B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells . Cells were infected with the indicated Mtb strains at an MOI of 1 . Non - infected cells were used as controls . After 4 hours , the cells were lysed and immunoprecipitated with the antibody against PknG . The immunoprecipitated proteins were immunoblotted with TRAF2 , TAK1 , TAB2 ( negative control ) , or TAB3 ( negative control ) antibodies .

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B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells. Cells were infected with the indicated Mtb strains at an MOI of 1. Non-infected cells were used as controls. After 4 hours, the cells were lysed and immunoprecipitated with the antibody against PknG. The immunoprecipitated proteins were immunoblotted with TRAF2, TAK1, TAB2 (negative control), or TAB3 (negative control) antibodies.


E Immunoblotting of TRAF2 , TAK1 , and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours . Cells were treated with CHX ( 50 μg / mL ) with or without MG132 ( 5 μM ) . Cell lysates were analyzed by immunoblotting for TRAF2 ( anti - TRAF2 ) , TAK1 ( anti - TAK1 ) , and PknG ( anti - PknG ) . GAPDH was used as the loading control . Densitometric quantification of immunoblots is indicated below the immunoblots .

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O

E Immunoblotting of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours. Cells were treated with CHX (50 μg/mL) with or without MG132 (5 μM). Cell lysates were analyzed by immunoblotting for TRAF2 (anti-TRAF2), TAK1 (anti-TAK1), and PknG (anti-PknG). GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots.


G , H In vivo ubiquitination assay of TRAF2 ( G ) and TAK1 ( H ) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours . Cells were lysed and immunoprecipitated using the antibody against Flag . Immunoprecipitated proteins were immunoblotted with the antibody against HA .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY O O O O B-GENEPROD O

G, H In vivo ubiquitination assay of TRAF2 (G) and TAK1 (H) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours. Cells were lysed and immunoprecipitated using the antibody against Flag. Immunoprecipitated proteins were immunoblotted with the antibody against HA.


A , B qPCR analysis of Tnf mRNA ( A ) and Il6 mRNA ( B ) in splenic cells from C57BL / 6 mice infected with WT or mutant Mtb stains for 0 - 20 days . Data are representative of one experiment with two independent biological replicates ( mean ± SEM of n = 6 mice per group ) . * P < 0 . 05 and * * P < 0 . 01 . Data were analyzed according to one - way ANOVA using GraphPad Prism 7 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O B-TISSUE O O O O O B-ORGANISM B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B qPCR analysis of Tnf mRNA (A) and Il6 mRNA (B) in splenic cells from C57BL/6 mice infected with WT or mutant Mtb stains for 0-20 days. Data are representative of one experiment with two independent biological replicates (mean ± SEM of n = 6 mice per group). *P < 0.05 and **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.


D H & E staining of lungs and livers of C57BL / 6 mice treated as in A . Arrows indicate puncta of cellular infiltration . Scale bars , 200 μm .

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D H&E staining of lungs and livers of C57BL/6 mice treated as in A. Arrows indicate puncta of cellular infiltration. Scale bars, 200 μm.


A . BAZ2A expression is higher in ESC + 2i than in differentiated cells ( neural progenitors , NPC ) . Left panel . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to ESC + 2i . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Right panel . Western blot showing BAZ2A protein levels in ESC + 2i and NPC . Tubulin is shown as a protein loading control .

O O B-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O

A. BAZ2A expression is higher in ESC+2i than in differentiated cells (neural progenitors, NPC). Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Right panel. Western blot showing BAZ2A protein levels in ESC+2i and NPC. Tubulin is shown as a protein loading control.


B . BAZ2A associates with chromatin of ESC and NPC . Chromatin - bound ( Chrom . ) and soluble ( Sol . ) fractions of equivalent cell number of ESC + 2i and NPCs were analyzed by western blot for BAZ2A levels . Total , total protein . Tubulin and histones are shown as loading and fractionation control .

O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O

B. BAZ2A associates with chromatin of ESC and NPC. Chromatin-bound (Chrom.) and soluble (Sol.) fractions of equivalent cell number of ESC+2i and NPCs were analyzed by western blot for BAZ2A levels. Total, total protein. Tubulin and histones are shown as loading and fractionation control.


C . siRNA - knockdown efficiency of BAZ2A shown by qRT - PCR and western blot . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to each ESC line . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * * < 0 . 0001 ) . Right panel . Western blot showing BAZ2A protein levels . Tubulin is shown as a protein loading control .

O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O

C. siRNA-knockdown efficiency of BAZ2A shown by qRT-PCR and western blot. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to each ESC line. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (**** < 0.0001). Right panel. Western blot showing BAZ2A protein levels. Tubulin is shown as a protein loading control.


D . BAZ2A knockdown affects proliferation of ESC + 2i but not of ESC + serum . Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * < 0 . 01 ; ns , non - significant ) .

O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_TYPE O O O O O B-CELL_TYPE O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. BAZ2A knockdown affects proliferation of ESC+2i but not of ESC+serum. Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01; ns, non-significant).


E . BAZ2A is expressed at similar levels in both ESC + 2i and ESC + serum . Left panel . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to ESC + 2i . Average values of three independent experiments . Error bars represent s . d . Right panel . Western blot showing BAZ2A protein levels in ESC + 2i and ESC + serum . Tubulin and histone H3 are shown as a protein loading controls .

O O B-GENEPROD O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O

E. BAZ2A is expressed at similar levels in both ESC+2i and ESC+serum. Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. Right panel. Western blot showing BAZ2A protein levels in ESC+2i and ESC+serum. Tubulin and histone H3 are shown as a protein loading controls.


F . BAZ2A is required for the differentiation of ESC + 2i but not of ESC + serum . Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10 % serum and in the absence of LIF .

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F. BAZ2A is required for the differentiation of ESC+2i but not of ESC+serum. Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10% serum and in the absence of LIF.


G . Quantification of differentiated cells . Values represent relative number of differentiated cells from three independent experiments relative to control cells . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ; ns , non - significant ) .

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G. Quantification of differentiated cells. Values represent relative number of differentiated cells from three independent experiments relative to control cells. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001; ns, non-significant).


H . Western blot showing BAZ2A protein levels in two BAZ2A - KO ESC lines obtained via CRISPr / Cas9 directly in ESC + serum . PARP1 is shown as a protein loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-TISSUE O B-GENEPROD O O O O O O O O

H. Western blot showing BAZ2A protein levels in two BAZ2A-KO ESC lines obtained via CRISPr/Cas9 directly in ESC+serum. PARP1 is shown as a protein loading control.


I . Cell proliferation is not affected in BAZ2A - KO ESC + serum . Data represent relative cell numbers 2 days culture starting with the same number of cells . Average values of four independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns , non - significant ) .

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I. Cell proliferation is not affected in BAZ2A-KO ESC+serum. Data represent relative cell numbers 2 days culture starting with the same number of cells. Average values of four independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns, non-significant).


J . Representative images of wt and Baz2a - KO ESC + serum before and after transition in 2i conditions .

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J. Representative images of wt and Baz2a-KO ESC+serum before and after transition in 2i conditions.


A . Volcano plot showing fold change ( log2 values ) in transcript level of ESC + 2i and ESC + serum with two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Gene expression values of three replicates were averaged and selected for 1 . 5 fold changes and P < 0 . 05 . Statistical significance ( P - values ) was calculated using R package DEseq2 .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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. Volcano plot showing fold change (log2 values) in transcript level of ESC+2i and ESC+serum with two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Gene expression values of three replicates were averaged and selected for 1.5 fold changes and P < 0.05. Statistical significance (P-values) was calculated using R package DEseq2.


