12 hrs. The cells were then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. **P < 0.01, and ***P < 0.001 as determined by Welch's t-test. (D) Role of NBR1 in the p62-liquid droplets and the p62-mediated Nrf2 activation. Oxidative stress up-regulates the level of NBR1, which enhances the phosphorylation and liquid-droplet formation of p62. The resulting p62-droplets are resistant to autophagic degradation and serve as signaling nodes for the activation of Nrf2. Since p62 is one of the targets of Nrf2, increased level of p62 in cooperation with NBR1 promote the formation of liquid droplets and contributes to the persistent activation of Nrf2 against oxidative stress.


( B ) Genotyping of Brd4fl / fl mice with ERT2 - Cre ( Cre ) or without ERT2 - Cre ( + / + ) . Tamoxifen treatment deleted 2 . 2 kb Brd4fl / fl fragment from Cre / + and Cre / Cre mice ( top ) . Cre / + , Cre / Cre and + / + mice were confirmed by the presence of ERT2 - Cre band ( Cre ) , or its absence ( WT ) ( bottom ) .

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(B) Genotyping of Brd4fl/fl mice with ERT2-Cre (Cre) or without ERT2 -Cre (+/+). Tamoxifen treatment deleted 2.2 kb Brd4fl/fl fragment from Cre/+ and Cre/Cre mice (top). Cre/+, Cre/Cre and +/+ mice were confirmed by the presence of ERT2 -Cre band (Cre), or its absence (WT) (bottom).


( C ) WT and Brd4 KO cells were tested for mRNA ( left ) and the protein ( right ) by qRT - PCR and immune blot assays . Values represent the average of three experiments + / - SD .

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(C) WT and Brd4 KO cells were tested for mRNA (left) and the protein (right) by qRT-PCR and immune blot assays. Values represent the average of three experiments +/- SD.


( D ) Vav - cre mediated Brd4 depletion was verified for cells from Brd4fl / fl Vav - Cre mice ( fetal liver or spleen ) by qRT - PCR and Immunoblot ( left and right ) . Values represent the average of three experiments + / - SD .

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(D) Vav-cre mediated Brd4 depletion was verified for cells from Brd4fl/fl Vav-Cre mice (fetal liver or spleen) by qRT-PCR and Immunoblot (left and right). Values represent the average of three experiments +/- SD.


( E ) Photographic images of E14 . 5 embryos from WT and KO mice .

(E) Photographic images of E14.5 embryos from WT and KO mice.


( F ) Corresponding Flow cytometric profile of Lin - fetal liver cells stained with C - Kit and Sca - 1 antibodies examining early hematopoietic progenitor population ( LSK ) ( left ) . LSK populations were further divided by the Flt3 marker to detect HSCs and MPPs ( middle ) . The number of total cells , that of LSKs , HSCs , MPPs in a fetal liver were estimated on indicated days of embryonic development ( E13 . 5 , E14 . 5 and E17 . 5 ) . Values are the average of 3 - 5 embryos + / - S . D ( right ) .

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(F) Corresponding Flow cytometric profile of Lin- fetal liver cells stained with C-Kit and Sca-1 antibodies examining early hematopoietic progenitor population (LSK) (left). LSK populations were further divided by the Flt3 marker to detect HSCs and MPPs (middle). The number of total cells, that of LSKs, HSCs, MPPs in a fetal liver were estimated on indicated days of embryonic development (E13.5, E14.5 and E17.5). Values are the average of 3-5 embryos +/- S.D (right).


( H ) Representative flow cytometric profile of CD71 - cells stained for the myeloid and lymphoid markers . Values are the average of 3 E17 . 5 embryos + / - S . D .

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(H) Representative flow cytometric profile of CD71- cells stained for the myeloid and lymphoid markers. Values are the average of 3 E17.5 embryos +/- S.D.


( A ) Flow cytometry profiles of peritoneal cells from WT and KO mice stained for CD19 and F4 / 80 ( left ) . FACS profile of CD19 - subset showing CD11b + and F4 / 80 + subsets of cells from WT and KO macrophage populations ( middle ) . Cell numbers ( right ) represent the average of 4 WT and 4 Brd4 KO mice + / - S . D . Note a reduction in F4 / 80 + cells from Brd4 KO peritoneum .

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(A) Flow cytometry profiles of peritoneal cells from WT and KO mice stained for CD19 and F4/80 (left). FACS profile of CD19- subset showing CD11b+ and F4/80+ subsets of cells from WT and KO macrophage populations (middle). Cell numbers (right) represent the average of 4 WT and 4 Brd4 KO mice +/- S.D. Note a reduction in F4/80+ cells from Brd4 KO peritoneum.


( B ) Flow cytometry profiles of peritoneal cells from IL - 4C injected WT and KO mice ( left ) . Cell numbers ( right ) represent the average of 4 WT and 4 Brd4 KO mice + / - S . D .

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(B) Flow cytometry profiles of peritoneal cells from IL-4C injected WT and KO mice (left). Cell numbers (right) represent the average of 4 WT and 4 Brd4 KO mice +/- S.D.


( C ) Flow cytometry profiles of BrdU incorporation and Ki67 , ARG1 expression in peritoneal cells ( gated for F4 / 80 + , CD19 - macrophages ) from IL - 4C treated WT and KO mice ( left ) . The numbers of cells positive for BrdU , Ki67 and ARG1 ( right ) . The numbers represent the average of three experiments + / - S . D .

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(C) Flow cytometry profiles of BrdU incorporation and Ki67, ARG1 expression in peritoneal cells (gated for F4/80+, CD19- macrophages) from IL-4C treated WT and KO mice (left). The numbers of cells positive for BrdU, Ki67 and ARG1 (right). The numbers represent the average of three experiments +/- S.D.


( D ) Microarray analysis of macrophage gene expression in IL - 4C treated WT or Brd4 KO mice . Macrophages were isolated from peritoneal cells by the MACS system . Differentially expressed genes were identified with a cut - off line of log 2fold change > 2 difference with p value of < 0 . 05 . Red dots indicate differentially expressed genes ( KO vs WT ) and gray dots are those below the cut - off line .

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(D) Microarray analysis of macrophage gene expression in IL-4C treated WT or Brd4 KO mice. Macrophages were isolated from peritoneal cells by the MACS system. Differentially expressed genes were identified with a cut-off line of log 2fold change >2 difference with p value of < 0.05. Red dots indicate differentially expressed genes (KO vs WT) and gray dots are those below the cut-off line.


( F ) qRT - PCR confirmation of representative genes downregulated in KO macrophages from IL - 4C treated mice . Values represent the average of three independent experiments + / - S . D .

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(F) qRT-PCR confirmation of representative genes downregulated in KO macrophages from IL-4C treated mice. Values represent the average of three independent experiments +/- S.D.


( B ) Flow cytometry profiles of CD11b + , F4 / 80 + macrophage population harvested on Day 7 from untreated ( WT ) or tamoxifen treated ( KO ) cultures ( left ) . Histogram depicting F4 / 80 expression levels in WT and KO macrophages ( middle ) . Total number of macrophage yields calculated from > 15 independent experiments . Values represent the average of 21 WT and 24 KO experiments + / - S . D .

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(B) Flow cytometry profiles of CD11b+, F4/80+ macrophage population harvested on Day 7 from untreated (WT) or tamoxifen treated (KO) cultures (left). Histogram depicting F4/80 expression levels in WT and KO macrophages (middle). Total number of macrophage yields calculated from > 15 independent experiments. Values represent the average of 21 WT and 24 KO experiments +/- S.D.


( C ) MA plots of RNA - seq data from untreated ( UT ) or LPS treated ( 4h ) WT and KO macrophages . Differentially expressed genes were identified by a cut off line of log2FC > 1 ( FDR < 0 . 1 ) . ( D ) Venn diagram depicting the number of LPS induced genes in WT and KO macrophages .

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(C) MA plots of RNA-seq data from untreated (UT) or LPS treated (4h) WT and KO macrophages. Differentially expressed genes were identified by a cut off line of log2FC>1 (FDR < 0.1). (D) Venn diagram depicting the number of LPS induced genes in WT and KO macrophages.


( E ) qRT - PCR analysis of genes representative of the BRD4 independent group ( unaffected by KO ) , dependent group ( downregulated by KO ) or upregulated by KO . Data were obtained from three independent macrophage samples . Values are the average of 3 + / - S . D .

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(E) qRT-PCR analysis of genes representative of the BRD4 independent group (unaffected by KO), dependent group (downregulated by KO) or upregulated by KO. Data were obtained from three independent macrophage samples. Values are the average of 3+/- S.D.


( B ) Heat maps of BRD4 occupancy on the promoter region ( TSS + / - 5kb ) , aligned by the degree of BRD4 signal intensity in UT and LPS treated macrophages .

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(B) Heat maps of BRD4 occupancy on the promoter region (TSS +/- 5kb), aligned by the degree of BRD4 signal intensity in UT and LPS treated macrophages.


( D ) Immunoblot detection of BRD4 protein levels in UT and LPS treated WT macrophages . Forty μg of whole cell extracts from UT or LPS treated ( 1h or 4h ) macrophages using antibody for BRD4 or β - actin ( loading control ) .

