( A ) ChIP - qPCR analysis of N - terminally V5 - tagged WT , F545L and M6 mutants of Rap1 . Untagged Rap1 was used as a control . Bars represent mean of percentage of DNA precipitated with anti - V5 antibody conjugated beads relative to input DNA . Error bars represent SEM . N = 4 biological replicates . Two - tailed unpaired Student ' s t - test was performed and significance is indicated . * * * : p < 0 . 001 , * * : 0 . 001 ≤ p < 0 . 01 , n . s . : p > 0 . 05 . ( B ) ChIP - qPCR analysis of N - terminally V5 - tagged WT , F545L and M6 mutants of Rap1 in poz1 ∆ background performed as in ( A ) . N = 4 biological replicates . Statistical analysis is the same as ( A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ChIP-qPCR analysis of N-terminally V5-tagged WT, F545L and M6 mutants of Rap1. Untagged Rap1 was used as a control. Bars represent mean of percentage of DNA precipitated with anti-V5 antibody conjugated beads relative to input DNA. Error bars represent SEM. N= 4 biological replicates. Two-tailed unpaired Student's t-test was performed and significance is indicated. ***: p < 0.001, **: 0.001 ≤ p < 0.01, n.s.: p>0.05. (B) ChIP-qPCR analysis of N-terminally V5-tagged WT, F545L and M6 mutants of Rap1 in poz1∆ background performed as in (A). N= 4 biological replicates. Statistical analysis is the same as (A).


( C ) Single - stranded overhang measured by denaturing gel electrophoresis of duplex - specific nuclease digested genomic DNA and Southern blot with oligonucleotide probes specific for the G - strand ( top panel ) and C - strand ( bottom panel ) . WT , F545L and M6 mutants of Rap1 in the presence or absence of poz1 or ppm1 were analyzed . A 21 - mer of ( GGTTACA ) 3 was used as a positive control and size marker for G - strand and a ( TGTAACC ) 3 oligo was used for the C - strand .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Single-stranded overhang measured by denaturing gel electrophoresis of duplex-specific nuclease digested genomic DNA and Southern blot with oligonucleotide probes specific for the G-strand (top panel) and C-strand (bottom panel). WT, F545L and M6 mutants of Rap1 in the presence or absence of poz1 or ppm1 were analyzed. A 21-mer of (GGTTACA)3 was used as a positive control and size marker for G-strand and a (TGTAACC)3 oligo was used for the C-strand.


( A ) Schematic of scoring matrix . Each nucleus was divided into three concentric zones of equal area with zone I corresponding to the nuclear periphery . The focal plane with the brightest GFP signal and well - defined NE was chosen for measurements . Distance of the GFP spot to NE ( X ) and diameter of the nucleus were measured for the calculation . GFP signals were allocated to one of the three concentric zones of equal surface area as described ( Hediger et al . , 2002 ) . Zone I : X < 0 . 184 x radius ( r ) ) ; Zone II : X between 0 . 184 and 0 . 422 times r ; and X > 0 . 422 x r is zone III . Representative images of cells expressing Nup44 - mCherry ( magenta ) and Taz1 - GFP or Pot1 - GFP ( green ) , respectively . The z - plane showing the brightest GFP spot is shown and was used for quantification . Scale bar : 2μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic of scoring matrix. Each nucleus was divided into three concentric zones of equal area with zone I corresponding to the nuclear periphery. The focal plane with the brightest GFP signal and well-defined NE was chosen for measurements. Distance of the GFP spot to NE (X) and diameter of the nucleus were measured for the calculation. GFP signals were allocated to one of the three concentric zones of equal surface area as described (Hediger et al., 2002). Zone I: X <0.184 x radius (r)); Zone II: X between 0.184 and 0.422 times r; and X >0.422 x r is zone III. Representative images of cells expressing Nup44-mCherry (magenta) and Taz1-GFP or Pot1-GFP (green), respectively. The z-plane showing the brightest GFP spot is shown and was used for quantification. Scale bar: 2μm.


( B ) Stacking bar chart showing telomere distribution ( labeled by Pot1 - GFP ) in each of the three zones . 100 telomere foci were quantified per sample . Chi square tests were performed by comparing the rap1 mutants to WT or bqt4 ∆ . Significance is indicated . * * * : p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stacking bar chart showing telomere distribution (labeled by Pot1-GFP) in each of the three zones. 100 telomere foci were quantified per sample. Chi square tests were performed by comparing the rap1 mutants to WT or bqt4∆. Significance is indicated. ***: p < 0.001.


( C ) Yeast two hybrid assay probing interaction between Rap1 and Bqt4 . WT and mutants of Rap1 were fused to the DNA - binding domain ( BD ) in pGBKT7 . Bqt4 and Taz1 were fused to the Gal4 activation domain ( AD ) in pGADT7 . Cells ( 5μl ) carrying both plasmids at 5 x106 cells / ml were spotted onto - Leu - Trp dropout plates and - Leu - Trp - Ade - His dropout plates to select for ADE2 and HIS3 markers . " - " : Empty BD or AD plasmid .

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(C) Yeast two hybrid assay probing interaction between Rap1 and Bqt4. WT and mutants of Rap1 were fused to the DNA-binding domain (BD) in pGBKT7. Bqt4 and Taz1 were fused to the Gal4 activation domain (AD) in pGADT7. Cells (5μl) carrying both plasmids at 5 x106 cells/ml were spotted onto -Leu-Trp dropout plates and -Leu-Trp-Ade-His dropout plates to select for ADE2 and HIS3 markers. "-": Empty BD or AD plasmid.


( D ) PFGE analysis of nitrogen - starved bqt4 ∆ and bqt3 ∆ cells in the presence or absence of poz1 .

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

(D) PFGE analysis of nitrogen-starved bqt4∆ and bqt3∆ cells in the presence or absence of poz1.


( E ) End fusions in bqt4 ∆ poz1 ∆ and bqt3 ∆ poz1 ∆ are dependent on the presence of ligase IV . PFGE analysis of nitrogen - starved cells in the presence or absence of lig4 .