B . Validation by qRT - PCR of genes regulated by BAZ2A in ESC + 2i but not in ESC + serum ( upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red ) . Nanog , Rex1 and Actin B ( ActB ) are shown as genes not regulated by BAZ2A . mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) . Data without P values are statistically non significant .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-CELL_TYPE O B-TISSUE O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Validation by qRT-PCR of genes regulated by BAZ2A in ESC+2i but not in ESC+serum (upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red). Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01, *** < 0.001). Data without P values are statistically non significant.


A . Western blot showing equal BAZ2A levels in wt - ESC and F / H - BAZ2A ESC line containing FLAG - HA sequences at the N - terminus of both Baz2a alleles . Whole cell lysates from equivalent amounts of cells were analysed . SNF2H , and Tubulin are shown as protein loading controls .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O O O B-GENEPROD B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O

A. Western blot showing equal BAZ2A levels in wt-ESC and F/H-BAZ2A ESC line containing FLAG-HA sequences at the N-terminus of both Baz2a alleles. Whole cell lysates from equivalent amounts of cells were analysed. SNF2H, and Tubulin are shown as protein loading controls.


B . Anti - FLAG ChIP of wt - ESCs and F / H - BAZ2A - ESCs cultured in 2i or serum . Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * < 0 . 05 , * * < 0 . 01 ) .

O O O O B-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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. Anti-FLAG ChIP of wt-ESCs and F/H-BAZ2A-ESCs cultured in 2i or serum. Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01).


C . Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3 . The grey rectangles highlighted some of BAZ2A associated regions .

O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O

C. Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3. The grey rectangles highlighted some of BAZ2A associated regions.


D . Pearson correlation heat map for the indicated ChIPseqs in ESC + 2i . BAZ2A ChIPseqs were performed with FLAG antibodies in F / H - BAZ2A - ESCs cultured in 2i or serum and with HA antibodies in F / H - BAZ2A - ESC + 2i . Data of H3K4me3 , H3K27ac and H3K27me3 are from this work . H3K36me3 , H3K9me3 and H3K4me1 values in ESC + 2i were taken from published data

O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-TISSUE O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O

D. Pearson correlation heat map for the indicated ChIPseqs in ESC+2i. BAZ2A ChIPseqs were performed with FLAG antibodies in F/H-BAZ2A-ESCs cultured in 2i or serum and with HA antibodies in F/H-BAZ2A-ESC+2i. Data of H3K4me3, H3K27ac and H3K27me3 are from this work. H3K36me3, H3K9me3 and H3K4me1 values in ESC+2i were taken from published data sets


E . Boxplot showing the mean of normalized BAZ2A occupancies in ESC + 2i and ESC + serum at BAZ2A - bound regions . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Boxplot showing the mean of normalized BAZ2A occupancies in ESC+2i and ESC+serum at BAZ2A-bound regions. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


F . Differential BAZ2A - binding in 2i and serum correlates with H3K27ac occupancies . Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC + 2i and ESC + serum at all BAZ2A - bound regions and BAZ2A - bound regions only in 2i were calculated and their log2 - fold changes are plotted . H3K27ac in ESC + serum Statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O O B-GENEPROD O B-EXP_ASSAY O O O B-TISSUE O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Differential BAZ2A-binding in 2i and serum correlates with H3K27ac occupancies. Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC+2i and ESC+serum at all BAZ2A-bound regions and BAZ2A-bound regions only in 2i were calculated and their log2-fold changes are plotted. H3K27ac in ESC+serum Statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


G . Representative images showing 2i - specific BAZ2A - bound regions and H3K27ac and H3K27me3 in ESC + 2i and ESC + serum . The grey box highlights strong TIP5 - bound regions and corresponding histone modifications .

O O O B-EXP_ASSAY O O O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O

G. Representative images showing 2i-specific BAZ2A-bound regions and H3K27ac and H3K27me3 in ESC+2i and ESC+serum. The grey box highlights strong TIP5-bound regions and corresponding histone modifications.


H . Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3 , depleted of H3K27ac and not bound by BAZ2A .

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H. Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3, depleted of H3K27ac and not bound by BAZ2A.


I . Heat map profiles of H3K27me3 and H3K27ac at ± 5 kb from the TSS of genes differentially regulated by BAZ2A in ESC + 2i and random genes . Differentially regulated genes are shown as upregulated or downregulated in ESC + 2i upon BAZ2A knockdown . Data were ranked by H3K27me3 levels . Transcription start site ( TSS ) .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O

I. Heat map profiles of H3K27me3 and H3K27ac at ±5 kb from the TSS of genes differentially regulated by BAZ2A in ESC+2i and random genes. Differentially regulated genes are shown as upregulated or downregulated in ESC+2i upon BAZ2A knockdown. Data were ranked by H3K27me3 levels. Transcription start site (TSS).


J . Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over + / - 1kb of TSSs from upregulated , downregulated and random genes . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over +/- 1kb of TSSs from upregulated, downregulated and random genes. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Number of BAZ2A - bound regions in A and B compartments of ESC + serum . B . Hi - C contact matrices for a zoomed in region on chromosome 3 at 100 - kb resolution showing the presence of strong long - distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC + serum . Low panels . HiC contacts of a zoomed region at 50 - kb resolution in ESC + serum , NPCs and CNs are shown . Data are from ( Bonev et al . , 2017 ) . HiC contacts were visualized using Juicebox C . BAZ2A - bound regions mark A subcompartments that are characterized by the strongest far - cis contacts in ESC + serum . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at near - cis and far - cis contacts in all genome or in A compartments . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . D . Long - range contacts marked by BAZ2A binding are specific for ESCs . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at far - cis contacts in all genome or in A compartments of ESC + serum , NPC and CN . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O B-TISSUE O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-CELL_TYPE O B-TISSUE O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O B-TISSUE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Number of BAZ2A-bound regions in A and B compartments of ESC+serum. B. Hi-C contact matrices for a zoomed in region on chromosome 3 at 100-kb resolution showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Low panels. HiC contacts of a zoomed region at 50-kb resolution in ESC+serum, NPCs and CNs are shown. Data are from (Bonev et al., 2017). HiC contacts were visualized using Juicebox C. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+serum. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (****< 0.0001). D. Long-range contacts marked by BAZ2A binding are specific for ESCs. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in all genome or in A compartments of ESC+serum, NPC and CN. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


E . Number of BAZ2A - bound regions in A and B compartments in ESC + 2i . F . BAZ2A - bound regions mark A subcompartments that are characterized by the strongest far - cis contacts in ESC + 2i . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at near - cis and far - cis contacts in all genome or in A compartments of ESC + 2i . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * < 0 . 001 , * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Number of BAZ2A-bound regions in A and B compartments in ESC+2i. F. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+2i. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments of ESC+2i. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (** < 0.001, ****< 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


G . BAZ2A binding correlates the best with eigenvector values . Heat map showing Pearson correlation values of ChIPseq data sets with the eigenvector values from HiC data in ESC + serum and ESC + 2i , respectively .

O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O B-TISSUE O B-CELL_TYPE O O O O O

G. BAZ2A binding correlates the best with eigenvector values. Heat map showing Pearson correlation values of ChIPseq data sets with the eigenvector values from HiC data in ESC+serum and ESC+2i, respectively.


