Figure 7A - C . mRNA expression of Sufu , Gli1 and Fbxl17 detected in 285 medulloblastoma samples ( GEO accession : GSE37382 ) , stratified by tumor subtype . Level of significance ( p ) for Kruskal - Wallis ( KW ) rank test is p < 10 - 8 . Analysis using one - way anova gave comparable results . D . Expression of Gli1 and Fbxl17 in the 285 patients is shown colored by subgroup ; level of significance ( p ) for Kruskal - Wallis ( KW ) rank test is p < 10 - 8 , and Spearman ' s rank correlation ( R = 0 . 5640 ) is indicated . E . Detection of HA - tagged Sufu wild type ( WT ) and HA - tagged Sufu S352F protein levels in NIH3T3 cells after cycloheximide ( CHX ) treatment . Where indicated , cells were transfected with a non - targeting siRNA ( Control ) or two siRNA to Fbxl17 ( 1 ) and ( 2 ) . F . Assessment of Hh pathway activation using Gli1 - luciferase reporter assay in MEFs Sufu - / - upon reintroduction of HA - tagged Sufu WT or Sufu mutant S352F using a retroviral system . ( mean ± SEM from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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

Figure 7A-C. mRNA expression of Sufu, Gli1 and Fbxl17 detected in 285 medulloblastoma samples (GEO accession: GSE37382), stratified by tumor subtype. Level of significance (p) for Kruskal-Wallis (KW) rank test is p<10-8. Analysis using one-way anova gave comparable results.D. Expression of Gli1 and Fbxl17 in the 285 patients is shown colored by subgroup; level of significance (p) for Kruskal-Wallis (KW) rank test is p<10-8, and Spearman's rank correlation (R=0.5640) is indicated.E. Detection of HA-tagged Sufu wild type (WT) and HA-tagged Sufu S352F protein levels in NIH3T3 cells after cycloheximide (CHX) treatment. Where indicated, cells were transfected with a non- targeting siRNA (Control) or two siRNA to Fbxl17 (1) and (2).F. Assessment of Hh pathway activation using Gli1-luciferase reporter assay in MEFs Sufu-/- upon reintroduction of HA-tagged Sufu WT or Sufu mutant S352F using a retroviral system. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


Figure 2B - E ) Expression of Hep - ID ( n = 13 genes ) , Hep - IDCONNECT ( n = 26 genes ) and remaining TF - encoding genes from cluster G ( Others ; n = 82 genes ) was monitored in indicated transcriptomic data Box plots show log2 fold changes between adult versus newborn mouse livers ( B ) , MPH of Hnf4ahep - / - ( Hnf4a KO ) versus wild - type mice ( C ) , a meta - analysis of severe mouse liver injuries versus control livers and microdissected hepatocytes from alcohol - related human liver cirrhosis ( alcoholic steatohepatitis ) versus control livers ( E ) . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line . Whiskers extent to the most extreme data point which is no more than 1 . 5 times the interquartile range from the box . Statistical significance was assessed using one - sided Wilcoxon rank sum test with Benjamini - Hochberg correction for multiple testing to determine if the mean log2 FC was statistically lower ( B , D , E ) or higher ( C ) than 0 . * q < 0 . 05 . I ) Binding of indicated Hep - ID and Hep - IDCONNECT TFs to the promoter of identity effector genes and a control group of non - TF - encoding genes of similar size ( n = 424 ) was monitored using mouse liver ChIP - seq data . A control group of non - TF - encoding genes ( n = 424 ) matched for their promoter mouse liver activity was also used The control groups used were selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of ChIP - seq signals is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line . Whiskers extent to the most extreme data point which is no more than 1 . 5 times the interquartile range from the box . Pairwise one - sided Wilcoxon Rank Sum Tests with Benjamini - Hochberg correction was used to define whether the binding at identity effector genes versus control genes was significantly greater for each analyzed TF recruitment . * q < 0 . 05 .

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

Figure 2B-E) Expression of Hep-ID (n=13 genes), Hep-IDCONNECT (n=26 genes) and remaining TF-encoding genes from cluster G (Others; n=82 genes) was monitored in indicated transcriptomic data Box plots show log2 fold changes between adult versus newborn mouse livers (B), MPH of Hnf4ahep-/- (Hnf4a KO) versus wild-type mice (C), a meta-analysis of severe mouse liver injuries versus control livers and microdissected hepatocytes from alcohol-related human liver cirrhosis (alcoholic steatohepatitis) versus control livers (E). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line. Whiskers extent to the most extreme data point which is no more than 1.5 times the interquartile range from the box. Statistical significance was assessed using one-sided Wilcoxon rank sum test with Benjamini-Hochberg correction for multiple testing to determine if the mean log2 FC was statistically lower (B, D, E) or higher (C) than 0. *q<0.05.I) Binding of indicated Hep-ID and Hep-IDCONNECT TFs to the promoter of identity effector genes and a control group of non-TF-encoding genes of similar size (n=424) was monitored using mouse liver ChIP-seq data. A control group of non-TF-encoding genes (n=424) matched for their promoter mouse liver activity was also used The control groups used were selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of ChIP-seq signals is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line. Whiskers extent to the most extreme data point which is no more than 1.5 times the interquartile range from the box. Pairwise one-sided Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction was used to define whether the binding at identity effector genes versus control genes was significantly greater for each analyzed TF recruitment. *q<0.05.


Figure 3A ) Binding of indicated Hep - ID and Hep - IDCONNECT TFs to the promoter of Hep - ID TF genes and a control group of non - Hep - ID TF - encoding genes matched for their promoter activity of similar size ( n = 13 ) was monitored using mouse liver ChIP - seq data . The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of ChIP - seq signals is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line . Whiskers extent to the most extreme data point which is no more than 1 . 5 times the interquartile range from the box . One - sided Wilcoxon rank sum tests with Benjamini - Hochberg correction was used to define whether the binding on Hep - ID TF gene promoters was greater than on control genes for each individual TF ChIP - seq dataset . * q < 0 . 05 . B ) Transcriptional modulation of Hep - ID TFs and a control group of non - Hep - ID TF - encoding genes matched for their promoter activity of similar size ( n = 13 ) in mouse liver / MPH of mice deleted for the indicated Hep - ID or Hep - IDCONNECT TF genes . The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of log2 fold changes is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line . Whiskers extent to the most extreme data point which is no more than 1 . 5 times the interquartile range from the box . One - sided Wilcoxon rank sum tests with Benjamini - Hochberg correction was used to define whether log2 fold changes for Hep - ID TF genes were lower than those of the control genes for each individual transcriptomic dataset . * q < 0 . 05 . D ) Average gene expression levels of Mlxipl in the livers of WT and XBP1hep - / - mice across circadian time ( n = 2 mice per group ) . Error bars show standard deviations .

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

Figure 3A) Binding of indicated Hep-ID and Hep-IDCONNECT TFs to the promoter of Hep-ID TF genes and a control group of non-Hep-ID TF-encoding genes matched for their promoter activity of similar size (n=13) was monitored using mouse liver ChIP-seq data. The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of ChIP-seq signals is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line. Whiskers extent to the most extreme data point which is no more than 1.5 times the interquartile range from the box. One-sided Wilcoxon rank sum tests with Benjamini-Hochberg correction was used to define whether the binding on Hep-ID TF gene promoters was greater than on control genes for each individual TF ChIP-seq dataset. *q<0.05.B) Transcriptional modulation of Hep-ID TFs and a control group of non-Hep-ID TF-encoding genes matched for their promoter activity of similar size (n=13) in mouse liver/MPH of mice deleted for the indicated Hep-ID or Hep-IDCONNECT TF genes. The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of log2 fold changes is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line. Whiskers extent to the most extreme data point which is no more than 1.5 times the interquartile range from the box. One-sided Wilcoxon rank sum tests with Benjamini-Hochberg correction was used to define whether log2 fold changes for Hep-ID TF genes were lower than those of the control genes for each individual transcriptomic dataset. *q<0.05.D) Average gene expression levels of Mlxipl in the livers of WT and XBP1hep-/- mice across circadian time (n=2 mice per group). Error bars show standard deviations.