O O O O O O O O O O O O O O O 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) End fusions in bqt4∆poz1∆ and bqt3∆poz1∆ are dependent on the presence of ligase IV. PFGE analysis of nitrogen-starved cells in the presence or absence of lig4.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 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 .

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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 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 ) .

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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 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 O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 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 .

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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 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 .

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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 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 .

(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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 the mouse colon are shown on the right , pointed by blue arrows on the 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 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 ) .

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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 ) 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

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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 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 ) .

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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 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 ) .

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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 ) 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 .

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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 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 .

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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 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 ) .

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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 - 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 .

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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 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 ) .

(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 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 .

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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.


( C ) STIM1 and STIM2 mRNA expression in PBMC of P1 , P2 , mother and a healthy donor ( HD ) that were left unstimulated or stimulated with anti - CD3 / CD28 for 24 hours . mRNA levels of STIM1 / 2 normalized to 18S . Graph shows the mean ± SEM of duplicates from one experiment .

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(C) STIM1 and STIM2 mRNA expression in PBMC of P1, P2, mother and a healthy donor (HD) that were left unstimulated or stimulated with anti-CD3/CD28 for 24 hours. mRNA levels of STIM1/2 normalized to 18S. Graph shows the mean ± SEM of duplicates from one experiment.


( D ) STIM1 protein expression in CD4 + and CD8 + T cells from P1 , P2 , the mother and a HD analyzed by flow cytometry . Shaded histograms : polyclonal rabbit IgG control antibody ; open histograms : polyclonal anti - STIM1 antibody . Bar graphs show the delta MFI calculated as MFISTIM1 - MFIIgG control that was normalized to HD T cells . Bar graphs are mean ± SEM of 2 independent repeat experiments .

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(D) STIM1 protein expression in CD4+ and CD8+ T cells from P1, P2, the mother and a HD analyzed by flow cytometry. Shaded histograms: polyclonal rabbit IgG control antibody; open histograms: polyclonal anti-STIM1 antibody. Bar graphs show the delta MFI calculated as MFISTIM1 - MFIIgG control that was normalized to HD T cells. Bar graphs are mean ± SEM of 2 independent repeat experiments.


( E ) STIM1 protein expression in expanded human T cells analyzed by immunoblotting . One representative Western blot of 3 is shown . Bar graphs are the mean ± SEM of STIM1 expression normalized to actin from 3 independent experiments .

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(E) STIM1 protein expression in expanded human T cells analyzed by immunoblotting. One representative Western blot of 3 is shown. Bar graphs are the mean ± SEM of STIM1 expression normalized to actin from 3 independent experiments.


( F ) Ca2 + influx in Fura - 2 loaded PBMC of P1 , P2 , the mother and a HD . T cells were stimulated with 1 μM thapsigargin ( TG ) in the absence of extracellular Ca2 + followed by addition of 1 mM extracellular Ca2 + . Bar graphs show the integrated Ca2 + influx response ( AUC , area under the curve ) from 400 - 800 sec and the peak Ca2 + influx normalized to baseline Ca2 + levels ( F340 / 380 ) at 400 sec . Data represent the mean ± SEM from 2 experiments . Statistical analysis by unpaired Student ' s t - test . * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) Ca2+ influx in Fura-2 loaded PBMC of P1, P2, the mother and a HD. T cells were stimulated with 1 μM thapsigargin (TG) in the absence of extracellular Ca2+ followed by addition of 1 mM extracellular Ca2+. Bar graphs show the integrated Ca2+ influx response (AUC, area under the curve) from 400-800 sec and the peak Ca2+ influx normalized to baseline Ca2+ levels (F340/380) at 400 sec. Data represent the mean ± SEM from 2 experiments. Statistical analysis by unpaired Student's t-test. **p<0.01, ***p<0.001.


GFP - ORAI1 expressing HEK293 cells were transfected with mCherry - STIM1 ( WT or L374P ) and left unstimulated or stimulated with 1 μM TG in Ca2 + - free Ringer ' s solution . ( D , E ) Representative TIRF microscopy images from one of two repeat experiments . Scale bars , 10 μm .

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GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (D,E) Representative TIRF microscopy images from one of two repeat experiments. Scale bars, 10 μm.


GFP - ORAI1 expressing HEK293 cells were transfected with mCherry - STIM1 ( WT or L374P ) and left unstimulated or stimulated with 1 μM TG in Ca2 + - free Ringer ' s solution . ( F ) Percentages of WT or mutant mCherry - STIM1 present at the plasma membrane ( i . e . visible in the TIRFM evanescent field ) before TG stimulation . Data are the mean ± SEM calculated from 550 ( WT ) and 830 ( L374P ) cells from two independent experiments . ( G ) Change of mCherry - STIM1 ( WT or L374P mutant ) fluorescence [ F ( t ) / F ( 0 ) ] in the TIRFM evanescence field . Fluorescence normalized to values at the time of TG addition in Ca2 + - free Ringer ' s buffer at 0 sec . Traces show mean ± SEM of 40 - 50 cells from two independent experiments . ( H ) Colocalization of mCherry - STIM1 ( WT or L374P mutant ) and GFP - ORAI1 from the time of TG addition at 0 sec . Colocalization measured using the Pearson ' s coefficient for GFP and mCherry .

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GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (F) Percentages of WT or mutant mCherry-STIM1 present at the plasma membrane (i.e. visible in the TIRFM evanescent field) before TG stimulation. Data are the mean ± SEM calculated from 550 (WT) and 830 (L374P) cells from two independent experiments. (G) Change of mCherry-STIM1 (WT or L374P mutant) fluorescence [F(t)/F(0)] in the TIRFM evanescence field. Fluorescence normalized to values at the time of TG addition in Ca2+-free Ringer's buffer at 0 sec. Traces show mean ± SEM of 40-50 cells from two independent experiments. (H) Colocalization of mCherry-STIM1 (WT or L374P mutant) and GFP-ORAI1 from the time of TG addition at 0 sec. Colocalization measured using the Pearson's coefficient for GFP and mCherry.