H . Boxplots showing H3K4me1 and H3K27ac content of BAZ2A - bound regions and loci not bound by BAZ2A at far - cis contacts in ESC + 2i . Data from regions with lowest 10 % and top 10 % contact strength are shown . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Boxplots showing H3K4me1 and H3K27ac content of BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in ESC+2i. Data from regions with lowest 10% and top 10% contact strength are shown. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Numbers of ATAC peaks called separately in ESC + 2i and ESC + serum including their localization according to compartmentalization . B . Numbers of significant changed ATAC peaks in ESC + 2i vs . ESC + serum and their localization according to compartmentalization . Increased chromatin accessibility sites ( IAS ) , decreased accessibility sites ( DAS ) . C . Average profile of normalized ATAC read counts over differential peaks in ESC + 2i and ESC + serum changed ATAC peaks . The signal is plotted over + / - 1kb on the center of the ATAC peak .

O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O B-CELL_TYPE O B-TISSUE O O B-EXP_ASSAY O 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-EXP_ASSAY O B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

A. Numbers of ATAC peaks called separately in ESC+2i and ESC+serum including their localization according to compartmentalization. B. Numbers of significant changed ATAC peaks in ESC+2i vs. ESC+serum and their localization according to compartmentalization. Increased chromatin accessibility sites (IAS), decreased accessibility sites (DAS). C. Average profile of normalized ATAC read counts over differential peaks in ESC+2i and ESC+serum changed ATAC peaks. The signal is plotted over +/- 1kb on the center of the ATAC peak.


D . H3K27ac correlates with differential accessible sites between ESC + 2i and ESC + serum . Boxplot showing the mean values of normalized H3K27ac at ATAC peaks ( + / - 500bp ) that are increased and decreased in ESC + 2i compared to ESC + serum , respectively . ESCs were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O B-GENEPROD O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-TISSUE O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. H3K27ac correlates with differential accessible sites between ESC+2i and ESC+serum. Boxplot showing the mean values of normalized H3K27ac at ATAC peaks (+/- 500bp) that are increased and decreased in ESC+2i compared to ESC+serum, respectively. ESCs were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


E . BAZ2A depletion increases chromatin accessibility only in ESC + 2i . Numbers of significant changed ATAC peaks in ESC + 2i and ESC + serum upon BAZ2A knockdown and their localization according to compartmentalization . F . Average density plots of normalized ATAC read counts over differential ATAC peaks of ESC + 2i and ESC + serum treated with siRNA - Baz2a and siRNA - control over changed ATAC peaks . The signal is plotted over + / - 1kb centered on the ATAC peak .

O O B-GENEPROD O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O

E. BAZ2A depletion increases chromatin accessibility only in ESC+2i. Numbers of significant changed ATAC peaks in ESC+2i and ESC+serum upon BAZ2A knockdown and their localization according to compartmentalization. F. Average density plots of normalized ATAC read counts over differential ATAC peaks of ESC+2i and ESC+serum treated with siRNA-Baz2a and siRNA-control over changed ATAC peaks. The signal is plotted over +/- 1kb centered on the ATAC peak.


G . The distances of increased ( IAS ) and decreased ( DAS ) chromatin accessibility sites in BAZ2A - depleted ESC + 2i to the nearest gene promoter ( + / - 5kb of TSS ) were calculated . Distances in bp to the nearest promoter of genes upregulated ( UP ) , downregulated ( DOWN ) or not affected ( NOT ) upon BAZ2A knockdown in ESC + 2i are plotted . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * < 0 . 01 ) . Error bars represent s . d . The mean is represented by a horizontal line .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. The distances of increased (IAS) and decreased (DAS) chromatin accessibility sites in BAZ2A-depleted ESC+2i to the nearest gene promoter (+/- 5kb of TSS) were calculated. Distances in bp to the nearest promoter of genes upregulated (UP), downregulated (DOWN) or not affected (NOT) upon BAZ2A knockdown in ESC+2i are plotted. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (** < 0.01). Error bars represent s.d. The mean is represented by a horizontal line.


H . Genomic annotation of IAS and DAS in ESC + 2i depleted of BAZ2A perfomed with

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O O B-GENEPROD O O

H. Genomic annotation of IAS and DAS in ESC+2i depleted of BAZ2A perfomed with ChIPseeker


I . Fold - enrichment relative to expected of regions with increased chromatin accessibility ( IAS ) and upregulated genes upon BAZ2A KD at sub - regions within compartment A and B ( center , inside and boundaries ) . Compartments smaller than 500 kb were excluded from the analysis . Statistical significance ( P - values ) was calculated with permutation test ( ns : non significant , * < 0 . 05 , * * * < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Fold-enrichment relative to expected of regions with increased chromatin accessibility (IAS) and upregulated genes upon BAZ2A KD at sub-regions within compartment A and B (center, inside and boundaries). Compartments smaller than 500 kb were excluded from the analysis. Statistical significance (P-values) was calculated with permutation test (ns: non significant, * < 0.05, *** < 0.001).


J . BAZ2A - upregulated genes are enriched in the B compartment . Numbers of BAZ2A - regulated genes upregulated and downregulated upon BAZ2A knockdown in ESC + 2i including their localization according to compartmentalization .

O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O

J. BAZ2A-upregulated genes are enriched in the B compartment. Numbers of BAZ2A-regulated genes upregulated and downregulated upon BAZ2A knockdown in ESC+2i including their localization according to compartmentalization.


K . Active histone marks increase in the B compartment upon BAZ2A knockdown . Boxplots indicating the log2 fold - changes of normalized H3K27ac and H3K4me3 read counts in ESC + 2i treated with siRNA - Baz2a and siRNA - control at boundaries and centers of A and B compartments . ) . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * < 0 . 001 , * < 0 . 05 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Active histone marks increase in the B compartment upon BAZ2A knockdown. Boxplots indicating the log2 fold-changes of normalized H3K27ac and H3K4me3 read counts in ESC+2i treated with siRNA-Baz2a and siRNA-control at boundaries and centers of A and B compartments. ). Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, * < 0.05). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Hi - C contact matrices for chromosome 1 of ESC + serum , ESC + 2i / siRNA - control and ESC + 2i / siRNA - Baz2a showing the presence of strong long - distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC + serum . Cells were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Eigenvector values are shown . Increase in far - cis contacts in ESC + 2i upon BAZ2A - KD are highlighted with a rectangle . B . Relative contact frequencies over genomic distances in ESCs treated with siRNA - control and siRNA - BAZ2A . Statistical significance ( P - values ) was calculated using the Kolmogorov - Smirnov test . C . Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments . Differences in eigenvector values were calculated from HiC data sets from ESC + 2i treated with siRNA - Baz2a and siRNA - control . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * < 0 . 001 , ns : non significant ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O B-TISSUE O B-CELL_TYPE O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Hi-C contact matrices for chromosome 1 of ESC+serum, ESC+2i/siRNA-control and ESC+2i/siRNA-Baz2a showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Cells were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Eigenvector values are shown. Increase in far-cis contacts in ESC+2i upon BAZ2A-KD are highlighted with a rectangle. B. Relative contact frequencies over genomic distances in ESCs treated with siRNA-control and siRNA-BAZ2A. Statistical significance (P-values) was calculated using the Kolmogorov-Smirnov test. C. Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments. Differences in eigenvector values were calculated from HiC data sets from ESC+2i treated with siRNA-Baz2a and siRNA-control. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, ns: non significant). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Western blot showing total levels of H3K27me3 in ESC + 2i transfected with siRNA - control and siRNA - Baz2a . Histone H3 is shown as a protein loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O

A. Western blot showing total levels of H3K27me3 in ESC+2i transfected with siRNA-control and siRNA-Baz2a. Histone H3 is shown as a protein loading control.