Figure 4B ) Overall survival of patients with HCC expressing low ( n = 289 ) or high ( n = 73 ) levels of the Hep - IDCONNECT TF encoding genes . Differential overall survival analysis was assessed by Kaplan - Meier ( KM ) log rank adjusted for 100 permutationsE ) Transcriptional modulation of Hep - ID TFs ( n = 13 genes ) and a control group of non - Hep - ID TF - encoding genes matched for their promoter activity of similar size ( n = 13 genes ) in Huh7 or HepG2 cells treated with GW3965 , T3 or GC - 1 , respectively . The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of log2 fold changes is shown using box plots . Kruskal - Wallis with two - sided Wilcoxon pairwise comparison tests followed by Benjamini - Hochberg correction was used to define whether transcriptional regulation of Hep - ID TF genes was different from that of the control genes for each individual transcriptomic dataset . * q < 0 . 05 . mRNA expression of the indicated genes was monitored using RT - qPCR in HepG2 cells treated with T3 or GC - 1 for 24h or 96h . Bar graphs show mean ± SD ( n = 3 biological replicates ) of log2 fold changes in treated versus untreated HepG2 cells . Gray dots show the results obtained from the 3 independent biological replicates ( each performed in technical triplicates ) . One - sample t - test with Benjamini - Hochberg correction for multiple testing was used to determine if the mean log2 FC was statistically different from 0 . * q < 0 . 05 . mRNA expression of the indicated genes was monitored using RT - qPCR in HepG2 cells treated with T3 or GC - 1 for 24h or 96h . For Hnf4a , the log2 fold change in the ratio of P1 over P2 promoter - derived isoforms is also shown . Gray dots show the results obtained from the 3 independent biological replicates ( each performed in technical triplicates ) . One - sample t - test with Benjamini - Hochberg correction for multiple testing was used to determine if the mean log2 FC was statistically different from 0 . * q < 0 . 05 . I ) Western blots assays performed using antibodies against the indicated proteins on extracts from HepG2 cells treated or not with T3 for 24h . Rep # 1 - 3 indicates the 3 independent biological replicates analyzed . L ) Identity effector genes significantly downregulated in Nodules / Control ( q < 0 . 05 ) were split in 3 groups according to their log2 fold changes ( i . e . low , intermediate and high repression ; pink boxes ) and then monitored for induction in Nodules + T3 / Nodules ( green boxes ) . Statistical differences between the High repression and the other groups regarding the Nodules / Control comparison , on the one hand , or the Nodules + T3 / Nodules comparison , on the other hand , were defined using Kruskal - Wallis with two - sided Wilcoxon pairwise comparison tests followed by Benjamini - Hochberg correction for multiple testing correction . * q < 0 . 05 .

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

Figure 4B) Overall survival of patients with HCC expressing low (n=289) or high (n=73) levels of the Hep-IDCONNECT TF encoding genes. Differential overall survival analysis was assessed by Kaplan-Meier (KM) log rank adjusted for 100 permutationsE) Transcriptional modulation of Hep-ID TFs (n=13 genes) and a control group of non-Hep-ID TF-encoding genes matched for their promoter activity of similar size (n=13 genes) in Huh7 or HepG2 cells treated with GW3965, T3 or GC-1, respectively. The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of log2 fold changes is shown using box plots. Kruskal-Wallis with two-sided Wilcoxon pairwise comparison tests followed by Benjamini-Hochberg correction was used to define whether transcriptional regulation of Hep-ID TF genes was different from that of the control genes for each individual transcriptomic dataset. *q<0.05.mRNA expression of the indicated genes was monitored using RT-qPCR in HepG2 cells treated with T3 or GC-1 for 24h or 96h. Bar graphs show mean ±SD (n=3 biological replicates) of log2 fold changes in treated versus untreated HepG2 cells. Gray dots show the results obtained from the 3 independent biological replicates (each performed in technical triplicates). One-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine if the mean log2 FC was statistically different from 0. *q<0.05.mRNA expression of the indicated genes was monitored using RT-qPCR in HepG2 cells treated with T3 or GC-1 for 24h or 96h. For Hnf4a, the log2 fold change in the ratio of P1 over P2 promoter-derived isoforms is also shown. Gray dots show the results obtained from the 3 independent biological replicates (each performed in technical triplicates). One-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine if the mean log2 FC was statistically different from 0. *q<0.05.I) Western blots assays performed using antibodies against the indicated proteins on extracts from HepG2 cells treated or not with T3 for 24h. Rep#1-3 indicates the 3 independent biological replicates analyzed.L) Identity effector genes significantly downregulated in Nodules/Control (q<0.05) were split in 3 groups according to their log2 fold changes (i.e. low, intermediate and high repression; pink boxes) and then monitored for induction in Nodules+T3/Nodules (green boxes). Statistical differences between the High repression and the other groups regarding the Nodules/Control comparison, on the one hand, or the Nodules+T3/Nodules comparison, on the other hand, were defined using Kruskal-Wallis with two-sided Wilcoxon pairwise comparison tests followed by Benjamini-Hochberg correction for multiple testing correction. *q<0.05.


Figure 5B - D ) mRNA expression of the indicated genes was monitored in mouse livers using RT - qPCR . Mice treated with IL1B + T3 were subdivided into tertiles based on the mean expression of the Dio1 and Hectd3 genes and defined as low , intermediate or high T3 responsiveness groups ( Low resp . , Int . resp . and High resp . , respectively ) . Fold change relative to the mean of the control group is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line ( n = 13 mice for the PBS group , 17 for the IL1B group and 10 for the other groups ) . Whiskers extend to the maximum and minimum values . Statistical differences between the High resp . and the other IL1B - treated groups were defined using Kruskal - Wallis with two - sided Wilcoxon pairwise comparison tests followed by Benjamini - Hochberg correction for multiple testing correction . * q < 0 . 05 .

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

Figure 5B-D) mRNA expression of the indicated genes was monitored in mouse livers using RT-qPCR. Mice treated with IL1B+T3 were subdivided into tertiles based on the mean expression of the Dio1 and Hectd3 genes and defined as low, intermediate or high T3 responsiveness groups (Low resp., Int. resp. and High resp., respectively). Fold change relative to the mean of the control group is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line (n=13 mice for the PBS group, 17 for the IL1B group and 10 for the other groups). Whiskers extend to the maximum and minimum values. Statistical differences between the High resp. and the other IL1B-treated groups were defined using Kruskal-Wallis with two-sided Wilcoxon pairwise comparison tests followed by Benjamini-Hochberg correction for multiple testing correction. *q<0.05.


Figure 1 ( C ) Flow cytometry analysis of CD8 T cell infiltration in B16F10 tumors from WT or KO mice 14 days post - injection ( DPI ) ( n = 7 per genotype ) . Statistical analysis was performed by 2 - way ANOVA with Sidak correction ( B , F ) , Mann - Whitney test The data are representative of 2 - 4 independent experimentssurvival curves ( G ) of Cre + or TKO mice injected subcutaneously with B16F10 OVA cells ( n = 7 per genotype ) . Survival time was defined as the time required for a tumor to reach a volume of 500 mm3 . ( J ) Representative plots ( left panels ) and quantification ( right panels ) of IFNγ - and TNFα - producing CD8 TILs of B16F10 OVA tumors from Cre + ( n = 10 ) and TKO ( n = 12 ) animals at day 14 .

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

Figure 1(C) Flow cytometry analysis of CD8 T cell infiltration in B16F10 tumors from WT or KO mice 14 days post-injection (DPI) (n = 7 per genotype). Statistical analysis was performed by 2-way ANOVA with Sidak correction (B, F), Mann-Whitney test The data are representative of 2-4 independent experimentssurvival curves (G) of Cre+ or TKO mice injected subcutaneously with B16F10 OVA cells (n = 7 per genotype). Survival time was defined as the time required for a tumor to reach a volume of 500 mm3.(J) Representative plots (left panels) and quantification (right panels) of IFNγ- and TNFα-producing CD8 TILs of B16F10 OVA tumors from Cre+ (n = 10) and TKO (n = 12) animals at day 14.