Cell size ( A ) of CD4 + T cells from P1 ( red ) , P2 ( blue ) , the mother ( grey ) and HD ( black ) stimulated with anti - CD3 ( 5 μg / ml ) and anti - CD28 ( 10 μg / ml ) in the presence or absence of 1 μM FK506 for 24 hours . ( A ) Representative histograms of FSC ( left panel ) and percentages of T cell blasts ( defined as cells to the right of the dotted vertical line ) analyzed by flow cytometry ( right panel ) .

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Cell size (A) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (A) Representative histograms of FSC (left panel) and percentages of T cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel).


proliferation ( B ) of CD4 + T cells from P1 ( red ) , P2 ( blue ) , the mother ( grey ) and HD ( black ) stimulated with anti - CD3 ( 5 μg / ml ) and anti - CD28 ( 10 μg / ml ) in the presence or absence of 1 μM FK506 for 24 hours . ( B ) Representative histograms of CFSE dilution ( left panel ) and percentages of proliferating cells ( defined as cells to the left of the dotted vertical line ) ( right panel ) . Bar graphs in A and B are the mean ± SEM from two independent experiments .

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proliferation (B) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments.


( C , D ) Cytokine production by PBMC from P1 , P2 , the mother and an unrelated HD after stimulation with PMA ( 40 ng / ml ) and ionomycin ( 500 ng / ml ) for 4 hours . Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3 , CD4 and CD45RO , permeabilization and intracellular cytokine staining for GM - CSF , IL - 22 and IL - 17A . Representative flow cytometry plots ( C ) and quantification of Th17 ( GM - CSF , IL - 22 , IL - 17A ) , Th1 ( TNF - α , IFN - γ ) and Th2 ( IL - 4 ) cytokines ( D ) . Data represent the mean ± SEM from two independent experiments .

(C,D) Cytokine production by PBMC from P1, P2, the mother and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 hours. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4 and CD45RO, permeabilization and intracellular cytokine staining for GM-CSF, IL-22 and IL-17A. Representative flow cytometry plots (C) and quantification of Th17 (GM-CSF, IL-22, IL-17A), Th1 (TNF-α, IFN-γ) and Th2 (IL-4) cytokines (D). Data represent the mean ± SEM from two independent experiments.


A ) Severe onychomycosis in P2 ( STIM1 p . L374P ) .

A) Severe onychomycosis in P2 (STIM1 p.L374P).


B ) PBMC of a healthy donor ( HD1 ) cultured for 6 days in the presence of 6 . 5 mg / ml C . albicans protein . At day 6 , PBMC were restimulated with PMA and ionomycin in the presence of 10 μM GSK - 7975A or DMSO for 6 h and CD4 + and CD8 + T cells were analyzed for cytokine production by flow cytometry . Bar graphs represent the mean ± SEM from two independent experiments .

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B) PBMC of a healthy donor (HD1) cultured for 6 days in the presence of 6.5 mg/ml C. albicans protein. At day 6, PBMC were restimulated with PMA and ionomycin in the presence of 10 μM GSK-7975A or DMSO for 6 h and CD4+ and CD8+ T cells were analyzed for cytokine production by flow cytometry. Bar graphs represent the mean ± SEM from two independent experiments.


Human T cells from two healthy donors , P1 and his mother were expanded and cultured in the presence of C . albicans and IL - 2 for 2 - 4 weeks . ( C ) T cells were restimulated with PMA and ionomycin for 6 h in the presence or absence of 10 μM GSK - 7975A and analyzed for cytokine production by flow cytometry . Bar graphs represent the mean ± SEM from two independent experiments .

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Human T cells from two healthy donors, P1 and his mother were expanded and cultured in the presence of C. albicans and IL-2 for 2-4 weeks. (C) T cells were restimulated with PMA and ionomycin for 6 h in the presence or absence of 10 μM GSK-7975A and analyzed for cytokine production by flow cytometry. Bar graphs represent the mean ± SEM from two independent experiments.


Human T cells from two healthy donors , P1 and his mother were expanded and cultured in the presence of C . albicans and IL - 2 for 2 - 4 weeks . ( D ) Fura - 2 loaded T cells were stimulated with thapsigargin ( TG ) in Ca2 + - free buffer followed by readdition of 2 mM Ca2 + to induce SOCE . Bar graphs represent the mean ± SEM from two independent experiments .

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Human T cells from two healthy donors, P1 and his mother were expanded and cultured in the presence of C. albicans and IL-2 for 2-4 weeks. (D) Fura-2 loaded T cells were stimulated with thapsigargin (TG) in Ca2+-free buffer followed by readdition of 2 mM Ca2+ to induce SOCE. Bar graphs represent the mean ± SEM from two independent experiments.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( A ) Body weight ( BW ) measured for 7 days and normalized to BW at day 0 of infection . Data are mean ± SEM measured from 4 repeat experiments and 13 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (A) Body weight (BW) measured for 7 days and normalized to BW at day 0 of infection. Data are mean ± SEM measured from 4 repeat experiments and 13 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( B ) Quantification of CFU of C . albicans per gram tongue tissue of the indicated mice at day 7 p . i . Data are mean ± SEM from 4 repeat experiments and 12 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (B) Quantification of CFU of C. albicans per gram tongue tissue of the indicated mice at day 7 p.i. Data are mean ± SEM from 4 repeat experiments and 12 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( C ) Macroscopic images of the C . albicans - infected tongues of mice at day 7 p . i . Arrows and dashes lines indicate C . albicans infiltrated areas .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (C) Macroscopic images of the C. albicans-infected tongues of mice at day 7 p.i. Arrows and dashes lines indicate C. albicans infiltrated areas.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( D ) Histological images of H & E ( left ) and PAS ( right ) stained tongue tissue at day 7 p . i . Clusters of polymorphonuclear ( PMN ) cells ( neutrophils , white arrows ) and Candida ( black arrows ) are indicated .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (D) Histological images of H&E (left) and PAS (right) stained tongue tissue at day 7 p.i. Clusters of polymorphonuclear (PMN) cells (neutrophils, white arrows) and Candida (black arrows) are indicated.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( E ) Frequencies of Cd11b + Gr1 + neutrophils in the tongues from the indicated mice at day 7 p . i . Bar graphs represent the mean ± SEM from 3 experiments and 9 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (E) Frequencies of Cd11b+Gr1+ neutrophils in the tongues from the indicated mice at day 7 p.i. Bar graphs represent the mean ± SEM from 3 experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


