Figure 6 ( A - B ) C57BL / 6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i . p . with 50 µg TNF , solved in 200 µL sterile PBS , per 20 g bodyweight or with PBS only ( N = 5 ) . 18h after TNF challenge , spleen and MLNs were isolated and lysates prepared and plated on TSA plates . Colonies were counted and expressed as bacterial colonies per ml of lysate . 40 colonies per plate were prepared for Maldi Tof identification and the relative amounts of bacterial species found on the plates are mentioned underneath the figure . Data are shown as means + / - SD . p - value was analyzed by means of a Student ' s t - test ( unpaired , two - tailed ) . ( E ) Impact of zinc concentration in bacterial TSA plates on the growth of colonies of 4 bacterial species commonly found in spleen and MLNs of mice injected with TNF ( see A - B ) . Pure colonies of Staphylococcus nepalensis ( orange ) , Staphylococcus sciuri ( red ) , Eschericchia coli ( purple ) and Enterococcus faecalis ( blue ) were grown in LB medium , then diluted and plated on TSA plates containing increasing concentrations of ZnCL2 . 24h later , the amounts of colonies were counted , and the amounts found in the 0 mM ZnCl2 condition expressed as 100 % . Data are shown as mean + / - SD pooled from three independent experiments . Monocolonization experiment . Mice ( N = 5 all groups ) were treated with H20 or ZnSO4 for 7 days , after which the ZnSO4 was removed and replaced by H20 . 12h and 24h later , the mice were transplanted with PBS , 108 Staphylococcus sciuri or 108 S . nepalensis ( in 100 ul ) , and 3h later a lethal dose of TNF ( 35 µg / mouse ) was injected , and 24h later body temperature ( insert in F ) and lethality ( F ) recorded and estimated by one - way ANOVA and Chi2 - tests resp . Survival significance was tested towards the H20 control group . Monocolonization experiment . Mice ( N = 5 all groups ) were treated with H20 or ZnSO4 for 7 days , after which the ZnSO4 was removed and replaced by H20 . 12h and 24h later , the mice were transplanted with PBS , 108 Staphylococcus sciuri ( in 100 ul ) , and 3h later a lethal dose of TNF ( 35 µg / mouse ) was injected In a second experiment ( G - I ) ( N = 4 all groups ) with S . sciuri colonization , Zbp1 and Stat1 , two key ISRE genes , and Mt2 , a Zn - induced gene , were measured in ileum biopsies by RT - qPCR .

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Figure 6(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only (N=5). 18h after TNF challenge, spleen and MLNs were isolated and lysates prepared and plated on TSA plates. Colonies were counted and expressed as bacterial colonies per ml of lysate. 40 colonies per plate were prepared for Maldi Tof identification and the relative amounts of bacterial species found on the plates are mentioned underneath the figure. Data are shown as means +/- SD. p-value was analyzed by means of a Student's t-test (unpaired, two-tailed).(E) Impact of zinc concentration in bacterial TSA plates on the growth of colonies of 4 bacterial species commonly found in spleen and MLNs of mice injected with TNF (see A-B). Pure colonies of Staphylococcus nepalensis (orange), Staphylococcus sciuri (red), Eschericchia coli (purple) and Enterococcus faecalis (blue) were grown in LB medium, then diluted and plated on TSA plates containing increasing concentrations of ZnCL2. 24h later, the amounts of colonies were counted, and the amounts found in the 0 mM ZnCl2 condition expressed as 100%. Data are shown as mean +/- SD pooled from three independent experiments.Monocolonization experiment. Mice (N=5 all groups) were treated with H20 or ZnSO4 for 7 days, after which the ZnSO4 was removed and replaced by H20. 12h and 24h later, the mice were transplanted with PBS, 108 Staphylococcus sciuri or 108 S. nepalensis (in 100 ul), and 3h later a lethal dose of TNF (35 µg/mouse) was injected, and 24h later body temperature (insert in F) and lethality (F) recorded and estimated by one-way ANOVA and Chi2-tests resp. Survival significance was tested towards the H20 control group.Monocolonization experiment. Mice (N=5 all groups) were treated with H20 or ZnSO4 for 7 days, after which the ZnSO4 was removed and replaced by H20. 12h and 24h later, the mice were transplanted with PBS, 108 Staphylococcus sciuri (in 100 ul), and 3h later a lethal dose of TNF (35 µg/mouse) was injected In a second experiment (G-I) (N=4 all groups) with S. sciuri colonization, Zbp1 and Stat1, two key ISRE genes, and Mt2, a Zn-induced gene, were measured in ileum biopsies by RT-qPCR.


Figure 7 ( A ) C57BL / 6J mice received 25 mM ZnSO4 or control water for 7 days . Mice were injected i . p . with 100 , 200 or 500 µg dexamethasone ( Dex , rapidexon ) , solved in sterile PBS , 30 min before a 50 µg TNF / 20 g bodyweight i . p . challenge . STAT1 + / + and STAT1 - / - mice were challenged with equal TNF dose , as controls . No mice died later than 120 h after TNF injection . Data show combined results of 2 experiments . Mice ( N = 4 per group ) treated as in ( A ) and 6h after challenge , ileum was taken and RT - qPCR performed to study gene expression of cell death ISRE genes Mlkl and Zbp1Mice ( N = 4 per group ) treated as in ( A ) and 6h after challenge , ileum was taken and RT - qPCR performed to study gene expression of cell death ISRE genes Stat1 .

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Figure 7(A) C57BL/6J mice received 25 mM ZnSO4 or control water for 7 days. Mice were injected i.p. with 100, 200 or 500 µg dexamethasone (Dex, rapidexon), solved in sterile PBS, 30 min before a 50 µg TNF/20 g bodyweight i.p. challenge. STAT1+/+ and STAT1-/- mice were challenged with equal TNF dose, as controls. No mice died later than 120 h after TNF injection. Data show combined results of 2 experiments.Mice (N=4 per group) treated as in (A) and 6h after challenge, ileum was taken and RT-qPCR performed to study gene expression of cell death ISRE genes Mlkl and Zbp1Mice (N=4 per group) treated as in (A) and 6h after challenge, ileum was taken and RT-qPCR performed to study gene expression of cell death ISRE genes Stat1.


Figure 1 ( B ) Fold - change ± standard error in baseline IL - 6 plasma levels at week 12 from RA patients treated with placebo ( n = 47 ) , baricitinib 1 - mg ( n = 23 ) , 2 - mg ( n = 24 ) , 4 - mg ( n = 23 ) , and 8 - mg ( n = 23 ) in the phase 2b randomized , placebo - controlled study NCT01469013 . p values are for comparisons of baricitinib 1 - mg , 2 - mg , 4 - mg , and 8 - mg change from baseline compared to the placebo change from baseline . Treatment effects were estimated using a mixed effects repeated measures model ( SAS Proc Mixed ) using log10 transformed IL - 6 levels with an unstructured covariance matrix .

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Figure 1(B) Fold-change ± standard error in baseline IL-6 plasma levels at week 12 from RA patients treated with placebo (n=47), baricitinib 1-mg (n=23), 2-mg (n=24), 4-mg (n=23), and 8-mg (n=23) in the phase 2b randomized, placebo-controlled study NCT01469013. p values are for comparisons of baricitinib 1-mg, 2-mg, 4-mg, and 8-mg change from baseline compared to the placebo change from baseline. Treatment effects were estimated using a mixed effects repeated measures model (SAS Proc Mixed) using log10 transformed IL-6 levels with an unstructured covariance matrix.


Figure 2 ( A and B ) Binding affinities in Kd are shown for baricitinib ( A and B ) , tofacitinib , upadacitinib , AZD7762 ( positive control ) , and broad - spectrum tyrosine kinase inhibitors sunitinib and erlotinib ( B ) . Kd values were estimated from an 11 - point concentration response curve with fixed top parameters and a four - parameter logistic model . Kd , equilibrium affinity constant ( n = 3 ) ( C ) Protein and mRNA expression of relevant NAKs in primary human liver organoids ( n = 3 ) ( p < 0 . 05 ) . The immunofluorescence shows the juxtaposition of 1A9 ( signal from the SARS - CoV - 2 ) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain ( n = 1 ) . Colocalization of 1A9 and ACE2 is depicted with an arrow . ( D , E ) Time course of days ( one , three , and six days ) post infection ( dpi ) using liver spheroids and SARS - CoV - 2 at 0 . 1 and 1 . 0 multiplicity of infection ( MOI ) ( n = 1 ) . ( F ) Viability of spheroids was quantified by measuring intracellular ATP concentrations . Note that no significant ( n . s . ; Student ' s t - test ) decrease in cell viability was observed for any concentration tested ( n = 10 ) . ( H ) Viral load in infected ( MOI 0 . 1 ) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection ( * p < 0 . 05 ; Student ' s t - test ) . Data are mean ± standard error of mean ( n = 3 ) .