Figure 2 ( D ) Representative flow cytometry plots ( left panels ) and quantification ( right panel ) of early activation markers CD25 and CD69 in CD8 cells activated ex vivo with CD3 / CD28 / IL - 2 for 24 h ( n = 7 mice per genotype ) . T cells were isolated as in A . ( E ) Representative flow cytometry plots ( left panels ) and quantification ( right panel ) of IFNγ + CD8 cells activated ex vivo as in B ( n = 9 mice per genotype ) . T cells were isolated as in A . ( F ) Oxygen consumption rate of Cre + ( n = 8 mice ) or TKO ( n = 7 mice ) CD8 T cells activated ex vivo as in B after mitochondrial stress test analysis . Two representative WT and TKO animals are shown ( left panel ) ; the corresponding quantification for basal and maximum respiration is shown ( right panels ) . Values are normalized to basal Cre + measurements . T cells were isolated as in A .

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

Figure 2(D) Representative flow cytometry plots (left panels) and quantification (right panel) of early activation markers CD25 and CD69 in CD8 cells activated ex vivo with CD3/CD28/IL-2 for 24 h (n = 7 mice per genotype). T cells were isolated as in A. (E) Representative flow cytometry plots (left panels) and quantification (right panel) of IFNγ+ CD8 cells activated ex vivo as in B (n = 9 mice per genotype). T cells were isolated as in A.(F) Oxygen consumption rate of Cre+ (n = 8 mice) or TKO (n = 7 mice) CD8 T cells activated ex vivo as in B after mitochondrial stress test analysis. Two representative WT and TKO animals are shown (left panel); the corresponding quantification for basal and maximum respiration is shown (right panels). Values are normalized to basal Cre+ measurements. T cells were isolated as in A.


Figure 3 ( C ) qPCR of Cpeb4 mRNA in CD8 T cells resting or activated ex vivo with CD3 / CD28 / IL - 2 at the indicated time - points ; Tbp is used as endogenous control ( n = 5 mice ) . CD8 T cells were isolated from the spleen and lymph nodes from WT mice . ( D ) Western blot ( left panel ) and quantification ( right panel ) of CPEB4 protein expression in CD8 resting or activated ex vivo with CD3 / CD28 / IL - 2 at the indicated time - points ; β - actin is used as loading control ( n = 7 mice ) . CD8 T cells were isolated as in C . ( F ) Western blot ( left panel ) and quantification ( right panel ) of CPEB4 protein expression in OT - I T cells resting or activated ex vivo with OVA / IL - 2 at the indicated time - points ; β - actin is used as loading control ( n = 4 ) .

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

Figure 3(C) qPCR of Cpeb4 mRNA in CD8 T cells resting or activated ex vivo with CD3/CD28/IL-2 at the indicated time-points; Tbp is used as endogenous control (n = 5 mice). CD8 T cells were isolated from the spleen and lymph nodes from WT mice.(D) Western blot (left panel) and quantification (right panel) of CPEB4 protein expression in CD8 resting or activated ex vivo with CD3/CD28/IL-2 at the indicated time-points; β-actin is used as loading control (n = 7 mice). CD8 T cells were isolated as in C.(F) Western blot (left panel) and quantification (right panel) of CPEB4 protein expression in OT-I T cells resting or activated ex vivo with OVA/IL-2 at the indicated time-points; β-actin is used as loading control (n = 4).


Figure 4 ( C ) Western blot analysis ( upper panel ) and quantification ( lower panel ) of eIF2α phosphorylation in WT ( n = 4 ) and TKO ( n = 4 ) CD8 T cells activated as in A ; total eiF2 α and β - actin are used as loading control . ( D ) qPCR analysis of Atf4 , Ddit3 , Xbp1s and Hspa5 mRNA levels in Cpeb4 + / + ( n = 5 ) and Cpeb4 - / - ( n = 6 ) CD8 cells activated as in a ; Tbp is used as endogenous control ( n = 5 ) . ( E ) Western blot analysis ( left panel ) and quantification ( right panels ) of BIP and CHOP proteins in WT ( n = 7 ) and TKO ( n = 6 ) CD8 T cells activated as in A . ( F ) Ddti3 mRNA levels in CD8 T cells from B16F10 tumors and spleen of WT ( n = 8 ) and TKO ( n = 10 ) mice . The data are shown as ratio between tumors and spleen . ( G ) Flow cytometry analysis of apoptotic AnnexinV + Cre + ( n = 4 ) or TKO ( n = 6 ) CD8 cells activated as in A .

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

Figure 4(C) Western blot analysis (upper panel) and quantification (lower panel) of eIF2α phosphorylation in WT (n=4) and TKO (n=4) CD8 T cells activated as in A; total eiF2 α and β-actin are used as loading control.(D) qPCR analysis of Atf4, Ddit3, Xbp1s and Hspa5 mRNA levels in Cpeb4+/+ (n = 5) and Cpeb4-/- (n = 6) CD8 cells activated as in a; Tbp is used as endogenous control (n = 5).(E) Western blot analysis (left panel) and quantification (right panels) of BIP and CHOP proteins in WT (n=7) and TKO (n=6) CD8 T cells activated as in A.(F) Ddti3 mRNA levels in CD8 T cells from B16F10 tumors and spleen of WT (n=8) and TKO (n=10) mice. The data are shown as ratio between tumors and spleen.(G) Flow cytometry analysis of apoptotic AnnexinV+ Cre+ (n = 4) or TKO (n = 6) CD8 cells activated as in A.


Figure 5 ( A ) Western blot of CPEB4 in input and immunoprecipitated fractions using anti - CPEB4 antibody of Cpeb4 + / + and Cpeb4 - / - CD8 T cells activated ex vivo with CD3 / CD28 / IL - 2 for 48 h ; β - actin is used as control ( n = 3 ) . ( D ) RIP - seq data from several 3 ′ UTRs depicting normalized RIP - seq coverage for inputs ( light grey and blue ) and IP ( dark grey and blue ) from Cpeb4 + / + and Cpeb4 - / - CD8 T cells . Image obtained using the integrated genome viewer ( IGV ) . ( E ) Western blot ( left panel ) and quantification ( right panel ) of DDIT4 protein levels in Cre + and TKO CD8 T cells activated as in A ; β - actin is used as loading control ( n = 7 ) . ( F ) Western blot ( left panel ) and quantification ( right panel ) of HERPUD2 protein levels in Cre + and TKO CD8 T cells activated as in A ; β - actin is used as loading control ( n = 8 ) . ( G ) Basal oxygen consumption rate ( left panel ) and maximal oxygen consumption rate ( right panel ) after mitochondrial stress test analysis . Cre + and TKO CD8 T cells were activated as in A , but were treated with either Tauroursodeoxycholic Acid ( TUDCA , T ) 250 μM or vehicle for the last 24 h . Values are normalized to basal Cre + measurements . n = 7 for all conditions in basal respiration ; n = 6 for all conditions in maximal respiration . ( I ) Quantification of IFNγ + CD8 cells activated and treated as in G ( n = 9 for Cre + and n = 12 for TKO in both conditions ) . * P = 0 . 0168 , * * P = 0 . 0014 for Cre + vs . TKO , P = 0 . 0029 for Cre + vs . Cre + TUDCA , * * * * P < 0 . 0001 .

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

Figure 5(A) Western blot of CPEB4 in input and immunoprecipitated fractions using anti-CPEB4 antibody of Cpeb4+/+ and Cpeb4-/- CD8 T cells activated ex vivo with CD3/CD28/IL-2 for 48 h; β-actin is used as control (n = 3).(D) RIP-seq data from several 3′ UTRs depicting normalized RIP-seq coverage for inputs (light grey and blue) and IP (dark grey and blue) from Cpeb4+/+ and Cpeb4-/- CD8 T cells. Image obtained using the integrated genome viewer (IGV).(E) Western blot (left panel) and quantification (right panel) of DDIT4 protein levels in Cre+ and TKO CD8 T cells activated as in A; β-actin is used as loading control (n = 7). (F) Western blot (left panel) and quantification (right panel) of HERPUD2 protein levels in Cre+ and TKO CD8 T cells activated as in A; β-actin is used as loading control (n = 8).(G) Basal oxygen consumption rate (left panel) and maximal oxygen consumption rate (right panel) after mitochondrial stress test analysis. Cre+ and TKO CD8 T cells were activated as in A, but were treated with either Tauroursodeoxycholic Acid (TUDCA, T) 250 μM or vehicle for the last 24 h. Values are normalized to basal Cre+ measurements. n = 7 for all conditions in basal respiration; n = 6 for all conditions in maximal respiration.(I) Quantification of IFNγ+ CD8 cells activated and treated as in G (n = 9 for Cre+ and n = 12 for TKO in both conditions). *P = 0.0168, ** P = 0.0014 for Cre+ vs. TKO, P = 0.0029 for Cre+ vs. Cre+ TUDCA, **** P <0.0001.