( F , G ) Cytokine production by CD4 + T cells isolated from submandibular lymph nodes of WT , Stim1fl / flVav - iCre , Stim1fl / flStim2fl / flVav - iCre and Stim1fl / flCd4Cre mice 7 days after sublingual infection with 2x107 CFU C . albicans . Representative contour plots of cytokine production ( F ) and percentages of CD4 + T cells producing TNF - α , IFN - γ , IL - 17A , IL - 17F and GM - CSF ( G ) . Data are the mean ± SEM from two independent experiments and 9 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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(F,G) Cytokine production by CD4+ T cells isolated from submandibular lymph nodes of WT, Stim1fl/flVav-iCre, Stim1fl/flStim2fl/flVav-iCre and Stim1fl/flCd4Cre mice 7 days after sublingual infection with 2x107 CFU C. albicans. Representative contour plots of cytokine production (F) and percentages of CD4+ T cells producing TNF-α, IFN-γ, IL-17A, IL-17F and GM-CSF (G). Data are the mean ± SEM from two independent experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


Candicidal function of neutrophils depend on SOCE . ( H ) Coculture of Cd11b + Gr - 1 + neutrophils from WT , Stim1fl / flMx1Cre and Stim1fl / flStim2fl / flMx1Cre mice with C . albicans in vitro for 0 , 30 , 60 and 90 min ( MOI 0 . 5 ) . Percentages of live C . albicans isolated from neutrophils that were lysed at the indicated time points . Data are the mean ± SEM from 6 mice per genotype and 3 independent repeat experiments .

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Candicidal function of neutrophils depend on SOCE. (H) Coculture of Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice with C.albicans in vitro for 0, 30, 60 and 90 min (MOI 0.5). Percentages of live C.albicans isolated from neutrophils that were lysed at the indicated time points. Data are the mean ± SEM from 6 mice per genotype and 3 independent repeat experiments.


ROS production of neutrophils depend on SOCE . ( I ) ROS production by Cd11b + Gr - 1 + neutrophils from WT , Stim1fl / flMx1Cre and Stim1fl / flStim2fl / flMx1Cre mice was measured after loading of cells with dihydrorhodamine 123 and stimulation with 10 nM PMA for 30 minutes . Representative histogram plots and bar graphs indicating the percentages of ROS + neutrophils normalized to WT neutrophils stimulated with PMA .

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ROS production of neutrophils depend on SOCE. (I) ROS production by Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice was measured after loading of cells with dihydrorhodamine 123 and stimulation with 10 nM PMA for 30 minutes. Representative histogram plots and bar graphs indicating the percentages of ROS+ neutrophils normalized to WT neutrophils stimulated with PMA.


Sublingual C . albicans infection of WT and Stim1fl / flCd4Cre mice as described in Figure 5 . ( A ) Body weight ( BW ) change measured for 7 days p . i . relative to BW at the day of infection . Data are mean ± SEM measured from 4 repeat experiments and 13 WT and 7 Stim1fl / flCd4Cre mice .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (A) Body weight (BW) change measured for 7 days p.i. relative to BW at the day of infection. Data are mean ± SEM measured from 4 repeat experiments and 13 WT and 7 Stim1fl/flCd4Cre mice.


Sublingual C . albicans infection of WT and Stim1fl / flCd4Cre mice as described in Figure 5 . ( B ) Quantification of CFU of C . albicans isolated from the tongues of mice at day 7 post - infection ( p . i . ) . Data are mean ± SEM from 4 repeat experiments and 7 - 13 mice per genotype .

Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (B) Quantification of CFU of C. albicans isolated from the tongues of mice at day 7 post-infection (p.i.). Data are mean ± SEM from 4 repeat experiments and 7-13 mice per genotype.


Sublingual C . albicans infection of WT and Stim1fl / flCd4Cre mice as described in Figure 5 . ( C ) Macroscopic images of the C . albicans - infected tongues of mice at day 7 p . i .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (C) Macroscopic images of the C. albicans-infected tongues of mice at day 7 p.i.


Sublingual C . albicans infection of WT and Stim1fl / flCd4Cre mice as described in Figure 5 . ( D ) Histological images of H & E ( left ) and PAS - stained ( right ) tongue tissue at day 7 p . i . Scale bars 100 μm . Arrows point to clusters of polymorphonuclear ( PMN ) cell ( neutrophil ) infiltrates .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (D) Histological images of H&E (left) and PAS-stained (right) tongue tissue at day 7 p.i. Scale bars 100 μm. Arrows point to clusters of polymorphonuclear (PMN) cell (neutrophil) infiltrates.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( E ) Kaplan - Meier survival plot showing survival of mice . Data from 5 Stim1fl / fl Cd4Cre mice and 12 WT mice is shown .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (E) Kaplan-Meier survival plot showing survival of mice. Data from 5 Stim1fl/fl Cd4Cre mice and 12 WT mice is shown.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( F ) Body weight ( BW ) of mice normalized to BW at the day of infection . Data are the mean ± SEM from 4 independent experiments and 8 WT and 7 Stim1fl / flCd4Cre mice .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (F) Body weight (BW) of mice normalized to BW at the day of infection. Data are the mean ± SEM from 4 independent experiments and 8 WT and 7 Stim1fl/flCd4Cre mice.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( G ) Representative macroscopic images of kidneys at day 6 p . i .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (G) Representative macroscopic images of kidneys at day 6 p.i.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( H ) CFUs of C . albicans per gram tissue isolated from the liver , the right kidney and the lung of mice at day 6 p . i . Each dot represents one mouse .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (H) CFUs of C. albicans per gram tissue isolated from the liver, the right kidney and the lung of mice at day 6 p.i. Each dot represents one mouse.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( I ) Representative histological images of H & E and PAS stained kidneys of mice at day 6 p . i . Magnification 6 . 4x ( left ) and 40x ( right ) . White arrows define areas infiltrated with leukocytes ( H & E ) and C . albicans ( PAS ) .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (I) Representative histological images of H&E and PAS stained kidneys of mice at day 6 p.i. Magnification 6.4x (left) and 40x (right). White arrows define areas infiltrated with leukocytes (H&E) and C. albicans (PAS).