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(D) Immunoblot detection of BRD4 protein levels in UT and LPS treated WT macrophages. Forty μg of whole cell extracts from UT or LPS treated (1h or 4h) macrophages using antibody for BRD4 or β-actin (loading control).


( E ) Binding of BRD4 and PolII and distribution of histone marks ( H3K27ac and H3K9ac ) on BRD4 dependent and independent genes ( from - 5kb of TSS to + 5kb of transcription end site ( TES ) ) in UT and LPS treated WT and KO macrophages . Normalized ChIP - seq signals ( rpm / bp ) are shown on the Y axis . Arrows show reduced signals in Brd4 KO macrophages .

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(E) Binding of BRD4 and PolII and distribution of histone marks (H3K27ac and H3K9ac) on BRD4 dependent and independent genes (from -5kb of TSS to +5kb of transcription end site (TES)) in UT and LPS treated WT and KO macrophages. Normalized ChIP-seq signals (rpm/bp) are shown on the Y axis. Arrows show reduced signals in Brd4 KO macrophages.


( F ) Gene tracks of normalized island - filtered ChIP - seq peaks for BRD4 , Pol II , and indicated histone modification marks , along with RNA - seq peaks for a BRD4 independent gene ( Ccl9 ) and a BRD4 dependent gene ( Fcgr2b ) . Values on the Y axis are normalized ChIP - seq signals ( rpm / bp ) .

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(F) Gene tracks of normalized island-filtered ChIP-seq peaks for BRD4, Pol II, and indicated histone modification marks, along with RNA-seq peaks for a BRD4 independent gene (Ccl9) and a BRD4 dependent gene (Fcgr2b). Values on the Y axis are normalized ChIP-seq signals (rpm/bp).


( A ) BRD4 bound enhancers within 12 . 5kb were stitched together and ranked by BRD4 ChIP - seq signals ( rpm ) in untreated ( UT , left ) and LPS treated ( middle ) WT macrophages . The dotted line distinguished SEs from TEs . Gene names with the ranking in parenthesis are among genes closest to SEs . Venn diagram ( right ) showing nearest genes common in UT and LPS treated macrophages .

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(A) BRD4 bound enhancers within 12.5kb were stitched together and ranked by BRD4 ChIP-seq signals (rpm) in untreated (UT, left) and LPS treated (middle) WT macrophages. The dotted line distinguished SEs from TEs. Gene names with the ranking in parenthesis are among genes closest to SEs. Venn diagram (right) showing nearest genes common in UT and LPS treated macrophages.


( B ) Genome - wide gain and loss of BRD4 SEs upon LPS stimulation . Based on SE coordinates ( rpm from LPS treated samples / rpm in untreated samples ) , changes greater or less than 2 folds ( Log2 ) were plotted as gain or loss .

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(B) Genome-wide gain and loss of BRD4 SEs upon LPS stimulation. Based on SE coordinates (rpm from LPS treated samples/rpm in untreated samples), changes greater or less than 2 folds (Log2) were plotted as gain or loss.


( C ) Examples of nearest genes that gained or lost SEs upon LPS treatment . Gene tracks for gained and lost loci ( near Tnf and Csf1r ) showing clusters of BRD4 , Pol II and H3K27ac signals . Red bar represents SE length .

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(C) Examples of nearest genes that gained or lost SEs upon LPS treatment. Gene tracks for gained and lost loci (near Tnf and Csf1r) showing clusters of BRD4, Pol II and H3K27ac signals. Red bar represents SE length.


( D ) Meta - gene alignment of BRD4 signals on BRD4 ranked SEs . Pol II and H3K27ac ChIP - seq reads were aligned centering on BRD4 SEs of LPS treated WT macrophages .

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(D) Meta-gene alignment of BRD4 signals on BRD4 ranked SEs. Pol II and H3K27ac ChIP-seq reads were aligned centering on BRD4 SEs of LPS treated WT macrophages.


( E ) Enhancer plots obtained by the H3K27ac - ranking in LPS stimulated WT and KO macrophages .

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(E) Enhancer plots obtained by the H3K27ac-ranking in LPS stimulated WT and KO macrophages.


( A ) Heat maps of p65 binding centered at the TSS on BRD4 enriched promoters .

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(A) Heat maps of p65 binding centered at the TSS on BRD4 enriched promoters.


( B ) Meta - gene profile of p65 ChIP - seq signals ( rpm / bp ) from the TSS to TES and + / - 5kb on BRD4 independent genes ( top ) and BRD4 dependent genes ( bottom ) .

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(B) Meta-gene profile of p65 ChIP-seq signals (rpm/bp) from the TSS to TES and +/- 5kb on BRD4 independent genes (top) and BRD4 dependent genes (bottom).


( C ) Gene tracks of BRD4 and p65 ChIP - seq peaks and RNA - seq profiles on a BRD4 independent gene ( Il1b ) , a gene upregulated in Brd4KO macrophages ( Ccl7 ) , and a BRD4 dependent gene ( Ptgs2 ) .

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(C) Gene tracks of BRD4 and p65 ChIP-seq peaks and RNA-seq profiles on a BRD4 independent gene (Il1b), a gene upregulated in Brd4KO macrophages (Ccl7), and a BRD4 dependent gene (Ptgs2).


( D ) Enhancers ranked by increasing p65 signals in WT ( left ) and Brd4 KO macrophages ( right ) .

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(D) Enhancers ranked by increasing p65 signals in WT (left) and Brd4 KO macrophages (right).


( E ) Meta - gene analysis of p65 occupancy on enhancers in WT ( red ) and KO macrophages ( Blue ) .

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(E) Meta-gene analysis of p65 occupancy on enhancers in WT (red) and KO macrophages (Blue).


a . b . c . d . Chest computed tomographic scans of Patient 1 were obtained on the day of admission ( day 6 after the onset of illness ) . Bilateral focal consolidation , lobar consolidation , and patchy consolidation were clearly observed , especially in the lower lung .

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a. b. c. d. Chest computed tomographic scans of Patient 1 were obtained on the day of admission (day 6 after the onset of illness). Bilateral focal consolidation, lobar consolidation, and patchy consolidation were clearly observed, especially in the lower lung.


e . Chest radiograph of patient 1 was obtained on day 5 after admission ( day 11 after the onset of illness ) . Bilateral diffuse patchy and fuzzy shadow were observed .

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e. Chest radiograph of patient 1 was obtained on day 5 after admission (day 11 after the onset of illness). Bilateral diffuse patchy and fuzzy shadow were observed.


d ) representative images of Ki67 and p16Ink4a immunofluorescence and SA - β - Gal staining ; e ) % of Ki67 positive cells ( 28 days after co - culture ) ; f ) % of p16INK4A positive cells ( 28days after co - culture ) ; g ) % of SA - β - Gal positive cells ( 28 days after co - culture ) ; h ) Western blotting for p21 and quantification ; i ) MitoSOX fluorescence ;

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d) representative images of Ki67 and p16Ink4a immunofluorescence and SA-β-Gal staining; e) % of Ki67 positive cells (28 days after co-culture); f) % of p16INK4A positive cells (28days after co-culture); g) % of SA-β-Gal positive cells (28 days after co-culture); h) Western blotting for p21 and quantification; i) MitoSOX fluorescence;


h ) Representative Immuno - FISH micrographs ( using telomere ‐ specific ( CCCTAA ) peptide nucleic acid probe and anti - 53BP1 antibody ) 20 days following co - culture ; i ) quantification of mean TAF and % TAF in MRC5 fibroblasts 8 and 20 days following neutrophil co - culture . Data are mean ± S . E . M . of 3 independent experiments ;

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h) Representative Immuno-FISH micrographs (using telomere‐specific (CCCTAA) peptide nucleic acid probe and anti-53BP1 antibody) 20 days following co-culture; i) quantification of mean TAF and % TAF in MRC5 fibroblasts 8 and 20 days following neutrophil co-culture. Data are mean±S.E.M. of 3 independent experiments;


J - L ) mRNA expression of p21 ( j ) , IL - 6 ( k ) and IL - 8 ( l ) 20 days after neutrophil co - culture ; Data are mean ± S . E . M . of 2 - 3 independent experiments .

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J-L) mRNA expression of p21 (j), IL-6 (k) and IL-8 (l) 20 days after neutrophil co-culture; Data are mean±S.E.M. of 2-3 independent experiments.


f ) mean number of TAF in hepatocytes ; g ) % of TAF positive hepatocytes ; h ) number of p21 positive hepatocytes per field ; i ) number of p16Ink4a positive hepatocytes per field .

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f) mean number of TAF in hepatocytes; g) % of TAF positive hepatocytes; h) number of p21 positive hepatocytes per field; i) number of p16Ink4a positive hepatocytes per field.


e ) Representative micrographs of neutrophil marker Ly6G and senescent - associated marker p21 ( 96h ) ; f ) number of Ly6G positive cells per field ( 96h ) ; g ) % of TAF positive hepatocytes ( 96h ) ; h ) mean number of TAF in hepatocytes ( 96h ) ; i ) total number of γH2A . X foci in hepatocytes ( 96h ) ; j ) Lactate dehydrogenase ( LD ) activity ( 96h ) ; k ) p21 positive cells per field ( 96h ) .