Figure 1C . RBM3 E3a regulation is conserved in humans . HEK293 cells were incubated at the indicated temperatures for 12h ( DMSO / CHX last 4h ) and investigated for E3a inclusion as in B ( mean ± s . d . , n = 3 , all individual data points are shown ) .

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

Figure 1C. RBM3 E3a regulation is conserved in humans. HEK293 cells were incubated at the indicated temperatures for 12h (DMSO/CHX last 4h) and investigated for E3a inclusion as in B (mean ± s.d., n= 3, all individual data points are shown).


Figure 2A . CRISPR / CAS9 mediated removal of RBM3 E3a . One of two guide RNAs targeting the upstream intron ( # 1 , # 2 ) were co - transfected with a guide RNA targeting the downstream intron ( # 3 ) . Below , genotyping PCR after clonal selection with px459 transfected cells serving as a negative control . RT - qPCR ( B ) analysis of RBM3 levels in edited cell lines . Clonal cell lines from A were incubated at the indicated temperatures for 24h . In B , isolated RNA was investigated by qPCR and rbm3 expression is shown relative to gapdh levels . ( C ) analysis of RBM3 levels in edited cell lines . Clonal cell lines from A were incubated at the indicated temperatures for 24h . In C , lysates from an independent experiment were investigated for RBM3 protein expression , hnRNP L served as a loading control , a representative gel ( left ) and quantification ( right ) are shown ( mean ± s . d . , n = 6 ( n = 3 per clone , indicated in black / grey ) , all individual data points are shown ) . Manipulation of RBM3 E3a splicing directly controls RBM3 expression levels . In D , HEK293 cells were transfected for 48h at 37 ° C with a MO blocking the 5 ' ss of E3a . RBM3 expression is shown relative to GAPDH levels and normalized to a non - targeting MO ( n = 4 - 6 , line indicates median , whiskers min to max , all individual data points are shown ) . Manipulation of RBM3 E3a splicing directly controls RBM3 expression levels . In E , primary hippocampal neurons were transfected with the indicated MOs for 48 hours and investigated for RBM3 by Western blotting . GAPDH served as a loading control ( n = 3 ; line indicates median , whiskers min to max , all individual data points are shown ) .

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

Figure 2A. CRISPR/CAS9 mediated removal of RBM3 E3a. One of two guide RNAs targeting the upstream intron (#1, #2) were co-transfected with a guide RNA targeting the downstream intron (#3). Below, genotyping PCR after clonal selection with px459 transfected cells serving as a negative control.RT-qPCR (B) analysis of RBM3 levels in edited cell lines. Clonal cell lines from A were incubated at the indicated temperatures for 24h. In B, isolated RNA was investigated by qPCR and rbm3 expression is shown relative to gapdh levels.(C) analysis of RBM3 levels in edited cell lines. Clonal cell lines from A were incubated at the indicated temperatures for 24h. In C, lysates from an independent experiment were investigated for RBM3 protein expression, hnRNP L served as a loading control, a representative gel (left) and quantification (right) are shown (mean ± s.d., n= 6 (n= 3 per clone, indicated in black/grey), all individual data points are shown).Manipulation of RBM3 E3a splicing directly controls RBM3 expression levels. In D, HEK293 cells were transfected for 48h at 37°C with a MO blocking the 5'ss of E3a. RBM3 expression is shown relative to GAPDH levels and normalized to a non-targeting MO (n= 4-6, line indicates median, whiskers min to max, all individual data points are shown).Manipulation of RBM3 E3a splicing directly controls RBM3 expression levels. In E, primary hippocampal neurons were transfected with the indicated MOs for 48 hours and investigated for RBM3 by Western blotting. GAPDH served as a loading control (n= 3; line indicates median, whiskers min to max, all individual data points are shown).


Figure 3D . ASOs targeting M2 - 9 , M4 - 7 or the 5 ' ss ( see Figure EV4A and Table EV2 ) prevent endogenous RBM3 E3a inclusion in human HeLa cells . ASO - transfected cells were kept for 24 hours at 40 ° C . Control samples at 37 ° C and 40 ° C are shown ; CHX was added for the last 4 hours . Exon 3a inclusion was investigated by splicing sensitive RT - PCR , a representative gel and phosphorimager quantification are shown ( mean ± s . d . , n = 3 ) . The hashtag marks the use of internal 5 ' and 3 ' ss that is promoted by all ASOs targeting the M4 region . ASOs targeting the 5 ' ss induced the usage of an internal 5 ' ss ( marked by two asterisks ) . M2D induces RBM3 mRNA ( E ) expression in human HeLa cells . ASOs were transfected for 24 hours at 37 ° C ( grey ) or at 40 ° C ( red ) . RBM3 induction was measured relative to GAPDH expression ( mean ± s . d . , n = 3 , all individual data points are shown ) . M2D induces RBM3 protein ( F ) expression in human HeLa cells . ASOs were transfected for 24 hours at 37 ° C ( grey ) or at 40 ° C ( red ) . RBM3 induction was measured relative to GAPDH expression ( mean ± s . d . , n = 3 , all individual data points are shown ) . G . M2D induces RBM3 protein expression in vivo . Hippocampus samples from two mice per condition were analyzed by Western blotting ( left ) and RBM3 protein was quantified relative to actin and PBS ( right , mean ± s . d . , n = 2 , all individual data points are shown ) .

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

Figure 3D. ASOs targeting M2-9, M4-7 or the 5'ss (see Figure EV4A and Table EV2) prevent endogenous RBM3 E3a inclusion in human HeLa cells. ASO-transfected cells were kept for 24 hours at 40°C. Control samples at 37°C and 40°C are shown; CHX was added for the last 4 hours. Exon 3a inclusion was investigated by splicing sensitive RT-PCR, a representative gel and phosphorimager quantification are shown (mean ± s.d., n= 3). The hashtag marks the use of internal 5' and 3'ss that is promoted by all ASOs targeting the M4 region. ASOs targeting the 5'ss induced the usage of an internal 5'ss (marked by two asterisks).M2D induces RBM3 mRNA (E) expression in human HeLa cells. ASOs were transfected for 24 hours at 37°C (grey) or at 40°C (red). RBM3 induction was measured relative to GAPDH expression (mean ± s.d., n= 3, all individual data points are shown).M2D induces RBM3 protein (F) expression in human HeLa cells. ASOs were transfected for 24 hours at 37°C (grey) or at 40°C (red). RBM3 induction was measured relative to GAPDH expression (mean ± s.d., n= 3, all individual data points are shown).G. M2D induces RBM3 protein expression in vivo. Hippocampus samples from two mice per condition were analyzed by Western blotting (left) and RBM3 protein was quantified relative to actin and PBS (right, mean ± s.d., n= 2, all individual data points are shown).


Figure 4B . Western blot of hippocampal lysates from prion - infected mice treated with non - targeting control ASO ( n = 5 mice ) or M2D ( n = 8 mice ) . M2D increases RBM3 expression by 2 - fold compared to control ASO - treated mice , 9 weeks after ASO injection at 12 w . p . i . * * p = 0 . 0058 , calculated using student ' s t - test . C . Representative images of haematoxylin and eosin stained brain slices from NBH ( control ) , and prion - infected control ASO - and M2D - treated mice at 12 w . p . i . when control ASO - treated mice were culled for prion signs . M2D confers marked neuroprotection in the hippocampus , with conservation of CA1 - 3 pyramidal layer , protection from shrinkage of the whole hippocampus , as well as reduced spongiform change . D . NeuN counts of pyramidal neurons in NBH ( n = 5 mice ) versus control ASO - treated ( n = 5 mice ) and M2D - treated prion mice ( n = 8 mice ) . M2D confers neuroprotection close to levels seen in NBH mice . control ASO - treated versus M2D - treated prion mice * * p = 0 . 0014 . One - way ANOVA . F . Total PrP and proteinase K - resistant PrPSc levels in NBH - injected mice and in prion - diseased mice injected with control - or M2D ASOs . PrPSc levels are unaffected by M2D - mediated RBM3 induction .

