B Representative plots showing hEGFRt expression in bulk edited CD4 + T cells derived from male HD before and 8 hr after Pma / Ionomycin stimulation . Cells were edited with the three constructs depicted in ( A ) .

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

B Representative plots showing hEGFRt expression in bulk edited CD4+ T cells derived from male HD before and 8 hr after Pma/Ionomycin stimulation. Cells were edited with the three constructs depicted in (A).


C Representative plots showing EGFR expression in bulk edited CD4 + T cells derived from male HD at 3 days after treatment with immunotoxin ( left ) , antibody ( middle ) or toxin ( right ) .

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

C Representative plots showing EGFR expression in bulk edited CD4+ T cells derived from male HD at 3 days after treatment with immunotoxin (left), antibody (middle) or toxin (right).


D Bar plot showing percentage of EGFR + T cells at 3 days after treatment with 5 nM or 1 nM of immunotoxin , antibody or toxin , measured by FACS Analysis . Friedman test with Dunn ' s multiple comparisons . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * * p = 0 . 0052 and * * p = 0 . 0024 for immunotoxin vs antibody and * * p = 0 . 0010 and * * p = 0 . 0024 for immunotoxin vs toxin , referring to EGFRmod1 and EGFRmod2 respectively ) . Different dose - conditions were used as a unified group for statistical analysis ( n = 10 for each group ) . Median ± IQR .

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

D Bar plot showing percentage of EGFR+ T cells at 3 days after treatment with 5 nM or 1 nM of immunotoxin, antibody or toxin, measured by FACS Analysis. Friedman test with Dunn's multiple comparisons. P-values were adjusted with Bonferroni's correction to account for multiple comparisons (**p=0.0052 and **p=0.0024 for immunotoxin vs antibody and **p=0.0010 and **p=0.0024 for immunotoxin vs toxin, referring to EGFRmod1 and EGFRmod2 respectively). Different dose-conditions were used as a unified group for statistical analysis (n=10 for each group). Median ± IQR.


B Percentage of HDR in HSPC subpopulations at 4 days after CD40LG editing , measured by molecular analysis ( ddPCR ) . Cells were edited with donor template either comprising only the corrective cDNA ( NR ; n = 4 ) , or carrying the selector cassette IRES . NGFR ( NGFR ; n = 5 ) and sorted according to surface markers : CD34 - ; CD34 + CD133 - CD90 - ; CD34 + CD133 + CD90 - ; CD34 + CD133 + CD90 + . CD34 + CD133 + CD90 + / CD90 - and CD34 + CD133 - / CD34 - conditions were used as unified groups for statistical analysis . The comparisons between the groups were performed with LME models accounting for the different subpopulations included in the analysis and with random effects defined to account for the same donor . In comparison about CD34 + CD133 + CD90 + / CD90 - , the percentages were used in square root scale to meet the assumption of normality of the residuals of the model . For CD34 + CD133 + CD90 + / CD90 - , * * p = 0 . 0035 , for CD34 + CD133 - / CD34 - p = 0 . 2051 . Median ± IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentage of HDR in HSPC subpopulations at 4 days after CD40LG editing, measured by molecular analysis (ddPCR). Cells were edited with donor template either comprising only the corrective cDNA (NR; n=4), or carrying the selector cassette IRES.NGFR (NGFR; n=5) and sorted according to surface markers: CD34-; CD34+CD133-CD90-; CD34+CD133+CD90-; CD34+CD133+CD90+. CD34+CD133+CD90+/CD90- and CD34+CD133-/CD34- conditions were used as unified groups for statistical analysis. The comparisons between the groups were performed with LME models accounting for the different subpopulations included in the analysis and with random effects defined to account for the same donor. In comparison about CD34+CD133+CD90+/CD90-, the percentages were used in square root scale to meet the assumption of normality of the residuals of the model. For CD34+CD133+CD90+/CD90-, **p= 0.0035, for CD34+CD133-/CD34- p=0.2051. Median ± IQR.


C Population composition in UT or bulk edited HSPC from ( B ) ( n = 8 for each group , except UT cells from NGFR group , n = 7 ) . Paired Wilcoxon ' s test , to account for the same donor , with p - values adjusted with Bonferroni ' s correction to account for multiple testing . Analysis was performed separately for NR and NGFR groups . Mean ± SEM .

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

C Population composition in UT or bulk edited HSPC from (B) (n=8 for each group, except UT cells from NGFR group, n=7). Paired Wilcoxon's test, to account for the same donor, with p-values adjusted with Bonferroni's correction to account for multiple testing. Analysis was performed separately for NR and NGFR groups. Mean ± SEM.


D Human CD45 + cell engraftment in PB after transplantation of HSPC edited with the two donor templates described in ( B ) and with ( n = 4 ) or without ( n = 5 ) GSE56 . Comparison between groups was performed by NLME model with an asymptotic model ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the first time - point ( i . e . 6 weeks ; * p = 0 . 0213 and * * p = 0 . 0069 ) . Group transplanted with HSPC treated without GSE56 needs significantly greater time for reaching the final plateau with respect to both GSE56 groups ( p = 0 . 0034 for NGFR + GSE56 and p = 0 . 0080 for NR + GSE56 ) . Median ± IQR .

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

D Human CD45+ cell engraftment in PB after transplantation of HSPC edited with the two donor templates described in (B) and with (n=4) or without (n=5) GSE56. Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the first time-point (i.e. 6 weeks; *p=0.0213 and **p=0.0069). Group transplanted with HSPC treated without GSE56 needs significantly greater time for reaching the final plateau with respect to both GSE56 groups (p=0.0034 for NGFR+GSE56 and p=0.0080 for NR+GSE56). Median ± IQR.


E Percentage of HDR within human cells over time , measured by ddPCR in PB of mice from ( D ) . Longitudinal comparisons were performed with an LME model with time treated as a continuous variable , and followed by an appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the last time - point ( i . e . 20 weeks ; * * * * p < 0 . 0001 in both comparisons ) . NR + GSE56 group shows a constant behavior over time ( p = 0 . 0588 ) . The slope of the linear trajectory over time is not significantly different between GSE56 groups ( p = 0 . 1624 ) , indicating gene editing stability of both groups over time . Mice transplanted with HSPC treated without GSE56 shows significantly lower slope than GSE56 groups ( p < 0 . 0001 in respect of NGFR group , p = 0 . 0054 in respect of NR group ) , thus highlighting decreased gene editing efficiency over time . Median ± IQR .

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

E Percentage of HDR within human cells over time, measured by ddPCR in PB of mice from (D). Longitudinal comparisons were performed with an LME model with time treated as a continuous variable, and followed by an appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the last time-point (i.e. 20 weeks; ****p<0.0001 in both comparisons). NR+GSE56 group shows a constant behavior over time (p=0.0588). The slope of the linear trajectory over time is not significantly different between GSE56 groups (p=0.1624), indicating gene editing stability of both groups over time. Mice transplanted with HSPC treated without GSE56 shows significantly lower slope than GSE56 groups (p<0.0001 in respect of NGFR group, p=0.0054 in respect of NR group), thus highlighting decreased gene editing efficiency over time. Median ± IQR.


Percentage of HDR within human cells in ( F ) PB , bone marrow , spleen , thymus measured by ddPCR in mice from ( D ) . Median .

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

Percentage of HDR within human cells in (F) PB, bone marrow, spleen, thymus measured by ddPCR in mice from (D). Median.


Percentage of HDR within human cells within BM - sorted human subpopulations ( CD19 + , CD33 + , CD3 + , CD34 + ) , measured by ddPCR in mice from ( D ) . Median .

Percentage of HDR within human cells within BM-sorted human subpopulations (CD19+, CD33+, CD3+, CD34+), measured by ddPCR in mice from (D). Median.


H Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by MFI on pooled CD4 + T cells retrieved from spleen of mice from ( D ) ( n = 4 NGFR + , 4 NGFR - ) . Mean ± SEM .