B . Scatterplot showing the changes in H3K27me3 upon BAZ2A depletion in ESC + 2i and ESC + serum . ESCs were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Mean of normalized H3K27me3 read counts in ESC + 2i and ESC + serum treated with siRNA - control or siRNA - Baz2a over all TSSs ( + / - 1kb ) were calculated . Log2 - fold changes of ( siRNA - Baz2a / siRNA - control ) are plotted relative to the mean of normalized H3K27me3 occupancies . The red line represents the mean of H3K27me3 fold - changes .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O

B. Scatterplot showing the changes in H3K27me3 upon BAZ2A depletion in ESC+2i and ESC+serum. ESCs were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Mean of normalized H3K27me3 read counts in ESC+2i and ESC+serum treated with siRNA-control or siRNA-Baz2a over all TSSs (+/- 1kb) were calculated. Log2-fold changes of (siRNA-Baz2a/siRNA-control) are plotted relative to the mean of normalized H3K27me3 occupancies. The red line represents the mean of H3K27me3 fold-changes.


C . Representative images showing the H3K27me3 occupancy .

C. Representative images showing the H3K27me3 occupancy.


D . Average density plot of ChIPseq read counts of H3K27me3 at - / + 5 kb from the TSS of refseq genes in ESCs treated with siRNA - control or siRNA - Baz2a Q1 : first quartile corresponding to sequences with high H3K27me3 content . Q2 - 4 : quartiles 2 - 4 corresponding to sequences with low H3K27me3 content .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O

D. Average density plot of ChIPseq read counts of H3K27me3 at -/+ 5 kb from the TSS of refseq genes in ESCs treated with siRNA-control or siRNA-Baz2a Q1: first quartile corresponding to sequences with high H3K27me3 content. Q2-4: quartiles 2-4 corresponding to sequences with low H3K27me3 content.


E . ChIP - qPCR of H3K27me3 in ESC + 2i and ESC + serum treated with siRNA - control or siRNA - Baz2a . Data were measured by qPCR and normalized to input and to Tshz1 value in control cells . Control represents an intergenic sequence that does not contain H3K27me3 . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) .

O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ChIP-qPCR of H3K27me3 in ESC+2i and ESC+serum treated with siRNA-control or siRNA-Baz2a. Data were measured by qPCR and normalized to input and to Tshz1 value in control cells. Control represents an intergenic sequence that does not contain H3K27me3. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001).


F . Boxplot showing changes in H3K27me3 in ESC + 2i upon BAZ2A at BAZ2A - regulated and random genes . Mean of normalized H3K27me3 read counts in ESC + 2i treated with siRNA - control or siRNA - Baz2a over TSSs ( + / - 1kb ) of upregulated , downregulated and random genes were calculated . Log2 - fold changes of ( siRNA - Baz2a / siRNA - control ) are plotted . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * < 0 . 01 and * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Boxplot showing changes in H3K27me3 in ESC+2i upon BAZ2A at BAZ2A-regulated and random genes. Mean of normalized H3K27me3 read counts in ESC+2i treated with siRNA-control or siRNA-Baz2a over TSSs (+/- 1kb) of upregulated, downregulated and random genes were calculated. Log2-fold changes of (siRNA-Baz2a/siRNA-control) are plotted. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01 and *** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


G . Average density and heatmap profile of H3K27me3 transition state regions marked by CTCF sites in ESC + 2i or ESC + serum transfected with siRNA - control or siRNA - Baz2a . Data were ranked by H3K27me3 content in the corresponding siRNA - control ESCs . Cluster 1 represents high to low H327me3 transition whereas Cluster 2 shows low to high H3K27me3 levels .

O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O O O O O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O

G. Average density and heatmap profile of H3K27me3 transition state regions marked by CTCF sites in ESC+2i or ESC+serum transfected with siRNA-control or siRNA-Baz2a. Data were ranked by H3K27me3 content in the corresponding siRNA-control ESCs. Cluster 1 represents high to low H327me3 transition whereas Cluster 2 shows low to high H3K27me3 levels.


H . H3K27me3 ChIP - qPCR in ESC + 2i ( top panel ) or ESC + serum ( bottom panel ) transfected with siRNA - control or siRNA - Baz2a . Values show H3K27me3 occupancy at four sequences ( # 1 - # 4 ) with elevated H3K27me3 at CTCF boundary ( H3K27me3hi / CTCF ) . Control represents an intergenic sequence that does not contain H3K27me3 . Data were measured by qPCR and normalized to input and to sequence # 1 value in control cells . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 ) .

O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. H3K27me3 ChIP-qPCR in ESC+2i (top panel) or ESC+serum (bottom panel) transfected with siRNA-control or siRNA-Baz2a. Values show H3K27me3 occupancy at four sequences (#1-#4) with elevated H3K27me3 at CTCF boundary (H3K27me3hi/CTCF). Control represents an intergenic sequence that does not contain H3K27me3. Data were measured by qPCR and normalized to input and to sequence #1 value in control cells. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01).


I . Hi - C contact matrices for a zoomed in region on chromosome 6 containing HoxA cluster ( 10 - kb resolution in upper panels , 5 kb resolution in lower panels ) . Blue arrows ( A , B , and C ) indicate gain or increase of contacts in ESC + siRNA - Baz2a compared to control cells ( siRNA - control ) . The orange arrow ( D ) indicates loss or decrease in contacts .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O 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-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

I. Hi-C contact matrices for a zoomed in region on chromosome 6 containing HoxA cluster (10-kb resolution in upper panels, 5 kb resolution in lower panels). Blue arrows (A, B, and C) indicate gain or increase of contacts in ESC+siRNA-Baz2a compared to control cells (siRNA-control). The orange arrow (D) indicates loss or decrease in contacts.


B . BAZ2A - interacting proteins on chromatin . Mass spectrometry analysis of FLAG immunoprecipitates from wt and F / H - BAZ2A ESC + 2i . Values of peptide number are shown as the difference between peptides obtained in FLAG - IP of F / H - BAZ2A and wt ESC chromatin extracts . Data are from three independent experiments . The proteins found enriched in FLAG - IP of F / H - BAZ2A ESCs in all three experiments are shown . Further BAZ2A - interacting proteins identified in only two or one of the immunoprecipitation ( IP ) experiments Error bars represent s . d .

O O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O

B. BAZ2A-interacting proteins on chromatin. Mass spectrometry analysis of FLAG immunoprecipitates from wt and F/H-BAZ2A ESC+2i. Values of peptide number are shown as the difference between peptides obtained in FLAG-IP of F/H-BAZ2A and wt ESC chromatin extracts. Data are from three independent experiments. The proteins found enriched in FLAG-IP of F/H-BAZ2A ESCs in all three experiments are shown. Further BAZ2A-interacting proteins identified in only two or one of the immunoprecipitation (IP) experiments Error bars represent s.d.


C . Anti - HA IP of wt - and F / H - BAZ2A - ESC + 2i and ESC + serum . Western blot shows the interaction of BAZ2A with SNF2H , TOP2A and SMC1a .

O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-CELL_TYPE O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O

C. Anti-HA IP of wt- and F/H-BAZ2A-ESC+2i and ESC+serum. Western blot shows the interaction of BAZ2A with SNF2H, TOP2A and SMC1a.