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Figure 2(A and B) Binding affinities in Kd are shown for baricitinib (A and B), tofacitinib, upadacitinib, AZD7762 (positive control), and broad-spectrum tyrosine kinase inhibitors sunitinib and erlotinib (B). Kd values were estimated from an 11-point concentration response curve with fixed top parameters and a four-parameter logistic model. Kd, equilibrium affinity constant (n=3)(C) Protein and mRNA expression of relevant NAKs in primary human liver organoids (n=3) (p<0.05). The immunofluorescence shows the juxtaposition of 1A9 (signal from the SARS-CoV-2) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain (n=1). Colocalization of 1A9 and ACE2 is depicted with an arrow.(D, E) Time course of days (one, three, and six days) post infection (dpi) using liver spheroids and SARS-CoV-2 at 0.1 and 1.0 multiplicity of infection (MOI) (n=1).(F) Viability of spheroids was quantified by measuring intracellular ATP concentrations. Note that no significant (n.s.; Student's t-test) decrease in cell viability was observed for any concentration tested (n=10).(H) Viral load in infected (MOI 0.1) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection (*p<0.05; Student's t-test). Data are mean ± standard error of mean (n=3).


Figure 3 ( A ) Detection of SARS - CoV - 2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID - 19 Plus RealAmp Kit assay ( ELITechGroup S . p . A . , Turin , Italy ) . Three viral target genes , RdRp , N , and E , together with the housekeeping gene GAPDH were simultaneously amplified . Here the most sensitive target gene , N , is shown . Samples with Ct values > 40 were defined as negative . A dashed horizontal line indicates the cut - off . IL - 6 values are shown in the same graph . Time of baricitinib treatment is highlighted ( Patients A , B , and D for 10 days , Patient C for 12 days ) with total time of evaluation on the X - axis . Fever and cough symptoms are indicated . The bars in grey indicate that cough has improved but has not resolved . ( B ) Levels of CD3 + , CD4 + , CD8 + , B cells ( CD19 + ) and NK cells ( CD3 - , CD16 + , CD56 + ) were determined using the AQUIOS CL Flow Cytometry System ( Beckman Coulter ) . ( C ) Total serum IgG , IgA , and IgM levels are shown for all patients .

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Figure 3(A) Detection of SARS-CoV-2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup S.p.A., Turin, Italy). Three viral target genes, RdRp, N, and E, together with the housekeeping gene GAPDH were simultaneously amplified. Here the most sensitive target gene, N, is shown. Samples with Ct values > 40 were defined as negative. A dashed horizontal line indicates the cut-off. IL-6 values are shown in the same graph. Time of baricitinib treatment is highlighted (Patients A, B, and D for 10 days, Patient C for 12 days) with total time of evaluation on the X-axis. Fever and cough symptoms are indicated. The bars in grey indicate that cough has improved but has not resolved.(B) Levels of CD3+, CD4+, CD8+, B cells (CD19+) and NK cells (CD3-, CD16+, CD56+) were determined using the AQUIOS CL Flow Cytometry System (Beckman Coulter).(C) Total serum IgG, IgA, and IgM levels are shown for all patients.


Figure 4Detection of IgG antibodies against the S1 / S2 antigens of SARS - CoV - 2 . Time of baricitinib treatment is indicated for all patients ( patients A , B , and D for 10 days , patient C for 12 days ) .

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Figure 4Detection of IgG antibodies against the S1/S2 antigens of SARS-CoV-2. Time of baricitinib treatment is indicated for all patients (patients A, B, and D for 10 days, patient C for 12 days).


Figure 5 ( A ) Levels of D - dimer , CRP , ferritin , white blood cells , and neutrophils are shown for all patients . ( B ) Levels of aspartate aminotransferase , alanine aminotransferase , and bilirubin are shown for all patients . ALT , alanine aminotransferase ; AST , aspartate aminotransferase ( C ) Chest CT scan for Patient C on day one and 19 from symptom onset showing clinical improvement over time . Day one CT scan shows ground glass opacity ( arrows ) sub - pleurally in the lower lobes bilaterally ( early stage one according to Pan et al ( Pan , Ye et al . , 2020 ) . Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis and emphysema bubbles ( arrows ) in the site of the early lesions ( absorption stage four according to Pan et al ( Pan et al . , 2020 ) .

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Figure 5(A) Levels of D-dimer, CRP, ferritin, white blood cells, and neutrophils are shown for all patients.(B) Levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin are shown for all patients. ALT, alanine aminotransferase; AST, aspartate aminotransferase(C) Chest CT scan for Patient C on day one and 19 from symptom onset showing clinical improvement over time. Day one CT scan shows ground glass opacity (arrows) sub-pleurally in the lower lobes bilaterally (early stage one according to Pan et al (Pan, Ye et al., 2020). Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis and emphysema bubbles (arrows) in the site of the early lesions (absorption stage four according to Pan et al (Pan et al., 2020).


Figure 1 , body weight trajectories of the mice . L , M , N , O The blood glucose level of mice after intraperitoneal injection of glucose or insulin for glucose ( GTT ) ( L ) and insulin tolerance tests ( ITT ) ( N ) , and AUC , area under the curve ( M , O ) .

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Figure 1, body weight trajectories of the mice.L, M, N, O The blood glucose level of mice after intraperitoneal injection of glucose or insulin for glucose (GTT) (L) and insulin tolerance tests (ITT) (N), and AUC, area under the curve (M, O).


Figure 2A ORO staining of SVF cells isolated from iWAT of male mice . Differentiation was induced with curcumin up to day 8 . C ORO staining of 3T3 - L1 cells . Differentiation was induced with curcumin up to day 8 . B mRNA expression of adipogenesis - related factors of SVF cells isolated from iWAT of male mice . D mRNA expression of PPARγ , C / EBPα and FABP4 expression of 3T3 - L1 cells treated with curcumin . protein expression levels of m6A regulator in iWAT from mice in HFD and HFD + CUR groups . protein expression levels of m6A regulator in 3T3 - L1 cells were analyzedK Oil Red O staining of Vector or OE - ALKBH5 transfected 3T3 - L1 cells . Differentiation was induced with curcumin up to day 8 .

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Figure 2A ORO staining of SVF cells isolated from iWAT of male mice. Differentiation was induced with curcumin up to day 8. C ORO staining of 3T3-L1 cells. Differentiation was induced with curcumin up to day 8.B mRNA expression of adipogenesis-related factors of SVF cells isolated from iWAT of male mice. D mRNA expression of PPARγ, C/EBPα and FABP4 expression of 3T3-L1 cells treated with curcumin.protein expression levels of m6A regulator in iWAT from mice in HFD and HFD+CUR groups. protein expression levels of m6A regulator in 3T3-L1 cells were analyzedK Oil Red O staining of Vector or OE-ALKBH5 transfected 3T3-L1 cells. Differentiation was induced with curcumin up to day 8.


Figure 3E Oil Red O staining of control , ALKBH5 - depleted and ALKBH5 + TRAF4 depleted cells after induced for 8 days . G Western blot analysis of PPARγ and C / EBPα protein levels in cells , and quantification of protein levels normalized to β - actin expression .

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Figure 3E Oil Red O staining of control, ALKBH5-depleted and ALKBH5+TRAF4 depleted cells after induced for 8 days.G Western blot analysis of PPARγ and C/EBPα protein levels in cells , and quantification of protein levels normalized to β-actin expression.


Figure 4Western blot analysis of YTHDF1 , ALKBH5 and TRAF4 in control , ALKBH5 - depleted and ALKBH5 + YTHDF1 depleted cells and quantification of protein levels normalized to β - actin expression . L Oil Red O staining of control , ALKBH5 - depleted and ALKBH5 + YTHDF1 depleted cells on day 8 of differentiation . M qPCR analysis of PPARγ , C / EBPα and FABP4 expression in cell β - Actin was used as an internal control .

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Figure 4Western blot analysis of YTHDF1, ALKBH5 and TRAF4 in control, ALKBH5-depleted and ALKBH5 + YTHDF1 depleted cells and quantification of protein levels normalized to β-actin expression.L Oil Red O staining of control, ALKBH5-depleted and ALKBH5+YTHDF1 depleted cells on day 8 of differentiation.M qPCR analysis of PPARγ, C/EBPα and FABP4 expression in cell β-Actin was used as an internal control.


Figure 5C , D Western blot analysis of PPARγ protein levels in control and TRAF4 overexpressing cells in the absence or presence of MG132 or 3MA and quantification of protein levels normalized to β - actin expression . E Ubiquitination of endogenous PPARγ in control and TRAF4 overexpressing cells were differentiated by treatment with DMI . Cells were treated with MG132 for 6 h .

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Figure 5C, D Western blot analysis of PPARγ protein levels in control and TRAF4 overexpressing cells in the absence or presence of MG132 or 3MA and quantification of protein levels normalized to β-actin expression.E Ubiquitination of endogenous PPARγ in control and TRAF4 overexpressing cells were differentiated by treatment with DMI. Cells were treated with MG132 for 6 h.