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

Figure 4B. Western blot of hippocampal lysates from prion-infected mice treated with non-targeting control ASO (n= 5 mice) or M2D (n= 8 mice). M2D increases RBM3 expression by 2-fold compared to control ASO-treated mice, 9 weeks after ASO injection at 12 w.p.i. ** p= 0.0058, calculated using student's t-test.C. Representative images of haematoxylin and eosin stained brain slices from NBH (control), and prion-infected control ASO- and M2D-treated mice at 12 w.p.i. when control ASO-treated mice were culled for prion signs. M2D confers marked neuroprotection in the hippocampus, with conservation of CA1-3 pyramidal layer, protection from shrinkage of the whole hippocampus, as well as reduced spongiform change.D. NeuN counts of pyramidal neurons in NBH (n= 5 mice) versus control ASO-treated (n= 5 mice) and M2D-treated prion mice (n= 8 mice). M2D confers neuroprotection close to levels seen in NBH mice. control ASO-treated versus M2D-treated prion mice ** p= 0.0014. One-way ANOVA.F. Total PrP and proteinase K-resistant PrPSc levels in NBH-injected mice and in prion-diseased mice injected with control - or M2D ASOs. PrPSc levels are unaffected by M2D-mediated RBM3 induction.


Figure 1A Specific CD8 + T - cell ( OT - I ) proliferation in response to immature BMDCs incubated with fdOVA , fdOVA / sc - αDEC or OVA257 - 264 peptide chemically coupled to anti - DEC - 205 antibody ( NLDC : pOVA ) . Percentage of CFSE - labeled proliferating T cells was evaluated on CD8 + - gated cells after 72 h of DC : T co - culture . The mean ± SD of three independent experiments is reported . B Proliferation of adoptively transferred OT - I T cells in C57BL / 6mice ( n = 3 / group ) immunized with vehicle ( PBS ) , NLDC : pOVA , NLDC : pOVA plus CpG , fd , fd / sc - αDEC or LPS - free ovalbuminprotein . Percentage of CFSE - labeled proliferating OT - I T cells was evaluated after three days on Vα2 + CD8 + - gated cells . The mean ± SD of two independent experiments is reported . Comparative analyses were performed using Student ' s t - test for unpaired samples . C Percentage of IFN - γ - producing CD8 + splenocytes isolated from mice immunized as in ( B ) . The mean ± SD of two independent experiments is reported . Comparative analyses were performed using Student ' s t - test for unpaired samples . D - F IL - 6 ( D ) , IL - 18 ( E ) and IFN - α ( F ) release in supernatants of BMDCs obtained from C57BL / 6 mice . Supernatants of BMDCs incubated with NLDC145 antibody , wild - type phage particles ( fdWT ) or scFv αDEC - 205 phage particles ( fdsc - αDEC ) for 20 h were analyzed by ELISA for cytokine production . Unstimulated culture ( medium ) and LPS - treated culture were , respectively , used as negative and positive controls . Bars represent mean values ± SD . Cumulative results are shown of three independent experiments assayed in duplicate . Comparative analyses were performed using Student ' s t - test for unpaired samples . D - F IL - 6 ( D ) , IL - 18 ( E ) and IFN - α ( F ) release in supernatants of BMDCs obtained from C57BL / 6mice . Supernatants of BMDCs incubated with NLDC145 antibody , wild - type phage particles ( fdWT ) or scFv αDEC - 205 phage particles ( fdsc - αDEC ) for 20 h were analyzed by ELISA for cytokine production . Unstimulated culture ( medium ) and LPS - treated culture were , respectively , used as negative and positive controls . Bars represent mean values ± SD . Cumulative results are shown of three independent experiments assayed in duplicate . Comparative analyses were performed using Student ' s t - test for unpaired samples .

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

Figure 1A Specific CD8+ T-cell (OT-I) proliferation in response to immature BMDCs incubated with fdOVA, fdOVA/sc-αDEC or OVA257-264 peptide chemically coupled to anti-DEC-205 antibody (NLDC:pOVA). Percentage of CFSE-labeled proliferating T cells was evaluated on CD8+-gated cells after 72 h of DC:T co-culture. The mean ±SD of three independent experiments is reported.B Proliferation of adoptively transferred OT-I T cells in C57BL/6mice (n = 3/group) immunized with vehicle (PBS), NLDC:pOVA, NLDC:pOVA plus CpG, fd, fd/sc-αDEC or LPS-free ovalbuminprotein. Percentage of CFSE-labeled proliferating OT-I T cells was evaluated after three days on Vα2+CD8+-gated cells. The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples.C Percentage of IFN-γ-producing CD8+ splenocytes isolated from mice immunized as in (B). The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples.D-F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6 mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.D-F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.


Figure 3IL - 6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6 , MYD88 , TLR9 or TLR4 KO mice and incubated for 20 h with wild - type or fdsc - αDEC phage particles . LPS or CpG - ODN were used as controls . IL - 6 release from DCs derived from MyD88 − / − mice was totally abolished and dramatically reduced in DCs derived from Tlr9 − / − mice , but not affected in Tlr4 − / − DCs . Bars represent mean values ± SD . Cumulative results are shown of three independent experiments assayed in duplicate . Comparative analyses were performed using Student ' s t - test for unpaired samples . IFN - α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL / 6 or KO mice and incubated for 20 h with wild - type or fdsc - αDEC phage particles . LPS or CpG - ODN were used as controls . MyD88 − / − and Tlr9 − / − but not Tlr4 − / − BMDCs were unable to produce IFN - α after fdsc - αDEC stimulation . Bars represent mean values ± SD . Cumulative results are shown of three independent experiments assayed in duplicate . Comparative analyses were performed using Student ' s t - test for unpaired samples . IL - 6 mRNA expression by DC cells . C57BL / 6 , MyD88 − / − and Tlr9 − / − mice were inoculated intraperitoneally with fdWT or fdsc - αDEC bacteriophages or , as a control , with LPS . Mice were sacrificed 2 h later , and purified spleen dendritic cells were analyzed for IL - 6 mRNA levels by quantitative real - time PCR . Bars represent the mean fold increase ± SD . The experiments were performed three times ( n = 2 per group ) . Comparative analyses were performed using Student ' s t - test for unpaired samples . Proliferation of OT - ICD8 + T cells after 72 h of co - culture with 1 × 104DCs isolated from C57BL / 6mice or from MyD88 − / − or Tlr9 − / − transgenic mice previously injected with fd / sc - αDECbacteriophage particles . The panel shows the percentage of divided , CFSE - low OT - ICD8 + T cells . As a control , the proliferation of OT - ICD8 + T cells co - cultured with DCs isolated from non - immunized ( NI ) C57BL / 6mice is reported . The mean ± SD of two independent experiments with n = 3 per group is reported . Comparative analyses were performed using Student ' s t - test for unpaired samples . IFN - γ production by OT - I CD8 + T cells co - cultured for 12 h in the presence of 3 . 3 × 103 DCs isolated from C57BL / 6 mice or MyD88 − / − and Tlr9 − / − transgenic mice injected as in ( D ) .

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

Figure 3IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/−mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/−mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples.Proliferation of OT-ICD8+T cells after 72 h of co-culture with 1 × 104DCs isolated from C57BL/6mice or from MyD88−/− or Tlr9−/− transgenic mice previously injected with fd/sc-αDECbacteriophage particles. The panel shows the percentage of divided, CFSE-low OT-ICD8+T cells. As a control, the proliferation of OT-ICD8+T cells co-cultured with DCs isolated from non-immunized (NI) C57BL/6mice is reported. The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-γ production by OT-I CD8+ T cells co-cultured for 12 h in the presence of 3.3 × 103 DCs isolated from C57BL/6 mice or MyD88−/− and Tlr9−/− transgenic mice injected as in (D).