D . TOP2A and BAZ2A regulate gene expression in ESC + 2i via a shared pathway . qRT - PCR of genes regulated by BAZ2A in ESC + 2i ( left panel ) and in ESC + serum ( right panel ) upon TOP2A or BAZ2A knockdown by siRNA . ESCs were treated with the corresponding RNAs for 4 days . Upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red . Nanog , Rex1 and Actin B ( ActB ) are shown as genes not regulated by BAZ2A . mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) .

O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. TOP2A and BAZ2A regulate gene expression in ESC+2i via a shared pathway. qRT-PCR of genes regulated by BAZ2A in ESC+2i (left panel) and in ESC+serum (right panel) upon TOP2A or BAZ2A knockdown by siRNA. ESCs were treated with the corresponding RNAs for 4 days. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001).


E . Inhibition of type 2 topoisomerase activity phenocopies the alterations in gene expression of BAZ2A - regulated genes in ESC + 2i observed upon TOP2A or BAZ2A knockdown . ESCs were treated with the TOP2 inhibitor ICF - 193 for 24 hours . mRNA levels were normalized to Rps12 mRNA and to ESCs treated with DMSO . Upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Inhibition of type 2 topoisomerase activity phenocopies the alterations in gene expression of BAZ2A-regulated genes in ESC+2i observed upon TOP2A or BAZ2A knockdown. ESCs were treated with the TOP2 inhibitor ICF-193 for 24 hours. mRNA levels were normalized to Rps12 mRNA and to ESCs treated with DMSO. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, ** < 0.01, *** < 0.001, **** < 0.0001).


F . ChIP analysis showing that TOP2A regulates H3K27me3 occupancy in ESC + 2i but not in ESC + serum . Data were measured by qPCR and normalized to input and to Tshz1 value in cells transfected with siRNA - control . # 1 - # 4 indicated sequences with elevated H3K27me3 at CTCF boundary ( H3K27me3hi / CTCF ) . Control represents an intergenic region that does not contain H3K27me3 . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-CELL_TYPE O B-TISSUE O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. ChIP analysis showing that TOP2A regulates H3K27me3 occupancy in ESC+2i but not in ESC+serum. Data were measured by qPCR and normalized to input and to Tshz1 value in cells transfected with siRNA-control. #1-#4 indicated sequences with elevated H3K27me3 at CTCF boundary (H3K27me3hi/CTCF). Control represents an intergenic region that does not contain H3K27me3. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001).


( B ) Changes in plasma level of each cocktail substances in both supplementation and control studies ( NR is detected in control study ) compared to time baseline based on untargeted metabolomics measurement . The grey shaded area represents the 95 % confidence level interval . For boxplots limits , the middle line represents the median . The upper and lower box limits represent the 25 % quantiles . The upper and lower error bars correspond to 75 % quantiles . The p - values are derived from one - way ANOVA ( FDR < 0 . 05 ) .

O O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Changes in plasma level of each cocktail substances in both supplementation and control studies (NR is detected in control study) compared to time baseline based on untargeted metabolomics measurement. The grey shaded area represents the 95% confidence level interval. For boxplots limits, the middle line represents the median. The upper and lower box limits represent the 25% quantiles. The upper and lower error bars correspond to 75% quantiles. The p-values are derived from one-way ANOVA (FDR< 0.05).


( A - G ) Changes in plasma level of key metabolites compared to time baseline ( solid line ) based on untargeted metabolomics measurement in metabolic cofactor supplementation study . The upper and lower box limits represent the 25 % quantiles . The upper and lower error bars correspond to 75 % quantiles . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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 - G) Changes in plasma level of key metabolites compared to time baseline (solid line) based on untargeted metabolomics measurement in metabolic cofactor supplementation study. The upper and lower box limits represent the 25% quantiles. The upper and lower error bars correspond to 75% quantiles. * Denotes significance (one-way ANOVA; FDR <0 .05).


( A ) A summary of significantly different plasma metabolites compared to time baseline in metabolic cofactor supplementation and control studies based on untargeted metabolomics measurements . Red and blue colors denote the increased and decreased plasma levels compared to baseline , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

(A) A summary of significantly different plasma metabolites compared to time baseline in metabolic cofactor supplementation and control studies based on untargeted metabolomics measurements. Red and blue colors denote the increased and decreased plasma levels compared to baseline, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05).


( B ) Metabolites significantly different between supplementation and control studies . Red and blue colors denote the increased and decreased plasma level in supplementation study compared to control study , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O 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) Metabolites significantly different between supplementation and control studies. Red and blue colors denote the increased and decreased plasma level in supplementation study compared to control study, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05).


( C - E ) Changes in plasma level of key metabolites compared to time baseline based on untargeted metabolomics measurement in supplementation and control studies . The grey shaded area represents the 95 % confidence level interval .

O O O O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(C-E) Changes in plasma level of key metabolites compared to time baseline based on untargeted metabolomics measurement in supplementation and control studies. The grey shaded area represents the 95% confidence level interval.


( A ) A summary of significantly different plasma inflammatory protein markers compared to time baseline in metabolic cofactor supplementation and control studies . Red and blue colors denote the increased and decreased plasma levels compared to baseline , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

O O O O O O O O B-TISSUE 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

(A) A summary of significantly different plasma inflammatory protein markers compared to time baseline in metabolic cofactor supplementation and control studies. Red and blue colors denote the increased and decreased plasma levels compared to baseline, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05).


( B ) Proteins significantly different between supplementation and control studies . Red and blue colors denote the increased and decreased plasma level in supplementation study compared to control study , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

(B) Proteins significantly different between supplementation and control studies. Red and blue colors denote the increased and decreased plasma level in supplementation study compared to control study, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05).


( C - F ) Changes in the plasma level of key proteins compared to time baseline based on untargeted metabolomics measurement in supplementation and control studies . The grey shaded area represents the 95 % confidence level interval . For boxplots limits , the middle line represents the median . The upper and lower box limits represent the 25 % quantiles . The upper and lower error bars correspond to 75 % quantiles . The p - values are derived from one - way ANOVA ( FDR < 0 . 05 ) .

O O O O O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Changes in the plasma level of key proteins compared to time baseline based on untargeted metabolomics measurement in supplementation and control studies. The grey shaded area represents the 95% confidence level interval. For boxplots limits, the middle line represents the median. The upper and lower box limits represent the 25% quantiles. The upper and lower error bars correspond to 75% quantiles. The p-values are derived from one-way ANOVA (FDR< 0.05).


( A ) Plasma serine interpolations for each of the nine subjects .

(A) Plasma serine interpolations for each of the nine subjects.


( B ) Model fit to the target concentration curve ( mean interpolation of plasma serine over 24 hours ) with standard deviation indicated in gray .

O O O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O

(B) Model fit to the target concentration curve (mean interpolation of plasma serine over 24 hours) with standard deviation indicated in gray.


( D ) Plasma L - carnitine interpolations for each of the nine subjects .

O O O B-TISSUE B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O

(D) Plasma L-carnitine interpolations for each of the nine subjects.


( E ) Model fit to the target concentration curve ( mean interpolation of plasma L - carnitine over 24 hours ) with standard deviation indicated in gray .

O O O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O

(E) Model fit to the target concentration curve (mean interpolation of plasma L-carnitine over 24 hours) with standard deviation indicated in gray.


( G ) Plasma NAC interpolations for each of the nine subjects .

O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O

(G) Plasma NAC interpolations for each of the nine subjects.