Figure 1 ( A ) Western blot of autophagy proteins LC3 and p62 at various time points after infection of human NSCs with strains MR766 or Paraiba . ( B - C ) Quantitative representation of LC3 - II and p62 protein levels during ZIKV infection at indicated time points . Mean ± SEM , n = 3 biological replicates . ( D ) GFP - LC3 puncta of mock - and ZIKV - infected hNSCs . Scale bar = 100 µm . ( E ) Tandem EGFP - LC3 and mCherry punctae co - localization in control and ZIKV infected hNSC . Scale bar = 100 µm , Boxed magnified images with scale bar = 50 µm ( F ) Number of GFP - LC3 puncta in MR766 and Paraiba infected hNSCs , n = > 50 cells . Box plots show the mean and the smallest ; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group ; and solid horizontal line indicates median . * * * p < 0 . 001 by Student ' s t test . ( G ) RT - qPCR of ZIKV RNA in hNSCs incubated with vehicle or 100 nM rapamycin , 20 μM chloroquine , 10 nM bafilomycin A1 , or 5 mM 3 - methyladenine for 24 h . Mean ± SEM of 3 biological replicates . * p < 0 . 05 , * * * p < 0 . 001 by Student ' s t test . ( H ) GFP - LC3 puncta in ZIKV with or without rapamycin and 3 - MA treated hNSC cells . Cells were treated with or without rapamycin ( 100 nM ) or 3 - methyladenine ( 5 μM ) . Box plots show the mean and the smallest ; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group ; and solid horizontal line indicates median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( I ) RT - qPCR of ZIKV RNA in infected hNSCs transfected with siRNAs targeting LC3 , BECN or ATG9A 48 h post infection . Mean ± SEM of 3 biological replicates . * p < 0 . 05 by Student ' s t test . ( L ) Transmission electron microscopy of hNSCs infected by ZIKV MR766 and Paraiba strains . Mitochondria ( MT ) , endoplasmic reticulum ( ER ) , Golgi bodies ( GB ) , ribosomes ( RS ) , phagophores ( PH ) , lipid droplet ( LD ) . Scale bars , first row , 1 μm ( left ) , 500 nm ( control right ) ; 200 nm ( Right ) .

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

Figure 1(A) Western blot of autophagy proteins LC3 and p62 at various time points after infection of human NSCs with strains MR766 or Paraiba. (B-C) Quantitative representation of LC3-II and p62 protein levels during ZIKV infection at indicated time points. Mean ± SEM, n=3 biological replicates. (D) GFP-LC3 puncta of mock- and ZIKV-infected hNSCs. Scale bar = 100 µm.(E) Tandem EGFP-LC3 and mCherry punctae co-localization in control and ZIKV infected hNSC. Scale bar = 100 µm, Boxed magnified images with scale bar = 50 µm(F) Number of GFP-LC3 puncta in MR766 and Paraiba infected hNSCs, n =>50 cells. Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test.(G) RT-qPCR of ZIKV RNA in hNSCs incubated with vehicle or 100 nM rapamycin, 20 μM chloroquine, 10 nM bafilomycin A1, or 5 mM 3-methyladenine for 24 h. Mean ± SEM of 3 biological replicates. *p < 0.05, ***p < 0.001 by Student's t test. (H) GFP-LC3 puncta in ZIKV with or without rapamycin and 3-MA treated hNSC cells. Cells were treated with or without rapamycin (100 nM) or 3-methyladenine (5 μM). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test.  (I) RT-qPCR of ZIKV RNA in infected hNSCs transfected with siRNAs targeting LC3, BECN or ATG9A 48 h post infection. Mean ± SEM of 3 biological replicates. *p < 0.05 by Student's t test.  (L) Transmission electron microscopy of hNSCs infected by ZIKV MR766 and Paraiba strains. Mitochondria (MT), endoplasmic reticulum (ER), Golgi bodies (GB), ribosomes (RS), phagophores (PH), lipid droplet (LD). Scale bars, first row, 1 μm (left), 500 nm (control right); 200 nm (Right).


Figure 2 Immunostaining and quantification of neural progenitor marker SOX2 ( red ) and ZIKV envelope flavivirus group antigen ( ZIKVE , green ) in the SVZ of uninfected or ZIKV Paraiba - infected wild - type mice treated with vehicle , rapamycin , or chloroquine at 6 days post - infection . n = 10 sections / group , mean ± SEM , * p < 0 . 05 , * * p < 0 . 001 . Nuclei were stained with DAPI ( blue ) . Right - most column shows enlargements of the boxed regions . STR = striatum , CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , SVZ = subventricular zone . Immunostaining and quantification of neural progenitor marker SOX2 ( red ) and ZIKV envelope flavivirus group antigen ( ZIKVE , green ) in the hippocampus of uninfected or ZIKV Paraiba - infected wild - type mice treated with vehicle , rapamycin , or chloroquine at 6 days post - infection . n = 10 sections / group , mean ± SEM , * p < 0 . 05 , * * p < 0 . 001 . Nuclei were stained with DAPI ( blue ) . Right - most column shows enlargements of the boxed regions . STR = striatum , CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , SVZ = subventricular zone . ( E ) qRT - PCR for ZIKV RNA expression in brain , mean ± SEM , n = 3 biological replicates , * p < 0 . 05 , * * p < 0 . 001 by Student ' s t test . ( F - G ) Western blot for autophagy protein LC3 - II , p62 and quantification normalized with GAPDH , mean ± SEM , n = 3 biological replicates , * p < 0 . 05 , * * p < 0 . 001 by 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the SVZ of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone.  Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the hippocampus of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone. (E) qRT-PCR for ZIKV RNA expression in brain, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test.(F-G) Western blot for autophagy protein LC3-II, p62 and quantification normalized with GAPDH, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test.


Figure 3 ( B ) RT - qPCR analysis of FA pathway genes at 48 h after ZIKV infection of hNSCs . Mean ± SEM of biological triplicates . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t test . ( C - D ) RT - qPCR analysis of ZIKV mRNA 48 h after ZIKV infection of HeLa cells overexpressing FANCC ( C ) or FANCC - specific siRNA ( D ) . Mean ± SEM of biological triplicates . * * p < 0 . 01 by Student ' s t test . ( E ) Flow cytometry analysis of ZIKVE expression in mock - infected ( red ) , ZIKV - infected ( blue ) , ZIKV - infected siFANCC - transfected ( orange ) , and ZIKV - infected siE2F4 - transfected ( green ) hNSCs at 48 h post - infection . ( F ) ZIKV NS1 and FANCC immunostaining 48 h after ZIKV infection of hNSCs . Scale bar = 100 µm . ( G ) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control siRNA ( NTC ) or siFANCC - targeting siRNA . Scale bar = 100 µm . ( H - I ) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control ( NTC ) or siE2F4 - targeting siRNA . Mean ± SEM of six imaging fields of biological triplicates , * * p < 0 . 01 by Student ' s t test , Scale bar = 100 µm , ( J ) Immunoblot of macroautophagy ( p62 , LC3I / LC3II ) and selective autophagy ( FANCC , TOMM20 ) proteins 24 h after overexpression of FLAG - tagged ZIKV NS1 , NS2B , NS3 , NS4A , NS4B and NS5 in 293FT cells . ( K ) Analysis of E2F4 ChIP - seq datasets ( Encode ) showing direct chromatin - binding interaction between E2F4 and FA pathway genes in HeLa ( GEO : GSM935365 , Farnham lab ) , K562 ( GEO : GSM935600 , Farnham lab ) , and MCF10A ( GEO : GSM : 935400 , Struhl lab ) cell lines . H3K9ac and H3K4me3 datasets identify promoter regions . The genomic region is displayed in log scale with the scale range identified in each panel ( 0 - 30 ) . The summed interaction ( Sum ) across all three cell types is represented as a heatmap . ( L - M ) Heatmaps of RT - qPCR analysis of FA pathway genes ( K ) or essential selective autophagy genes ( L ) in hNSCs 48 h after transfection with control ( NTC ) or E2F4 - targeting siRNAs or after infection with ZIKV MR766 and Paraiba strains . Color patterns in heat map indicate as red showing the highest expression and blue showing the lowest expression of genes .