Figure 4BMDCs were incubated with TRITC - conjugated fdWT ( A , C ) or fdsc - αDECbacteriophages ( B , D ) for 6 h . Cells were then fixed , permeabilized , immunostained with anti - EEA - 1 ( A , B ) or anti - LAMP - 1 ( C , D ) ( green ) antibody and analyzed by confocalmicroscopy . Representative images are shown . Numbers at the bottom of the right panels indicate the percentage of EEA - 1 ( A , B ) or LAMP - 1 ( C , D ) bacteriophage co - localization ( mean values ± SD , N = 2 , n = 100 ) . Scale bars , 10 mm . White arrows indicate LAMP - 1 - positive structures that also are positive or contain fdsc - αDEC or fdWT bacteriophages .

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

Figure 4BMDCs were incubated with TRITC-conjugated fdWT (A, C) or fdsc-αDECbacteriophages (B, D) for 6 h. Cells were then fixed, permeabilized, immunostained with anti-EEA-1 (A, B) or anti-LAMP-1 (C, D) (green) antibody and analyzed by confocalmicroscopy. Representative images are shown. Numbers at the bottom of the right panels indicate the percentage of EEA-1 (A, B) or LAMP-1 (C, D) bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm. White arrows indicate LAMP-1-positive structures that also are positive or contain fdsc-αDEC or fdWT bacteriophages.


Figure 5A - D BMDCs from C57BL / 6mice were stably transduced with YFP - tagged TLR9 . Cells grown on coverslips were then incubated with TRITC - conjugated wild - type ( A , B ) or fdsc - αDEC ( C , D ) bacteriophages for 6 h . Cells were then washed , fixed and analyzed by confocal microscopy . Nuclei were counterstained with Hoechst . ( B , D ) are enlargements of a single cell . Representative images are shown . Numbers at bottom of right panels indicate the percentage of TLR9bacteriophage co - localization ( mean values ± SD , N = 2 , n = 100 ) . Scale bars , 10 mm .

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

Figure 5A-D BMDCs from C57BL/6mice were stably transduced with YFP-tagged TLR9. Cells grown on coverslips were then incubated with TRITC-conjugated wild-type (A, B) or fdsc-αDEC (C, D) bacteriophages for 6 h. Cells were then washed, fixed and analyzed by confocal microscopy. Nuclei were counterstained with Hoechst. (B, D) are enlargements of a single cell. Representative images are shown. Numbers at bottom of right panels indicate the percentage of TLR9bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm.


Figure 1 A - C . Positioning of Upf1 , Upf2 and Upf3 in the abundance distribution of all yeast proteins , based on the data compiled by Ho et al . , 2018 ( A ) The distribution of abundance for the proteins identified by mass spectrometry in input ( B ) show the expected bias for proteins present in high copy numbersThe distribution of abundance for the proteins identified by mass spectrometry i purified samples ( C ) , show the expected bias for proteins present in high copy numbers . Light grey colour highlights the positions of proteins that were only identified in the Upf1 - TAP samples ( 6 replicates ) and not in the total extract ( 3 replicates ) D . Correlation of LTOP2 - based protein level estimates in a total yeast protein extract with published protein abundance data ( Ho et al , 2018 ) E . Distribution of the intensity for the proteins quantified in association with Upf1 ( LTOP2 score , log2 scale ) . Positioning of Upf1 is highlighted as a white rectangle while Upf2 and Upf3 are highlighted in red . The dark grey line indicates the region of intensities corresponding to ribosomal proteins F . Distribution of the protein enrichment values for Upf1 associated proteins based on LTOP2 scores and known protein abundance in a total yeast extract . Upf1 , Upf2 and Upf3 positions are also highlighted as white rectangles G . A combination of the data presented in ( E ) and ( F ) as a scatter plot , to see both the amount and enrichment of proteins in Upf1 - TAP purified samples . The horizontal axis represents the LTOP2 score , with the vertical axis showing enrichment over input values . Both axes use log2 transformed values

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

Figure 1 A-C. Positioning of Upf1, Upf2 and Upf3 in the abundance distribution of all yeast proteins, based on the data compiled by Ho et al., 2018 (A) The distribution of abundance for the proteins identified by mass spectrometry in input (B) show the expected bias for proteins present in high copy numbersThe distribution of abundance for the proteins identified by mass spectrometry i purified samples (C), show the expected bias for proteins present in high copy numbers. Light grey colour highlights the positions of proteins that were only identified in the Upf1-TAP samples (6 replicates) and not in the total extract (3 replicates) D. Correlation of LTOP2-based protein level estimates in a total yeast protein extract with published protein abundance data (Ho et al, 2018)  E. Distribution of the intensity for the proteins quantified in association with Upf1 (LTOP2 score, log2 scale). Positioning of Upf1 is highlighted as a white rectangle while Upf2 and Upf3 are highlighted in red. The dark grey line indicates the region of intensities corresponding to ribosomal proteins F. Distribution of the protein enrichment values for Upf1 associated proteins based on LTOP2 scores and known protein abundance in a total yeast extract. Upf1, Upf2 and Upf3 positions are also highlighted as white rectangles G. A combination of the data presented in (E) and (F) as a scatter plot, to see both the amount and enrichment of proteins in Upf1-TAP purified samples. The horizontal axis represents the LTOP2 score, with the vertical axis showing enrichment over input values. Both axes use log2 transformed values


Figure 2 A - F . Enrichment values for proteins identified in tagged Upf1 ( A ) , Upf2 ( B ) , Upf3 ( C ) , Nmd4 ( D ) , Ebs1 ( E ) , and Dcp1 ( F ) purifications . Tagged proteins are indicated in bold for each experiment . Black bars correspond to average enrichment values obtained in purifications done without RNase and orange bars with an RNase A and RNase T1 treatment . Error bars represent SD . Dots represent individual enrichment values for each protein in the 6 replicates for Upf1 - TAP , 3 for Upf1 - TAP and RNase , 6 for Upf2 - TAP , 3 for Upf2 - TAP and RNase , 5 for Upf3 - TAP , 3 for Upf3 - TAP and RNase , 4 for Nmd4 - TAP , 3 for Nmd4 - TAP and RNase , 3 for Ebs1 - TAP , with and without RNase , and 3 for Dcp1 - TAP with or without RNase . Only proteins enriched by a factor of 16 or more in one of the purifications presented here are shown . For clarity , groups of related proteins were combined ( the group of Lsm1 to Lsm7 is marked as " Lsm1 - 7 " and the values correspond to the mean of all the values found in purifications ; CK2 group corresponds to Cka1 , Cka2 , Ckb1 and Ckb2 ) G . Immunoblot validation of Upf1 - TAP interactions with HA tagged Nmd4 and Ebs1 . Control strains did not express Upf1 - TAP H . Immunoblot validation of Upf1 - TAP interaction with Pat1 - HA , Lsm1 - HA and Edc3 - HA . The purification was performed with a mix of three strains , expressing Upf1 - TAP or not ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 A-F. Enrichment values for proteins identified in tagged Upf1 (A), Upf2 (B), Upf3 (C), Nmd4 (D), Ebs1 (E), and Dcp1 (F) purifications. Tagged proteins are indicated in bold for each experiment. Black bars correspond to average enrichment values obtained in purifications done without RNase and orange bars with an RNase A and RNase T1 treatment. Error bars represent SD. Dots represent individual enrichment values for each protein in the 6 replicates for Upf1-TAP, 3 for Upf1-TAP and RNase, 6 for Upf2-TAP, 3 for Upf2-TAP and RNase, 5 for Upf3-TAP, 3 for Upf3-TAP and RNase, 4 for Nmd4-TAP, 3 for Nmd4-TAP and RNase, 3 for Ebs1-TAP, with and without RNase, and 3 for Dcp1-TAP with or without RNase. Only proteins enriched by a factor of 16 or more in one of the purifications presented here are shown. For clarity, groups of related proteins were combined (the group of Lsm1 to Lsm7 is marked as "Lsm1-7" and the values correspond to the mean of all the values found in purifications; CK2 group corresponds to Cka1, Cka2, Ckb1 and Ckb2) G. Immunoblot validation of Upf1-TAP interactions with HA tagged Nmd4 and Ebs1. Control strains did not express Upf1-TAP H. Immunoblot validation of Upf1-TAP interaction with Pat1-HA, Lsm1-HA and Edc3-HA. The purification was performed with a mix of three strains, expressing Upf1-TAP or not (control)