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e) Representative micrographs of neutrophil marker Ly6G and senescent-associated marker p21 (96h); f) number of Ly6G positive cells per field (96h); g) % of TAF positive hepatocytes (96h); h) mean number of TAF in hepatocytes (96h); i) total number of γH2A.X foci in hepatocytes (96h); j) Lactate dehydrogenase (LD) activity (96h); k) p21 positive cells per field (96h).


m ) Representative immunoFISH ( Telomere - FISH and 8 - oxodG ) ; n ) mean number of 8 - oxodG co - localizing with telomeres in hepatocytes ; o ) Mean telomere FISH intensity in hepatocytes .

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

m) Representative immunoFISH (Telomere-FISH and 8-oxodG); n) mean number of 8-oxodG co-localizing with telomeres in hepatocytes; o) Mean telomere FISH intensity in hepatocytes.


a ) RNA - sequencing data showing expression of genes associated with neutrophil recruitment during liver aging ;

a) RNA-sequencing data showing expression of genes associated with neutrophil recruitment during liver aging;


i ) Comparison between p16Ink4a mRNA levels of young ( 3 months old ) and old INK - ATTAC mice ( 28 - 29 month old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 - 8 mice per age group ; j ) % of TAF positive hepatocytes in old INK - ATTAC mice ( 28 - 29 months old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 - 6 mice per group ; k ) Liver neutrophils ( Ly6G ) in old INK - ATTAC mice ( 28 - 29 months old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 - 7 mice per group ;

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

i) Comparison between p16Ink4a mRNA levels of young (3 months old) and old INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-8 mice per age group; j) % of TAF positive hepatocytes in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-6 mice per group; k) Liver neutrophils (Ly6G) in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-7 mice per group;


p ) Quantification of liver neutrophils ( Ly6G ) in mice injected with vehicle or Cxcl1 . Data are mean ± SEM of n = 7 - 10 mice per group ; q ) Quantification of the % of TAF positive hepatocytes in mice injected with vehicle or Cxcl1 . Data are mean ± SEM of n = 6 - 7 mice per group ;

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

p) Quantification of liver neutrophils (Ly6G) in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 7-10 mice per group ; q) Quantification of the % of TAF positive hepatocytes in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 6-7 mice per group;


Histology : all metastatic lesions are composed of poorly differentiated cells with an epithelial ' flavour ' . The neoplastic population is mainly arranged in solid nests and sheets with focal rudimental gland formation . Scale bar : 50µm .

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

Histology: all metastatic lesions are composed of poorly differentiated cells with an epithelial 'flavour'. The neoplastic population is mainly arranged in solid nests and sheets with focal rudimental gland formation. Scale bar: 50µm.


Triplicate samples of L _ 03 , L _ 04A , L _ 07A , L _ 09 , L _ 10 ; L _ 12C are compared with the expression profiles deposited in the TCGA dataset of a spectrum of primary tumors or metastases ( meta ) from known origin . Two ovarian cancers analysed in house ( CTR _ OV1 and CTR _ OV2 ) were used as controls and have expression profiles matching the profiles displayed by the ovarian cancers listed in TCGA ( purple box ) . The acronyms are as follow : ACC : adrenocortical carcinoma ; BLCA : bladder urothelial carcinoma ; BRCA : breast invasive carcinoma ; CESC : cervical squamous cell carcinoma and endocervical adenocarcinoma ; CHOL : cholangiocarcinoma ; COAD : colon adenocarcinoma ; DLBC : lymphoid neoplasm diffuse large B - cell lymphoma ; ESCA : oesophageal carcinoma ; GBM : glioblastoma multiforme ; HNSC : head and neck squamous cell carcinoma ; KICH : kidney chromophobe ; KIRC : kidney renal clear cell carcinoma ; KIRP : kidney renal papillary cell carcinoma ; LAML : acute myeloid leukaemia ; LGG : brain lower grade glioma ; LIHC : liver hepatocellular carcinoma ; LUAD : lung adenocarcinoma ; LUSC : lung squamous cell carcinoma ; MESO : mesothelioma ; OV : ovarian serous cystadenocarcinoma ; PAAD : pancreatic adenocarcinoma ; PCPG : pheochromocytoma and paraganglioma ; PRAD : prostate adenocarcinoma ; READ : rectum adenocarcinoma ; SARC : sarcoma ; SKCM : skin cutaneous melanoma ; STAD : stomach adenocarcinoma ; TGCT : testicular germ cell tumors ; THCA : thyroid carcinoma ; THYM : thymoma ; UCEC : uterine corpus endometrial carcinoma ; UCS : uterine carcinosarcoma ; UVM : uveal melanoma .

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

Triplicate samples of L_03, L_04A, L_07A, L_09, L_10; L_12C are compared with the expression profiles deposited in the TCGA dataset of a spectrum of primary tumors or metastases (meta) from known origin. Two ovarian cancers analysed in house (CTR_OV1 and CTR_OV2) were used as controls and have expression profiles matching the profiles displayed by the ovarian cancers listed in TCGA (purple box). The acronyms are as follow: ACC: adrenocortical carcinoma; BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; DLBC: lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: oesophageal carcinoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LAML: acute myeloid leukaemia; LGG: brain lower grade glioma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; MESO: mesothelioma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; STAD: stomach adenocarcinoma; TGCT: testicular germ cell tumors; THCA: thyroid carcinoma; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma; UCS: uterine carcinosarcoma; UVM: uveal melanoma.


Somatic mutations distribution : SNVs and InDels displayed by each metastasis ( listed in the Y axis ) were detected by Strelka2 tool ( Kim et al . , 2018 ) . Brown traits represent single mutations . The number of fully shared ( red ) , partially shared ( orange ) or private ( yellow ) mutations is indicated in the horizontal bar below . Five cancer - associated genes ( TP53 , ARID2 , NTRK1 , SMARCA4 and ZFHX3 ) mutated in all metastases are highlighted .

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

Somatic mutations distribution: SNVs and InDels displayed by each metastasis (listed in the Y axis) were detected by Strelka2 tool (Kim et al., 2018). Brown traits represent single mutations. The number of fully shared (red), partially shared (orange) or private (yellow) mutations is indicated in the horizontal bar below. Five cancer-associated genes (TP53, ARID2, NTRK1, SMARCA4 and ZFHX3) mutated in all metastases are highlighted.


Genetic similarity among metastases : the heatmap has been drawn according to the Jaccard index . The similarity ranges from 58 % to 82 % among all pairs with the exception of L _ 08 due to the scarce number of mutations .

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

Genetic similarity among metastases: the heatmap has been drawn according to the Jaccard index. The similarity ranges from 58% to 82% among all pairs with the exception of L_08 due to the scarce number of mutations.


Variant allele frequency of fully shared mutations present in copy neutral regions . The box plot represents the fluctuation of the VAF ( in the Y axis ) of single mutations ( grey dots ) in each metastasis ; each box represents the upper and lower quartiles , while the central short black line within each box represents the median ; whiskers indicate variability outside the upper and lower quartiles .

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

Variant allele frequency of fully shared mutations present in copy neutral regions. The box plot represents the fluctuation of the VAF (in the Y axis) of single mutations (grey dots) in each metastasis; each box represents the upper and lower quartiles, while the central short black line within each box represents the median; whiskers indicate variability outside the upper and lower quartiles.


( A ) WT , Irgm1 - / - , Irgm2 - / - and Irgm3 - / - mice ( n = 4 mice / genotype ) were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hours post injection ( hpi ) and concentration of various cytokines determined via a preconfigured Luminex multiplex panel . Relative concentration ( fold change relative mean of WT ) is shown for the indicated cytokines ( absolute cytokine concentrations of same experiment are shown in Fig EV1 ) .

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

(A) WT, Irgm1-/-, Irgm2-/- and Irgm3-/- mice (n = 4 mice/genotype) were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hours post injection (hpi) and concentration of various cytokines determined via a preconfigured Luminex multiplex panel. Relative concentration (fold change relative mean of WT) is shown for the indicated cytokines (absolute cytokine concentrations of same experiment are shown in Fig EV1).


( B ) WT ( n = 9 ) , Irgm1 - / - ( n = 7 ) , Irgm2 - / - ( n = 9 ) and Casp1 - / - Casp11 - / - ( n = 7 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hpi and concentration of IL - 1β , IL - 18 , and TNFα was measured via ELISA .

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

(B) WT (n = 9), Irgm1-/- (n = 7), Irgm2-/- (n = 9) and Casp1-/-Casp11-/- (n = 7) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, IL-18, and TNFα was measured via ELISA.


( A ) qPCR measurement of IL - 1β , and TNF - α mRNA levels in WT and Irgm2 - / - BMMs following 8 - hour stimulation with LPS ( 1 µg / ml ) .

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

(A) qPCR measurement of IL-1β, and TNF-α mRNA levels in WT and Irgm2-/- BMMs following 8-hour stimulation with LPS (1 µg/ml).


( B ) WT , Irgm2 - / - and Casp1 - / - Casp11 - / - BMMs were treated for 24 hours with LPS at indicated doses and supernatant TNFα , IL - 1β , and IL - 18 was measured by ELISA .

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

(B) WT, Irgm2-/- and Casp1-/-Casp11-/- BMMs were treated for 24 hours with LPS at indicated doses and supernatant TNFα, IL-1β, and IL-18 was measured by ELISA.