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

H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by MFI on pooled CD4+ T cells retrieved from spleen of mice from (D) (n=4 NGFR+, 4 NGFR-). Mean ± SEM.


B Total counts of recipient CD3 + cells gated within CD45 . 2 + cells in PB of experimental mice ( n = 4 CPA 300 , 9 NC ) . Comparison between groups was performed by NLME model with an asymptotic model ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to day 1 ( * * * * p < 0 . 0001 ) . Mean ± SEM .

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

B Total counts of recipient CD3+ cells gated within CD45.2+ cells in PB of experimental mice (n=4 CPA 300, 9 NC). Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 1 (****p<0.0001). Mean ± SEM.


C Total counts of engrafted donor CD45 . 1 . 2 + cells in PB of mice from ( B ) . Comparison between groups was performed by NLME model with an asymptotic model ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to plateau ( * * * p = 0 . 0001 ) . Mean ± SEM .

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

C Total counts of engrafted donor CD45.1.2+ cells in PB of mice from (B). Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to plateau (***p=0.0001). Mean ± SEM.


D , E Percentage of donor CD4 + and CD8 + cells gated within CD3 + CD45 . 1 . 2 + cells in PB of CPA treated ( D ) or NC mice ( E ) from ( B ) . Mean ± SEM .

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D, E Percentage of donor CD4+ and CD8+ cells gated within CD3+CD45.1.2+ cells in PB of CPA treated (D) or NC mice (E) from (B). Mean ± SEM.


F TNP - KLH specific IgG in sera of transplanted mice collected at the times indicated in ( A ) ( n = 7 CPA 300 , 13 NC for first boost and 4 CPA 300 , 9 NC for second boost ) . Sera from vaccinated WT ( n = 5 ) and CD40lg - / - ( n = 5 ) mice were used as positive and negative controls , respectively . Two independent experiments . Comparisons of first boost data were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups ( * * * * p < 0 . 0001 in all comparisons ) . Median ± IQR .

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

F TNP-KLH specific IgG in sera of transplanted mice collected at the times indicated in (A) (n=7 CPA 300, 13 NC for first boost and 4 CPA 300, 9 NC for second boost). Sera from vaccinated WT (n=5) and CD40lg-/- (n=5) mice were used as positive and negative controls, respectively. Two independent experiments. Comparisons of first boost data were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.


G Average area of PNA + foci in splenic sections of experimental mice from ( B ) , calculated as ratio between total PNA + area and number of PNA + foci / cells . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test ( n = 9 CD40LG , 6 WT , 18 NC , 8 CPA300 ) . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * p = 0 . 0149 , * * p = 0 . 0065 and * * * * p < 0 . 0001 ) . Mean ± SEM .

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

G Average area of PNA+ foci in splenic sections of experimental mice from (B), calculated as ratio between total PNA+ area and number of PNA+ foci/cells. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test (n=9 CD40LG, 6 WT, 18 NC, 8 CPA300). P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0149, **p=0.0065 and ****p<0.0001). Mean ± SEM.


H Detection of splenic TNP - KLH specific IgM ( left ) and IgG ( right ) - secreting cells by ELISPOT assay ( n = 7 NC , 4 CPA300 , 3 WT , 2 CD40LG ) . Spots were counted by an ELISPOT Reader using a size range of 0 . 005 - 1 mm . Median ± IQR .

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

H Detection of splenic TNP-KLH specific IgM (left) and IgG (right) -secreting cells by ELISPOT assay (n=7 NC, 4 CPA300, 3 WT, 2 CD40LG). Spots were counted by an ELISPOT Reader using a size range of 0.005-1 mm. Median ± IQR.


B Total counts of recipient CD3 + CD45 . 2 + cells in PB of mice NC ( n = 13 ) or pre - conditioned with 300 mg / kg CPA ( n = 19 ) , 200 mg / kg CPA ( n = 6 ) , anti - lymphocyte serum ( ALS ; n = 5 ) or anti - CD4 antibody ( n = 6 ) . Three independent experiments . Comparisons at day 3 \ 4 were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ; * * * p = 0 . 0009 and * * * * p < 0 . 0001 in all comparisons ) . Mean ± SEM .

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

B Total counts of recipient CD3+ CD45.2+ cells in PB of mice NC (n=13) or pre-conditioned with 300 mg/kg CPA (n=19), 200 mg/kg CPA (n=6), anti-lymphocyte serum (ALS; n=5) or anti-CD4 antibody (n=6). Three independent experiments. Comparisons at day 3\4 were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods; ***p=0.0009 and ****p<0.0001 in all comparisons). Mean ± SEM.


C Total counts of engrafted donor CD45 . 1 + cells in PB of mice from ( B ) . Three independent experiment . Comparisons at day 18 \ 24 were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ; * * * p = 0 . 0002 and * * * * p < 0 . 0001 in all comparisons ) . CPA200 was not included in the analysis because n = 4 . Mean ± SEM .

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

C Total counts of engrafted donor CD45.1+ cells in PB of mice from (B). Three independent experiment. Comparisons at day 18\24 were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods; ***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. Mean ± SEM.


D Analysis of the relationship between total counts of recipient CD3 + cells gated within CD45 . 2 + cells at day 3 , 4 and engrafted donor CD45 . 1 + T cells at 18 \ 24 days . This relationship was modeled with an asymptotic NLME model ( Appendix Supplementary Statistical Methods ) , accounting for the different groups and multiple experiments . From the estimated model , a lower value of recipient CD3 + T cells corresponds to a higher value of engrafted donor CD4 + T cells ( p = 0 . 0006 ) .

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

D Analysis of the relationship between total counts of recipient CD3+ cells gated within CD45.2+ cells at day 3, 4 and engrafted donor CD45.1+ T cells at 18\24 days. This relationship was modeled with an asymptotic NLME model (Appendix Supplementary Statistical Methods), accounting for the different groups and multiple experiments. From the estimated model, a lower value of recipient CD3+ T cells corresponds to a higher value of engrafted donor CD4+ T cells (p=0.0006).


E TNP - KLH specific IgG concentration in sera of mice from ( B ) collected before ( day 35 ; pre ) and after ( day 49 ; post ) the first boost ( n = 13 NC , 19 CPA 300 , 4 CPA 200 , 5 ALS , 6 ANTI - CD4 ; three independent experiments ) and at day 81 ( pre ) and 89 ( post ) for the second boost ( n = 4 NC , 4 CPA 300 , 4 CPA 200 , 5 ALS , 6 ANTI - CD4 ) . Sera from vaccinated WT ( n = 5 ) and Cd40lg - / - ( n = 5 ) mice were used as positive and negative controls , respectively . For early challenge data , comparisons were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups ( * * * p = 0 . 0002 and * * * * p < 0 . 0001 in all comparisons ) . CPA200 was not included in the analysis because n = 4 . WT group , not indicated in the figure , is significantly different from all groups ( p < 0 . 0001 in all comparisons ) . Of note , regarding the comparisons between time - points , the groups CPA 300 and WT show a significant increase between pre and post values ( p < 0 . 0001 for both ) , while all other groups show a significant decrease between pre and post values ( p < 0 . 0001 for all except p = 0 . 0022 for ANTI - CD4 ) . Median ± IQR .

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

E TNP-KLH specific IgG concentration in sera of mice from (B) collected before (day 35; pre) and after (day 49; post) the first boost (n=13 NC, 19 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4; three independent experiments) and at day 81 (pre) and 89 (post) for the second boost (n=4 NC, 4 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4). Sera from vaccinated WT (n=5) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. For early challenge data, comparisons were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. WT group, not indicated in the figure, is significantly different from all groups (p<0.0001 in all comparisons). Of note, regarding the comparisons between time-points, the groups CPA 300 and WT show a significant increase between pre and post values (p<0.0001 for both), while all other groups show a significant decrease between pre and post values (p<0.0001 for all except p=0.0022 for ANTI-CD4). Median ± IQR.


