B IP of TRAF2 TAK1 by Mtb PknG in U937 . were infected with indicated Mtb strains at an MOI of 1 . Non - infected were used as controls . After 4 hours , were lysed immunoprecipitated with antibody against PknG . immunoprecipitated 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 , PknG from lysates of U937 infected with indicated Mtb strains at an MOI of 1 for 8 hours . were treated with CHX ( 50 μg / mL ) with or without MG132 ( 5 μM ) . lysates were analyzed by immunoblotting for TRAF2 ( anti - TRAF2 ) , TAK1 ( anti - TAK1 ) , PknG ( anti - PknG ) . GAPDH was used as loading control . Densitometric of immunoblots is indicated below immunoblots .

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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 of TRAF2 ( G ) TAK1 ( H ) in absence or presence of PknG in HEK293T transfected with indicated plasmids for 24 hours . were lysed immunoprecipitated using antibody against Flag . Immunoprecipitated were immunoblotted with antibody against HA .

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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 of Tnf mRNA ( A ) Il6 mRNA ( B ) in splenic 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 ± of n = 6 mice per group ) . * P < 0 . 05 * * P < 0 . 01 . Data were analyzed according to one - way ANOVA using GraphPad Prism 7 .

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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 of lungs livers of C57BL / 6 mice treated as in A . Arrows indicate of 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 ( neural progenitors , NPC ) . Left panel . BAZ2A mRNA were measured by qRT - PCR normalized to Rps12 mRNA to ESC + 2i . Average values of three independent experiments . Error bars represent s . d . statistical significance ( P - values ) was calculated using paired two - t - ( * * * < 0 . 001 ) . Right panel . Western blot showing BAZ2A in ESC + 2i NPC . Tubulin is shown as a loading control .

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

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

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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 numbers after 3 days of siRNA treatment were normalized to ESC transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for experiments was calculated using paired two - t - ( * * < 0 . 01 ; ns , non - significant ) .

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

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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 differentiation of ESC + 2i but not of ESC + serum . Representative of alkaline phosphatase of ESC after 3 days of differentiation upon culture in completed medium containing 10 % serum in 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 . of differentiated . Values represent relative number of differentiated from three independent experiments relative to control . Error bars represent s . d . Statistical significance ( P - values ) for experiments was calculated using paired two - t - ( * * * < 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 in two BAZ2A - KO ESC lines obtained via CRISPr / Cas9 directly in ESC + serum . PARP1 is shown as a loading control .

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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 . proliferation is not affected in BAZ2A - KO ESC + serum . Data represent relative numbers 2 days culture starting with same number of . Average values of four independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for experiments was calculated using paired two - t - ( 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 of wt Baz2a - KO ESC + serum before 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 ESC + serum with two consecutive treatments with siRNA - control siRNA - Baz2a , each one lasting for 4 days . Gene expression values of three replicates were averaged selected for 1 . 5 fold changes P < 0 . 05 . Statistical significance ( P - values ) was calculated using R package DEseq2 .

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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 Actin B ( ActB ) are shown as genes not regulated by BAZ2A . mRNA were normalized to Rps12 mRNA to ESCs transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for experiments was calculated using paired two - t - ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) . Data without P values are statistically non significant .

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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 in wt - ESC F / H - BAZ2A ESC containing FLAG - HA sequences at N - terminus of both Baz2a alleles . Whole lysates from equivalent amounts of were analysed . SNF2H , Tubulin are shown as loading controls .

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

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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 showing association of BAZ2A at regions enriched in H3K27ac low in H3K27me3 . grey rectangles highlighted some of BAZ2A associated regions .

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

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

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

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

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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 showing that genes regulated by BAZ2A are enriched in H3K27me3 , depleted of H3K27ac 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 H3K27ac at ± 5 kb from TSS of genes differentially regulated by BAZ2A in ESC + 2i random genes . Differentially regulated genes are shown as upregulated or downregulated in ESC + 2i upon BAZ2A knockdown . Data were ranked by H3K27me3 . Transcription start site ( TSS ) .