Figure 3 B - D . Results of purifications using Upf1 - FL ( B ) , Upf1 - CH ( C as bait with the x - axis showing the average enrichment value ( log2 scale ) for Upf1 - decapping and Upf1 - 23 components B - D . Results of purifications usin Upf1 - Cter ( D ) as bait with the x - axis showing the average enrichment value ( log2 scale ) for Upf1 - decapping and Upf1 - 23 components . The colour of the bars illustrates the treatment used during the purification , black without RNase , and orange with an RNase treatment . Error bars represent SD . White dots correspond to enrichment values obtained in individual experiments E . Comparison of the enrichment of Upf1 , Nmd4 and Ebs1 in the purification of the Upf1 fragments . The Upf1 C - terminal region ( 854 - 971 ) affected the association with Nmd4 or Ebs1 ( Student t - test with a one - sided alternative hypothesis ) . We compared the enrichment of each of Ebs1 and Nmd4 between purifications of Upf1 fragments having this region ( Upf1 - FL and Upf1 - HD - Cter , 6 experiments ) and purifications of Upf1 fragments lacking the extension ( Upf1 - CH - HD and Upf1 - HD , 4 experiments ) . The C - terminal region had no effect on Nmd4 enrichment ( p - value ≈ 0 . 98 ) , whereas there was significantly less associated Ebs1 on this small region ( p - value ≈ 0 . 0013 ) F . Western blot of input and eluates of Upf1 domains purification in a Nmd4 - HA strain . The band with the # might corresponds to a dimer of Upf1 - CH , bands marked with a star correspond to residual signal with the anti - HA antibodies ( Nmd4 ) . Fragments in the eluate have a smaller size because the protein A part of the tag was removed by digestion with the TEV protease . G6PDH served as a loading control in the input

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

Figure 3 B-D. Results of purifications using Upf1-FL (B), Upf1-CH (C as bait with the x-axis showing the average enrichment value (log2 scale) for Upf1-decapping and Upf1-23 components B-D. Results of purifications usin Upf1-Cter (D) as bait with the x-axis showing the average enrichment value (log2 scale) for Upf1-decapping and Upf1-23 components. The colour of the bars illustrates the treatment used during the purification, black without RNase, and orange with an RNase treatment. Error bars represent SD. White dots correspond to enrichment values obtained in individual experiments E. Comparison of the enrichment of Upf1, Nmd4 and Ebs1 in the purification of the Upf1 fragments. The Upf1 C-terminal region (854-971) affected the association with Nmd4 or Ebs1 (Student t-test with a one-sided alternative hypothesis). We compared the enrichment of each of Ebs1 and Nmd4 between purifications of Upf1 fragments having this region (Upf1-FL and Upf1-HD-Cter, 6 experiments) and purifications of Upf1 fragments lacking the extension (Upf1-CH-HD and Upf1-HD, 4 experiments). The C-terminal region had no effect on Nmd4 enrichment (p-value ≈ 0.98), whereas there was significantly less associated Ebs1 on this small region (p-value ≈ 0.0013)  F. Western blot of input and eluates of Upf1 domains purification in a Nmd4-HA strain. The band with the # might corresponds to a dimer of Upf1-CH, bands marked with a star correspond to residual signal with the anti-HA antibodies (Nmd4). Fragments in the eluate have a smaller size because the protein A part of the tag was removed by digestion with the TEV protease. G6PDH served as a loading control in the input samples


Figure 4 A . Average enrichment of Upf1 - 23 and Upf1 - decapping components in purified Upf1 - TAP in the presence ( black bars ) and absence ( blue bars ) of NMD4 . Pink and dark blue vertical lines highlight proteins of the Upf1 - 23 and Upf1 - decapping complexes respectively . White dots correspond to individual enrichment values obtained in replicate experiments , 6 for Upf1 - TAP and 3 for the nmd4Δ condition . Error bars correspond to SD B . Average enrichment of Upf1 - decapping components in Nmd4 - TAP in the presence ( black bars ) and absence ( blue bars ) of UPF1 . White dots correspond to individual enrichment values obtained in replicate experiments , 4 for Nmd4 - TAP and 2 for the upf1Δ condition . Error bars correspond to SD D . CBP - Nmd4 was mixed with hUPF1 - HD or yUPF1 - HD ( all the proteins overexpressed in E . coli and purified ) . Protein mixtures before ( input , 20 % of total ) or after purification on calmodulin affinity beads were separated on 10 % SDS - PAGE ( w / v ) acrylamide gels E . Upf1 interaction with a 30 - mer 3 ' biotinylated RNA fragment was tested by mixing purified Upf1 helicase domain with Nmd4 and testing the fraction of recovered protein on streptavidin beads after washes with 150 mM and 300 mM NaCl containing buffer solution

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

Figure 4 A. Average enrichment of Upf1-23 and Upf1-decapping components in purified Upf1-TAP in the presence (black bars) and absence (blue bars) of NMD4. Pink and dark blue vertical lines highlight proteins of the Upf1-23 and Upf1-decapping complexes respectively. White dots correspond to individual enrichment values obtained in replicate experiments, 6 for Upf1-TAP and 3 for the nmd4Δ condition. Error bars correspond to SD  B. Average enrichment of Upf1-decapping components in Nmd4-TAP in the presence (black bars) and absence (blue bars) of UPF1. White dots correspond to individual enrichment values obtained in replicate experiments, 4 for Nmd4-TAP and 2 for the upf1Δ condition. Error bars correspond to SD  D. CBP-Nmd4 was mixed with hUPF1-HD or yUPF1-HD (all the proteins overexpressed in E. coli and purified). Protein mixtures before (input, 20% of total) or after purification on calmodulin affinity beads were separated on 10% SDS-PAGE (w/v) acrylamide gels  E. Upf1 interaction with a 30-mer 3' biotinylated RNA fragment was tested by mixing purified Upf1 helicase domain with Nmd4 and testing the fraction of recovered protein on streptavidin beads after washes with 150 mM and 300 mM NaCl containing buffer solution


Figure 5B . Scatter plot of the mean fold change of transcripts in nmd4 ∆ RNAseq experiment against transcript mean fold change in ebs1 ∆ ( Pearson correlation , r = 0 . 66 , p - value < 2 . 10 - 16 ) . Black dots correspond to NMD substrate examples for which individual traces are shown in Fig . EV3 . Dashed lines represent boundaries of five bins of equal numbers of transcripts ( 2046 transcripts or notable features per bin ) , with bin 5 containing the transcripts that were most increased in the mutant condition compared with wild typeC . Percentage of transcripts affected by UPF1 deletion ( increase by at least 1 . 4 fold ) among nmd4 ∆ bins , as defined in B . Differences between percentages of upf1 ∆ affected transcripts in bin 4 and 5 and bin 3 and 4 were significant ( binomial test , p < 10 - 24 ) . D . Same as in C , for ebs1 ∆ . Differences between distribution of transcripts in bin n and bin n + 1 were significant ( binomial test , p < 10 - 9 ) E . NMD efficiency of WT , upf1 ∆ or double mutant strains complemented with Upf1 - FL , Upf1 - HD - Cter or an empty plasmid . The NMD efficiency for each strain is based on reverse - transcription followed by quantitative PCR for RPL28 pre - mRNA . A wild - type strain has 100 % NMD efficiency and a upf1 ∆ strain has 0 % NMD efficiency , by definition . Each value is an average of replicate experiments , 3 for the WT condition , 5 for upf1Δ , 3 for ufp1Δ / upf2Δ , upf1Δ / ufp3Δ and upf1Δ / ebs1Δ and 4 for the upf1Δ / nmd4Δ condition . Error bars represent SD . Dots represent values obtained in the various replicate experimentsG . Changes in the steady - state level of the reporter encoded HA - tagged protein in upf1Δ , nmd4Δ , ebs1Δ and the nmd4Δ / ebs1Δ strains were estimated by immunoblot from three independent experiments , with one example shownH . RNA decay in a wild - type and nmd4Δ / ebs1Δ strains was tested by reverse - transcription quantitative PCR specific to the 5 " end of the reporter RNA at different times after transcription shut - off . The quantifications represent mean RNA amounts and standard deviation of the average ( three independent