F Total counts of recipient CD3 + cells gated within CD45 . 2 + cells after transfer of primed donor T cells in PB of mice treated with 300 mg / kg CPA ( n = 11 ) , 200 mg / kg CPA ( n = 11 ) or NC ( n = 6 ) . Longitudinal comparisons were performed by LME model followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to day 4 ( * * * * p < 0 . 0001 in all comparisons ) . Mean ± SEM .

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

F Total counts of recipient CD3+ cells gated within CD45.2+ cells after transfer of primed donor T cells in PB of mice treated with 300 mg/kg CPA (n=11), 200 mg/kg CPA (n=11) or NC (n=6). Longitudinal comparisons were performed by LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 4 (****p<0.0001 in all comparisons). Mean ± SEM.


G Total counts of engrafted donor CD45 . 1 + cells in PB of mice from ( F ) . Longitudinal comparisons were performed with an LME model followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to day 25 . At day 215 a significant difference was observed between CPA 300 vs NC and CPA 300 vs CPA 200 ( * * * * p < 0 . 0001 for both ) . Mean ± SEM .

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

G Total counts of engrafted donor CD45.1+ cells in PB of mice from (F). Longitudinal comparisons were performed with an LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 25. At day 215 a significant difference was observed between CPA 300 vs NC and CPA 300 vs CPA 200 (****p<0.0001 for both). Mean ± SEM.


H TNP - KLH specific IgG concentration in sera of mice from ( F ) collected before ( day 36 , pre ) and after ( day 50 , post ) the first boost ( n = 6 NC , 11 CPA 300 , 11 CPA 200 ) and at day 212 ( pre ) and 219 ( post ) for the second boost ( n = 6 NC , 11 CPA 300 , 9 CPA 200 ) . Sera from vaccinated WT ( n = 11 ) and Cd40lg - / - ( n = 5 ) mice were used as positive and negative controls , respectively . Comparisons were performed with an LME model followed by an appropriate post - hoc analysis , separately for each challenge data ( Appendix Supplementary Statistical Methods ) . Reported statistical comparisons refer to the overall difference among groups , for early challenge data ( * p = 0 . 0282 and * * p = 0 . 0037 ) , while to differences between time - points within each group , for late challenge data ( * * p = 0 . 0018 and * * * * p < 0 . 0001 in both comparisons ) . Of note , in the late challenge data , no significant overall differences were observed among the groups . Median ± IQR .

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

H TNP-KLH specific IgG concentration in sera of mice from (F) collected before (day 36, pre) and after (day 50, post) the first boost (n=6 NC, 11 CPA 300, 11 CPA 200) and at day 212 (pre) and 219 (post) for the second boost (n=6 NC, 11 CPA 300, 9 CPA 200). Sera from vaccinated WT (n=11) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. Comparisons were performed with an LME model followed by an appropriate post-hoc analysis, separately for each challenge data (Appendix Supplementary Statistical Methods). Reported statistical comparisons refer to the overall difference among groups, for early challenge data (*p=0.0282 and **p=0.0037), while to differences between time-points within each group, for late challenge data (**p=0.0018 and ****p<0.0001 in both comparisons). Of note, in the late challenge data, no significant overall differences were observed among the groups. Median ± IQR.


B Total counts of WT donor CD4 + T cells in mice from ( A ) ( n = 13 100 % WT , 8 25 % WT , 14 10 % WT , 6 1 % , 14 0 % WT ) . Two representative experiments shown out of 3 . Mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Total counts of WT donor CD4+ T cells in mice from (A) (n=13 100% WT, 8 25% WT, 14 10% WT, 6 1%, 14 0% WT). Two representative experiments shown out of 3. Mean ± SEM.


C TNP - KLH specific IgG concentration in sera of mice from ( A ) collected seven days before ( pre ) and after ( post ) TNP - KLH vaccination ( n = 13 100 % WT , 13 25 % WT , 14 10 % WT , 6 1 % WT , 14 0 % WT ) . Three independent experiments . Comparisons were performed with an LME model , accounting for multiple experiments , followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups ( * * * * p < 0 . 0001 in all comparisons ) . Median ± IQR .

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

C TNP-KLH specific IgG concentration in sera of mice from (A) collected seven days before (pre) and after (post) TNP-KLH vaccination (n=13 100% WT, 13 25% WT, 14 10% WT, 6 1% WT, 14 0% WT). Three independent experiments. Comparisons were performed with an LME model, accounting for multiple experiments, followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.


D Percentage of PNA + GL7 + GC B cells within the spleen of mice from ( A ) ( n = 16 100 % WT , 6 25 % WT , 16 10 % WT , 11 1 % WT , 18 0 % WT ) . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * p = 0 . 0385 for 25 % WT vs 1 % WT , * p = 0 . 0129 for 100 % WT vs 1 % WT , * * * p = 0 . 0001 and * * * * p < 0 . 0001 in both comparisons ) . Median ± IQR .

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

D Percentage of PNA+GL7+ GC B cells within the spleen of mice from (A) (n=16 100% WT, 6 25% WT, 16 10% WT, 11 1% WT, 18 0% WT). Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0385 for 25% WT vs 1% WT, *p=0.0129 for 100% WT vs 1% WT, ***p=0.0001 and ****p<0.0001 in both comparisons). Median ± IQR.


E Quantitation of P . murina rRNA in lung homogenate of mice from ( A ) transplanted with different ratios of WT HSPC cells ( n = 8 100 % WT , 7 25 % WT , 7 10 % WT , 8 0 % WT ) and infected with the pathogen . Results are expressed in P . murina / HPRT RNA copies . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons . Only the groups 100 % WT and 0 % WT resulted to be significantly different ( * * p = 0 . 0028 ) . Median ± IQR .

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

E Quantitation of P. murina rRNA in lung homogenate of mice from (A) transplanted with different ratios of WT HSPC cells (n=8 100% WT, 7 25% WT, 7 10% WT, 8 0% WT) and infected with the pathogen. Results are expressed in P.murina/HPRT RNA copies. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (**p=0.0028). Median ± IQR.


F Lung weight of mice from ( E ) . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons . Only the groups 100 % WT and 0 % WT resulted to be significantly different ( * p = 0 . 0254 ) . Median ± IQR .

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

F Lung weight of mice from (E). Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (*p=0.0254). Median ± IQR.


G Left : Representative pictures of lung tissue sections stained immunohistochemically with a rabbit primary antibody ( upper left : negative sample ; lower left : positive sample ) . Brown areas represent intra - alveolar aggregates of P . murina organisms . Right : Representative pictures of P . murina organisms detected by immunofluorescence in lungs homogenate ( upper right : negative sample ; lower right : positive sample ) .

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

G Left: Representative pictures of lung tissue sections stained immunohistochemically with a rabbit primary antibody (upper left: negative sample; lower left: positive sample). Brown areas represent intra-alveolar aggregates of P. murina organisms. Right: Representative pictures of P. murina organisms detected by immunofluorescence in lungs homogenate (upper right: negative sample; lower right: positive sample).


H Quantitation of P . Murina rRNA in lung homogenate of mice adoptively transferred ( n = 9 CD40LG + T cells ) or not ( n = 8 CD40LG ) with in vivo primed CD4 + T cells in absence of conditioning and infected with the pathogen . Results are expressed in P . Murina / HPRT RNA copies . Mann - Whitney test . * * * P - value of the comparison = 0 . 0003 . Median ± IQR .

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

H Quantitation of P. Murina rRNA in lung homogenate of mice adoptively transferred (n=9 CD40LG + T cells) or not (n=8 CD40LG) with in vivo primed CD4+ T cells in absence of conditioning and infected with the pathogen. Results are expressed in P.Murina/HPRT RNA copies. Mann-Whitney test. ***P-value of the comparison = 0.0003. Median ± IQR.