( C ) IFNγ - primed WT and Irgm2 - / - BMMs were treated with LPS , Pam3CSK4 , poly ( I : C ) or a combination of Pam3CSK4 and poly ( I : C ) ( 1 µg / ml for all treatments ) for 24 hours and cell supernatant IL - 1β and IL - 18 concentrations were assessed by ELISA .

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

(C) IFNγ-primed WT and Irgm2-/- BMMs were treated with LPS, Pam3CSK4, poly(I:C) or a combination of Pam3CSK4 and poly(I:C) (1 µg/ml for all treatments) for 24 hours and cell supernatant IL-1β and IL-18 concentrations were assessed by ELISA.


( D ) WT , Irgm2 - / - , Casp1 - / - Casp11 - / - and Irgm2 - / - Casp1 - / - Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) and IL - 1β , IL - 18 , and LDH release were assessed at 24 hours post treatment ( hpt ) .

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

(D) WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and IL-1β, IL-18, and LDH release were assessed at 24 hours post treatment (hpt).


( E ) IFNγ - primed WT , Irgm2 - / - , Casp1 - / - Casp11 - / - and Irgm2 - / - Casp1 - / - Casp11 - / - BMMs were treated with LPS ( 5 µg / ml ) for 4 hours and subsequently stained with anti - ASC antibody and Hoechst stain ( DNA / nuclei ) . Representative images are shown with white arrows pointing at ASC specks . Number of ASC specks per nuclei was quantified . Scale bars : 20 µm .

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

(E) IFNγ-primed WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (5 µg/ml) for 4 hours and subsequently stained with anti-ASC antibody and Hoechst stain (DNA/nuclei). Representative images are shown with white arrows pointing at ASC specks. Number of ASC specks per nuclei was quantified. Scale bars: 20 µm.


( F ) IFNγ - primed WT , Irgm2 - / - , Casp1 - / - Casp11 - / - and Irgm2 - / - Casp1 - / - Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) for 24 h and cell lysates and supernatants collected . Protein levels in cell lysates ( caspase - 1 , and actin ) and supernatants ( caspase - 1 p20 ) were visualized via immunoblotting .

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

(F) IFNγ-primed WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) for 24 h and cell lysates and supernatants collected. Protein levels in cell lysates (caspase-1, and actin) and supernatants (caspase-1 p20) were visualized via immunoblotting.


( A ) IFNγ primed WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 5 µg / ml ) for 4 h . Following treatment , cells were stained with anti - ASC antibody and Hoechst ( DNA / nuclei ) . Representative images of ASC specks ( white arrows point at specks ) are shown and number of ASC specks per nuclei quantified . Scale bars : 20 µm .

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

(A) IFNγ primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with LPS (5 µg/ml) for 4 h. Following treatment, cells were stained with anti-ASC antibody and Hoechst (DNA/nuclei). Representative images of ASC specks (white arrows point at specks) are shown and number of ASC specks per nuclei quantified. Scale bars: 20 µm.


( B ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) . IL - 1β , IL - 18 , and LDH release were assessed at 24 hours - post treatment ( hpt ) .

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

(B) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with LPS (1 µg/ml). IL-1β, IL-18, and LDH release were assessed at 24 hours-post treatment (hpt).


( C ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were infected with E . coli K - 12 at indicated MOIs and cell viability assessed via CellTiter - Glo . Cell death was calculated as a function of relative viability to uninfected cells .

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

(C) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were infected with E. coli K-12 at indicated MOIs and cell viability assessed via CellTiter-Glo. Cell death was calculated as a function of relative viability to uninfected cells.


( D ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were infected with E . coli K - 12 ( MOI 25 ) and 24 hpi supernatant IL - 1β and IL - 18 levels were measured by ELISA .

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

(D) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were infected with E. coli K-12 (MOI 25) and 24 hpi supernatant IL-1β and IL-18 levels were measured by ELISA.


( E ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with OMVs at indicated concentrations for 24 h . Cell viability was assessed via CellTiter - Glo and cell death was calculated as a function of relative viability to untreated cells .

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

(E) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with OMVs at indicated concentrations for 24 h. Cell viability was assessed via CellTiter-Glo and cell death was calculated as a function of relative viability to untreated cells.


( F ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with OMVs ( 1 µg / ml ) and 24 hpt cell supernatant IL - 1β and IL - 18 levels were measured via ELISA .

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

(F) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with OMVs (1 µg/ml) and 24 hpt cell supernatant IL-1β and IL-18 levels were measured via ELISA.


( A ) WT , and Irgm2 - / - BMMs were stimulated overnight with IFNγ or left untreated and cell lysates were collected . Lysates were assessed for Casp11 , Gbp2 , and actin protein levels via immunoblotting .

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

(A) WT, and Irgm2-/- BMMs were stimulated overnight with IFNγ or left untreated and cell lysates were collected. Lysates were assessed for Casp11, Gbp2, and actin protein levels via immunoblotting.


( B ) IFNγ - primed and unprimed WT , Irgm2 - / - , Gbpchr3 - / - and Aim2 - / - BMMs were infected with Francisella novicida ( MOI 10 ) and LDH release measured at 4 hpi .

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

(B) IFNγ-primed and unprimed WT, Irgm2-/-, Gbpchr3-/- and Aim2-/- BMMs were infected with Francisella novicida (MOI 10) and LDH release measured at 4 hpi.


( C ) WT , Irgm2 - / - , Gbpchr3 - / - and Irgm2 - / - Gbpchr3 - / - BMMs were treated with LPS ( 1 µg / ml ) . IL - 1β , IL - 18 , and LDH release were assessed at 24 hpt .

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

(C) WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml). IL-1β, IL-18, and LDH release were assessed at 24 hpt.


( D ) IFNγ - primed WT , Irgm2 - / - , Gbpchr3 - / - and Irgm2 - / - Gbpchr3 - / - BMMs were treated with LPS ( 1 µg / ml ) for 24 hours and cell lysates and supernatants collected . Protein levels in cell lysates ( Caspase - 1 , and actin ) and supernatants ( Caspase - 1 p20 ) were visualized via immunoblotting .

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

(D) IFNγ-primed WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml) for 24 hours and cell lysates and supernatants collected. Protein levels in cell lysates (Caspase-1, and actin) and supernatants (Caspase-1 p20) were visualized via immunoblotting.


( A ) WT , Irgm2 - / - , and Nlrp3 - / - BMMs were treated with LPS ( 0 . 1 µg / ml ) for 3 h followed by nigericin for 1 h and IL - 1β / LDH release was measured .

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(A) WT, Irgm2-/-, and Nlrp3-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin for 1 h and IL-1β/LDH release was measured.


( B ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 0 . 1 µg / ml ) for 3 h followed by nigericin and / or MCC950 for 1 h and IL - 1β / LDH release was measured .

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(B) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin and/or MCC950 for 1 h and IL-1β/LDH release was measured.


( C ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) and / or MCC950 for 24 h and IL - 1β / LDH release was measured .

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(C) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and/or MCC950 for 24 h and IL-1β/LDH release was measured.


( D ) IFNγ - primed WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were transfected with LPS using lipofectamine LTX and LDH release was measured at 2 hpt .

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(D) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were transfected with LPS using lipofectamine LTX and LDH release was measured at 2 hpt.


( E ) IFNγ - primed WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were co - treated with LPS ( indicated doses ) and Listeria monocytogenes ( MOI 10 ) and LDH release measured at 4 hpt .

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(E) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were co-treated with LPS (indicated doses) and Listeria monocytogenes (MOI 10) and LDH release measured at 4 hpt.


( A ) WT ( n = 9 ) , Irgm2 - / - ( n = 10 ) , Casp11 - / - ( n = 7 ) and Irgm2 - / - Casp11 - / - ( n = 8 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Morbidity and mortality were observed for 48 h at 3 hour intervals .

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(A) WT (n = 9), Irgm2-/- (n = 10), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 8) mice were injected i.p. with LPS (8 mg/kg). Morbidity and mortality were observed for 48 h at 3 hour intervals.


( B ) WT ( n = 9 ) , Irgm2 - / - ( n = 8 ) , Casp11 - / - ( n = 7 ) and Irgm2 - / - Casp11 - / - ( n = 10 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hpi and concentration of IL - 1β , and IL - 18 was measured by ELISA . Data shown are from 2 pooled experiments .

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(B) WT (n = 9), Irgm2-/- (n = 8), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 10) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, and IL-18 was measured by ELISA. Data shown are from 2 pooled experiments.


( B ) WT ( n = 9 ) , Irgm2 - / - ( n = 8 ) , Casp11 - / - ( n = 7 ) and Irgm2 - / - Casp11 - / - ( n = 10 ) mice were injected i . p . with LPS ( 8 mg / kg ) .

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(B) WT (n = 9), Irgm2-/- (n = 8), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 10) mice were injected i.p. with LPS (8 mg/kg). Serum


( A ) WT , Irgm2 - / - , Irgm3 - / - , Irgm2 - / - Irgm3 - / - , panIrgm - / - , and Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) or infected with E . coli K - 12 ( MOI 25 ) and IL - 1β , and LDH release were assessed at 24 hpt ( n = 3 independent experiments ) . ( B ) IFNγ - primed WT , Irgm2 - / - , Irgm3 - / - , Irgm2 - / - Irgm3 - / - , panIrgm - / - , and Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) or infected with E . coli K - 12 ( MOI 25 ) and IL - 1β , and LDH release were assessed at 8 hpt / hpi ( n = 3 independent experiments ) .