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

Figure 3(B) RT-qPCR analysis of FA pathway genes at 48 h after ZIKV infection of hNSCs. Mean ± SEM of biological triplicates. *p < 0.05, **p < 0.01 by Student's t test.(C-D) RT-qPCR analysis of ZIKV mRNA 48 h after ZIKV infection of HeLa cells overexpressing FANCC (C) or FANCC-specific siRNA (D). Mean ± SEM of biological triplicates. **p < 0.01 by Student's t test.(E) Flow cytometry analysis of ZIKVE expression in mock-infected (red), ZIKV-infected (blue), ZIKV-infected siFANCC-transfected (orange), and ZIKV-infected siE2F4-transfected (green) hNSCs at 48 h post-infection.(F) ZIKV NS1 and FANCC immunostaining 48 h after ZIKV infection of hNSCs. Scale bar = 100 µm.(G) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control siRNA (NTC) or siFANCC-targeting siRNA. Scale bar = 100 µm. (H-I) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control (NTC) or siE2F4-targeting siRNA. Mean ± SEM of six imaging fields of biological triplicates, **p < 0.01 by Student's t test, Scale bar = 100 µm, (J) Immunoblot of macroautophagy (p62, LC3I/LC3II) and selective autophagy (FANCC, TOMM20) proteins 24 h after overexpression of FLAG-tagged ZIKV NS1, NS2B, NS3, NS4A, NS4B and NS5 in 293FT cells.(K) Analysis of E2F4 ChIP-seq datasets (Encode) showing direct chromatin-binding interaction between E2F4 and FA pathway genes in HeLa (GEO: GSM935365, Farnham lab), K562 (GEO: GSM935600, Farnham lab), and MCF10A (GEO: GSM: 935400, Struhl lab) cell lines. H3K9ac and H3K4me3 datasets identify promoter regions. The genomic region is displayed in log scale with the scale range identified in each panel (0-30). The summed interaction (Sum) across all three cell types is represented as a heatmap.(L-M) Heatmaps of RT-qPCR analysis of FA pathway genes (K) or essential selective autophagy genes (L) in hNSCs 48 h after transfection with control (NTC) or E2F4-targeting siRNAs or after infection with ZIKV MR766 and Paraiba strains. Color patterns in heat map indicate as red showing the highest expression and blue showing the lowest expression of genes.


Figure 4 ( A ) Representative images of mock - or ZIKV - infected human neurospheres expressing control ( NTC ) or FANCC - targeting siRNAs at 1 - 4 days post - infection . Scale bar = 250 µm . ( B - C ) Violin plot ( B ) and box plot ( C ) of relative size of neurospheres treated as described in ( A ) and measured on day 4 ( B ) or days 1 - 4 ( C ) . Box plots show the mean and the smallest ; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group ; and solid horizontal line indicates median . * * * p < 0 . 001 by Student ' s t test .

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

Figure 4(A) Representative images of mock- or ZIKV-infected human neurospheres expressing control (NTC) or FANCC-targeting siRNAs at 1-4 days post-infection. Scale bar = 250 µm. (B-C) Violin plot (B) and box plot (C) of relative size of neurospheres treated as described in (A) and measured on day 4 (B) or days 1-4 (C). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test.


Figure 5Immunostaining of neural progenitor marker SOX2 ( green ) and ZIKV envelope flavivirus group antigen ( ZIKVE , red ) in the hippocampus of uninfected or ZIKV Paraiba - infected Ifnar - / - mice six days post - infection . Nuclei were stained with DAPI ( gray ) . Right - most column in ( A ) shows enlargements of the regions . CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , STR = striatum , SVZ = subventricular zone . Scale bars , 100 μm . Immunostaining of neural progenitor marker SOX2 ( green ) and ZIKV envelope flavivirus group antigen ( ZIKVE , red ) in the SVZ of uninfected or ZIKV Paraiba - infected Ifnar - / - mice six days post - infection . Nuclei were stained with DAPI ( gray ) . Right - most column in ( A ) shows enlargements of the regions . CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , STR = striatum , SVZ = subventricular zone . Scale bars , 100 μm . ( C ) RT - qPCR analysis of relative ZIKV RNA in different brain regions of Ifnar - / - mice . mean ± SEM of n = 3 biological replicates , * * p < 0 . 005 , * * * p < 0 . 001 by Student ' s t test . ( D ) RT - qPCR analysis of relative Fancc mRNA levels from whole brains or testes in mock - and ZIKV Paraiba infected Ifnar - / - mice six days post - infection . Mean ± SEM of n = 3 biological replicates , * p < 0 . 05 and ns by Student ' s t test . ( E ) RT - qPCR analysis of relative Fancc or Fancl mRNA levels from whole brains from mock - and ZIKV Paraiba infected Ifnar - / - newborn mice . Mean ± SEM of n = 3 biological replicates , * * * p < 0 . 001 by Student ' s t test . ( F ) Western blot analysis of Fancc protein in brain regions from mock - and ZIKV Paraiba infected Ifnar - / - mice six days post - infection . HP = hippocampus , CT = cortex , ST = striatum and OB = Olfactory Bulb . ( G ) Bar graph showing the quantification of Fancc proteins , mean ± SEM of n = 3 biological replicates , * p < 0 . 05 and ns by Student ' s t test . ( H ) RT - qPCR analysis of relative ZIKV RNA in different brain regions of Fancc KO neonatal mice six days post - infection . mean ± SEM of n = 3 biological replicates , * * p < 0 . 005 , * * * p < 0 . 001 by Student ' s t test . ( I ) qRT - PCR for Ifnab gene expression in wild type and Fancc KO mice , Mean ± SEM of n = 3 biological replicates , ns by Student ' s t test .

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

Figure 5Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the hippocampus of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm.Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the SVZ of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm.(C) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Ifnar-/- mice. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.(D) RT-qPCR analysis of relative Fancc mRNA levels from whole brains or testes in mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. Mean ± SEM of n=3 biological replicates, *p < 0.05 and ns by Student's t test.(E) RT-qPCR analysis of relative Fancc or Fancl mRNA levels from whole brains from mock- and ZIKV Paraiba infected Ifnar-/- newborn mice. Mean ± SEM of n=3 biological replicates, ***p < 0.001 by Student's t test.(F) Western blot analysis of Fancc protein in brain regions from mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. HP = hippocampus, CT= cortex, ST = striatum and OB= Olfactory Bulb.(G) Bar graph showing the quantification of Fancc proteins, mean ± SEM of n=3 biological replicates, *p<0.05 and ns by Student's t test.(H) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Fancc KO neonatal mice six days post-infection. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.(I) qRT-PCR for Ifnab gene expression in wild type and Fancc KO mice, Mean ± SEM of n=3 biological replicates, ns by Student's t test.


Figure 6 ( A ) Immunostaining of immature neuronal marker DCX ( green ) and ZIKV envelope ( ZIKVE , red ) in the hippocampus of uninfected or ZIKV Paraiba - infected Ifnar - / - mice six days post - infection . Nuclei were stained with DAPI ( gray ) . Bottom row shows enlargements of the hippocampus region . GCL = granular cell layer , SGZ = subgranular zone , DG = dentate gyrus , InGr = internal granular layer , Me = medulla , OB = olfactory bulb . Scale bars , 50 and 100 μm . Immunostaining of immature neuronal marker DCX ( green ) and ZIKV envelope ( ZIKVE , red ) in the olfactory bulb of uninfected or ZIKV Paraiba - infected Ifnar - / - mice six days post - infection . Nuclei were stained with DAPI ( gray ) . InGr = internal granular layer , Me = medulla , OB = olfactory bulb . Scale bars , 50 and 100 μm . ( C - E ) Western blot of autophagy protein LC3 and p62 in different brain region of Fancc KO neonatal mice six days post - infection of ZIKV ( MR766 ) . Mean ± SEM of n = 3 biological replicates , * p < 0 . 05 , * * p < 0 . 005 , by Student ' s t test . HP = hippocampus , CT = cortex , ST = striatum and OB = Olfactory Bulb . ( F ) Representative immunofluorescent images showing immunoreactivity of neural progenitor marker ( Sox2 ) with apoptosis marker ( Cleaved - caspase - 3 , CC3 ) in hippocampus region of Fancc KO mice . Scale bar = 50µm . ( G ) Quantitative analysis of number of Sox2 + cells and Sox2 / CC3 + co - labelled cells in Fancc KO mice hippocampal brain region . Mean ± SEM , n = 3 biological replicates , * p < 0 . 05 by Student ' s t test .

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

Figure 6(A) Immunostaining of immature neuronal marker DCX (green) and ZIKV envelope (ZIKVE, red) in the hippocampus of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Bottom row shows enlargements of the hippocampus region. GCL = granular cell layer, SGZ = subgranular zone, DG = dentate gyrus, InGr = internal granular layer, Me = medulla, OB = olfactory bulb. Scale bars, 50 and 100 μm.Immunostaining of immature neuronal marker DCX (green) and ZIKV envelope (ZIKVE, red) in the olfactory bulb of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). InGr = internal granular layer, Me = medulla, OB = olfactory bulb. Scale bars, 50 and 100 μm.(C-E) Western blot of autophagy protein LC3 and p62 in different brain region of Fancc KO neonatal mice six days post-infection of ZIKV (MR766). Mean ± SEM of n=3 biological replicates, *p<0.05, **p<0.005, by Student's t test. HP = hippocampus, CT= cortex, ST = striatum and OB= Olfactory Bulb.(F) Representative immunofluorescent images showing immunoreactivity of neural progenitor marker (Sox2) with apoptosis marker (Cleaved-caspase-3, CC3) in hippocampus region of Fancc KO mice. Scale bar=50µm. (G) Quantitative analysis of number of Sox2+ cells and Sox2/CC3+ co-labelled cells in Fancc KO mice hippocampal brain region. Mean ± SEM, n=3 biological replicates, *p < 0.05 by Student's t test.