I Lung weight of experimental mice from ( H ) . Mann - Whitney test . * * P - value of the comparison = 0 . 0055 . Median ± IQR .

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

I Lung weight of experimental mice from (H). Mann-Whitney test. **P-value of the comparison = 0.0055. Median ± IQR.


Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , Xnr1 ( 50 pg )

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), Xnr1 (50 pg)


Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , BMP4 ( 100 pg ) .

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), BMP4 (100 pg).


Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng )

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng)


( D wt Wip1 , but not Wip1 ( D277A ) , down - regulates the activity of Activin - or BMP - responsive promoters in HEK293T cells . FAST1 , were co - transfected as indicated for efficient activation of the promoters . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired 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

(D wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. FAST1, were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


E ) wt Wip1 , but not Wip1 ( D277A ) , down - regulates the activity of Activin - or BMP - responsive promoters in HEK293T cells . Smad1 and Smad4 were co - transfected as indicated for efficient activation of the promoters . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired 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

E) wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. Smad1 and Smad4 were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


( F - Q ) Dorsal ( F - N ) or ventral ( O - Q ) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10 . 25 as analyzed by in situ hybridization . Embryos are shown in vegetal views with dorsal to the top . Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos . Control , an embryo injected with LacZ only . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) and LacZ ( 100 pg ) . Scale bar , 150 μm .

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

(F-Q) Dorsal (F-N) or ventral (O-Q) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10.25 as analyzed by in situ hybridization. Embryos are shown in vegetal views with dorsal to the top. Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos. Control, an embryo injected with LacZ only. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng) and LacZ (100 pg). Scale bar, 150 μm.


( A , Nodal - induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT - PCR . Animal caps ere treated with - ) , no injection of Co MO , Wip1 MO and / or Wip1 mRNA . The dose of reagent injected : Xnr1 ( 5 pg ) , Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) and Wip1 ( 1 ng ) .

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

(A, Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps ere treated with -), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Xnr1 (5 pg), Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).


B ) Activin / Nodal - induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT - PCR . Animal caps in ( B ) were treated with Activin protein ( 5 ng / ml ) . ( - ) , no injection of Co MO , Wip1 MO and / or Wip1 mRNA . The dose of reagent injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) and Wip1 ( 1 ng ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Activin/Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps in (B) were treated with Activin protein (5 ng/ml). (-), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).


( C ) Knockdown of Wip1 up - regulates the activity of Activin / Nodal - responsive promoter . HEK293T cells transfected with Co siRNA ( 50 nM ) or Wip1 siRNA ( 50 nM ) were stimulated with Activin protein ( 10 ng / ml ) for 10 h and then subjected to luciferase assays . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * * * P < 0 . 001 by unpaired 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

(C) Knockdown of Wip1 up-regulates the activity of Activin/Nodal-responsive promoter. HEK293T cells transfected with Co siRNA (50 nM) or Wip1 siRNA (50 nM) were stimulated with Activin protein (10 ng/ml) for 10 h and then subjected to luciferase assays. Data are expressed as the mean ± SEM (n=3 biological replicates). ***P<0.001 by unpaired Student's t-test.


Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate . ( - ) , no injection of Noggin mRNA . The amount of MO and mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G and J ; 30 ng for F and I ) and Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm .

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

Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. (-), no injection of Noggin mRNA. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.


Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate . Embryos in ( E - J ) are shown in dorsal views with anterior to the top . The amount of MO and mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G and J ; 30 ng for F and I ) and Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm .

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

Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. Embryos in (E-J) are shown in dorsal views with anterior to the top. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.


( K , Activin induction of p21 is up - regulated upon Wip1 knockdown . HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection , were stimulated with Activin ( 10 ng / ml ) for 3 to 12 h as indicated t = 0 in ( K ) is 48 h after siRNA transfection . P - Smad2 serves as an indicator of active Activin / Nodal signaling . Arrows ndicate non - specific bands .

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

(K, Activin induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with Activin (10 ng/ml) for 3 to 12 h as indicated t=0 in (K) is 48 h after siRNA transfection. P-Smad2 serves as an indicator of active Activin/Nodal signaling. Arrows ndicate non-specific bands.


L ) TGF - β induction of p21 is up - regulated upon Wip1 knockdown . HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection , were stimulated with increasing concentration of TGF - β1 ( 10 , 20 ng / ml ) for 3 h .

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

L) TGF-β induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with increasing concentration of TGF-β1 (10, 20 ng/ml) for 3 h.


( A , Effects of the gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T cells were transfected or not with wt Wip1 , Wip1 ( D277A ) , as indicated and subjected to CCK - 8 assays before ( 0 h ) and after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - test . n . s . , not significant .

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

(A, Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with wt Wip1, Wip1(D277A), as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.


B ) Effects of the gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T cells were transfected or not with Co siRNA or Wip1 siRNA as indicated and subjected to CCK - 8 assays before ( 0 h ) and after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - test . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with Co siRNA or Wip1 siRNA as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.


Depletion of Wip1 augments TGF - β - induced migration and invasion of MDA - MB231 breast cancer cells . MDA - MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20 - μL pipette tip and then incubated in the absence or presence of TGF - β1 for the indicated times ( C , D )

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

Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20-μL pipette tip and then incubated in the absence or presence of TGF-β1 for the indicated times (C, D)


Depletion of Wip1 augments TGF - β - induced migration and invasion of MDA - MB231 breast cancer cells . MDA - MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF - β1 for 20 h ( E , F ) .

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

Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF-β1 for 20 h (E, F).


( A - D ) Knockdown of Wip1 expands the expression of dorsal mesodermal markers . Embryos injected dorsally with Co MO ( 40 ng ) or Wip1 MO ( 40 ng ) were subjected to in situ hybridization at stage 10 . 25 . Embryos are shown in dorso - vegetal views with dorsal to the top . Scale bar , 150 μm .

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(A-D) Knockdown of Wip1 expands the expression of dorsal mesodermal markers. Embryos injected dorsally with Co MO (40 ng) or Wip1 MO (40 ng) were subjected to in situ hybridization at stage 10.25. Embryos are shown in dorso-vegetal views with dorsal to the top. Scale bar, 150 μm.


( E - I ) Morphological phenotypes of Wip1 morphants . Embryos were injected radially in the marginal zone with Co MO ( 80 ng ) , Wip1 MO ( 80 ng ) , wt Wip1 ( 1 ng ) and Wip1 ( D277A ) mRNA as indicated and cultured to tadpole stages . Embryos are shown in lateral views with anterior to the left . Un Co , uninjected control . Scale bar , 1 mm . ( J ) Quantification of the phenotypes shown in ( E - I ) . Severe defects include microcephaly , shortened and kinked body axis , and no eye . Mild defects indicate normal body axis with malformed eyes .

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(E-I) Morphological phenotypes of Wip1 morphants. Embryos were injected radially in the marginal zone with Co MO (80 ng), Wip1 MO (80 ng), wt Wip1 (1 ng) and Wip1(D277A) mRNA as indicated and cultured to tadpole stages. Embryos are shown in lateral views with anterior to the left. Un Co, uninjected control. Scale bar, 1 mm. (J) Quantification of the phenotypes shown in (E-I). Severe defects include microcephaly, shortened and kinked body axis, and no eye. Mild defects indicate normal body axis with malformed eyes.


( A Wip1 and Smad4 form an endogenous protein complex . Cell lysates of HEK293T cells , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) ( - ) , no treatment with protein ligand . IgG , control IgG . An arrow in ( A ) indicates non - specific bands .

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(A Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) (-), no treatment with protein ligand. IgG, control IgG. An arrow in (A) indicates non-specific bands.


B ) Wip1 and Smad4 form an endogenous protein complex . Cell lysates of HEK293T cells , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) and vice versa ( B ) . ( - ) , no treatment with protein ligand . IgG , control IgG .