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(A) WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 24 hpt (n = 3 independent experiments). (B) IFNγ-primed WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 8 hpt/ hpi (n = 3 independent experiments).


( A ) WT , Irgm2 - / - , Irgm3 - / - , Irgm2 - / - Irgm3 - / - , panIrgm - / - , and Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) or infected with E . coli K - 12 ( MOI 25 ) and IL - 1β , and LDH release were assessed at 24 hpt ( n = 3 independent experiments ) .

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(A) WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 24 hpt (n = 3 independent experiments).


( C ) WT ( n = 15 ) , Irgm2 - / - ( n = 12 ) , and panIrgm - / - ( n = 13 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hpi and concentration of IL - 1β and TNFα was measured via ELISA . Data shown are from 3 pooled experiments .

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(C) WT (n = 15), Irgm2-/- (n = 12), and panIrgm-/- (n = 13) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β and TNFα was measured via ELISA. Data shown are from 3 pooled experiments.


( D ) WT , Irgm2 - / - , panIrgm - / - and Casp11 - / - mice ( n = 8 mice / genotype ) were injected i . p . with LPS ( 8 mg / kg body weight ) . Morbidity and mortality were observed for 48 h at 3 - h intervals .

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(D) WT, Irgm2-/-, panIrgm-/- and Casp11-/- mice (n = 8 mice/genotype) were injected i.p. with LPS (8 mg/kg body weight). Morbidity and mortality were observed for 48 h at 3-h intervals.


Immunostaining of H3K27ac in mouse germinal vesicle ( GV ) oocytes ( n = 8 ) , metaphase II ( MII ) oocytes ( n = 7 ) , zygotes ( n = 7 ) , early 2 - cell ( 2C ) ( n = 7 ) and late 2C ( n = 9 ) embryos . Right panel is the quantification of H3K27ac immunostaining relative signal intensities at different stages . Scale bar : 20 μm . Data information : For boxplots in A the central band represents the median . The lower and upper edges of the box represent the first and third quartiles , respectively . The whiskers of the boxplot extend to 1 . 5 times inter - quartile range ( IQR ) .

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Immunostaining of H3K27ac in mouse germinal vesicle (GV) oocytes (n = 8), metaphase II (MII) oocytes (n = 7), zygotes (n = 7), early 2-cell (2C) (n = 7) and late 2C (n = 9) embryos. Right panel is the quantification of H3K27ac immunostaining relative signal intensities at different stages. Scale bar: 20 μm. Data information: For boxplots in A the central band represents the median. The lower and upper edges of the box represent the first and third quartiles, respectively. The whiskers of the boxplot extend to 1.5 times inter-quartile range (IQR).


Immunostaining of H3K27ac in A - 485 treated embryos versus control ( DMSO treated ) at different time points . Scale bar : 20 μm .

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Immunostaining of H3K27ac in A-485 treated embryos versus control (DMSO treated) at different time points. Scale bar: 20 μm.


Developmental rate of embryos treated with A - 485 versus control .

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Developmental rate of embryos treated with A-485 versus control.


Immunostaining of H3K27ac in TSA treated embryos under different concentrations versus control ( DMSO treated ) at 28 hpi . Scale bar : 20 μm .

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Immunostaining of H3K27ac in TSA treated embryos under different concentrations versus control (DMSO treated) at 28 hpi. Scale bar: 20 μm.


Developmental rate of embryos treated with TSA at different time points ( 0 - 20 hpi and 8 - 28 hpi ) versus control .

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Developmental rate of embryos treated with TSA at different time points (0-20 hpi and 8-28 hpi) versus control.


A Live cell imaging reveals that expression of GFP ‐ KSHV ‐ TK , but not GFP induces contraction of HeLa cells ( see Supplementary Movies S1 and S2 ) . The time from the start of imaging is indicated above each panel , and the scale bars represent 18 μm . Quantification of GFP ‐ KSHV ‐ TK ‐ induced cell contraction . The graphs show cell area ( μm2 ) of cells expressing the indicated protein , and error bars represent SEM from three independent experiments in which n = 224 for GFP and n = 203 for GFP ‐ KSHV ‐ TK . * * * P 0 . 001 .

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A Live cell imaging reveals that expression of GFP‐KSHV‐TK, but not GFP induces contraction of HeLa cells (see Supplementary Movies S1 and S2). The time from the start of imaging is indicated above each panel, and the scale bars represent 18 μm. Quantification of GFP‐KSHV‐TK‐induced cell contraction. The graphs show cell area (μm2) of cells expressing the indicated protein, and error bars represent SEM from three independent experiments in which n = 224 for GFP and n = 203 for GFP‐KSHV‐TK. ***P 0.001.


B Phase images taken from Supplementary Movie S3 illustrating GFP ‐ KSHV ‐ TK induced blebbing of contracted cells . The time from the start of imaging is indicated above each panel , and the scale bars represent 18 μm .

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B Phase images taken from Supplementary Movie S3 illustrating GFP‐KSHV‐TK induced blebbing of contracted cells. The time from the start of imaging is indicated above each panel, and the scale bars represent 18 μm.


C Scanning electron microscopy of HeLa cells expressing GFP or GFP ‐ KSHV ‐ TK .

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C Scanning electron microscopy of HeLa cells expressing GFP or GFP‐KSHV‐TK.


D Immunofluorescence microscopy images illustrating the association of GFP ‐ KSHV ‐ TK , but not GFP , with actinstress fibres . Scale bar , 10 μm .

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D Immunofluorescence microscopy images illustrating the association of GFP‐KSHV‐TK, but not GFP, with actinstress fibres. Scale bar, 10 μm.


A Co ‐ expression of GFP ‐ RhoN19 but not GFP inhibits RFP ‐ KSHV ‐ TK ‐ induced cell contraction . Graphs represent cell area ( μm2 ) of KSHV ‐ TK , RhoN19 or KSHV ‐ TK and RhoN19 expressing cells . Error bars represent SEM from 3 independent experiments , n = 204 for KSHV ‐ TK , n = 223 for RhoN19 and n = 189 for KSHV ‐ TK and RhoN19 .

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A Co‐expression of GFP‐RhoN19 but not GFP inhibits RFP‐KSHV‐TK‐induced cell contraction. Graphs represent cell area (μm2) of KSHV‐TK, RhoN19 or KSHV‐TK and RhoN19 expressing cells. Error bars represent SEM from 3 independent experiments, n = 204 for KSHV‐TK, n = 223 for RhoN19 and n = 189 for KSHV‐TK and RhoN19.


B GFP ‐ KSHV ‐ TK ‐ induced cell contraction does not occur in cells treated with the ROCK and myosin II inhibitors Y ‐ 27632 and blebbistatin , respectively . Error bars represent SEM from three independent experiments , n = 213 for DMSO , n = 189 for Y ‐ 27632 and n = 245 . * * * P 0 . 001 .

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B GFP‐KSHV‐TK‐induced cell contraction does not occur in cells treated with the ROCK and myosin II inhibitors Y‐27632 and blebbistatin, respectively. Error bars represent SEM from three independent experiments, n = 213 for DMSO, n = 189 for Y‐27632 and n = 245. ***P 0.001.


C Immunoblot analysis of rhotekinpull ‐ down ( P . D . ) assays reveals that GFP ‐ KSHV ‐ TK increases the level of GTP ‐ bound RhoA ( GTP ‐ RhoA ) . Actin is included as a loading control , and the input lysates are indicated .

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C Immunoblot analysis of rhotekinpull‐down (P.D.) assays reveals that GFP‐KSHV‐TK increases the level of GTP‐bound RhoA (GTP‐RhoA). Actin is included as a loading control, and the input lysates are indicated.


D GFP ‐ KSHV ‐ TK but not GFP induces the loss of phosphotyrosine , phospho ‐ FAK Y925 and phospho ‐ paxillin Y31 epitopes from focal adhesions .

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D GFP‐KSHV‐TK but not GFP induces the loss of phosphotyrosine, phospho‐FAK Y925 and phospho‐paxillin Y31 epitopes from focal adhesions.


B Immunoblot analysis of BHK cells infected with MuHV ‐ 4 and MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] or uninfected cells using antibodies against phosphotyrosine ( p ‐ Tyr ) , GFP , MuHV ‐ 4 TK , ORF17 ( capsid ) and glycoprotein N .

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B Immunoblot analysis of BHK cells infected with MuHV‐4 and MuHV‐4 [g‐KSHV‐TK] or uninfected cells using antibodies against phosphotyrosine (p‐Tyr), GFP, MuHV‐4 TK, ORF17 (capsid) and glycoprotein N.


C Immunofluorescence microscopy analysis of MuHV ‐ 4 ‐ and MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] ‐ infected cells with the indicated antibodies reveals GFP ‐ KSHV ‐ TK expressed in the context of lytic infection ( MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] ) induces the loss of phospho ‐ FAK Y925 and paxillin Y31 from focal adhesions . White arrowheads indicate examples of infected cells expressing GFP ‐ KSHV ‐ TK or GFP ( indicating WT MuHV ‐ 4 infection , BAC + virus ) .