Figure 1Jurkat T cells were transfected with control , FIP3 . 1 or FIP3 . 2 siRNA oligonucleotides . A : Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence . A , E , G : Three - dimensional ( 3D ) confocal images were post - treated by deconvolution . A 0 . 4 - µ - thick medial stack is shown . The pericentrosomal vesicular compartment is zoomed at the bottom right - hand corner . B , F , H : Population analysis of the co - localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment . Each dot represents one cell . Horizontal bars represent the mean ± SEM , Mann - Whitney test was used . Images are representative of three experiments . Bar , 5 µmA - C : JurkatT cells were transfected with control , FIP3 . 1 or FIP3 . 2 siRNA oligonucleotides . A : Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence . C : FIP3 expression was analyzed by Western blot . Quantification of bands was performed with ImageJ and normalized to a non - specific band of 35 kDa used as loading control . E , F : Jurkat cells were transfected with expression vectors encoding GFP , GFP - FIP3 - WT or GFP - FIP3 - I738E . Endogenous Rac1 was detected by immunofluorescence . G : Jurkat cells were transfected with expression vectors encoding GFP , Rab11WT - GFP , or Rab11Q70L - GFP ( constitutively active ) or Rab11S25N - GFP ( dominant negative ) mutants . Endogenous Rac1 was detected by immunofluorescence . A , E , G : Three - dimensional ( 3D ) confocal images were post - treated by deconvolution . A 0 . 4 - µ - thick medial stack is shown . The pericentrosomal vesicular compartment is zoomed at the bottom right - hand corner . B , F , H : Population analysis of the co - localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment . Each dot represents one cell . Horizontal bars represent the mean ± SEM , Mann - Whitney test was used . Images are representative of three experiments . Bar , 5

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

Figure 1Jurkat T cells were transfected with control, FIP3.1 or FIP3.2 siRNA oligonucleotides. A: Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence.A, E, G: Three-dimensional (3D) confocal images were post-treated by deconvolution. A 0.4-µ-thick medial stack is shown. The pericentrosomal vesicular compartment is zoomed at the bottom right-hand corner.B, F, H: Population analysis of the co-localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment. Each dot represents one cell. Horizontal bars represent the mean ± SEM, Mann-Whitney test was used. Images are representative of three experiments. Bar, 5 µmA-C: JurkatT cells were transfected with control, FIP3.1 or FIP3.2 siRNA oligonucleotides. A: Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence.C: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control.E, F: Jurkat cells were transfected with expression vectors encoding GFP, GFP-FIP3-WT or GFP-FIP3-I738E. Endogenous Rac1 was detected by immunofluorescence.G: Jurkat cells were transfected with expression vectors encoding GFP, Rab11WT-GFP, or Rab11Q70L-GFP (constitutively active) or Rab11S25N-GFP (dominant negative) mutants. Endogenous Rac1 was detected by immunofluorescence.A, E, G: Three-dimensional (3D) confocal images were post-treated by deconvolution. A 0.4-µ-thick medial stack is shown. The pericentrosomal vesicular compartment is zoomed at the bottom right-hand corner.B, F, H: Population analysis of the co-localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment. Each dot represents one cell. Horizontal bars represent the mean ± SEM, Mann-Whitney test was used. Images are representative of three experiments. Bar, 5 µm


Figure 2A , B : Effect of FIP3 silencing on Rac1 expression . Jurkat cells were transfected with control , FIP3 . 1 or FIP3 . 2 siRNA . Three days later cells were lysed and the expression of FIP3 , Rac1 and β - tubulin analyzed by western blot . The intensity of bands corresponding to FIP3 and to Rac1 was quantified by ImageJ and normalized to the β - tubulin band used as loading control . The mean ± SD of the percentage of band intensity in silenced cells with respect to controls of three independent experiments is shown . B : Rac1 interaction with FIP3 : HEK293T cells were transfected with GFP , GFP - FIP3 WT or GFP - FIP3I738E expression vectors . 24 h later , cells were lysed and immunoprecipitated with anti - GFP Ab . Immunoprecipitates were analyzed by electrophoresis and western blot using anti - GFP Ab ( top panel ) or anti - Rac1 Ab ( bottom panel ) . Total cell lysates were analyzed ( 100 , 000 cells ) . The position of Rac1 ( 20 kDa ) and Ig light chain ( 25 kDa ) polypeptides is depicted on the right side . Arrowheads at the bottom point the bands corresponding to Rac . Quantification of bands was performed with ImageJ as the percentage of intensity with respect to the total lysate bands on the left , shown below . A representative experiment out of three carried out is shown .

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

Figure 2A, B: Effect of FIP3 silencing on Rac1 expression. Jurkat cells were transfected with control, FIP3.1 or FIP3.2 siRNA. Three days later cells were lysed and the expression of FIP3, Rac1 and β-tubulin analyzed by western blot. The intensity of bands corresponding to FIP3 and to Rac1 was quantified by ImageJ and normalized to the β-tubulin band used as loading control. The mean ± SD of the percentage of band intensity in silenced cells with respect to controls of three independent experiments is shown.B: Rac1 interaction with FIP3: HEK293T cells were transfected with GFP, GFP-FIP3 WT or GFP-FIP3I738E expression vectors. 24 h later, cells were lysed and immunoprecipitated with anti-GFP Ab. Immunoprecipitates were analyzed by electrophoresis and western blot using anti-GFP Ab (top panel) or anti-Rac1 Ab (bottom panel). Total cell lysates were analyzed (100,000 cells). The position of Rac1 (20 kDa) and Ig light chain (25 kDa) polypeptides is depicted on the right side. Arrowheads at the bottom point the bands corresponding to Rac. Quantification of bands was performed with ImageJ as the percentage of intensity with respect to the total lysate bands on the left, shown below. A representative experiment out of three carried out is shown.


Figure 3JurkatT cells were transfected with expression vectors encoding : A , C : GFP , GFP - FIP3WT , or GFP - FIP3I738E . B , C : Rab11WT - GFP , Rab11Q70L - GFP ( constitutively active ) , or Rab11S25N - GFP ( dominant negative ) . C : Rab11WT - GFP , Rab11Q70L - GFP ( constitutively active ) , or Rab11S25N - GFP ( dominant negative ) . D , E : or transfected with control , or FIP3 . 1 siRNA . Cells were allowed to form immunological synapses with SEE - pulsed Raji cells for 30 min . Intracellular distribution of endogenous Rac1 and surface distribution of CD3 were detected by immunofluorescence . 3D confocalimages were post - treated by deconvolution . A 0 . 4 - µm - thick medial stack is shown . C , E : Population analyses of the amount of Rac1 at the immunological synapse relative to the total cellular Rac1 . Each dot corresponds to one cell . Horizontal bars represent the mean ± SEM . Mann - Whitney test . Images are representative of three experiments . Bar , 5 µm .

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

Figure 3JurkatT cells were transfected with expression vectors encoding: A, C: GFP, GFP-FIP3WT, or GFP-FIP3I738E. B, C: Rab11WT-GFP, Rab11Q70L-GFP (constitutively active), or Rab11S25N-GFP (dominant negative).C: Rab11WT-GFP, Rab11Q70L-GFP (constitutively active), or Rab11S25N-GFP (dominant negative).D, E: or transfected with control, or FIP3.1 siRNA. Cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min. Intracellular distribution of endogenous Rac1 and surface distribution of CD3 were detected by immunofluorescence. 3D confocalimages were post-treated by deconvolution. A 0.4-µm-thick medial stack is shown. C, E: Population analyses of the amount of Rac1 at the immunological synapse relative to the total cellular Rac1. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.


Figure 4JurkatT cells were transfected with either control or FIP3 . 1 siRNA . A : Cells were allowed to form immunological synapses with SEE - pulsed Raji cells for 30 min and endogenous Lck was detected by immunofluorescence . 3D confocalimages were post - treated by deconvolution . A 0 . 4 - µm - thick medial stack is shown . B , C : The minimal angle formed by the axis connecting the synapse edges and the axis connecting the mass centers of the T cell and the APC nuclei was measured , as schematized in B . D : Transfected Jurkat T cells were allowed to spread on anti - CD3 - coated coverslips for the indicated time , and F - actin was detected with FITC - Phalloidin . A single optical section at the level of the contact surface is shown . E , F : Population analyses of the spreading area of cells at the level of the contact surface . Each dot corresponds to one cell . Horizontal bars represent the mean ± SEM . Mann - Whitney test . Images are representative of three experiments . Bar , 5 µm . G : FIP3 expression was analyzed by Western blot . Quantification of bands was performed with ImageJ and normalized to a non - specific band of 35 kDa used as loading control .