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( J ) Frequencies of CD4 + T cells isolated from the spleens of mice at day 6 p . i . producing the indicated cytokines after restimulation with 20 nM PMA and 1 μM ionomycin for 6 hours and analyzed by flow cytometry .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (J) Frequencies of CD4+ T cells isolated from the spleens of mice at day 6 p.i. producing the indicated cytokines after restimulation with 20 nM PMA and 1 μM ionomycin for 6 hours and analyzed by flow cytometry.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( K ) Frequencies CD45 + Cd11b + Gr - 1 + neutrophils in the blood of mice at day 6 post - infection analyzed by flow cytometry . Data in J are the mean ± SEM from 2 independent experiments and 6 WT and 4 Stim1fl / flCd4Cre mice ; data in K are the mean ± SEM from one experiment and 2 WT and 3 Stim1fl / flCd4Cre mice .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (K) Frequencies CD45+Cd11b+Gr-1+ neutrophils in the blood of mice at day 6 post-infection analyzed by flow cytometry. Data in J are the mean ± SEM from 2 independent experiments and 6 WT and 4 Stim1fl/flCd4Cre mice; data in K are the mean ± SEM from one experiment and 2 WT and 3 Stim1fl/flCd4Cre mice.


RNA - seq analysis of CD4 + T cells isolated from 3 WT and 3 Stim1fl / flCd4Cre mice that were differentiated into pathogenic Th17 cells ( pTh17 : IL - 1β , IL - 6 , IL - 23 ) and non - pathogenic Th17 cells ( npTh17 : IL - 6 , TGFβ1 ) . ( B ) Volcano plots of DEGs in npTh17 cells ( gray dots ) . Highlighted in red ( upregulated in Stim1fl / flCd4Cre ) and blue ( downregulated in Stim1fl / flCd4Cre ) are DEGs that belong to a gene expression signature of npTh17 cells and pTh17 cells defined previously ( Gaublomme et al . , 2015 , Lee et al . , 2012 ) .

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RNA-seq analysis of CD4+ T cells isolated from 3 WT and 3 Stim1fl/flCd4Cre mice that were differentiated into pathogenic Th17 cells (pTh17: IL-1β, IL-6, IL-23) and non-pathogenic Th17 cells (npTh17: IL-6, TGFβ1). (B) Volcano plots of DEGs in npTh17 cells (gray dots). Highlighted in red (upregulated in Stim1fl/flCd4Cre) and blue (downregulated in Stim1fl/flCd4Cre) are DEGs that belong to a gene expression signature of npTh17 cells and pTh17 cells defined previously (Gaublomme et al., 2015, Lee et al., 2012).


( C ) Normalized expression of Th17 cell - associated cytokines and receptors in npTh17 cells derived from WT and Stim1fl / flCd4Cre mice . Bar graphs represent the mean ± SEM from 3 mice per group . Statistical analysis by unpaired Student ' s t - test with the following significance levels : * p < 0 . 05 , * * * p < 0 . 001 .

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(C) Normalized expression of Th17 cell-associated cytokines and receptors in npTh17 cells derived from WT and Stim1fl/flCd4Cre mice. Bar graphs represent the mean ± SEM from 3 mice per group. Statistical analysis by unpaired Student's t-test with the following significance levels: *p<0.05, ***p<0.001.


( F ) Heat map of DEGs ( p ( adj . ) < 0 . 1 ) within the KEGG pathways " Foxo signaling " and " HIF - 1 signaling " that are specifically deregulated in npTh17 cells . Numerical values are relative gene expression .

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(F) Heat map of DEGs (p (adj.) < 0.1) within the KEGG pathways "Foxo signaling" and "HIF-1 signaling" that are specifically deregulated in npTh17 cells. Numerical values are relative gene expression.


( G ) Gene set enrichment analysis ( GSEA ) of DEGs in WT and STIM1 - deficient npTh17 cells . Normalized enrichment score ( NES ) and p - values are as indicated for the following genesets : Myc targets : " Menssen _ myc _ targets " ; Glycolysis : " Humancyc _ mm _ glycolysis _ i " ; Mitochondrial function : " Wong _ mitochondria _ gene _ module " ; TCA cycle : " Kegg _ mm _ citrate _ cycle " .

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(G) Gene set enrichment analysis (GSEA) of DEGs in WT and STIM1-deficient npTh17 cells. Normalized enrichment score (NES) and p-values are as indicated for the following genesets: Myc targets: "Menssen_myc_targets"; Glycolysis: "Humancyc_mm_glycolysis_i"; Mitochondrial function: "Wong_mitochondria_gene_module"; TCA cycle: "Kegg_mm_citrate_cycle".


( A ) Heat map of metabolism - associated DEGs ( p ( adj . ) < 0 . 10 ) in npTh17 cells of WT and Stim1fl / flCd4Cre mice determined by RNA - Seq as described in Figure 7 . Heatmap shows relative minimum and maximum values per gene ( row min / max ) . Numerical values are relative gene expression . TCA , tricarboxylic acid .

(A) Heat map of metabolism-associated DEGs (p (adj.) < 0.10) in npTh17 cells of WT and Stim1fl/flCd4Cre mice determined by RNA-Seq as described in Figure 7. Heatmap shows relative minimum and maximum values per gene (row min/max). Numerical values are relative gene expression. TCA, tricarboxylic acid.


Glucose and mitochondrial metabolism in npTh17 cells from WT ( grey or black ) and Stim1fl / flCd4Cre ( blue ) mice differentiated for 3 days in vitro . ( B ) Glucose uptake by npTh17 cells were loaded with 2 - NBDG ( + ) or not ( - ) and analyzed by flow cytometry 90 min later . Bar graphs show the delta MFI of 2 - NBDG fluorescence normalized to unlabeled cells . Data represent the mean ± SEM from 3 independent experiments .