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

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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 B compartments of ESC + serum . B . Hi - C contact matrices for a zoomed in region on chromosome 3 at 100 - kb resolution showing presence of strong long - 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 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 strongest far - cis contacts in ESC + serum . Boxplots indicating observed / expected contact values between BAZ2A - bound regions loci not bound by BAZ2A at near - cis far - cis contacts in genome or in A compartments . Statistical significance ( P - values ) was calculated using unpaired two - t - ( * * * * < 0 . 0001 ) . D . Long - range contacts marked by BAZ2A binding are specific for ESCs . Boxplots indicating observed / expected contact values between BAZ2A - bound regions loci not bound by BAZ2A at far - cis contacts in genome or in A compartments of ESC + serum , NPC CN . Statistical significance ( P - values ) was calculated using unpaired two - t - ( * * * * < 0 . 0001 ) . Box plots depict minimum maximum values . mean is represented by a horizontal within boxes .

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

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

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

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

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

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

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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 . distances of increased ( IAS ) decreased ( DAS ) accessibility sites in BAZ2A - depleted ESC + 2i to nearest gene promoter ( + / - 5kb of TSS ) were calculated . Distances in bp to 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 experiments was calculated using unpaired two - t - ( * * < 0 . 01 ) . Error bars represent s . d . mean is represented by a horizontal .

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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 DAS in ESC + 2i depleted of BAZ2A perfomed with

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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 accessibility ( IAS ) upregulated genes upon BAZ2A KD at sub - regions within compartment A B ( center , inside boundaries ) . Compartments smaller than 500 kb were excluded from . Statistical significance ( P - values ) was calculated with permutation ( ns : non significant , * < 0 . 05 , * * * < 0 . 001 ) .

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

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

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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 ESC + 2i / siRNA - Baz2a showing presence of strong long - contacts corresponding to regions in A compartment bound by BAZ2A in ESC + serum . were subjected to two consecutive treatments with siRNA - control 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 siRNA - BAZ2A . Statistical significance ( P - values ) was calculated using Kolmogorov - Smirnov . C . Boxplot indicating differences in eigenvector values at boundaries centers of A B compartments . Differences in eigenvector values were calculated from HiC data sets from ESC + 2i treated with siRNA - Baz2a siRNA - control . Statistical significance ( P - values ) for experiments was calculated using unpaired two - t - ( * * * < 0 . 001 , ns : non significant ) . Box plots depict minimum maximum values . mean is represented by a horizontal within boxes .

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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 of H3K27me3 in ESC + 2i transfected with siRNA - control siRNA - Baz2a . Histone H3 is shown as a loading control .

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

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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 showing H3K27me3 occupancy .

C. Representative images showing the H3K27me3 occupancy.


D . Average density plot of ChIPseq read of H3K27me3 at - / + 5 kb from 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 .

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

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

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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 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 corresponding siRNA - control ESCs . Cluster 1 represents high to low H327me3 transition whereas Cluster 2 shows low to high H3K27me3 .

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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 that does not contain H3K27me3 . Data were measured by qPCR normalized to input to # 1 value in control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for experiments was calculated using paired two - t - ( ns : non significant , * < 0 . 05 , * * < 0 . 01 ) .

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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 , C ) indicate gain or increase of contacts in ESC + siRNA - Baz2a compared to control ( siRNA - control ) . orange arrow ( D ) indicates loss or decrease in contacts .

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

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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 - F / H - BAZ2A - ESC + 2i ESC + serum . Western blot shows interaction of BAZ2A with SNF2H , TOP2A SMC1a .

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

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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 alterations in gene expression of BAZ2A - regulated genes in ESC + 2i observed upon TOP2A or BAZ2A knockdown . ESCs were treated with TOP2 inhibitor ICF - 193 for 24 hours . mRNA were normalized to Rps12 mRNA 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 experiments was calculated using paired two - t - ( ns : non significant , * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) .

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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 showing that TOP2A regulates H3K27me3 occupancy in ESC + 2i but not in ESC + serum . Data were measured by qPCR normalized to input to Tshz1 value in 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 experiments was calculated using paired two - t - ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) .