( H ) Model fit to the target concentration curve ( mean interpolation of plasma serine over 24 hours ) with standard deviation indicated in gray .

(H) Model fit to the target concentration curve (mean interpolation of plasma serine over 24 hours) with standard deviation indicated in gray.


( A ) Lung cancer cells PC - 9 , prostate cancer cells 22RV1 , bladder cancer cells UMUC3 , breast cancer cells MCF - 7 , colonic adenoma - derived non - cancerous cells AA / C1 ( aka C1 ) and AA / C1 / SB ( aka SB ) , or cancerous cells AA / C1 / SB / 10C ( aka 10C ) were incubated with wild - type F . nucleatum 12230 ( Fn ) , the fadA - deletion mutant US1 ( US1 ) , or E . coli DH5α ( E . coli ) at multiplicity of infection ( MOI ) of 1 , 000 : 1 . Cells numbers are mean values ± SEM . The experiment was performed in triplicates and repeated three times . * * p < 0 . 01 , * * * p < 0 . 001 , compared to untreated controls ; # p < 0 . 05 , # # # p < 0 . 001 , compared to US1 - treated cells ( two - way ANOVA ) .

O O O B-DISEASE I-DISEASE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE O B-DISEASE I-DISEASE O B-CELL_LINE O B-DISEASE I-DISEASE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-DISEASE I-DISEASE O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lung cancer cells PC-9, prostate cancer cells 22RV1, bladder cancer cells UMUC3, breast cancer cells MCF-7, colonic adenoma-derived non-cancerous cells AA/C1 (aka C1) and AA/C1/SB (aka SB), or cancerous cells AA/C1/SB/10C (aka 10C) were incubated with wild-type F. nucleatum 12230 (Fn), the fadA-deletion mutant US1 (US1), or E. coli DH5α (E. coli) at multiplicity of infection (MOI) of 1,000:1. Cells numbers are mean values ± SEM. The experiment was performed in triplicates and repeated three times. **p<0.01, ***p<0.001, compared to untreated controls; #p<0.05, ### p<0.001, compared to US1-treated cells (two-way ANOVA).


( B ) Attachment ( left panel ) and invasion ( right panel ) of wild - type F . nucleatum 12230 ( Fn ) to the non - cancerous SB cells , either untreated or transfected with antisense or sense ANXA1 , and to the cancerous 10C cells , either untreated or transfected with control or ANXA1 - or CDH1 - specific siRNA or both ( MOI 50 : 1 ) . F . nucleatum attachment and invasion to the untreated SB cells were designated as 100 % , respectively ; all other values were expressed as relative to those obtained with untreated SB . Data are mean values ± SEM . The experiment was performed in triplicates and repeated four times . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( one - way ANOVA ) .

O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O B-DISEASE O O O O O O O O O O O B-GENEPROD O O O O B-DISEASE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Attachment (left panel) and invasion (right panel) of wild-type F. nucleatum 12230 (Fn) to the non-cancerous SB cells, either untreated or transfected with antisense or sense ANXA1, and to the cancerous 10C cells, either untreated or transfected with control or ANXA1- or CDH1-specific siRNA or both (MOI 50:1). F. nucleatum attachment and invasion to the untreated SB cells were designated as 100%, respectively; all other values were expressed as relative to those obtained with untreated SB. Data are mean values ± SEM. The experiment was performed in triplicates and repeated four times. *p<0.05, **p<0.01 and ***p<0.001 (one-way ANOVA).


( C ) Left panel : qPCR analysis of Villin1 ( VIL1 ) mRNA levels in 10C cells treated with control siRNA or VIL1 - specific siRNA , demonstrating knocking down of Villin1 . Right panel : attachment of F . nucleatum 12230 to 10C cells treated with control siRNA or VIL1 - specific siRNA . Data are mean values ± SD . The experiment was performed in triplicates and repeated twice . * p < 0 . 05 ( student ' s t - test ) .

O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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) Left panel: qPCR analysis of Villin1 (VIL1) mRNA levels in 10C cells treated with control siRNA or VIL1-specific siRNA, demonstrating knocking down of Villin1. Right panel: attachment of F. nucleatum 12230 to 10C cells treated with control siRNA or VIL1-specific siRNA. Data are mean values ± SD. The experiment was performed in triplicates and repeated twice. *p<0.05 (student's t-test).


Relationship between ANXA1 mRNA expression levels and disease - free survival ( DFS ) in colon cancer patients . The relationship between ANXA1 mRNA expression levels and DFS was investigated in a database of 466 primary colon carcinomas , assembled by pooling four independent gene - expression array datasets from the NCBI - GEO online repository ( GSE14333 , GSE17538 , GSE31595 , GSE37892 ) The association between ANXA1 expression levels and DFS was tested using Kaplan - Meier survival curves , after patient stratification in groups with high , medium and low ANXA1 expression , using three different methods : ( A ) based on the median of ANXA1 mRNA expression levels ( high 50 % vs . low 50 % ) ; ( B ) based on the quartile distribution of ANXA1 mRNA expression levels ( high 25 % vs . middle 50 % vs . low 25 % ) ; and ( C ) based on ANXA1 mRNA expression thresholds calculated using the StepMiner algorithm ( low vs . high ) Overall , high ANXA1 mRNA expression levels were associated with a statistically significant reduction in DFS ( p < 0 . 001 , log - rank test ) , irrespective of the method used for the stratification . Differences in ANXA1 mRNA expression levels did not appear to correlate with differences in each tumor ' s relative content of epithelial cells in the analyzed biospecimens ( i . e . tumor cell density ) as revealed by the lack of visual correlations with the epithelial cell marker Desmoplakin ( DSP ) .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-DISEASE I-DISEASE B-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-DISEASE O B-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O

Relationship between ANXA1 mRNA expression levels and disease-free survival (DFS) in colon cancer patients. The relationship between ANXA1 mRNA expression levels and DFS was investigated in a database of 466 primary colon carcinomas, assembled by pooling four independent gene-expression array datasets from the NCBI-GEO online repository (GSE14333, GSE17538, GSE31595, GSE37892) The association between ANXA1 expression levels and DFS was tested using Kaplan-Meier survival curves, after patient stratification in groups with high, medium and low ANXA1 expression, using three different methods: (A) based on the median of ANXA1 mRNA expression levels (high 50% vs. low 50%); (B) based on the quartile distribution of ANXA1 mRNA expression levels (high 25% vs. middle 50% vs. low 25%); and (C) based on ANXA1 mRNA expression thresholds calculated using the StepMiner algorithm (low vs. high) Overall, high ANXA1 mRNA expression levels were associated with a statistically significant reduction in DFS (p<0.001, log-rank test), irrespective of the method used for the stratification. Differences in ANXA1 mRNA expression levels did not appear to correlate with differences in each tumor's relative content of epithelial cells in the analyzed biospecimens (i.e. tumor cell density) as revealed by the lack of visual correlations with the epithelial cell marker Desmoplakin (DSP).