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C Immunofluorescence microscopy analysis of MuHV‐4‐ and MuHV‐4 [g‐KSHV‐TK]‐infected cells with the indicated antibodies reveals GFP‐KSHV‐TK expressed in the context of lytic infection (MuHV‐4 [g‐KSHV‐TK]) induces the loss of phospho‐FAK Y925 and paxillin Y31 from focal adhesions. White arrowheads indicate examples of infected cells expressing GFP‐KSHV‐TK or GFP (indicating WT MuHV‐4 infection, BAC+ virus).


D Immunofluorescence microscopy analysis reveals that GFP ‐ KSHV ‐ TK expressed in the context of lytic infection ( MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] ) does not alter focal adhesion integrity ( paxillin ) in the presence of the ROCK inhibitor Y ‐ 27632 .

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D Immunofluorescence microscopy analysis reveals that GFP‐KSHV‐TK expressed in the context of lytic infection (MuHV‐4 [g‐KSHV‐TK]) does not alter focal adhesion integrity (paxillin) in the presence of the ROCK inhibitor Y‐27632.


E Immunofluorescence microscopy analysis of paxillin in the lytically induced rKSHV . 219 / Vero cell line in the presence or absence of Y ‐ 27632 .

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E Immunofluorescence microscopy analysis of paxillin in the lytically induced rKSHV.219/Vero cell line in the presence or absence of Y‐27632.


A Cellular localisation and immunoblot analysis of phosphotyrosine ( upper panel ) and GFP ( lower panel ) in cells expressing wild ‐ type or kinase ‐ dead GFP ‐ KSHV ‐ TK .

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A Cellular localisation and immunoblot analysis of phosphotyrosine (upper panel) and GFP (lower panel) in cells expressing wild‐type or kinase‐dead GFP‐KSHV‐TK.


B Scanning electron microscopy of HeLa cells expressing GFP or GFP ‐ KSHV ‐ TK ‐ DEAD .

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B Scanning electron microscopy of HeLa cells expressing GFP or GFP‐KSHV‐TK‐DEAD.


C Immunofluorescence microscopy analysis of HeLa cells expressing GFP ‐ KSHV ‐ TK ‐ DEAD ( white arrowheads ) with the indicated phospho antibodies .

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C Immunofluorescence microscopy analysis of HeLa cells expressing GFP‐KSHV‐TK‐DEAD (white arrowheads) with the indicated phospho antibodies.


D Immunofluorescence microscopy images of the localisation of FAK and zyxin in HeLa cells expressing GFP ‐ KSHV ‐ TK ‐ DEAD ( white arrowheads ) .

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D Immunofluorescence microscopy images of the localisation of FAK and zyxin in HeLa cells expressing GFP‐KSHV‐TK‐DEAD (white arrowheads).


E Purified recombinant GST ‐ EBV ‐ TK , GST ‐ MuHV ‐ 4 - TK and GST ‐ KSHV ‐ TKimmunoblotted for phosphotyrosine and GST following a sequential dephosphorylation and kinase reaction .

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E Purified recombinant GST‐EBV‐TK, GST‐MuHV‐4-TK and GST‐KSHV‐TKimmunoblotted for phosphotyrosine and GST following a sequential dephosphorylation and kinase reaction.


F Purified recombinant GST ‐ KSHV ‐ TK and GST ‐ KSHV ‐ TK ‐ KINASE ‐ DEAD immunoblotted for phosphotyrosine and GST .

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F Purified recombinant GST‐KSHV‐TK and GST‐KSHV‐TK‐KINASE‐DEAD immunoblotted for phosphotyrosine and GST.


G Immunoblot analysis demonstrating that the N ‐ terminus of KSHV ‐ TK is only auto ‐ phosphorylated by the kinase domain in cis .

G Immunoblot analysis demonstrating that the N‐terminus of KSHV‐TK is only auto‐phosphorylated by the kinase domain in cis.


A Immunoblot analysis of N ‐ terminal deletions of KSHV ‐ TK demonstrates that the first 100 amino acids are required for tyrosine phosphorylation .

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A Immunoblot analysis of N‐terminal deletions of KSHV‐TK demonstrates that the first 100 amino acids are required for tyrosine phosphorylation.


B Immunoblot analysis reveals that mutation of tyrosines 65 , 85 and 120 ablates tyrosine phosphorylation of the KSHV ‐ TK .

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B Immunoblot analysis reveals that mutation of tyrosines 65, 85 and 120 ablates tyrosine phosphorylation of the KSHV‐TK.


C - E Immunofluorescence microscopy analysis with the indicated antibodies reveals that mutation of tyrosines 65 , 85 and 120 to phenylalanine abrogates the ability of GFP ‐ KSHV ‐ TK to induce loss of phosphotyrosine epitopes , phospho ‐ paxillin and phospho ‐ FAK from focal adhesions .

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C-E Immunofluorescence microscopy analysis with the indicated antibodies reveals that mutation of tyrosines 65, 85 and 120 to phenylalanine abrogates the ability of GFP‐KSHV‐TK to induce loss of phosphotyrosine epitopes, phospho‐paxillin and phospho‐FAK from focal adhesions.


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F Immunoblot analysis of level of GTP ‐ bound and total RhoA in cells expressing the indicated GFP ‐ tagged KSHV ‐ TK proteins .

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F Immunoblot analysis of level of GTP‐bound and total RhoA in cells expressing the indicated GFP‐tagged KSHV‐TK proteins.


G Immunofluorescence microscopy analysis reveals that localisation of paxillin at focal adhesions is unaffected by KSHV ‐ TK ‐ Y3F expressed in the context of lytic infection .

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G Immunofluorescence microscopy analysis reveals that localisation of paxillin at focal adhesions is unaffected by KSHV‐TK‐Y3F expressed in the context of lytic infection.


A Immunoblot analysis reveals that GFP ‐ KSHV ‐ TK WT , Y2F and Y3F but not GFP ‐ KSHV ‐ TK ‐ DEAD associate with FAK .

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A Immunoblot analysis reveals that GFP‐KSHV‐TK WT, Y2F and Y3F but not GFP‐KSHV‐TK‐DEAD associate with FAK.


B GFP ‐ KSHV ‐ TK does not displace paxillin or zyxin from focal adhesions ( white arrowheads ) or induce contraction in the absence of FAK . Scale bar , 20 μm .

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B GFP‐KSHV‐TK does not displace paxillin or zyxin from focal adhesions (white arrowheads) or induce contraction in the absence of FAK. Scale bar, 20 μm.


C GFP ‐ KSHV ‐ TK does not induce loss of zyxin and p ‐ Tyr from focal adhesions or contraction of HeLa cells depleted of paxillin . Error bars represent SEM from three independent experiments , n = 196 for mock siRNA and n = 229 for paxillin siRNA ; * * * P 0 . 001 . Scale bar , 20 μm .

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C GFP‐KSHV‐TK does not induce loss of zyxin and p‐Tyr from focal adhesions or contraction of HeLa cells depleted of paxillin. Error bars represent SEM from three independent experiments, n = 196 for mock siRNA and n = 229 for paxillin siRNA; ***P 0.001. Scale bar, 20 μm.


B Immunoblot analysis of GFP antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK but not Y2F or GFP associates with CrkI , II and L .

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B Immunoblot analysis of GFP antibody immunoprecipitates reveals that GFP‐KSHV‐TK but not Y2F or GFP associates with CrkI, II and L.


C Immunofluorescence microscopy analysis reveals CrkII associates with GFP ‐ KSHV ‐ TK but not the Y2F mutant . Scale bars , 20 μm .

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C Immunofluorescence microscopy analysis reveals CrkII associates with GFP‐KSHV‐TK but not the Y2F mutant. Scale bars, 20 μm.


D Immunoblot analysis of RFP antibody immunoprecipitates reveals that RFP ‐ KSHV ‐ TK specifically associates with the SH2 domain of CrkII and CrkL .

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D Immunoblot analysis of RFP antibody immunoprecipitates reveals that RFP‐KSHV‐TK specifically associates with the SH2 domain of CrkII and CrkL.


E Immunofluorescence microscopy analysis of phosphotyrosine epitopes in cells expressing GFP ‐ CrkII SH2 , GFP ‐ CrkL SH2 , RFP ‐ KSHV ‐ TK or a combination of CrkII / LSH2 and RFP ‐ KSHV ‐ TK . Arrowheads represent contracted KSHV ‐ TK ‐ expressing cells . The SH2 domains of CrkII or L block KSHV ‐ TK ‐ induced cell rounding . Scale bars , 20 μm .

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E Immunofluorescence microscopy analysis of phosphotyrosine epitopes in cells expressing GFP‐CrkII SH2, GFP‐CrkL SH2, RFP‐KSHV‐TK or a combination of CrkII/LSH2 and RFP‐KSHV‐TK. Arrowheads represent contracted KSHV‐TK‐expressing cells. The SH2 domains of CrkII or L block KSHV‐TK‐induced cell rounding. Scale bars, 20 μm.


B Immunoblot analysis reveals that GFP ‐ KSHV ‐ TK WT and Y2F but not Y120F associate with p85 of PI3 ‐ Kinase .

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B Immunoblot analysis reveals that GFP‐KSHV‐TK WT and Y2F but not Y120F associate with p85 of PI3‐Kinase.