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

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 control studies ( NR is detected in control study ) compared to time baseline on untargeted metabolomics measurement . grey shaded represents 95 % confidence level interval . For boxplots limits , middle represents median . upper lower box limits represent 25 % quantiles . upper lower error bars correspond to 75 % quantiles . p - values are derived from one - way ANOVA ( FDR < 0 . 05 ) .

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(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 ) on untargeted metabolomics measurement in metabolic cofactor supplementation study . upper lower box limits represent 25 % quantiles . upper lower error bars correspond to 75 % quantiles . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

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(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 control studies on untargeted metabolomics measurements . Red blue colors denote increased decreased plasma compared to baseline , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

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

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(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 on untargeted metabolomics measurement in supplementation control studies . grey shaded represents 95 % confidence level interval .

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(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 markers compared to time baseline in metabolic cofactor supplementation control studies . Red blue colors denote increased decreased plasma compared to baseline , respectively . * Denotes significance ( one - way ANOVA ; FDR < 0 . 05 ) .

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

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

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(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 nine subjects .

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


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

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(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 nine subjects .

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


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

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(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 nine subjects .

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


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

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

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

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

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

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

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(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 of SB 10C grown to 20 % ( top panels ) or 100 % ( bottom panels ) confluency followed by immuno - of Annexin A1 ( green ) . nuclei were stained with DAPI ( blue ) . A series of 20 - 50 consecutive in z - axis were stacked together to generate 3 - D figure at 400x magnification . Annexin A1 is most abundantly expressed on 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 . experiment was repeated at least twice .

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

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

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

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

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(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 of Annexin A1 expression in 10C 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 . experiment was performed in triplicates repeated more than three times . * * p < 0 . 001 ( one - way ANOVA ) .

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(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 of Annexin A1 in 10C transfected with control siRNA ( dotted black ) or CDH1 - specific siRNA ( dotted red ) 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 . experiment was performed in triplicates repeated twice . * * * p < 0 . 001 ( two - way ANOVA ) .

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(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 of SB 10C 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 E - cadherin blue . Imagines are 800x magnification . Note enhanced expression of Annexin A1 in 10C compared to SB its location on outer layer of mass . experiment was repeated three times . Scale bar , 250 nm .

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

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(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 lysates were incubated with FadAc for 15 or 120 min 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 ) β - catenin in eluates were detected by Western blot . C , untreated control . experiment was repeated three times .

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

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

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

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

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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 mouse colon are shown on right , pointed by blue arrows on right . * p < 0 . 05 , * * p < 0 . 01 ( one - way ANOVA ) .

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(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).


( B ) ANXA1 mRNA in Apcmin / + mouse colonic tumors normal colonic tissues as measured by real - time qPCR . Each symbol represents one mouse . Horizontal lines represent mean values . * p < 0 . 05 , * * p < 0 . 01 ( two - way ANOVA ) .

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(B) ANXA1 mRNA levels in Apcmin/+ mouse colonic tumors and normal colonic tissues as measured by real-time qPCR. Each symbol represents one mouse. Horizontal lines represent mean values. *p<0.05, **p<0.01 (two-way ANOVA).


( C ) Positive correlation between fadA gene copy numbers ( x - axis ) ANXA1 mRNA ( y - axis ) in F . nucleatum - induced Apcmin / + mouse colonic tumors ( n = 34 ; Pearson ' s correlation ) . Each dot represents average of qPCR results performed in

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(C) Positive correlation between fadA gene copy numbers (x-axis) and ANXA1 mRNA levels (y- axis) in F. nucleatum-induced Apcmin/+ mouse colonic tumors (n=34; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicates


( D ) Abundance of fadA in paired normal adenocarcinoma tissues from human CRC ( n = 18 ) expressed as of fadA over total 16S rRNA genes determined by qPCR . Each symbol shows average of duplicate qPCR results from one . Horizontal lines represent mean values . * * p < 0 . 01 ( paired t - ) .