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Glucose and mitochondrial metabolism in npTh17 cells from WT (grey or black) and Stim1fl/flCd4Cre (blue)mice differentiated for 3 days in vitro. (B) Glucose uptake by npTh17 cells were loaded with 2-NBDG (+) or not (-) and analyzed by flow cytometry 90 min later. Bar graphs show the delta MFI of 2-NBDG fluorescence normalized to unlabeled cells. Data represent the mean ± SEM from 3 independent experiments.


( C ) Extracellular acidification rate ( ECAR ) of npTh17 cells before and after addition of 25 mM glucose , 5 μM oligomycin and 100 mM 2 - deoxy - D - glucose ( 2 - DG ) . Seahorse graphs and bar graphs depicting glycolysis , glycolytic capacity and reserve the mean ± SEM from of 2 - 3 mice per genotype from one representative out of three independent experiments .

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(C) Extracellular acidification rate (ECAR) of npTh17 cells before and after addition of 25 mM glucose, 5 μM oligomycin and 100 mM 2-deoxy-D-glucose (2-DG). Seahorse graphs and bar graphs depicting glycolysis, glycolytic capacity and reserve the mean ± SEM from of 2-3 mice per genotype from one representative out of three independent experiments.


Glucose and mitochondrial metabolism in npTh17 cells from WT ( grey or black ) and Stim1fl / flCd4Cre ( blue ) mice differentiated for 3 days in vitro . ( D ) Oxygen consumption rate ( OCR ) of npTh17 cells before and after addition of 1 μM oligomycin , 1 . 5 μM FCCP and 100 nM rotenone / 1 μM antimycin . Seahorse graphs and bar graphs depicting basal and maximal respiration , coupling efficiency and ATP production represent the mean ± SEM from of 2 - 3 mice per genotype from one representative out of three independent experiments .

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Glucose and mitochondrial metabolism in npTh17 cells from WT (grey or black) and Stim1fl/flCd4Cre (blue)mice differentiated for 3 days in vitro. (D) Oxygen consumption rate (OCR) of npTh17 cells before and after addition of 1 μM oligomycin, 1.5 μM FCCP and 100 nM rotenone / 1 μM antimycin. Seahorse graphs and bar graphs depicting basal and maximal respiration, coupling efficiency and ATP production represent the mean ± SEM from of 2-3 mice per genotype from one representative out of three independent experiments.


Glucose and mitochondrial metabolism in human CD4 + T cells isolated from PBMC of a HD and P1 and stimulated with 5 μg / ml anti - CD3 and 10 μg / ml anti - CD28 for 24 hours before analysis . Some HD T cells were stimulated in the presence of 1μM FK506 . ( E , F ) ECAR before and after addition of 25 mM glucose , 5 μM oligomycin and 100 mM 2 - DG .

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Glucose and mitochondrial metabolism in human CD4+ T cells isolated from PBMC of a HD and P1 and stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 for 24 hours before analysis. Some HD T cells were stimulated in the presence of 1μM FK506. (E,F) ECAR before and after addition of 25 mM glucose, 5 μM oligomycin and 100 mM 2-DG.


Glucose and mitochondrial metabolism in human CD4 + T cells isolated from PBMC of a HD and P1 and stimulated with 5 μg / ml anti - CD3 and 10 μg / ml anti - CD28 for 24 hours before analysis . Some HD T cells were stimulated in the presence of 1μM FK506 . ( G , H ) OCR before and after addition of 1 μM oligomycin , 1 . 5 μM FCCP and 100 nM rotenone / 1 μM antimycin . Seahorse graphs ( G ) and bar graphs ( H ) depicting basal and maximal respiration , SRC and ATP production ( right ) represent the mean ± SEM of 2 technical replicates from isolated T cells from one experiment .

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

Glucose and mitochondrial metabolism in human CD4+ T cells isolated from PBMC of a HD and P1 and stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 for 24 hours before analysis. Some HD T cells were stimulated in the presence of 1μM FK506. (G,H) OCR before and after addition of 1 μM oligomycin, 1.5 μM FCCP and 100 nM rotenone / 1 μM antimycin. Seahorse graphs (G) and bar graphs (H) depicting basal and maximal respiration, SRC and ATP production (right) represent the mean ± SEM of 2 technical replicates from isolated T cells from one experiment.


THP - 1 cells were stimulated with ( 50 000 particles / cell ) AdV - GFP and infection after 24 h measured by flow cytometry .

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THP-1 cells were stimulated with (50 000 particles/cell) AdV-GFP and infection after 24 h measured by flow cytometry.


Cells were stimulated as in ( A ) with AdV - GFP or AdV - GFP complexed with 20 μg / ml h9C12 or 20 mg / ml human IVIg . Cell supernatants were harvested at indicated time - points and cytokines measured by ELISA ( B )

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Cells were stimulated as in (A) with AdV-GFP or AdV-GFP complexed with 20 μg/ml h9C12 or 20 mg/ml human IVIg. Cell supernatants were harvested at indicated time-points and cytokines measured by ELISA (B)


Cells were stimulated as in ( A ) with AdV - GFP or AdV - GFP complexed with 20 μg / ml h9C12 or 20 mg / ml human IVIg . pro - and cleaved IL - 1β was measured in whole cell lysates or in supernatants by western blot ( C ) .

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Cells were stimulated as in (A) with AdV-GFP or AdV-GFP complexed with 20 μg/ml h9C12 or 20 mg/ml human IVIg. pro- and cleaved IL-1β was measured in whole cell lysates or in supernatants by western blot (C).


HMDM were primed with 10 μg / ml pI : C for 2h , then stimulated for 16h with virus + / - antibody . Cytokines in the supernatants were measured by ELISA .

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HMDM were primed with 10 μg/ml pI:C for 2h, then stimulated for 16h with virus +/- antibody. Cytokines in the supernatants were measured by ELISA.


AdV - GFP was incubated with 20 ug / ml h9C12 antibody or 20 mg / ml IVIg in PBS . These AdV - Ab complexes were then diluted 1 : 2 to give final doses of viral particles being added to WT THP - 1 cells ( with antibody complexed ) as indicated . A single experiment representative of 3 independent experiments is shown .