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B) Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) and vice versa (B). (-), no treatment with protein ligand. IgG, control IgG.


( C ) Upon ligand stimulation , Wip1 forms a complex with Smad4 in the nucleus . HeLa cells were treated or not with TGF - β1 ( 40 ng / ml ) for 2 h , fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4 . Blue : DAPI ; Red : PLA signal . Scale bar , 20 μm . ( D ) Quantitative analysis of Wip1 / Smad4 interactions shown in ( C ) . Data are represented as the mean ± SEM ( n = 3 biological replicates ) . Twenty cells were analyzed per sample . * * * P < 0 . 001 by Student ' s t - test .

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(C) Upon ligand stimulation, Wip1 forms a complex with Smad4 in the nucleus. HeLa cells were treated or not with TGF-β1 (40 ng/ml) for 2 h, fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4. Blue: DAPI; Red: PLA signal. Scale bar, 20 μm. (D) Quantitative analysis of Wip1/Smad4 interactions shown in (C). Data are represented as the mean ± SEM (n=3 biological replicates). Twenty cells were analyzed per sample. ***P<0.001 by Student's t-test.


( E ) Wip1 interferes with the ability of Smad4 to bind to Smad2 . HEK293T cells co - transfected with the indicated constructs were treated or not with TGF - β1 for 1 h and then harvested . An arrow denotes Myc - tagged Wip1 protein .

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(E) Wip1 interferes with the ability of Smad4 to bind to Smad2. HEK293T cells co-transfected with the indicated constructs were treated or not with TGF-β1 for 1 h and then harvested. An arrow denotes Myc-tagged Wip1 protein.


( F ) Wip1 has a negative effect on nuclear accumulation of Smad4 . HEK293T cells were transfected with Co siRNA or Wip1 siRNA , treated with TGF - β1 ( 40 ng / ml , 1 h ) 48 h after siRNA transfection and subjected to nuclear / cytoplasmic fractionation and western blotting analysis . β - tubulin and lamin A / C serve as cytoplasmic and nuclear markers , respectively . C , cytoplasm ; N , nucleus . The experiment was repeated two times with similar results . ( G ) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in ( F ) .

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(F) Wip1 has a negative effect on nuclear accumulation of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA, treated with TGF-β1 (40 ng/ml, 1 h) 48 h after siRNA transfection and subjected to nuclear/cytoplasmic fractionation and western blotting analysis. β-tubulin and lamin A/C serve as cytoplasmic and nuclear markers, respectively. C, cytoplasm; N, nucleus. The experiment was repeated two times with similar results. (G) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in (F).


( A ) Overexpression of Wip1 down - regulates Smad4 phosphorylation at Thr277 . MOCK - or Flag - Wip1 - transfected HEK293T cells were treated with 1 h pulse of FGF2 ( 10 ng / ml ) , followed by addition of U0126 ( 40 μM ) and harvesting for western blotting at the indicated times . - FGF2 , no treatment with FGF2 .

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(A) Overexpression of Wip1 down-regulates Smad4 phosphorylation at Thr277. MOCK- or Flag-Wip1-transfected HEK293T cells were treated with 1 h pulse of FGF2 (10 ng/ml), followed by addition of U0126 (40 μM) and harvesting for western blotting at the indicated times. - FGF2, no treatment with FGF2.


( B ) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control and Wip1 - depleted HEK293T cells , showing that knockdown of Wip1 up - regulates Smad4 phosphorylation at the MAPK site . A bracket denotes phospho - Smad4 .

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(B) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control and Wip1-depleted HEK293T cells, showing that knockdown of Wip1 up-regulates Smad4 phosphorylation at the MAPK site. A bracket denotes phospho-Smad4.


( C ) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay . Myc - tagged Smad4 was expressed in HEK293T cells , which were then treated or not with FGF2 ( 40 ng / ml , 2 h ) , and the Myc - Smad4 was immunoprecipitated using anti - Myc antibody and subsequently incubated with or without recombinant human Wip1 protein ( 0 . 5 μg ) in the absence or presence of Mg2 + as indicated .

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(C) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay. Myc-tagged Smad4 was expressed in HEK293T cells, which were then treated or not with FGF2 (40 ng/ml, 2 h), and the Myc-Smad4 was immunoprecipitated using anti-Myc antibody and subsequently incubated with or without recombinant human Wip1 protein (0.5 μg) in the absence or presence of Mg2+ as indicated.


( D ) FGF - induced destabilization of Smad4 is enhanced further in Wip1 - depleted animal cap cells as analyzed by western blotting . The amount of reagent injected : Myc - Smad4 ( 400 pg ) , FGF8 ( 100 pg ) , Wnt8 ( 400 pg ) , Co MO ( 60 ng ) and Wip1 MO ( 60 ng ) .

(D) FGF-induced destabilization of Smad4 is enhanced further in Wip1-depleted animal cap cells as analyzed by western blotting. The amount of reagent injected: Myc-Smad4 (400 pg), FGF8 (100 pg), Wnt8 (400 pg), Co MO (60 ng) and Wip1 MO (60 ng).


( E ) Polyubiquitination of wt Smad4 is promoted in Wip1 - silenced cells . HEK293T cells were transfected with the indicated combinations of Flag - ubiquitin , Co siRNA , Wip1 siRNA , Myc - Smad4 and Myc - Smad4 ( T277A ) and then treated with FGF2 ( 10 ng / ml , 4 h ) in the presence of the proteasome inhibitor MG132 , and Myc - Smad4 and its phosphorylation - resistant mutant were immunoprecipitated with anti - Myc antibody . Ubiquitin - conjugated Smad4 ( Smad4 - Ubn ) was detected by western blotting with anti - Flag antibody .

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(E) Polyubiquitination of wt Smad4 is promoted in Wip1-silenced cells. HEK293T cells were transfected with the indicated combinations of Flag-ubiquitin, Co siRNA, Wip1 siRNA, Myc-Smad4 and Myc-Smad4(T277A) and then treated with FGF2 (10 ng/ml, 4 h) in the presence of the proteasome inhibitor MG132, and Myc-Smad4 and its phosphorylation-resistant mutant were immunoprecipitated with anti-Myc antibody. Ubiquitin-conjugated Smad4 (Smad4-Ubn) was detected by western blotting with anti-Flag antibody.


( F ) Wip1 prolongs the half - life of Smad4 . HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide ( CHX , 100 ng / ml ) for the indicated times and harvested for western blotting . An arrow indicates non - specific bands .

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(F) Wip1 prolongs the half-life of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide (CHX, 100 ng/ml) for the indicated times and harvested for western blotting. An arrow indicates non-specific bands.


( A - J ) Embryos injected dorsally with wt Wip1 ( 1 ng ) , wt Smad4 ( 1 ng ) , Smad4 ( T277A ) ( 1 ng ) and Smad4 ( T277E ) ( 1 ng ) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10 . 25 . Control , control embryo injected with LacZ mRNA only . Scale bar , 150 μm . ( K ) Quantification of the results from in situ hybridization analysis in ( A - J ) .

(A-J) Embryos injected dorsally with wt Wip1 (1 ng), wt Smad4 (1 ng), Smad4(T277A) (1 ng) and Smad4 (T277E) (1 ng) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10.25. Control, control embryo injected with LacZ mRNA only. Scale bar, 150 μm. (K) Quantification of the results from in situ hybridization analysis in (A-J).


( L ) Wip1 - mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho - mimetic amino acid at the MAPK site . ( - ) , no co - injection of wt or mutated Smad4 .

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(L) Wip1-mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho-mimetic amino acid at the MAPK site. (-), no co-injection of wt or mutated Smad4.


( M ) MDA - MB231 and MDA - MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4 ( T277A ) and then subjected to CCK - 8 assays to measure cell growth . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test .