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(D) Abundance of fadA in the paired normal and adenocarcinoma tissues from human CRC patients (n=18) expressed as ratio of fadA over total 16S rRNA genes determined by qPCR. Each symbol shows the average of duplicate qPCR results from one patient. Horizontal lines represent mean values. **p<0.01 (paired t-test).


( E ) ANXA1 mRNA in paired normal adenocarcinoma tissues from CRC ( n = 18 ) . Each symbol shows average of duplicate qPCR results from one . Horizontal lines represent mean values . * * p < 0 . 01 ( paired t - ) .

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(E) ANXA1 mRNA levels in paired normal and adenocarcinoma tissues from CRC patients (n=18). Each symbol shows the average of duplicate qPCR results from one patient. Horizontal lines represent mean values. **p<0.01 (paired t-test).


( F ) Positive correlation between fadA gene copy numbers ( x - axis ) ANXA1 mRNA ( y - axis ) in human colorectal adenocarcinoma tissues ( n = 18 ; Pearson ' s correlation ) . Each dot represents average of qPCR results performed in duplicates .

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(F) Positive correlation between fadA gene copy numbers (x-axis) and ANXA1 mRNA levels (y-axis) in human colorectal adenocarcinoma tissues (n=18; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicates.


( G ) Confocal microscopy of paired normal carcinoma tissues from two colon cancer . frozen sections were incubated with rabbit anti - AnnexinA1 polyclonal antibodies 5G11 mouse anti - FadA monoclonal antibodies . slides were then stained with Alexa Fluor ® 680 - conjugated donkey anti - rabbit Alexa Fluor ® 555 - conjugated goat anti - mouse , washed , covered in mounting medium containing DAPI . scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for normal tissues 400x for carcinomas . Scale bar , 50 μm . Co - localization of FadA ( red ) Annexin A1 ( green ) was observed in carcinomas but not in paired normal tissues .

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(G) Confocal microscopy analysis of paired normal and carcinoma tissues from two colon cancer patients. The frozen sections were incubated with rabbit anti-AnnexinA1 polyclonal antibodies and 5G11 mouse anti-FadA monoclonal antibodies. The slides were then stained with Alexa Fluor®680-conjugated donkey anti-rabbit and Alexa Fluor®555-conjugated goat anti-mouse, washed, and covered in mounting medium containing DAPI. The scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for the normal tissues and 400x for the carcinomas. Scale bar, 50 μm. Co-localization of FadA (red) and Annexin A1 (green) was observed in carcinomas but not in the paired normal tissues.


( A ) Flow cytometry of β - catenin expression in 10C , HCT116 DLD1 following transfection with control siRNA ( clear bars ) or ANXA1 - specific siRNA ( solid bars ) incubation with F . nucleatum 12230 at MOI of ~ 20 : 1 for indicated time periods . geometric means of treated with control siRNA at time 0 was designated as 1 . Data are mean values ± SD . experiment was performed in duplicates or triplicates repeated 1 - 3 times . * * p < 0 . 01 , * * * p < 0 . 001 ( two - way ANOVA ) .

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(A) Flow cytometry analysis of β-catenin expression in 10C, HCT116 and DLD1 cells following transfection with control siRNA (clear bars) or ANXA1-specific siRNA (solid bars) and incubation with F. nucleatum 12230 at MOI of ~20:1 for indicated time periods. The geometric means of cells treated with control siRNA at time 0 was designated as 1. Data are mean values ± SD. The experiment was performed in duplicates or triplicates and repeated 1-3 times. **p<0.01, *** p<0.001 (two-way ANOVA).


( B ) Immuno - of β - catenin in 10C following transfection with control siRNA or ANXA1 - specific siRNA incubation with F . nucleatum 12230 ( Fn ) at MOI of ~ 100 : 1 for 2 hrs . β - catenin was stained with Alexa Fluor ® 680 ( red ) nuclei with DAPI ( blue ) . were captured with confocal microscope at 800x magnification . - , no added . Note increased expression of β - catenin its nucleus translocation in response to F . nucleatum in control siRNA - treated , but not in ANXA1 siRNA - treated . experiment was repeated twice . Scale bar , 200 nm .