( A ) Real - time qPCR analysis of ANXA1 expression using mRNA extracted from the non - cancerous SB , cancerous 10C , and human CRC cell lines HCT116 , DLD1 and RKO , each grown to 50 % or 100 % confluency . All results were normalized to the ANXA1 mRNA levels in SB cells of 100 % confluency , which was designated as 1 . Data are mean values ± SEM . The experiment was performed in triplicates and repeated twice . * * p < 0 . 01 and * * * p < 0 . 001 ( student ' s t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-DISEASE O O B-DISEASE O O O B-ORGANISM B-DISEASE O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Real-time qPCR analysis of ANXA1 expression using mRNA extracted from the non-cancerous SB, cancerous 10C, and human CRC cell lines HCT116, DLD1 and RKO, each grown to 50% or 100% confluency. All results were normalized to the ANXA1 mRNA levels in SB cells of 100% confluency, which was designated as 1. Data are mean values ± SEM. The experiment was performed in triplicates and repeated twice. **p<0.01 and ***p<0.001 (student's t-test).


( B ) Confocal microscopy analysis of SB and 10C cells grown to 20 % ( top panels ) or 100 % ( bottom panels ) confluency followed by immuno - fluorescence staining of Annexin A1 ( green ) . The nuclei were stained with DAPI ( blue ) . A series of 20 - 50 consecutive images in the z - axis were stacked together to generate the 3 - D figure at 400x magnification . Annexin A1 is most abundantly expressed on the outer layer of 20 % confluent 10C ( pointed by arrows compared to 100 % confluent 10C , or SB of either confluency . Scale bars , x = 1 μm , y = 1 μm , z = 1 . 6 μm . The experiment was repeated at least twice .

O O O B-EXP_ASSAY I-EXP_ASSAY 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy analysis of SB and 10C cells grown to 20% (top panels) or 100% (bottom panels) confluency followed by immuno-fluorescence staining of Annexin A1 (green). The nuclei were stained with DAPI (blue). A series of 20-50 consecutive images in the z-axis were stacked together to generate the 3-D figure at 400x magnification. Annexin A1 is most abundantly expressed on the outer layer of 20% confluent 10C (pointed by arrows compared to 100% confluent 10C, or SB of either confluency. Scale bars, x = 1 μm, y = 1 μm, z = 1.6 μm. The experiment was repeated at least twice.


( C ) Cell proliferation assay of adenoma - derived non - cancerous SB and cancerous 10C and human CRC cell lines HCT116 , DLD1 , SW480 , HT29 and RKO either untreated ( black lines ) or following treatment with control siRNA ( gray lines ) or ANXA1 - specific siRNA ( green lines ) . Data are mean values ± SEM . The experiment was performed in triplicates and repeated three times . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 , compared to the untreated cells ( two - way ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-DISEASE O O O O B-DISEASE O O B-DISEASE O O B-ORGANISM B-DISEASE O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell proliferation assay of adenoma-derived non-cancerous SB and cancerous 10C and human CRC cell lines HCT116, DLD1, SW480, HT29 and RKO either untreated (black lines) or following treatment with control siRNA (gray lines) or ANXA1-specific siRNA (green lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *p<0.05, ** p<0.01 and *** p<0.001, compared to the untreated cells (two-way ANOVA).


( D ) Cell proliferation assay of SB ( left panel ) and RKO ( right panel ) cells transfected with ANXA1 ( red line ) , as compared to the control cells ( black lines ) . Data are mean values ± SEM . The experiment was performed in triplicates and repeated three times . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( two - way ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Cell proliferation assay of SB (left panel) and RKO (right panel) cells transfected with ANXA1 (red line), as compared to the control cells (black lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *p<0.05, **p<0.01 and ***p<0.001 (two-way ANOVA).


( E ) Xenografted tumor growth in nude mice following subcutaneous and bilateral inoculation of HCT116 cells transfected with control siRNA or ANXA1 - specific siRNA . The tumor volumes were measured after 7 days post injection ( left panel , n = 4 ) . For each mouse , the tumor resulting from ANXA1 - specific siRNA treated cells was normalized to that from control siRNA - treated cells , which was designated 100 % . The line represents the average . * p < 0 . 05 ( paired t - test ) . The individual tumor pairs are shown on the right panel : top , tumors arising from control siRNA treated cells ; bottom , tumors arising from ANXA1 - specific siRNA treated cells .

O O O B-EXP_ASSAY B-DISEASE B-EXP_ASSAY O O B-ORGANISM O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O B-DISEASE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-DISEASE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-DISEASE O O O O O O O O O O B-DISEASE O O O O O O O O O B-DISEASE O O B-GENEPROD O O O O O O

(E) Xenografted tumor growth in nude mice following subcutaneous and bilateral inoculation of HCT116 cells transfected with control siRNA or ANXA1-specific siRNA. The tumor volumes were measured after 7 days post injection (left panel, n=4). For each mouse, the tumor resulting from ANXA1-specific siRNA treated cells was normalized to that from control siRNA-treated cells, which was designated 100%. The line represents the average. *p<0.05 (paired t-test). The individual tumor pairs are shown on the right panel: top, tumors arising from control siRNA treated cells; bottom, tumors arising from ANXA1-specific siRNA treated cells.


( A ) Real - time qPCR analysis of ANXA1 mRNA levels in SB , 10C , HCT116 and DLD1 cells incubated with wild - type F . nucleatum 12230 ( Fn ) or fadA - deletion mutant US1 ( US1 ) at MOI of 50 : 1 for the indicated time periods . The results were normalized to those obtained with untreated cells and were the mean of 3 independent experiments each performed in triplicates . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( two - way ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O B-CELL_LINE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Real-time qPCR analysis of ANXA1 mRNA levels in SB, 10C, HCT116 and DLD1 cells incubated with wild-type F. nucleatum 12230 (Fn) or fadA-deletion mutant US1 (US1) at MOI of 50:1 for the indicated time periods. The results were normalized to those obtained with untreated cells and were the mean of 3 independent experiments each performed in triplicates. *p<0.05, **p<0.01 and ***p<0.001 (two-way ANOVA).


( B ) Flow cytometry analysis of Annexin A1 expression in 10C cells either untreated , or incubated with BSA ( 1000 µg / ml ) , or mFadA ( 1000 µg / ml ) , or FadAc ( 100 , 300 , or 1000 µg / ml ) for 1 hr . Data are mean values ± SD . The experiment was performed in triplicates and repeated more than three times . * * p < 0 . 001 ( one - way ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometry analysis of Annexin A1 expression in 10C cells either untreated, or incubated with BSA (1000 µg/ml), or mFadA (1000 µg/ml), or FadAc (100, 300, or 1000 µg/ml) for 1 hr. Data are mean values ± SD. The experiment was performed in triplicates and repeated more than three times. **p<0.001 (one-way ANOVA).


( C ) Flow cytometry analysis of Annexin A1 in 10C cells transfected with control siRNA ( dotted black line ) or CDH1 - specific siRNA ( dotted red line ) followed by no treatment , or incubation with BSA ( 1000 µg / ml ) or FadAc ( 1000 µg / ml ) for 1 hr . C , untreated control . Data are mean values ± SD . The experiment was performed in triplicates and repeated twice . * * * p < 0 . 001 ( two - way ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometry analysis of Annexin A1 in 10C cells transfected with control siRNA (dotted black line) or CDH1-specific siRNA (dotted red line) followed by no treatment, or incubation with BSA (1000 µg/ml) or FadAc (1000 µg/ml) for 1 hr. C, untreated control. Data are mean values ± SD. The experiment was performed in triplicates and repeated twice. ***p<0.001 (two-way ANOVA).