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(M) MDA-MB231 and MDA-MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4(T277A) and then subjected to CCK-8 assays to measure cell growth. Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


( N ) Anchorage - independent colony - formation assays were performed in MDA - MB231 and MDA - MB468 cells transfected with Wip1 or negative control constructs . Scale bar , 100 μm . ( O ) Quantification of the assays shown in ( N ) . The number of colonies larger than 50 μm in diameter was scored . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test .

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(N) Anchorage-independent colony-formation assays were performed in MDA-MB231 and MDA-MB468 cells transfected with Wip1 or negative control constructs. Scale bar, 100 μm. (O) Quantification of the assays shown in (N). The number of colonies larger than 50 μm in diameter was scored. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


E - H Experimental glucose uptake flux ( E ) , acetate flux ( F ) , and growth rate ( G ) of E . coli grown on glucose ( 15 mM ) with different concentrations of αMG ( 0 - 100 mM ) , at low ( 0 mM ) and high ( 30 mM ) acetate concentrations . These data were used to quantify the total carbon uptake flux as a function of the αMG concentration ( H ) . Each data point represents an independent biological replicate , and lines represent the best polynomial fits .

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E-H Experimental glucose uptake flux (E), acetate flux (F), and growth rate (G) of E. coli grown on glucose (15 mM) with different concentrations of αMG (0-100 mM), at low (0 mM) and high (30 mM) acetate concentrations. These data were used to quantify the total carbon uptake flux as a function of the αMG concentration (H). Each data point represents an independent biological replicate, and lines represent the best polynomial fits.


I , J Total carbon uptake ( I ) and growth rate ( J ) as a function of the glucose uptake flux , at low ( 0 mM ) and high ( 30 mM ) acetate concentrations . Each data point represents an independent biological replicate , and lines represent the best linear fits . K , L Relationships between the acetate flux and glucose uptake ( K ) , and between the contribution of acetate to carbon uptake and glucose uptake ( L ) . Each data point represents an independent biological replicate , and lines represent the best polynomial fits .

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I, J Total carbon uptake (I) and growth rate (J) as a function of the glucose uptake flux, at low (0 mM) and high (30 mM) acetate concentrations. Each data point represents an independent biological replicate, and lines represent the best linear fits. K, L Relationships between the acetate flux and glucose uptake (K), and between the contribution of acetate to carbon uptake and glucose uptake (L). Each data point represents an independent biological replicate, and lines represent the best polynomial fits.


C Experimental acetate flux of E . coli K - 12 MG1655 grown on glucose ( 15 mM ) at different acetate concentrations in the presence of αMG ( 0 , 20 , 40 or 100 mM ) . Each data point represents an independent biological replicate , and the lines represent best fits using a logistic function .

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C Experimental acetate flux of E. coli K-12 MG1655 grown on glucose (15 mM) at different acetate concentrations in the presence of αMG (0, 20, 40 or 100 mM). Each data point represents an independent biological replicate, and the lines represent best fits using a logistic function.


D Experimental acetate concentration threshold at which the acetate flux reverses as a function of the αMG concentration , determined from the best fits shown in panel ( C ) .

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D Experimental acetate concentration threshold at which the acetate flux reverses as a function of the αMG concentration, determined from the best fits shown in panel (C).


B Experimental growth rate of E . coli K - 12 MG1655 grown on glucose ( 15 mM ) as a function of the acetate concentration at high ( 0 mM αMG , light blue , low ( 100 mM αMG , dark blue ) glycolytic flux . Each data point represents an independent biological replicate and the lines represent best polynomial fits .

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B Experimental growth rate of E. coli K-12 MG1655 grown on glucose (15 mM) as a function of the acetate concentration at high (0 mM αMG, light blue, low (100 mM αMG, dark blue) glycolytic flux. Each data point represents an independent biological replicate and the lines represent best polynomial fits.


C Relative change in the growth rate of E . coli K - 12 BW25113 wild - type in the presence of 10 mM acetate at high ( 0 mM αMG ) and low ( 100 mM αMG ) glycolytic flux . Mean values ± standard deviations ( error bars ) were estimated from n independent biological replicates , as indicated on the figure .

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C Relative change in the growth rate of E. coli K-12 BW25113 wild-type in the presence of 10 mM acetate at high (0 mM αMG) and low (100 mM αMG) glycolytic flux. Mean values ± standard deviations (error bars) were estimated from n independent biological replicates, as indicated on the figure.


D Relative change in the growth rates of E . coli K - 12 BW25113 Δpgi and ΔpfkA strains in the presence of 10 mM acetate . Mean values ± standard deviations ( error bars ) were estimated from three independent biological replicates .

D Relative change in the growth rates of E. coli K-12 BW25113 Δpgi and ΔpfkA strains in the presence of 10 mM acetate. Mean values ± standard deviations (error bars) were estimated from three independent biological replicates.


Relative change in the growth rates of wild - type , Δpta , and Δacs E . coli K - 12 BW25113 strains in the presence of 10 mM acetate at low glycolytic flux ( 100 mM αMG ) . Mean values and standard deviations ( error bars ) were estimated from n independent biological replicates , as indicated on the figure .

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Relative change in the growth rates of wild-type, Δpta, and Δacs E. coli K-12 BW25113 strains in the presence of 10 mM acetate at low glycolytic flux (100 mM αMG). Mean values and standard deviations (error bars) were estimated from n independent biological replicates, as indicated on the figure.


A , B Impact of the presence of 10 mM acetate on the growth rate of wild - type , Δpta , and Δacs E . coli K - 12 BW25113 strains grown on ( A ) 30 mM glycerol and ( B ) 15 mM galactose . Mean values and standard deviations ( error bars ) were estimated from three independent biological replicates .

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A,B Impact of the presence of 10 mM acetate on the growth rate of wild-type, Δpta, and Δacs E. coli K-12 BW25113 strains grown on (A) 30 mM glycerol and (B) 15 mM galactose. Mean values and standard deviations (error bars) were estimated from three independent biological replicates.


WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Lethality was followed - up . LPS - treated mice ( n = 5 ) are depicted as black circles and DEX / LPS - treated mice ( n = 5 ) are depicted as white circles . P - values for survival curves were analyzed with a Fisher ' s exact test .

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WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Lethality was followed-up. LPS-treated mice (n=5) are depicted as black circles and DEX/LPS-treated mice (n=5) are depicted as white circles. P-values for survival curves were analyzed with a Fisher's exact test.


WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Blood samples were taken 2 hours after LPS and TNF levels were measured in serum ( n = 4 - 5 ) .

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WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Blood samples were taken 2 hours after LPS and TNF levels were measured in serum (n=4-5).


WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Three hours after LPS challenge , dextran labeled with FITC was administered by oral gavage . Five hours after gavaging , blood plasma was collected and fluorescence was measured as a degree of intestinal permeability ( λexc / λem = 488 / 520 nm ) ( PBS : n = 3 , LPS and DEX / LPS : n = 5 ) .

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WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Three hours after LPS challenge, dextran labeled with FITC was administered by oral gavage. Five hours after gavaging, blood plasma was collected and fluorescence was measured as a degree of intestinal permeability (λexc/λem = 488/520 nm) (PBS: n=3, LPS and DEX/LPS: n=5).


RNA sequencing ( RNAseq ) results of IECs of WT mice ( n = 3 ) treated with PBS or 12 . 5 mg / kg LPS for 6 hours . Heatmap with selection of LPS - induced genes . Color key shows the log2 of normalized counts .

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RNA sequencing (RNAseq) results of IECs of WT mice (n=3) treated with PBS or 12.5 mg/kg LPS for 6 hours. Heatmap with selection of LPS-induced genes. Color key shows the log2 of normalized counts.


WT mice ( n = 5 ) were treated with PBS or 12 . 5 mg / kg LPS with or without 10 mg / kg DEX pre - treatment . Ileum was isolated and gene expression was measured via qPCR .