C Immunoblot analysis of p85PI3 ‐ Kinase antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK and its Y2F mutant but not Y120F associate with p85 of PI3 ‐ Kinase .

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C Immunoblot analysis of p85PI3‐Kinase antibody immunoprecipitates reveals that GFP‐KSHV‐TK and its Y2F mutant but not Y120F associate with p85 of PI3‐Kinase.


D Immunoblot analysis of CrkII and CrkL antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK and the p85PI3 ‐ Kinase associate with CrkII and CrkL . No association is observed with the Y2F , Y3F or kinase ‐ dead TK .

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D Immunoblot analysis of CrkII and CrkL antibody immunoprecipitates reveals that GFP‐KSHV‐TK and the p85PI3‐Kinase associate with CrkII and CrkL. No association is observed with the Y2F, Y3F or kinase‐dead TK.


E Tyrosine phosphorylation of CrkII and CrkL is only upregulated in cells expressing GFP ‐ KSHV ‐ TK .

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E Tyrosine phosphorylation of CrkII and CrkL is only upregulated in cells expressing GFP‐KSHV‐TK.


F Immunoblot analysis reveals that recombinant His ‐ CrkII binds to and is phosphorylated by KSHV ‐ TK but not the Y2F or Y3F mutants .

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F Immunoblot analysis reveals that recombinant His‐CrkII binds to and is phosphorylated by KSHV‐TK but not the Y2F or Y3F mutants.


( A ) The expression pattern of FOXM1 in fetal human cortex at GW15 . VZ , ventricular zone ; ISVZ , inner subventricular zone ; OSVZ , outer subventricular zone . Right panels show higher magnification images . Arrows show that FOXM1 + cells were colocalized with SOX2 + cells . Scale bars , 100μm ( left ) ; 50μm ( right ) .

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(A) The expression pattern of FOXM1 in fetal human cortex at GW15. VZ, ventricular zone; ISVZ, inner subventricular zone; OSVZ, outer subventricular zone. Right panels show higher magnification images. Arrows show that FOXM1+cells were colocalized with SOX2+ cells. Scale bars, 100μm (left); 50μm (right).


( B ) Staining for NESTIN and FOXM1 in human neural progenitor cells cultured in vitro . Down panels show higher magnification images . Scale bars , 50μm ( up ) ; 10μm ( down ) .

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(B) Staining for NESTIN and FOXM1 in human neural progenitor cells cultured in vitro. Down panels show higher magnification images. Scale bars, 50μm(up); 10μm(down).


( D ) Staining for Pax6 in E15 . 5 mice after IUE at E13 . 5 . Arrowheads indicate Pax6 + cell in enlarged SVZ . Right panels show higher magnification images . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bars , 50μm ( left ) ; 20μm ( right ) .

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(D) Staining for Pax6 in E15.5 mice after IUE at E13.5. Arrowheads indicate Pax6+ cell in enlarged SVZ. Right panels show higher magnification images. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars,50μm(left); 20μm (right).


( G ) Staining for upper neuron marker Cux1 in P3 mice after IUE at E13 . 5 . Arrowhead and asterisk indicate deformation of the cortical surface at regions containing electroporated cells . Right panels show higher magnification images . Arrows show GFP + Cux1 + double - positive cells . Scale bars , 100μm ( left ) ; 20μm ( right ) .

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(G) Staining for upper neuron marker Cux1 in P3 mice after IUE at E13.5. Arrowhead and asterisk indicate deformation of the cortical surface at regions containing electroporated cells. Right panels show higher magnification images. Arrows show GFP+ Cux1+ double-positive cells. Scale bars,100μm(left); 20μm (right).


( A ) Representative images of whole brains from wild - type ( WT ) and hFOXM1f / + - NesCre ( hFOXM1 - cKI ) mice pups at P0 . Scale bar , 100μm . ( B ) Quantification of cortical width and length at P0 ( WT n = 7 ; hFOXM1 - cKI n = 7 ; two - tailed unpaired t - test , P = 0 . 0361for width , P = 0 . 2866 for length ) . ( C ) Quantification of brain / body weight at P0 ( WT n = 9 ; hFOXM1 - cKI n = 9 ; two - tailed unpaired t - test , P = 0 . 0004 ) . ( D ) Quantification of cortex thickness at P0 ( WT n = 7 ; hFOXM1 - cKI n = 11 ; two - tailed unpaired t - test , P = 0 . 0002 ) . ( E )

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(A) Representative images of whole brains from wild-type (WT) and hFOXM1f/+-NesCre (hFOXM1-cKI) mice pups at P0. Scale bar,100μm. (B) Quantification of cortical width and length at P0 (WT n=7; hFOXM1-cKI n=7; two-tailed unpaired t-test, P= 0.0361for width, P= 0.2866 for length). (C) Quantification of brain/body weight at P0 (WT n=9; hFOXM1-cKI n=9; two-tailed unpaired t-test, P= 0.0004). (D) Quantification of cortex thickness at P0 (WT n=7; hFOXM1-cKI n=11; two-tailed unpaired t-test, P= 0.0002). (E)


( E ) Staining for Satb2 and Ctip2 in P0 WT and hFOXM1 - cKI mice folding structure . Asterisks indicate gyrus and arrows indicate sulcus structures . A1 , A2 , B1and B2 ( DAPI ) are high - magnification images . Scale bars , 200μm ( left ) ; 100μm ( right ) . ( F ) Quantification of GI of WT and hFOXM1 - cKI mice pups at P0 . The quantification of the GI was analyzed in pre - selected hFOXM1 - cKI mice with folding . ( n = 6 each group ; two - tailed u unpaired t - test , P = 0 . 0010 ) .

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(E) Staining for Satb2 and Ctip2 in P0 WT and hFOXM1-cKI mice folding structure. Asterisks indicate gyrus and arrows indicate sulcus structures. A1, A2, B1and B2 (DAPI) are high-magnification images. Scale bars, 200μm(left); 100μm(right). (F) Quantification of GI of WT and hFOXM1-cKI mice pups at P0. The quantification of the GI was analyzed in pre-selected hFOXM1-cKI mice with folding. (n = 6 each group; two-tailed u unpaired t-test, P= 0.0010).


( H ) Graph shows the number of Satb2 + cells in 4 × 104µm2 at P0 ( WT n = 5 ; hFOXM1 - cKI n = 5 ; two - tailed unpaired t - test , P = 0 . 0086 ) . ( I ) Graph shows the number of Ctip2 + cells in 4 × 104µm2 at P0 ( WT n = 5 ; hFOXM1 - cKI n = 5 ; two - tailed unpaired t - test , P = 0 . 0025 ) . (

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(H) Graph shows the number of Satb2+cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0086). (I) Graph shows the number of Ctip2 +cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0025). (


( K ) Staining for Cux1 in P0 WT and hFOXM1 - cKI mice . Scale bar , 100μm . ( I ) Graph shows the number of Cux1 + cells in 4 × 104µm2 at P0 ( WT n = 3 ; hFOXM1 - cKI n = 3 ; two - tailed unpaired t - test , P = 0 . 0451 ) .

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(K) Staining for Cux1 in P0 WT and hFOXM1-cKI mice. Scale bar,100μm. (I) Graph shows the number of Cux1+cells in 4×104µm2 at P0 (WT n=3; hFOXM1-cKI n=3; two-tailed unpaired t-test, P= 0.0451). D


( A ) Staining for Pax6 and p - Vim in E15 WT and hFOXM1 - cKI mice . The right A and B are high - magnification images . Arrows indicate double positive of Pax6 + p - Vim + cells with cells with a basal process ( arrowheads ) . Scale bars , 50μm ( left ) ; 5μm ( right ) . ( B ) Graph shows the percentage of Pax6 + cells in enlarged SVZ ( n = 6 each group ; two - tailed unpaired t - test , P = 0 . 0002 ) . ( C ) Graph shows the number of Pax6 + p - Vim + cells in 4x10 ^ 4 μm2 in enlarged SVZ ( n = 6 each group ; two - tailed unpaired t - test , P = 0 . 0003 ) . (

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(A) Staining for Pax6 and p-Vim in E15 WT and hFOXM1-cKI mice. The right A and B are high-magnification images. Arrows indicate double positive of Pax6+ p-Vim+ cells with cells with a basal process (arrowheads). Scale bars, 50μm (left); 5μm (right). (B) Graph shows the percentage of Pax6+ cells in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0002). (C) Graph shows the number of Pax6+ p-Vim+ cells in 4x10^4 μm2 in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0003). (D


( D ) Staining for Sox2 in E15 WT and hFOXM1 - cKI mice . Scale bar , 50μm . ( E ) Graph shows the number of Sox2 + cells in 4x104 μm2 in enlarged SVZ ( WT n = 3 ; hFOXM1 - cKI n = 4 ; two - tailed unpaired t - test , P = 0 . 0292 ) . (

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(D) Staining for Sox2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (E) Graph shows the number of Sox2+ cells in 4x104 μm2 in enlarged SVZ (WT n=3; hFOXM1-cKI n=4; two-tailed unpaired t-test, P=0.0292). (


( F ) Staining for Tbr2 in E15 WT and hFOXM1 - cKI mice . Scale bar , 50μm . ( G ) Graph shows the number of Tbr2 + cells in 4x104 μm2 in enlarged SVZ ( WT n = 6 ; hFOXM1 - cKI n = 9 ; two - tailed unpaired t - test , P = 0 . 0003 ) . (

(F) Staining for Tbr2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (G) Graph shows the number of Tbr2+ cells in 4x104 μm2 in enlarged SVZ (WT n=6; hFOXM1-cKI n=9; two-tailed unpaired t-test, P=0.0003). (


( H ) Staining for HOPX in E15 WT and hFOXM1 - cKI mice . Scale bar , 50μm . ( I ) Graph shows the number of HOPX + cells in 4 × 104 μm2 in enlarged SVZ ( n = 4 each group ; two - tailed unpaired t - test , P = 0 . 0044 ) . (

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(H) Staining for HOPX in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (I) Graph shows the number of HOPX+ cells in 4×104 μm2 in enlarged SVZ (n = 4 each group; two-tailed unpaired t-test, P= 0.0044). (


( J ) Staining for Ki67 in E15 WT and hFOXM1 - cKI mice . Scale bar , 50μm .