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(B) Immuno-staining of β-catenin in 10C cells following transfection with control siRNA or ANXA1-specific siRNA and incubation with F. nucleatum 12230 (Fn) at MOI of ~100:1 for 2 hrs. β-catenin was stained with Alexa Fluor®680 (red) and the nuclei with DAPI (blue). The images were captured with confocal microscope at 800x magnification. -, no bacteria added. Note the increased expression of β-catenin and its nucleus translocation in response to F. nucleatum in control siRNA-treated cells, but not in ANXA1 siRNA-treated cells. The experiment was repeated twice. Scale bar, 200 nm.


( C ) Real - time qPCR of CCND1 expression using mRNA extracted from cancerous 10C , human CRC HCT116 RKO , each grown to 50 % or 100 % confluency . Data are mean values ± . experiment was performed in triplicates repeated twice . * p < 0 . 05 , * * * p < 0 . 001 ( student ' s t - ) .

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(C) Real-time qPCR analysis of CCND1 expression using mRNA extracted from the cancerous 10C, and human CRC cells HCT116 and RKO, each grown to 50% or 100% confluency. Data are mean values ± SEM. The experiment was performed in triplicates and repeated twice. *p<0.05, ***p<0.001 (student's t-test).


( D ) Western - blot of Cyclin D1 , Annexin A1 , β - actin in RKO transfected with control vector or ANXA1 . Induction of Cyclin D1 was observed in response to increased Annexin A1 . experiment was repeated twice .

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(D) Western-blot analysis of Cyclin D1, Annexin A1, and β-actin in RKO cells transfected with control vector or ANXA1. Induction of Cyclin D1 was observed in response to increased Annexin A1. The experiment was repeated twice.


( A ) Tru - Seq of E12 . 5 midbrain floor - plate ( mFP ) , midbrain roof - plate ( mRP ) , anterior ( A , adjacent anterior FP ) posterior ( P , adjacent posterior FP ) . Pbx1 is enriched in midbrain FP , together with Th Pitx3 . Lower of Pbx3 are also expressed in mFP . Gdf7 Wnt1 are restricted to mRP at E12 . 5 .

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(A) Tru-Seq RNA sequence analysis of E12.5 midbrain floor-plate (mFP), midbrain roof-plate (mRP), anterior (A, adjacent anterior FP) and posterior (P, adjacent posterior FP). Pbx1 is enriched in the midbrain FP, together with Th and Pitx3. Lower levels of Pbx3 are also expressed in the mFP. Gdf7 and Wnt1 are restricted to the mRP at E12.5.


( B ) Pbx1 is expressed in intermediate zone ( IZ ) marginal zones ( MZ ) , but not ventricular zone ( VZ ) , of mFP at E12 . 5 , as detected by in situ hybridization .

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(B) Pbx1 is expressed in the intermediate zone (IZ) and marginal zones (MZ), but not the ventricular zone (VZ), of the mFP at E12.5, as detected by in situ hybridization.


( C ) PBX1 is first detected in ventro - lateral part of LMX1a + domain at E10 , preceding birth of first ( TH + ) mDA neurons at E10 . 5 . At E12 . 5 , PBX1 is present in mDA neurons , but not PBX1 + are TH + . boxes indicate showed in higher magnification ( right ) .

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(C) PBX1 is first detected in the ventro-lateral part of the LMX1a+ domain at E10, preceding the birth of the first (TH+) mDA neurons at E10.5. At E12.5, PBX1 is present in all mDA neurons, but not all PBX1+ cells are TH+. White boxes indicate the area showed in higher magnification (right).


( D ) At E11 . 5 , PBX1 defines a subpopulation of NURR1 + neuroblasts labels NURR1 + TH + mDA neurons .

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(D) At E11.5, PBX1 protein defines a subpopulation of NURR1+ neuroblasts and labels all NURR1+TH+ mDA neurons.