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

Figure 4JurkatT cells were transfected with either control or FIP3.1 siRNA. A: Cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min and endogenous Lck was detected by immunofluorescence. 3D confocalimages were post-treated by deconvolution. A 0.4-µm-thick medial stack is shown.B, C: The minimal angle formed by the axis connecting the synapse edges and the axis connecting the mass centers of the T cell and the APC nuclei was measured, as schematized in B.D: Transfected Jurkat T cells were allowed to spread on anti-CD3-coated coverslips for the indicated time, and F-actin was detected with FITC-Phalloidin. A single optical section at the level of the contact surface is shown. E, F: Population analyses of the spreading area of cells at the level of the contact surface. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.G: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control.


Figure 5A , B , C : JurkatT cells transfected with siRNA control , FIP3 . 1 or FIP3 . 2 were allowed to spread on poly - lysine - coated coverslips for the indicated times . Intracellular actin was detected with FITC - coupled Phalloidin . A : A single optical section at the level of the contact surface is shown . Bar , 5 µm . B : Population analyses of the spreading area of cells at the level of the contact surface . Each dot represents one cell . Horizontal bars represent the mean ± SEM . C : Time course representation of B , showing mean ± SEM . D - F : Primary humanCD4T cells were transfected with control or FIP3 . 1 siRNA . D , E : Cells were allowed to spread on poly - lysine - coated coverslips for 5 min . Intracellular Lck was detected by immunofluoresecence . D : A single optical section at the level of the contact surface is shown . Bar , 10 µm . E : Population analyses of the spreading area of cells at the level of the contact surface . F : Cells were stained with CMFDA dye , suspended in a paraformaldehyde solution and submitted to centrifugation at 3 , 724 xg onto poly - lysine - coated coverslips . Confocal optical sections separated by 0 . 2 µm were acquired . X - Z images were built with ImageJ . The maximal sections in ' x ' ( width ) and ' z ' ( thickness ) were determined for each cell ( upper panel ) . Plots represent the deformability index ( x / z ) . Each dot corresponds to one cell . Horizontal bars represent the mean ± SEM . Mann - Whitney test . Images are representative of three experiments . G : FIP3 expression was analyzed by Western blot . Quantification of bands was performed with ImageJ and normalized to a non - specific band of 35 kDa used as loading control .

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

Figure 5A, B, C: JurkatT cells transfected with siRNA control, FIP3.1 or FIP3.2 were allowed to spread on poly-lysine-coated coverslips for the indicated times. Intracellular actin was detected with FITC-coupled Phalloidin. A: A single optical section at the level of the contact surface is shown. Bar, 5 µm. B: Population analyses of the spreading area of cells at the level of the contact surface. Each dot represents one cell. Horizontal bars represent the mean ± SEM. C:Time course representation of B, showing mean ± SEM.D-F: Primary humanCD4T cells were transfected with control or FIP3.1 siRNA. D, E: Cells were allowed to spread on poly-lysine-coated coverslips for 5 min. Intracellular Lck was detected by immunofluoresecence. D: A single optical section at the level of the contact surface is shown. Bar, 10 µm. E: Population analyses of the spreading area of cells at the level of the contact surface.F: Cells were stained with CMFDA dye, suspended in a paraformaldehyde solution and submitted to centrifugation at 3,724 xg onto poly-lysine-coated coverslips. Confocal optical sections separated by 0.2 µm were acquired. X-Z images were built with ImageJ. The maximal sections in 'x' (width) and 'z' (thickness) were determined for each cell (upper panel). Plots represent the deformability index (x/z). Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments.G: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control.


Figure 6Jurkat T cells were transfected with control or FIP3 . 1 siRNA , then transfected again with empty vector or with expression vectors encoding Myc - tagged - Rac1WT , - Rac1G12V ( constitutively active mutant ) , or - Rac1Rac1T17N ( dominant negative mutant ) . Cells were allowed to spread on poly - lysine - coated coverslips for the indicated time and the spreading area of cells measured . A - C , H : Population analyses of the spreading area of cells at the level of the contact surface . A - C : Time course plots representing the mean ± SEM of the data shown as in I . D - G : Staining for nucleus ( blue = Dapi ) , F - actin ( green = phalloidin ) and Rac1 - Myc ( red = anti - Myc ) is shown . A single optical section at the level of the contact surface is shown . Bar , 5 µm . H : Dot plots corresponding to measurements at 15 min of spreading : Each dot represents one cell . Horizontal bars represent the mean ± SEM . Mann - Whitney test . Data are representative of three experiments .

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

Figure 6Jurkat T cells were transfected with control or FIP3.1 siRNA, then transfected again with empty vector or with expression vectors encoding Myc-tagged-Rac1WT, -Rac1G12V (constitutively active mutant), or -Rac1Rac1T17N (dominant negative mutant). Cells were allowed to spread on poly-lysine-coated coverslips for the indicated time and the spreading area of cells measured. A-C, H: Population analyses of the spreading area of cells at the level of the contact surface. A-C: Time course plots representing the mean ± SEM of the data shown as in I. D-G: Staining for nucleus (blue = Dapi), F-actin (green = phalloidin) and Rac1-Myc (red = anti-Myc) is shown. A single optical section at the level of the contact surface is shown. Bar, 5 µm.H: Dot plots corresponding to measurements at 15 min of spreading: Each dot represents one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Data are representative of three experiments.


Figure 7A : Jurkat T cells transfected with control or FIP3 . 1 were treated 1 h with the Rac1 inhibitor NSC23766 at 100 µM and allowed to spread on poly - lysine - coated coverslips for 15 min . F - actin was detected with FITC - Phalloidin and the spreading area of cells at the contact surface was measured . B : Transfected cells were allowed to form immunological synapses with SEE - pulsed Raji cells for 30 min and intracellular distribution of Lck was detected by immunofluorescence . Synapse angle was measured as in figure 4 . Each dot corresponds to one cell . Horizontal bars represent the mean ± SEM . Mann - Whitney test .

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

Figure 7A: Jurkat T cells transfected with control or FIP3.1 were treated 1 h with the Rac1 inhibitor NSC23766 at 100 µM and allowed to spread on poly-lysine-coated coverslips for 15 min. F-actin was detected with FITC-Phalloidin and the spreading area of cells at the contact surface was measured.B: Transfected cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min and intracellular distribution of Lck was detected by immunofluorescence. Synapse angle was measured as in figure 4. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test.


Figure 8A : Jurkat T cells transfected with control or FIP3 . 1 were activated on anti - CD3 - coated plastic plates in the presence of soluble anti - CD28 during 16 h . Concentration of IL2 released in the supernatant was measured by ELISA . Histograms represent the mean ± SD of three experiments . Mann - Whitney test . B : T cells were activated with soluble anti - CD3 and Erk1 / 2 phosphorylation was analyzed by Western blot . Graph represents the percentage of each band intensity with respect to that of cells transfected with control siRNA and left non - stimulated ( t = 0 ) . The intensity each band from Western blot was normalized with respect to the intensity of β - actin for each time point . Representative experiment out of three performed .

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

Figure 8A: Jurkat T cells transfected with control or FIP3.1 were activated on anti-CD3-coated plastic plates in the presence of soluble anti-CD28 during 16 h. Concentration of IL2 released in the supernatant was measured by ELISA. Histograms represent the mean ± SD of three experiments. Mann-Whitney test.B: T cells were activated with soluble anti-CD3 and Erk1/2 phosphorylation was analyzed by Western blot. Graph represents the percentage of each band intensity with respect to that of cells transfected with control siRNA and left non-stimulated (t=0). The intensity each band from Western blot was normalized with respect to the intensity of β-actin for each time point. Representative experiment out of three performed.


Figure 1a . Fold change in mRNA expression of members of the PI3K / AKT / Foxo pathway in IFN + as compared to IFN - Tregs in the microarray analysis . b . mRNA expression of members of the PI3K / AKT / Foxo signaling pathway in sorted IFN + and IFN - Tregs . c . Sorted CD4 + CD25highCD127 - Tregs were stimulated in the presence of IL - 2 ( white ) or IL - 2 + IL - 12 ( black dots ) and RNA was isolated every 24 hours for 4 days . mRNA expression of members of the PI3K / AKT / Foxo signaling pathway . d . Representative example of the protein expression of AKT1 , AKT2 , AKT3 , PTEN and FOXO3 on sorted CD4 + CD25highCD127 - Tregs stimulated in the presence of IL - 2 ( grey line ) or IL - 2 + IL - 12 ( black line ) and day 2 for AKT1 , AKT2 , AKT3 and PTEN and day 3 for FOXO3 . Grey histogram represents the isotype . e . Statistical analysis of AKT1 , AKT2 , AKT3 , FOXO3 and PTEN gMFI of n = 6 experiments performed . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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

Figure 1a. Fold change in mRNA expression of members of the PI3K/AKT/Foxo pathway in IFN+ as compared to IFN- Tregs in the microarray analysis.b. mRNA expression of members of the PI3K/AKT/Foxo signaling pathway in sorted IFN+ and IFN- Tregs.c. Sorted CD4+CD25highCD127- Tregs were stimulated in the presence of IL-2 (white) or IL-2+IL-12 (black dots) and RNA was isolated every 24 hours for 4 days. mRNA expression of members of the PI3K/AKT/Foxo signaling pathway.d. Representative example of the protein expression of AKT1, AKT2, AKT3, PTEN and FOXO3 on sorted CD4+CD25highCD127- Tregs stimulated in the presence of IL-2 (grey line) or IL-2+IL-12 (black line) and day 2 for AKT1, AKT2, AKT3 and PTEN and day 3 for FOXO3. Grey histogram represents the isotype. e. Statistical analysis of AKT1, AKT2, AKT3, FOXO3 and PTEN gMFI of n=6 experiments performed. * p<0.05, ** p<0.005, *** p<0.0005.