( D ) Confocal microscopy analysis of SB and 10C cells either untreated or following incubation with CFSE - labeled F . nucleatum 12230 ( Fn ) for 1 hr at MOI of 5 : 1 . Annexin A1 was stained green and E - cadherin blue . Imagines are 800x magnification . Note the enhanced expression of Annexin A1 in 10C compared to SB and its location on the outer layer of the cell mass . The experiment was repeated three times . Scale bar , 250 nm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O

(D) Confocal microscopy analysis of SB and 10C cells either untreated or following incubation with CFSE-labeled F. nucleatum 12230 (Fn) for 1 hr at MOI of 5:1. Annexin A1 was stained green and E-cadherin blue. Imagines are 800x magnification. Note the enhanced expression of Annexin A1 in 10C compared to SB and its location on the outer layer of the cell mass. The experiment was repeated three times. Scale bar, 250 nm.


( A ) Western blotting analysis of FadA , E - cadherin ( CDH1 ) , Annexin A1 ( ANXA1 ) and β - catenin in DLD1 cells following incubation with FadAc for 15 or 120 min . C , untreated cells . β - actin was included as an internal control . The experiment was repeated three times .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

(A) Western blotting analysis of FadA, E-cadherin (CDH1), Annexin A1 (ANXA1) and β-catenin in DLD1 cells following incubation with FadAc for 15 or 120 min. C, untreated cells. β-actin was included as an internal control. The experiment was repeated three times.


( B ) Co - immunoprecipitation with Annexin A1 . DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with rabbit anti - Annexin A1 polyclonal antibody ( α - Annexin A1 ) or control rabbit IgG . FadA , E - cadherin ( CDH1 ) , Annexin A1 ( ANXA1 ) and β - catenin in the eluates were detected by Western blot . C , untreated control . The experiment was repeated three times .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

(B) Co-immunoprecipitation with Annexin A1. DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with rabbit anti-Annexin A1 polyclonal antibody (α-Annexin A1) or control rabbit IgG. FadA, E-cadherin (CDH1), Annexin A1 (ANXA1) and β-catenin in the eluates were detected by Western blot. C, untreated control. The experiment was repeated three times.


( C ) Co - immunoprecipiation with FadA . DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with mouse anti - FadA monoclonal antibody ( α - FadA ) or control mouse IgG . FadA , E - cadherin ( CDH1 ) , Annexin A1 ( ANXA1 ) and β - catenin in the eluates were detected by Western blot . The experiment was repeated three times .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O B-GENEPROD O O O O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(C) Co-immunoprecipiation with FadA. DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with mouse anti-FadA monoclonal antibody (α-FadA) or control mouse IgG. FadA, E-cadherin (CDH1), Annexin A1 (ANXA1) and β-catenin in the eluates were detected by Western blot. The experiment was repeated three times.


( D ) Confocal microscopy analysis of 10C cells either untreated ( top panel ) or following incubation with CFSE - labeled F . nucleatum 12230 ( red , bottom panel ) for 3 hrs and then immunofluorescent - stained for Annexin A1 ( green ) and E - cadherin ( blue ) . Images are 1200x magnification . A side view of the enlarged image is shown on the far right . Note the enhanced expression of Annexin A1 in the F . nucleatum - bound cells and the co - localization of Annexin A1 , E - cadherin and F . nucleatum on the cell membranes ( arrows ) . The experiment was repeated more than three times . Scale bar , 500 nm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

(D) Confocal microscopy analysis of 10C cells either untreated (top panel) or following incubation with CFSE-labeled F. nucleatum 12230 (red, bottom panel) for 3 hrs and then immunofluorescent-stained for Annexin A1 (green) and E-cadherin (blue). Images are 1200x magnification. A side view of the enlarged image is shown on the far right. Note the enhanced expression of Annexin A1 in the F. nucleatum-bound cells and the co-localization of Annexin A1, E-cadherin and F. nucleatum on the cell membranes (arrows). The experiment was repeated more than three times. Scale bar, 500 nm.


( E ) Confocal microscopy analysis of 10C cells following incubation with Alexa Fluor ™ 488 - conjugated BSA , mFadA , or FadAc ( red ) for 1 hr and immuno - staining of Annexin A1 ( green ) and E - cadherin ( blue ) . Images are 1200x magnification . Note the enhanced expression of Annexin A1 and its co - localization with E - cadherin in the presence of FadAc ( arrows ) , but not with BSA and mFadA . The experiment was repeated twice . The side views are shown to the right and bottom of each image . Scale bar , 500 nm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

(E) Confocal microscopy analysis of 10C cells following incubation with Alexa Fluor™ 488-conjugated BSA, mFadA, or FadAc (red) for 1 hr and immuno-staining of Annexin A1 (green) and E-cadherin (blue). Images are 1200x magnification. Note the enhanced expression of Annexin A1 and its co-localization with E-cadherin in the presence of FadAc (arrows), but not with BSA and mFadA. The experiment was repeated twice. The side views are shown to the right and bottom of each image. Scale bar, 500 nm.


Flow cytometry analysis of SB ( A ) , 10C ( B ) , DLD1 ( C ) and HCT116 ( D ) cells incubated with CFSE - labeled F . nucleatum 12230 ( Fn ) or its fadA - deletion mutant US1 ( US1 ) at MOI of 10 - 20 : 1 for the indicated time and immuno - stained with anti - Annexin A1 antibodies . Shown on the top panels are the density plots . x - axis , CFSE - labeled F . nucleatum or US1 ( CFSE - Fn ) ; y - axis , Annexin A1 ( ANXA1 ) . Shown on the bottom panels are the percentages of Annexin A1 - positive ( solid bars ) or negative ( clear bars ) cells bound by F . nucleatum or US1 out of the total number of cells analyzed . Data are mean values ± SD . The experiments were performed in triplicates and repeated 2 - 3 times . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - way ANOVA ) .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometry analysis of SB (A), 10C (B), DLD1 (C) and HCT116 (D) cells incubated with CFSE-labeled F. nucleatum 12230 (Fn) or its fadA-deletion mutant US1 (US1) at MOI of 10-20:1 for the indicated time and immuno-stained with anti-Annexin A1 antibodies. Shown on the top panels are the density plots. x-axis, CFSE-labeled F. nucleatum or US1 (CFSE-Fn); y-axis, Annexin A1 (ANXA1). Shown on the bottom panels are the percentages of Annexin A1-positive (solid bars) or negative (clear bars) cells bound by F. nucleatum or US1 out of the total number of cells analyzed. Data are mean values ± SD. The experiments were performed in triplicates and repeated 2-3 times. *p<0.05, **p<0.01, ***p<0.001 (two-way ANOVA).


( A ) Colorectal tumors generated in Apcmin / + mice following treatment with PBS , E . coli DH5α ( E . coli ) , fadA - deletion mutant US1 ( US1 ) or F . nucleatum 12230 ( Fn ) . Each symbol represents one mouse . Horizontal lines represent mean values . Representative tumors formed in the mouse colon are shown on the right , pointed by blue arrows on the right . * p < 0 . 05 , * * p < 0 . 01 ( one - way ANOVA ) .

O O O B-DISEASE I-DISEASE O O B-GENEPROD O O B-ORGANISM O O O B-SMALL_MOLECULE O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O B-DISEASE O O O B-ORGANISM B-TISSUE O O O O O O O O O O O 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) Colorectal tumors generated in Apcmin/+ mice following treatment with PBS, E. coli DH5α (E. coli), fadA-deletion mutant US1 (US1) or F. nucleatum 12230 (Fn). Each symbol represents one mouse. Horizontal lines represent mean values. Representative tumors formed in the mouse colon are shown on the right, pointed by blue arrows on the right. *p<0.05, **p<0.01 (one-way ANOVA).