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AdV-GFP was incubated with 20 ug/ml h9C12 antibody or 20 mg/ml IVIg in PBS. These AdV-Ab complexes were then diluted 1:2 to give final doses of viral particles being added to WT THP-1 cells (with antibody complexed) as indicated. A single experiment representative of 3 independent experiments is shown.


THP - 1s were stimulated with a constant dose of AdV - GFP ( 50 000 pp / cell ) , and complexed with decreasing antibody concentrations as indicated . A single experiment representative of 2 independent experiments is shown .

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THP-1s were stimulated with a constant dose of AdV-GFP (50 000 pp/cell), and complexed with decreasing antibody concentrations as indicated. A single experiment representative of 2 independent experiments is shown.


HMDM were stimulated directly ( TNF ) or primed ( IL - 1β , LDH ) with AdV - Ab complexes as in ( B ) . Data for each individual donor ( 2 total ) is shown .

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HMDM were stimulated directly (TNF) or primed (IL-1β, LDH) with AdV-Ab complexes as in (B). Data for each individual donor (2 total) is shown.


THP - 1 were stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell death was measured by LDH

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THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release


THP - 1 were stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell viability measured by PrestoBlue

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THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assay


HMDM were primed , or not , and stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell death was measured by LDH

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HMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release


HMDM were primed , or not , and stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell viability measured by PrestoBlue

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

HMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assay


HMDM were stimulated with AdV ( 50 000 pp / cell ) complexed with antibody at indicated doses for 2h and viral genomes were measured by qPCR . ( n = 4 , mean ± s . e . m )

HMDM were stimulated with AdV (50 000 pp/cell) complexed with antibody at indicated doses for 2h and viral genomes were measured by qPCR. (n = 4, mean ± s.e.m)


AdV - mCherry was added to AdV - GFP at a ratio of 1 : 200 and 50 000 pp / cell of this mix was complexed with h9C12 and IVIg and indicated doses . HMDM were stimulated with these complexes for 16h and infection measured as mCherry fluorescence by flow cytometry . In virus alone the percentage of mCherry positive cells was ~ 20 % , the relative infection with antibodies was normalised to virus alone as 100 % .

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AdV-mCherry was added to AdV-GFP at a ratio of 1:200 and 50 000 pp/cell of this mix was complexed with h9C12 and IVIg and indicated doses. HMDM were stimulated with these complexes for 16h and infection measured as mCherry fluorescence by flow cytometry. In virus alone the percentage of mCherry positive cells was ~ 20%, the relative infection with antibodies was normalised to virus alone as 100%.


HMDM were stimulated for 3 h with AdV - Ab complexes as in ( A , B ) or with LeuLeu at 10μM , and lysosomal damage measured by loss of acridine orange red fluorescence using flow cytometry .

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HMDM were stimulated for 3 h with AdV-Ab complexes as in (A,B) or with LeuLeu at 10μM, and lysosomal damage measured by loss of acridine orange red fluorescence using flow cytometry.


HMDM were stimulated as in ( C ) and cell death was assessed by an increase in e780 fluorescence . The mean of 3 independent donors is shown .

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HMDM were stimulated as in (C) and cell death was assessed by an increase in e780 fluorescence. The mean of 3 independent donors is shown.


( E ) The MFI of the AO - red channel ( 685nM ) for the populations determined as e780 positive ( dying ) or e780 negative ( live ) for the condition of AdV + IVIg 20 was measured and plotted as mean + SEM of the three donors shown in ( D ) .

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(E)The MFI of the AO- red channel (685nM) for the populations determined as e780 positive (dying) or e780 negative (live) for the condition of AdV + IVIg 20 was measured and plotted as mean + SEM of the three donors shown in (D).


Primed or ( C ) un - primed HMDM were stimulated with AdV ( 50 000 pp / cell ) + / - h9C12 ( 20 μg / ml ) for 16h .

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Primed or (C) un- primed HMDM were stimulated with AdV (50 000 pp/cell) +/- h9C12 (20 μg/ml) for 16h.


Ctrl or Trim21 - deficient THP - 1s were stimulated for 4 h with 1000 U / ml IFNα and TRIM21 expression measured by immunoblot .

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

Ctrl or Trim21- deficient THP-1s were stimulated for 4 h with 1000 U/ml IFNα and TRIM21 expression measured by immunoblot.


WT or TRIM21 - deficient THP - 1s were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 or 20 mg / ml IVIg or with 200 ng / well HT - DNA or 10 μM Nigericin for 16h , and IL - 1β measured in the supernatant by ELISA .

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WT or TRIM21- deficient THP-1s were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT-DNA or 10 μM Nigericin for 16h, and IL-1β measured in the supernatant by ELISA.


WT or TRIM21 - deficient THP - 1s were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 or 20 mg / ml IVIg or with 200 ng / well HT - DNA or 10 μM Nigericin for 16h , and TNF measured in the supernatant by ELISA .

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WT or TRIM21- deficient THP-1s were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT-DNA or 10 μM Nigericin for 16h, and TNF measured in the supernatant by ELISA.


WT mice were injected i . v . with 2 . 5ug WT or H433A h9C12 antibody , then the next day injected i . v . with 2 . 5 x 1011 pp AdV - GFP . 4h later spleens were harvested and neutrophil influx measured by flow cytometry .

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WT mice were injected i.v. with 2.5ug WT or H433A h9C12 antibody, then the next day injected i.v. with 2.5 x 1011 pp AdV-GFP. 4h later spleens were harvested and neutrophil influx measured by flow cytometry.


HMDM were stimulated with AdV - GFP ( 250 pp / cell ) in the presence of h9C12 antibodies at indicated doses . Infection was measured after 24h by flow cytometry . Infection relative to AdV alone is shown graphed on the left ( n = 4 mean ± s . e . m ) . Representative plots and a comparison of the different h9C12 mutants at 0 . 2 ug / ml are also shown in the graph on the right ( n = 4 , mean ± s . e . m . ) .

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

HMDM were stimulated with AdV-GFP (250 pp/cell) in the presence of h9C12 antibodies at indicated doses. Infection was measured after 24h by flow cytometry. Infection relative to AdV alone is shown graphed on the left (n = 4 mean ± s.e.m). Representative plots and a comparison of the different h9C12 mutants at 0.2 ug/ml are also shown in the graph on the right(n = 4,mean ± s.e.m.).