Figure 2Sorted CD4 + CD25hiCD127 - Tregs were stimulated with anti - CD3 , anti - CD28 and IL - 2 in the presence or absence of IL - 12 . a . Tregs were stimulated for 50 minutes with IL - 12 or vehicle . Histograms show a representative example of n = 8 independent experiments performed of the expression of phosphorylated molecules at 30 minutes after stimulation for pAKT and pFoxo1 , and at 40 minutes for pFoxo3 in vehicle - ( grey open histogram ) or IL - 12 - treated ( dark open histogram ) Tregs . Grey histograms represent the isotype . The time points selected were based on kinetic experiments performed for a total of 50 minutesSorted CD4 + CD25hiCD127 - Tregs were stimulated with anti - CD3 , anti - CD28 and IL - 2 in the presence or absence of IL - 12 . b . Tregs were stimulated in the presence ( black ) or absence ( grey ) of IL - 12 and the expression of phosphorylated molecules was examined 16 hours later . c . gMFI of pAKT , pFoxo1 and pFoxo3 of n = 6 independent experiments performed . d . Foxo1 and Foxo3 transcriptional targets expression on sorted CD4 + CD25hiCD127 - Tregs stimulated in the presence of vehicle or IL - 12 at day 2 after stimulation ( n = 8 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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

Figure 2Sorted CD4+CD25hiCD127- Tregs were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence or absence of IL-12. a. Tregs were stimulated for 50 minutes with IL-12 or vehicle. Histograms show a representative example of n=8 independent experiments performed of the expression of phosphorylated molecules at 30 minutes after stimulation for pAKT and pFoxo1, and at 40 minutes for pFoxo3 in vehicle- (grey open histogram) or IL-12-treated (dark open histogram) Tregs. Grey histograms represent the isotype. The time points selected were based on kinetic experiments performed for a total of 50 minutesSorted CD4+CD25hiCD127- Tregs were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence or absence of IL-12. b. Tregs were stimulated in the presence (black) or absence (grey) of IL-12 and the expression of phosphorylated molecules was examined 16 hours later. c. gMFI of pAKT, pFoxo1 and pFoxo3 of n=6 independent experiments performed.d. Foxo1 and Foxo3 transcriptional targets expression on sorted CD4+CD25hiCD127-Tregs stimulated in the presence of vehicle or IL-12 at day 2 after stimulation (n=8). *p < 0.05, **p < 0.005, ***p < 0.0005.


Figure 3a . Sorted CD4 + CD25hiCD127 - Tregs were stimulated in the presence of vehicle ( white dots ) or either SF1670 ( first row ) , SC - 79 ( second row ) , 740 Y - P ( third row ) or AS1842856 ( fourth row , black dots ) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours . Statistical analysis of n = 4 independent experiments performed . b . Representative example of the frequency of IFN + Foxp3 + cells in Tregs stimulated in the presence of vehicle , SF1670 , SC - 79 , 740 Y - P or AS1842856 for 3 days after 4 hours of stimulation with PMA and ionomycin . Numbers in brackets represent total cell numbers c . Statistical analysis of the frequency of IFN + Foxp3 + cells in Tregs stimulated in the presence of vehicle , SF1670 , SC - 79 , 740 Y - P or AS1842856 of n = 5 experiments performed . d . Proliferation of CFSE - labeled responder T cells alone ( left histogram ) or co - cultured with Tregs that have been pre - incubated with either vehicle , SF1670 , SC - 79 or 740 Y - P at day 4 after co - culture at a 1 : 2 Treg : Tresp ratio . e . Statistical analysis of the percentage of suppression at different Treg : Tresp ratios of n = 6 experiments performed .

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

Figure 3a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white dots) or either SF1670 (first row), SC-79 (second row), 740 Y-P (third row) or AS1842856 (fourth row, black dots) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=4 independent experiments performed.b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 for 3 days after 4 hours of stimulation with PMA and ionomycin. Numbers in brackets represent total cell numbers c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 of n=5 experiments performed.d. Proliferation of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that have been pre-incubated with either vehicle, SF1670, SC-79 or 740 Y-P at day 4 after co-culture at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression at different Treg:Tresp ratios of n=6 experiments performed.


Figure 4a . Sorted CD4 + CD25hiCD127 - were pre - incubated with increasing concentrations of AS1842856 , washed and co - cultured with CFSE - labeled responder T cells . Representative histograms of proliferation of Tresp alone ( left ) or in co - culture with Tregs treated with either vehicle or different concentrations of AS1842856 measured at day 4 at a 1 : 2 Treg : Tresp ratio . b . Statistical analysis of the suppression of proliferation at different Treg : Tresp ratios ( n = 6 ) on Tregs pretreated with vehicle ( white dots ) or 10 nM ( black dots , left diagram ) or 50 nM AS1842856 ( black dots , right diagram ) . c . Representative dot plots of the frequency of IFN + Foxp3 + Tregs in the co - cultures in a at a 1 : 2 Treg : Tresp ratio after adding GolgiStop for the last 6 hours . d . Statistical analysis of the frequency of IFN + Foxp3 + Tregs in the co - cultures in a of n = 6 experiments performed . e . Correlation between frequency of IFN + Foxp3 + Tregs in co - cultures and percentage of suppression in Tregs pre - incubated with different doses of AS1842856 at a 1 : 4 Treg : Tresp ratio . f . IFNG and TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to the NT - shRNA control , measured at day 6 after 4 hours of stimulation with PMA and ionomycin . g . Percentage of suppression of CFSE - labeled Tresp proliferation co - cultured with Tregs transduced with a non - target ( white dots ) , Foxo1 ( left diagram , black dots ) or Foxo3 shRNA ( right diagram , black dots ) at different Treg : Tresp ratios ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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

Figure 4a. Sorted CD4+CD25hiCD127- were pre-incubated with increasing concentrations of AS1842856, washed and co-cultured with CFSE-labeled responder T cells. Representative histograms of proliferation of Tresp alone (left) or in co-culture with Tregs treated with either vehicle or different concentrations of AS1842856 measured at day 4 at a 1:2 Treg:Tresp ratio. b. Statistical analysis of the suppression of proliferation at different Treg:Tresp ratios (n=6) on Tregs pretreated with vehicle (white dots) or 10 nM (black dots, left diagram) or 50 nM AS1842856 (black dots, right diagram).c. Representative dot plots of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a at a 1:2 Treg:Tresp ratio after adding GolgiStop for the last 6 hours.d. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a of n=6 experiments performed.e. Correlation between frequency of IFN+Foxp3+ Tregs in co-cultures and percentage of suppression in Tregs pre-incubated with different doses of AS1842856 at a 1:4 Treg:Tresp ratio.f. IFNG and TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to the NT-shRNA control, measured at day 6 after 4 hours of stimulation with PMA and ionomycin.g. Percentage of suppression of CFSE-labeled Tresp proliferation co-cultured with Tregs transduced with a non-target (white dots), Foxo1 (left diagram, black dots) or Foxo3 shRNA (right diagram, black dots) at different Treg:Tresp ratios (n=4). *p < 0.05, **p < 0.005, ***p < 0.0005.


Figure 5Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 and shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control and resorted based on GFP expression at day 5 . a . IFNG , TBX21 , IL10 , FOXP3 gene expression after sort at day 5 ( n = 5 ) . Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 and shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control and resorted based on GFP expression at day 5 . b . Representative example of staining on resorted Tregs stimulated with PMA and ionomicin for 4 hours at day 5 ( n = 4 ) . Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 and shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control and resorted based on GFP expression at day 5 . c . Statistical analysis of the frequency of IFN + Foxp3 + Tregs after AKT silencing ( n = 4 ) . Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 and shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control and resorted based on GFP expression at day 5 . d . shRNA transduced cells were co - cultured with CFSE - labeled Tresp and Tresp proliferation was measured after 4 days . e . Statistical analysis of the percentage of suppression by AKT1 - , AKT2 - and AKT3 - silenced Tregs as compared to NT - transduced cells of n = 6 experiments performed . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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

Figure 5Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. a. IFNG, TBX21, IL10, FOXP3 gene expression after sort at day 5 (n=5).Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. b. Representative example of staining on resorted Tregs stimulated with PMA and ionomicin for 4 hours at day 5 (n=4).Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. c. Statistical analysis of the frequency of IFN+Foxp3+ Tregs after AKT silencing (n=4).Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. d. shRNA transduced cells were co-cultured with CFSE-labeled Tresp and Tresp proliferation was measured after 4 days. e. Statistical analysis of the percentage of suppression by AKT1-, AKT2- and AKT3-silenced Tregs as compared to NT-transduced cells of n=6 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.