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WT mice (n=5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated and gene expression was measured via qPCR.


WT mice ( n = 3 - 5 ) were treated with PBS or 12 . 5 mg / kg LPS with or without 10 mg / kg DEX pre - treatment . Ileum was isolated 6 hours after LPS challenge . ( A ) p - MLKL and MLKL protein levels and ( B ) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control . Expression levels were quantified using FIJI and normalized to ACTIN levels . P - values were calculated using 1 - way ANOVA .

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WT mice (n=3-5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated 6 hours after LPS challenge. (A) p-MLKL and MLKL protein levels and (B) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control. Expression levels were quantified using FIJI and normalized to ACTIN levels. P-values were calculated using 1-way ANOVA.


Gene expression ( Defa6 , lysozyme ) was measured via qPCR . P - values were calculated using 1 - way ANOVA . All bars represent mean ± SEM . * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P ≤ 0 . 01 ; * P ≤ 0 . 05 .

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Gene expression (Defa6, lysozyme) was measured via qPCR. P-values were calculated using 1-way ANOVA. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.


H & E staining was performed on ileum samples . Scale bar : 50µm .

H&E staining was performed on ileum samples. Scale bar: 50µm.


Lysozyme expression ( in red ) was detected via IHC , with Hoechst counterstaining . Scale bars : 50µm . Quantification of lysozyme expression . For every picture ( n = 3 - 4 per treatment ) , a selection of 10 crypts was made . Lysozyme positive signal was quantified in this region of interest ( ROI ) using an automated script in QuPath . Values are the positive area relative to total area of ROI .

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Lysozyme expression (in red) was detected via IHC, with Hoechst counterstaining. Scale bars: 50µm. Quantification of lysozyme expression. For every picture (n=3-4 per treatment), a selection of 10 crypts was made. Lysozyme positive signal was quantified in this region of interest (ROI) using an automated script in QuPath. Values are the positive area relative to total area of ROI.


TNFR1 + / + ( n = 9 ) and TNFR1 - / - mice ( n = 11 ) were treated with 12 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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TNFR1+/+ (n=9) and TNFR1-/- mice (n=11) were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TLR4fl / fl mice ( n = 7 ) and TLR4VillinKO ( n = 9 ) mice were treated with 12 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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TLR4fl/fl mice (n=7) and TLR4VillinKO (n=9) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNFR1fl / fl mice ( n = 13 ) and TNFR1VillinKO ( n = 10 ) mice were treated with 12 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

TNFR1fl/fl mice (n=13) and TNFR1VillinKO (n=10) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNFR1 + / + and TNFR1 - / - mice ( n = 4 - 6 ) were treated with 12 . 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR . Fold changes are shown on the graph .

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TNFR1+/+ and TNFR1-/- mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.


TNFR1fl / fl and TNFR1VillinKO mice ( n = 4 - 6 ) were treated with 12 . 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR . Fold changes are shown on the graph .

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

TNFR1fl/fl and TNFR1VillinKO mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.


GRfl / fl ( n = 8 , ) and GRLysMKO mice ( n = 4 ) were treated with 2 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

GRfl/fl (n=8,) and GRLysMKO mice (n=4) were treated with 2.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


GRLysMKO mice were treated with PBS ( n = 12 ) or 10 mg / kg DEX ( n = 13 ) before a lethal dose of LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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

GRLysMKO mice were treated with PBS (n=12) or 10 mg/kg DEX (n=13) before a lethal dose of LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNF measurement in the serum of GRfl / fl ( n = 4 ) and GRLysMKO mice ( n = 3 ) . Mice were treated with 2 . 5 mg / kg LPS and blood was drawn 2 hours later . TNF concentration was measured with ELISA .

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

TNF measurement in the serum of GRfl/fl (n=4) and GRLysMKO mice (n=3). Mice were treated with 2.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.


TNF measurement in the serum of GRfl / fl and GRLysMKO ( n = 4 ) . Mice were treated with PBS or a lethal dose of LPS ( 12 . 5 and 2 . 5 mg / kg for GRfl / fl and GRLysMKO mice respectively ) , with or without pre - treatment with 10 mg / kg DEX and blood was drawn 2 hours later . TNF concentration was measured with ELISA .

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

TNF measurement in the serum of GRfl/fl and GRLysMKO (n=4). Mice were treated with PBS or a lethal dose of LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) , with or without pre-treatment with 10 mg/kg DEX and blood was drawn 2 hours later. TNF concentration was measured with ELISA.


GRfl / fl ( n = 4 ) and GRLysMKO mice ( n = 3 - 4 ) were treated with 2 . 5 mg / kg LPS . Ileum was isolated after 6 hours and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

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

GRfl/fl (n=4) and GRLysMKO mice (n=3-4) were treated with 2.5 mg/kg LPS. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


GRfl / fl and GRLysMKO mice ( n = 4 ) were treated with a PBS or LPS ( 12 . 5 and 2 . 5 mg / kg for GRfl / fl and GRLysMKO mice respectively ) with or without 10 mg / kg DEX pre - treatment of 30 minutes . Ileum was isolated after 6 hours and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

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

GRfl/fl and GRLysMKO mice (n=4) were treated with a PBS or LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) with or without 10 mg/kg DEX pre-treatment of 30 minutes. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


GRfl / fl ( n = 8 ) and GRVillinKO mice ( n = 10 ) were treated with 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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

GRfl/fl (n=8) and GRVillinKO mice (n=10) were treated with 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


GRVillinKO mice were treated with PBS ( n = 9 ) or 10 mg / kg DEX ( n = 10 ) before 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

GRVillinKO mice were treated with PBS (n=9) or 10 mg/kg DEX (n=10) before 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNF measurement in the serum of GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) . Mice were treated with 5 mg / kg LPS and blood was drawn 2 hours later .

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

TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n= 4). Mice were treated with 5 mg/kg LPS and blood was drawn 2 hours later.


TNF measurement in the serum of GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) . Mice were treated with PBS or LPS ( 12 . 5 and 5 mg / kg for GRfl / fl and GRVillinKO mice respectively ) with or without pre - treatment with 10 mg / kg DEX for 30 minutes . Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA .

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

TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n=4). Mice were treated with PBS or LPS (12.5 and 5 mg/kg for GRfl/fl and GRVillinKO mice respectively) with or without pre-treatment with 10 mg/kg DEX for 30 minutes. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.


GRVillinKO mice were treated with PBS ( n = 8 ) or a neutralizing P55 ( TNFR1 ) antibody ( n = 9 ) 2 hours before and 8 hours after 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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GRVillinKO mice were treated with PBS (n=8) or a neutralizing P55 (TNFR1) antibody (n=9) 2 hours before and 8 hours after 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


Ileum was isolated from non - stimulated GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) and gene expression was measured via qPCR . Expression levels are shown as fold induction with GRfl / fl expression values set at 1 . P - values were calculated using a 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

Ileum was isolated from non-stimulated GRfl/fl (n=4) and GRVillinKO mice (n=4) and gene expression was measured via qPCR. Expression levels are shown as fold induction with GRfl/fl expression values set at 1. P-values were calculated using a student's t-test.


GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) were treated with 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR .

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

GRfl/fl (n=4) and GRVillinKO mice (n=4) were treated with 5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR.


Left : LPS dose - response curves of GRwt / wt ( n = 3 - 7 per LPS dose ) and GRdim / dim mice ( n = 3 - 7 per LPS dose ) treated with 1 . 5 mg / kg LPS . Right : LD50 values of LPS are depicted on top of each bar for each group .

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Left: LPS dose-response curves of GRwt/wt (n=3-7 per LPS dose) and GRdim/dim mice (n=3-7 per LPS dose) treated with 1.5 mg/kg LPS. Right: LD50 values of LPS are depicted on top of each bar for each group.