HMDM were stimulated with AdV ( 50 000 pp / cell ) and antibody ( h9C12 ; 20 ug / ml , IVIg : 20 mg / ml ) or 10 μg / ml pI : C for 3h and gene expression was measured by qPCR ( n = 4 mean ± s . e . m ) .

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HMDM were stimulated with AdV (50 000 pp/cell) and antibody (h9C12; 20 ug/ml, IVIg: 20 mg/ml) or 10 μg/ml pI:C for 3h and gene expression was measured by qPCR (n = 4 mean ± s.e.m).


HMDM were stimulated as in ( A ) , or with 10 ng / ml LPS for 6 h and pro - IL - 1β levels in the cytosol measured by western blot . Blot is representative of three independent donors .

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HMDM were stimulated as in (A), or with 10 ng/ml LPS for 6 h and pro-IL-1β levels in the cytosol measured by western blot. Blot is representative of three independent donors.


HMDM were stimulated as in ( A ) , or with 10 ng / ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR . Data shows average ± s . d . of 2 independent

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

HMDM were stimulated as in (A), or with 10 ng/ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR. Data shows average ± s.d. of 2 independent donors


HMDM were stimulated as in ( B ) and NLRP3 levels in the cytosol measured by western blot . Blot is representative of two independent donors .

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HMDM were stimulated as in (B) and NLRP3 levels in the cytosol measured by western blot. Blot is representative of two independent donors.


TLR9 mRNA levels from PBMCs , CD19 + ve B cells , CD14 + ve monocytes or HMDM derived from CD14 + ve monocytes were assessed by qPCR and copy number was determined relative to actin copy number ( n = 5 mean ± s . e . m ) .

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TLR9 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve monocytes or HMDM derived from CD14+ve monocytes were assessed by qPCR and copy number was determined relative to actin copy number (n = 5 mean ± s.e.m).


THP - 1s expressing ASC - GFP were stimulated for 6 h with virus and antibody or 200 ng / well HT - DNA or 10 μM Nigericin , in the presence of the pan - caspase inhibitor zVAD - fmk . A representative image ( scale bar 100 μm ) and quantification of number of cells with ASC specks from three independent experiments ( mean ± s . e . m , * P - value ≤ 0 . 05 , paired , two - tailed t - test ) is shown .

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THP-1s expressing ASC-GFP were stimulated for 6 h with virus and antibody or 200 ng/well HT-DNA or 10 μM Nigericin, in the presence of the pan-caspase inhibitor zVAD-fmk. A representative image (scale bar 100 μm) and quantification of number of cells with ASC specks from three independent experiments (mean ± s.e.m, *P-value ≤ 0.05, paired, two-tailed t-test) is shown.


Primed HMDM were treated with MCC950 ( 1 μM ) for 30 mins before stimulation overnight with AdV ( 50 000 pp / cell ) and h9C12 ( 20 μg / ml ) or IVIg ( 20 mg / ml ) or 10 μM Nigericin . IL - 1β ( A ) or TNF ( B ) or LDH release ( C ) were measured in cell supernatants . Data shows average ± s . d . of two independent donors and is representative of three independent experiments .

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

Primed HMDM were treated with MCC950 (1 μM) for 30 mins before stimulation overnight with AdV (50 000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin. IL-1β (A) or TNF (B) or LDH release (C) were measured in cell supernatants. Data shows average ± s.d. of two independent donors and is representative of three independent experiments.


WT , NLRP3 , ASC or Caspase - 1 - deficient THP - 1s were stimulated as in ( A ) and IL - 1β in the supernatant measured by ELISA . Data is representative of two independent experiments .

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WT, NLRP3, ASC or Caspase-1 - deficient THP-1s were stimulated as in (A) and IL-1β in the supernatant measured by ELISA. Data is representative of two independent experiments.


HMDM were primed with 10 ug / ml pI : C for 2h , treated with 100 μM VX - 765 for 30 mins before stimulation for 16 h as in ( A ) . IL - 1β or LDH release was measured in cell supernatants . Data shows average ± s . d . of two independent

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HMDM were primed with 10 ug/ml pI:C for 2h, treated with 100 μM VX-765 for 30 mins before stimulation for 16 h as in (A). IL-1β or LDH release was measured in cell supernatants. Data shows average ± s.d. of two independent donors


Primed HMDM were treated with KCl as indicated for 1h , before being stimulated for a further 6h as in ( A ) . IL - 1β and TNF were measured in cell supernatants by ELISA . Data is representative of three independent donors .

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

Primed HMDM were treated with KCl as indicated for 1h, before being stimulated for a further 6h as in (A). IL-1β and TNF were measured in cell supernatants by ELISA. Data is representative of three independent donors.


Primed HMDM were stimulated with AdV and 20 μg / ml h9C12 or 10 μM Nigericin for 3h , and then immunostained for ASC or intracellular antibody . Co - localisation was assessed by confocal microscopy . Data is representative of two independent donors .

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Primed HMDM were stimulated with AdV and 20 μg/ml h9C12 or 10 μM Nigericin for 3h, and then immunostained for ASC or intracellular antibody. Co-localisation was assessed by confocal microscopy. Data is representative of two independent donors.


AIM2 mRNA levels from PBMCs , CD19 + ve B cells , CD14 + ve Monocytes or HMDM derived from CD14 + ve monocytes . were assessed by qPCR and copy number was determined relative to Actin copy number ( n = 5 mean ± s . e . m ) .

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AIM2 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve Monocytes or HMDM derived from CD14+ve monocytes. were assessed by qPCR and copy number was determined relative to Actin copy number (n = 5 mean ± s.e.m).


HMDM were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 for 1h , washed 2x with SFM , then whole cell lysates harvested after a further 5h . Viral hexon and transgene ( GFP ) expression in the cytosol was assessed by western blot . Data is representative of two independent experiments .

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HMDM were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 for 1h, washed 2x with SFM, then whole cell lysates harvested after a further 5h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by western blot. Data is representative of two independent experiments.