Figure 6a . Sorted CD4 + CD25hiCD127 - Tregs were stimulated in the presence of vehicle ( white squares ) and IL - 12 either alone ( white circles ) or in the presence of LY294002 or MK2206 ( black circles ) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours . Statistical analysis of n = 8 independent experiments performed . b . Representative example of the frequency of IFN + Foxp3 + cells in Tregs stimulated in the presence of vehicle or IL - 12 either alone or in the presence of LY294002 or MK2206 for 3 days after a 4 hour stimulation with PMA and ionomycin . c . Statistical analysis of the frequency of IFN + Foxp3 + cells in Tregs stimulated as in c of n = 8 experiments performed . d . Proliferation measured at day 4 of CFSE - labeled responder T cells alone ( left histogram ) or co - cultured with Tregs that had been pre - incubated with either vehicle or IL - 12 alone or with LY294002 or MK2206 at a 1 : 2 Treg : Tresp ratio . e . Statistical analysis of the percentage of suppression as in d at different Treg : Tresp ratios of n = 6 experiments performed . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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

Figure 6a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white squares) and IL-12 either alone (white circles) or in the presence of LY294002 or MK2206 (black circles) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=8 independent experiments performed.b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle or IL-12 either alone or in the presence of LY294002 or MK2206 for 3 days after a 4 hour stimulation with PMA and ionomycin.c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated as in c of n=8 experiments performed.d. Proliferation measured at day 4 of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that had been pre-incubated with either vehicle or IL-12 alone or with LY294002 or MK2206 at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression as in d at different Treg:Tresp ratios of n=6 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.


Figure 7a . mRNA expression on ex vivo isolated Tregs from MS patients and healthy controls ( n = 15 ) . b . Representative example of the expression of AKT3 , Foxo3 and PTEN on ex vivo sorted Tregs from MS patients ( black open histogram ) and HC ( grey open histogram ) . Grey histograms represent isotype . c . Ex vivo expression of pAKT , pFoxo1 and pFoxo3 in Tregs from RRMS patients ( black open histogram ) and healthy controls ( white open histograms ) . Grey histograms represent isotype . d . Representative example of the frequency of IFN + Foxp3 + Tregs isolated ex vivo from RRMS patients ( lower row ) and healthy controls ( HC , upper row ) and stimulated in the presence of vehicle ( left histogram ) , LY294002 ( middle histogram ) or MK2206 ( right histogram ) for 3 days . Staining was performed after a 4 - hour stimulation with PMA and ionomycin . e . Statistical analysis of the frequency of IFN + Foxp3 + Tregs in HC ( white bars ) and RRMS patients ( black bars ) stimulated in the presence of either vehicle , LY294002 or MK2206 ( n = 10 ) . f . Representative example of the proliferation of Tresp cells from HC ( upper row ) and RRMS patients ( lower row ) either alone ( first column ) or in co - culture with Tregs that had been previously incubated with either vehicle , LY294002 or MK2206 for one hour and washed to remove drug traces . g . Statistical analysis of the percentage of suppression as in h at different Treg : Tresp ratios of n = 8 experiments performed . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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

Figure 7a. mRNA expression on ex vivo isolated Tregs from MS patients and healthy controls (n=15).b. Representative example of the expression of AKT3, Foxo3 and PTEN on ex vivo sorted Tregs from MS patients (black open histogram) and HC (grey open histogram). Grey histograms represent isotype. c. Ex vivo expression of pAKT, pFoxo1 and pFoxo3 in Tregs from RRMS patients (black open histogram) and healthy controls (white open histograms). Grey histograms represent isotype.d. Representative example of the frequency of IFN+Foxp3+ Tregs isolated ex vivo from RRMS patients (lower row) and healthy controls (HC, upper row) and stimulated in the presence of vehicle (left histogram), LY294002 (middle histogram) or MK2206 (right histogram) for 3 days. Staining was performed after a 4-hour stimulation with PMA and ionomycin. e. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in HC (white bars) and RRMS patients (black bars) stimulated in the presence of either vehicle, LY294002 or MK2206 (n=10).f. Representative example of the proliferation of Tresp cells from HC (upper row) and RRMS patients (lower row) either alone (first column) or in co-culture with Tregs that had been previously incubated with either vehicle, LY294002 or MK2206 for one hour and washed to remove drug traces. g. Statistical analysis of the percentage of suppression as in h at different Treg:Tresp ratios of n=8 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.


Figure 1 ( A - B ) Representative Hematoxylin and Eosin ( H & E ) staining , and immunohistochemistry of phosphorylated FAK on tyrosine ( Y ) 397 ( pY397 FAK ) , alpha - Smooth Muscle Actin ( α - SMA ) , and Fibroblast Activated Protein alpha ( FAP - α ) in serial sections of human normal pancreas ( Ct1 ) , of human normal adjacent PDAC tissue ( Ct2 ) and of human PDAC tissues ( PDAC1 - 3 ) ; scale bar , 100 μm ( A ) , and 300 µm ( B ) . ( C ) Quantification of pY397 FAK level in cancer - associated fibroblasts from human PDAC microarray : 40 different PDAC patient samples and 10 normal human pancreas have been stained for pY397 FAK and double blinded quantified ( based on intensity and positive cell number ) on a scale between 0 ( no staining ) and 6 ( Y397 FAK highly positive ) in whole cell ( left ) , cytoplasm ( middle ) and nucleus ( right ) .

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

Figure 1(A-B) Representative Hematoxylin and Eosin (H&E) staining, and immunohistochemistry of phosphorylated FAK on tyrosine (Y) 397 (pY397 FAK), alpha-Smooth Muscle Actin (α-SMA), and Fibroblast Activated Protein alpha (FAP-α) in serial sections of human normal pancreas (Ct1), of human normal adjacent PDAC tissue (Ct2) and of human PDAC tissues (PDAC1-3); scale bar, 100 μm (A), and 300 µm (B).(C) Quantification of pY397 FAK level in cancer-associated fibroblasts from human PDAC microarray: 40 different PDAC patient samples and 10 normal human pancreas have been stained for pY397 FAK and double blinded quantified (based on intensity and positive cell number) on a scale between 0 (no staining) and 6 (Y397 FAK highly positive) in whole cell (left), cytoplasm (middle) and nucleus (right).


Figure 2 ( A ) Representative images of Definiens ® Tissue studio multi - resolution segmentation algorithm on two PDAC TMA examples ( X , Y ) . From left to right : IHC for pY397 FAK ; ROI detection of tumour cells ( dark blue ) , stroma ( orange ) and other ( light blue / grey ) ; Nuclear / cytoplasm detection in blue and green respectively ; IHC staining classification for pY397 FAK intensity signal ( dark red for high signal , orange for medium signal , yellow for low signal and white for negative signal ) . ( B - C ) Disease Free Survival ( B ) and Overall Survival ( C ) from 120 patient cohort according to pY397 FAK expression in CAFs ; HR : Hazard Ratio . Survival curves were estimated with the Kaplan - Meier method and compared using log - rank test , non - parametric tests ( chi2 - test ) to compare independent groups for categorical data . ( D - E ) Disease Free Survival ( D ) and Overall Survival ( E ) from 120 patient cohort according to pY397 FAK expression in tumour cells . Survival curves were estimated with the Kaplan - Meier method and compared using log - rank test , non - parametric tests ( chi2 - test ) to compare independent groups for categorical data .

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

Figure 2(A) Representative images of Definiens® Tissue studio multi-resolution segmentation algorithm on two PDAC TMA examples (X, Y). From left to right: IHC for pY397 FAK; ROI detection of tumour cells (dark blue), stroma (orange) and other (light blue/grey); Nuclear/ cytoplasm detection in blue and green respectively; IHC staining classification for pY397 FAK intensity signal (dark red for high signal, orange for medium signal, yellow for low signal and white for negative signal).(B-C) Disease Free Survival (B) and Overall Survival (C) from 120 patient cohort according to pY397 FAK expression in CAFs; HR: Hazard Ratio. Survival curves were estimated with the Kaplan-Meier method and compared using log-rank test, non-parametric tests (chi2-test) to compare independent groups for categorical data.(D-E) Disease Free Survival (D) and Overall Survival (E) from 120 patient cohort according to pY397 FAK expression in tumour cells. Survival curves were estimated with the Kaplan-Meier method and compared using log-rank test, non-parametric tests (chi2-test) to compare independent groups for categorical data.