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(J) Staining for Ki67 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm.


( L ) The experimental scheme and staining for BrdU and EdU in E15 WT and hFOXM1 - cKI mice . White arrows represent BrdU and EdU double - positive cells . Scale bars , 50μm ( left ) ; 20μm ( right ) . ( M ) Graph shows the percentage of BrdU and EdU double - positive cells in E15 WT and hFOXM1 - cKI mice ( n = 6 each group ; two - tailed unpaired t - test , P = 0 . 0413 ) .

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(L) The experimental scheme and staining for BrdU and EdU in E15 WT and hFOXM1-cKI mice. White arrows represent BrdU and EdU double-positive cells. Scale bars, 50μm(left);20μm(right). (M) Graph shows the percentage of BrdU and EdU double-positive cells in E15 WT and hFOXM1-cKI mice (n = 6 each group; two-tailed unpaired t-test, P=0.0413). D


( B ) Cell immunostaining for HOPX in control , hFOXM1 - FL and mutants in mice primary neural progenitors 3 days following lentiviral infection . White arrows represent GFP HOPX double - positive cells . Scale bar , 50μm . ( C ) Graph shows the percentage of HOPX + cells among GFP + cells ( Control n = 3 ; hFOXM1 - FL n = 6 ; hFLΔexon9 n = 5 ; one - way ANOVA , F ( 2 , 12 ) = 29 . 13 , control vs . hFOXM1 , P = 0 . 0002 ; control vs . hFOXM1Δexon9 , P = 0 . 9080 ) . ( D ) Cell immunostaining for HOPX in control , mFoxm1 - FL and mutants in mice primary neural progenitors 3 days following lentiviral infection . White arrows represent GFP HOPX double - positive cells . Scale bar , 50μm . ( E ) ( E ) Graph shows the percentage of HOPX + cells among GFP + cells ( Control n = 4 ; mFoxm1 - FL n = 6 ; mFL + h exon9 n = 4 ; one - way ANOVA , F ( 2 , 11 ) = 8 . 163 , control vs . mFoxm1 , P = 0 . 4747 , control vs . mFoxm1 + h exon9 , P = 0 . 0066 ) . (

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(B) Cell immunostaining for HOPX in control, hFOXM1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm. (C) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=3; hFOXM1-FL n=6; hFLΔexon9 n=5; one-way ANOVA, F (2, 12) = 29.13, control vs. hFOXM1, P=0.0002; control vs. hFOXM1Δexon9, P=0.9080). (D) Cell immunostaining for HOPX in control, mFoxm1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm. (E) (E) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=4; mFoxm1-FL n=6; mFL+h exon9 n=4; one-way ANOVA, F (2, 11) = 8.163, control vs. mFoxm1, P= 0.4747, control vs. mFoxm1+h exon9, P= 0.0066). (


( J ) Staining for Tbr2 in E15 . 5 mice after IUE at E13 . 5 . Down panels show higher magnification images . White arrows represent GFP and TBR2 double - positive cells . Scale bars , 50μm . ( K ) Graph shows the percentage of GFP + Tbr2 + / GFP + in enlarged SVZ ( n = 3 each group ; one - way ANOVA , F ( 3 , 8 ) = 8 . 787 , P = 0 . 0065 ) .

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(J) Staining for Tbr2 in E15.5 mice after IUE at E13.5. Down panels show higher magnification images. White arrows represent GFP and TBR2 double-positive cells. Scale bars, 50μm. (K) Graph shows the percentage of GFP+Tbr2+/GFP+ in enlarged SVZ (n = 3 each group; one-way ANOVA, F (3,8) =8.787, P=0.0065). D


( C ) Volcano plots shows the gene expression states . The yellow dots are the up - regulated genes , and the blue dots are the down - regulated genes . The Lin28a gene ( red dot ) is significantly up - regulated .

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(C) Volcano plots shows the gene expression states. The yellow dots are the up-regulated genes, and the blue dots are the down-regulated genes. The Lin28a gene (red dot) is significantly up-regulated.


( D ) Western blot analysis for Lin28a in cortex of E15 WT and hFOXM1 - cKI mice . β - actin was detected as loading control .

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(D) Western blot analysis for Lin28a in cortex of E15 WT and hFOXM1-cKI mice. β-actin was detected as loading control.


( F ) Staining for Sox2 in E15 . 5 mice after IUE at E13 . 5 . Arrowheads indicate Sox2 + cell in enlarged SVZ . Scale bar , 50μm . ( G ) Graph shows the percentage of Sox2 + cells among GFP + cells in enlarged SVZ ( n = 3 each group ; two - tailed unpaired t - test , P = 0 . 0154 ) . ( H ) Graph shows the percentage of Sox2 + cells among GFP + cells ( n = 3 each group ; two - tailed unpaired t - test , P = 0 . 0018 ) .

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(F) Staining for Sox2 in E15.5 mice after IUE at E13.5. Arrowheads indicate Sox2+ cell in enlarged SVZ. Scale bar, 50μm. (G) Graph shows the percentage of Sox2+ cells among GFP+ cells in enlarged SVZ (n = 3 each group; two-tailed unpaired t-test, P=0.0154). (H) Graph shows the percentage of Sox2+ cells among GFP+ cells (n = 3 each group; two-tailed unpaired t-test, P=0.0018).


( I ) LIN28A - shRNA1 rescues the neuronal progenitor cell increase in hFOXM1 - cKI mice . Brain slice staining for Sox2 two days after IUE at E13 . Scale bar , 50μm . ( J ) Graph shows the percentage of Sox2 + cells among GFP + cells ( n = 3 each group ; one - way

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(I) LIN28A-shRNA1 rescues the neuronal progenitor cell increase in hFOXM1-cKI mice. Brain slice staining for Sox2 two days after IUE at E13. Scale bar, 50μm. (J) Graph shows the percentage of Sox2+ cells among GFP+ cells (n=3 each group; one-way A


( B ) Representative cases of FOXM1 + / + and FOXM1 - / - NPCs clones . Scale bar , 20μm . ( C ) Graph shows the number of cells per clone in FOXM1 + / + and FOXM1 - / - human NPCs ( FOXM1 + / + n = 12 ; FOXM1 - / - n = 13 ; at least three biological replicates . two - tailed unpaired t - test , P = 0 . 0014 ) . (

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(B) Representative cases of FOXM1+/+ and FOXM1-/- NPCs clones. Scale bar, 20μm. (C) Graph shows the number of cells per clone in FOXM1+/+ and FOXM1-/- human NPCs (FOXM1+/+ n=12; FOXM1-/- n=13; at least three biological replicates. two-tailed unpaired t-test, P= 0.0014). (


( D ) Overexpression of FOXM1 or LIN28A rescues the human neural progenitor cell reduction in FOXM1 - / - cell lines . Western blot analysis for SOX2 , PAX6 and HOPX . β - actin was detected as loading control . ( E ) Statistics of relative intensity of SOX2 , PAX6 and HOPX ( PAX6 : n = 3 biological replicates ; one - way ANOVA , F ( 2 , 6 ) = 8 . 716 , FOXM1 - / - ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 0173 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 0476 . SOX2 : n = 5 biological replicates ; one - way ANOVA , F ( 2 , 12 ) = 14 . 72 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( Control ) , P = 0 . 0015 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 9906 . HOPX : n = 4 biological replicates , one - way ANOVA F ( 2 , 9 ) = 25 . 28 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( Control ) , P = 0 . 0018 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 2211 ) . (

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(D) Overexpression of FOXM1 or LIN28A rescues the human neural progenitor cell reduction in FOXM1-/- cell lines. Western blot analysis for SOX2, PAX6 and HOPX. β-actin was detected as loading control. (E) Statistics of relative intensity of SOX2, PAX6 and HOPX (PAX6: n=3 biological replicates; one-way ANOVA, F (2,6) = 8.716, FOXM1-/-(Control) vs. FOXM1-/-(hFOXM1), P= 0.0173, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.0476. SOX2: n=5 biological replicates; one-way ANOVA, F (2, 12) = 14.72, FOXM1+/+(Control) vs. FOXM1-/-(Control), P= 0.0015, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P=0.9906. HOPX: n=4 biological replicates, one-way ANOVA F (2, 9) = 25.28, FOXM1+/+(Control) vs. FOXM1-/- (Control), P= 0.0018, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.2211). (