GRdim / dim mice were treated with PBS ( n = 5 ) or 10 mg / kg DEX ( n = 5 ) before 1 . 5 mg / kg LPS and survival was monitored . P - values were analyzed with a Fisher ' s exact test .

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GRdim/dim mice were treated with PBS (n=5) or 10 mg/kg DEX (n=5) before 1.5 mg/kg LPS and survival was monitored. P-values were analyzed with a Fisher's exact test.


TNF measurement in the serum of GRwt / wt ( n = 5 ) and GRdim / dim mice ( n = 5 ) . Mice were treated with 1 . 5 mg / kg LPS and blood was drawn 2 hours later . TNF concentration was measured with ELISA .

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

TNF measurement in the serum of GRwt/wt (n=5) and GRdim/dim mice (n= 5). Mice were treated with 1.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.


TNF measurement in the serum of GRwt / wt and GRdim / dim mice ( n = 4 ) treated with PBS or LPS ( 12 . 5 and 1 . 5 mg / kg in GRwt / wt and GRdim / dim mice respectively ) with or without pretreatment with 10 mg / kg DEX . Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA .

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

TNF measurement in the serum of GRwt/wt and GRdim/dim mice (n=4) treated with PBS or LPS (12.5 and 1.5 mg/kg in GRwt/wt and GRdim/dim mice respectively) with or without pretreatment with 10 mg/kg DEX. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.


GRdim / dim mice were treated with PBS ( n = 6 ) or a neutralizing P55 ( TNFR1 ) antibody ( n = 8 ) 2 hours before and 8 hours after 1 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

GRdim/dim mice were treated with PBS (n=6) or a neutralizing P55 (TNFR1) antibody (n=8) 2 hours before and 8 hours after 1.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


Tofacitinib ( n = 6 ) or vehicle ( n = 7 ) was given orally to GRdim / dim mice . After 1 . 5 mg / kg LPS injection survival was monitored and p - values were analyzed with a Fisher ' s exact test .

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

Tofacitinib (n=6) or vehicle (n=7) was given orally to GRdim/dim mice. After 1.5 mg/kg LPS injection survival was monitored and p-values were analyzed with a Fisher's exact test.


GRwt / wt ( n = 4 ) and GRdim / dim ( n = 4 ) mice were treated with 1 . 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

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

GRwt/wt (n=4) and GRdim/dim (n=4) mice were treated with 1.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


GRdim / dim mice ( n = 4 ) were treated with PBS or 1 . 5 mg / kg LPS with or without 10 mg / kg DEX pretreatment . Ileum was isolated and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

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

GRdim/dim mice (n=4) were treated with PBS or 1.5 mg/kg LPS with or without 10 mg/kg DEX pretreatment. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


d / e : Density gradient centrifugation analysis of virus - containing supernatant . Forty milliliters of culture supernatant was harvested at 72 hours post ZIKV infection ( MOI = 0 . 01 ) and concentrated to 1 ml , followed by gradient centrifugation as described in the methods . Fractions were analyzed by Western blot with the indicated antibodies ( d ) . The ZIKV titer of each fraction was measured by plaque assay , and the viral genome was determined via RT - PCR ( e ) . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Dashed line represents infectivity detection limit .

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d/e: Density gradient centrifugation analysis of virus-containing supernatant. Forty milliliters of culture supernatant was harvested at 72 hours post ZIKV infection (MOI=0.01) and concentrated to 1 ml, followed by gradient centrifugation as described in the methods. Fractions were analyzed by Western blot with the indicated antibodies (d). The ZIKV titer of each fraction was measured by plaque assay, and the viral genome was determined via RT-PCR (e). Data represent the means from three biological replicates; error bars represent standard deviations from the means. Dashed line represents infectivity detection limit.


f / g : Density gradient centrifugation analysis of UC - EVs . UC - EVs ( 0 . 1 ml ) were loaded onto the top of a density gradient in a centrifugation tube as described in the methods . The fractions were obtained from the top of gradient and analyzed by Western blot with the indicated antibodies ( f ) , the ZIKV titer of each fraction was measured by plaque assay , and the viral genome was quantified via RT - PCR ( g ) . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Dashed line represents infectivity detection limit .

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

f/g: Density gradient centrifugation analysis of UC-EVs. UC-EVs (0.1 ml) were loaded onto the top of a density gradient in a centrifugation tube as described in the methods. The fractions were obtained from the top of gradient and analyzed by Western blot with the indicated antibodies (f), the ZIKV titer of each fraction was measured by plaque assay, and the viral genome was quantified via RT-PCR (g). Data represent the means from three biological replicates; error bars represent standard deviations from the means. Dashed line represents infectivity detection limit.


b : Western blots of ZIKV - infected cell - derived UC - EVs or IC - EVs . Samples during purification were subjected to western blot analysis with the indicated antibodies against cellular proteins . Total lysate of ZIKV - infected cells was served as positive control .

O O O O O O O O O O O O O O O O O O O O O O O O O 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: Western blots of ZIKV-infected cell-derived UC-EVs or IC-EVs. Samples during purification were subjected to western blot analysis with the indicated antibodies against cellular proteins. Total lysate of ZIKV-infected cells was served as positive control.


d : Density gradient centrifugation analysis of IC - EVs . ZIKV - infected cell - derived UC - EVs ( 0 . 1 ml ) were subjected to density gradient analysis similarly . Fractions were obtained from the top of gradient and analyzed by Western blot with the indicated antibodies .

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

d: Density gradient centrifugation analysis of IC-EVs. ZIKV-infected cell-derived UC-EVs (0.1 ml) were subjected to density gradient analysis similarly. Fractions were obtained from the top of gradient and analyzed by Western blot with the indicated antibodies.


Detection of infectivity ( d e ) of UC - EVs pre - and post CD9 - capture or E - capture . UC - EV was subjected to CD9 - capture and E - capture respectively . After 1hour incubation , ZIKV infectivity of pre - incubation and post - incubation samples was measured .

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

Detection of infectivity(d e) of UC-EVs pre- and post CD9-capture or E-capture. UC-EV was subjected to CD9-capture and E-capture respectively. After 1hour incubation, ZIKV infectivity of pre-incubation and post-incubation samples was measured.


f : The ZIKV infectious titer of each fraction was measured by plaque assay , and the number of viral genomes was determined via RT - PCR . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Dashed line represents infectivity detection limit .

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

f: The ZIKV infectious titer of each fraction was measured by plaque assay, and the number of viral genomes was determined via RT-PCR. Data represent the means from three biological replicates; error bars represent standard deviations from the means. Dashed line represents infectivity detection limit.


d : Western blots of ZIKV - infected cell - derived UC - EVs or IC - EVs using indicated antibodies against viral proteins and EV proteins . Total lysate of ZIKV - infected cells was the positive control .

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

d: Western blots of ZIKV-infected cell-derived UC-EVs or IC-EVs using indicated antibodies against viral proteins and EV proteins. Total lysate of ZIKV-infected cells was the positive control.


a : ZIKV E and CD9 distribution in a density gradient . After density gradient centrifugation , all fractions were analyzed by Western blotting with anti - ZIKV E and anti - CD9 antibodies . CD9 blots was reused from Figure 2d .

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

a: ZIKV E and CD9 distribution in a density gradient. After density gradient centrifugation, all fractions were analyzed by Western blotting with anti-ZIKV E and anti-CD9 antibodies. CD9 blots was reused from Figure 2d.


d : Quantification of the number of ZIKV E proteins on the EV surface . The percentage of 10nm gold sphere - labeled IC - EVs was counted based on the immuno - EM images of IC - EVs . N indicates the total number of counted IC - EVs .

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

d: Quantification of the number of ZIKV E proteins on the EV surface. The percentage of 10nm gold sphere-labeled IC-EVs was counted based on the immuno-EM images of IC-EVs. N indicates the total number of counted IC-EVs.