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

Figure 5B. Scatter plot of the mean fold change of transcripts in nmd4∆ RNAseq experiment against transcript mean fold change in ebs1∆ (Pearson correlation, r = 0.66, p-value < 2.10-16). Black dots correspond to NMD substrate examples for which individual traces are shown in Fig. EV3. Dashed lines represent boundaries of five bins of equal numbers of transcripts (2046 transcripts or notable features per bin), with bin 5 containing the transcripts that were most increased in the mutant condition compared with wild typeC. Percentage of transcripts affected by UPF1 deletion (increase by at least 1.4 fold) among nmd4∆ bins, as defined in B. Differences between percentages of upf1∆ affected transcripts in bin 4 and 5 and bin 3 and 4 were significant (binomial test, p<10-24). D. Same as in C, for ebs1∆. Differences between distribution of transcripts in bin n and bin n+1 were significant (binomial test, p<10-9)E. NMD efficiency of WT, upf1∆ or double mutant strains complemented with Upf1-FL, Upf1-HD-Cter or an empty plasmid. The NMD efficiency for each strain is based on reverse-transcription followed by quantitative PCR for RPL28 pre-mRNA. A wild-type strain has 100% NMD efficiency and a upf1∆ strain has 0% NMD efficiency, by definition. Each value is an average of replicate experiments, 3 for the WT condition, 5 for upf1Δ, 3 for ufp1Δ/upf2Δ, upf1Δ/ufp3Δ and upf1Δ/ebs1Δ and 4 for the upf1Δ/nmd4Δ condition. Error bars represent SD. Dots represent values obtained in the various replicate experimentsG. Changes in the steady-state level of the reporter encoded HA-tagged protein in upf1Δ, nmd4Δ, ebs1Δ and the nmd4Δ/ebs1Δ strains were estimated by immunoblot from three independent experiments, with one example shownH. RNA decay in a wild-type and nmd4Δ/ebs1Δ strains was tested by reverse-transcription quantitative PCR specific to the 5" end of the reporter RNA at different times after transcription shut-off. The quantifications represent mean RNA amounts and standard deviation of the average (three independent experim


Figure 6 A - B . Comparison between average enrichment values for Upf1 - decapping and Upf1 - 23 components , Lsm proteins and CK subunits in Upf1 - TAP purified samples in the presence ( grey bars ) or absence ( blue bars ) of UPF2 ( AError bars represent SD . White dots represent enrichment values for individual replicates , 6 for Upf1 - TAP , 3 for the upf2Δ an conditions A - B . Comparison between average enrichment values for Upf1 - decapping and Upf1 - 23 components , Lsm proteins and CK subunits in Upf1 - TAP purified samples in the presence ( grey bars ) or absence ( blue bars ) o UPF3 ( B ) . Error bars represent SD . White dots represent enrichment values for individual replicates , 6 for Upf1 - TAP , 3 for th upf3Δ conditions C - D . Evaluation of the effects of UPF2 deletion on Upf3 - TAP levels ( C in total extracts , in comparison with a loading control ( G6PDH ) was done by immunoblot C - D . Evaluation of the effect of UPF3 deletion on Upf2 - TAP levels ( D ) in total extracts , in comparison with a loading control ( G6PDH ) was done by immunoblot E . Comparison between average enrichment values for Upf1 - decapping and Upf1 - 23 components , Lsm proteins and CK subunits in Upf2 - TAP purified samples in the presence ( grey bars , 6 experiments ) or absence ( blue bars , 2 experiments ) of UPF1 . Error bars represent SD . The levels of expression of Upf2 - TAP and Upf3 - TAP proteins have been verified in this condition ( Appendix Fig . S2 )

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

Figure 6 A-B. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf1 -TAP purified samples in the presence (grey bars) or absence (blue bars) of UPF2 (AError bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1-TAP, 3 for the upf2Δ an conditions A-B. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf1-TAP purified samples in the presence (grey bars) or absence (blue bars) o UPF3 (B). Error bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1-TAP, 3 for th upf3Δ conditions C-D. Evaluation of the effects of UPF2 deletion on Upf3-TAP levels (C in total extracts, in comparison with a loading control (G6PDH) was done by immunoblot C-D. Evaluation of the effect of UPF3 deletion on Upf2-TAP levels (D) in total extracts, in comparison with a loading control (G6PDH) was done by immunoblot E. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf2-TAP purified samples in the presence (grey bars, 6 experiments) or absence (blue bars, 2 experiments) of UPF1. Error bars represent SD. The levels of expression of Upf2-TAP and Upf3-TAP proteins have been verified in this condition (Appendix Fig. S2)


Figure 7 A . Unspliced RPL28 , an NMD substrate , was enriched in Nmd4 - TAP purification in comparison with a control untagged strain , as measured by reverse transcription followed by quantitative PCR . Bars correspond to the mean of pre - RPL28 enrichment for 3 independent experiments , as compared with RIM1 , a non - NMD mRNA . Error bars correspond to SD . The indicated p - value was computed using the Welch t - test , single - sided comparison B - D . Distribution of Nmd4 - TAP , Nog1 ( marker of free 60S subunits ) and Rps8 ( marker of 40S , 80S and polysome fractions ) in wild type ( B ) , upf1 ∆ ( C ) and upf2 ∆ ( D ) strains , tested by immunoblot . Fractions 1 , 2 , 3 are light fractions , fractions 4 , 5 and 6 correspond respectively to ribosomal 40S , 60S and 80S positions , fractions 7 to 12 correspond to polysomes . The percent of total signal for Nmd4 - TAP in three independent replicates along with standard deviation of the values are indicated for each fraction E . Quantified relative changes in Nmd4 - TAP average levels in light and monosome / polysome fractions in upf1 ∆ and upf2 ∆ strains ( 100 % correspond to levels in the wild type strain ) . Indicated p - values correspond to the Welch t - test , single - sided comparison of 4 replicate experiments ( individual values are indicated as dots ) . Error bars correspond to SD

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

Figure 7 A. Unspliced RPL28, an NMD substrate, was enriched in Nmd4-TAP purification in comparison with a control untagged strain, as measured by reverse transcription followed by quantitative PCR. Bars correspond to the mean of pre-RPL28 enrichment for 3 independent experiments, as compared with RIM1, a non-NMD mRNA. Error bars correspond to SD. The indicated p-value was computed using the Welch t-test, single-sided comparison  B-D. Distribution of Nmd4-TAP, Nog1 (marker of free 60S subunits) and Rps8 (marker of 40S, 80S and polysome fractions) in wild type (B), upf1∆ (C) and upf2∆ (D) strains, tested by immunoblot. Fractions 1, 2, 3 are light fractions, fractions 4, 5 and 6 correspond respectively to ribosomal 40S, 60S and 80S positions, fractions 7 to 12 correspond to polysomes. The percent of total signal for Nmd4-TAP in three independent replicates along with standard deviation of the values are indicated for each fraction E. Quantified relative changes in Nmd4-TAP average levels in light and monosome/polysome fractions in upf1∆ and upf2∆ strains (100% correspond to levels in the wild type strain). Indicated p-values correspond to the Welch t-test, single-sided comparison of 4 replicate experiments (individual values are indicated as dots). Error bars correspond to SD


Figure 1 ( A , B ) A brightfield image showing the zebrafish thyroid gland along with surrounding tissue . The thyroid follicles reside in the soft tissue surrounding the ventral aorta , which extends from the outflow tract of the heart into the gills towards the basibranchial cartilage in the lower jaw . The thyroid follicular cells , or thyrocytes , are labeled in green in the Tg ( tg : nls - mVenus - NTR ) transgenic line ( B ' ) . Scale bars : 500 µm ( C ) Maximum intensity projection of 3D confocal stack obtained from the dissected thyroid gland of Tg ( tg : nls - EGFP ) animal and labeled with DAPI . ( D ) Confocal scan of a transverse section across the dissected thyroid gland from Tg ( tg : nls - EGFP ) animal at 3 mpf . Sections were stained with DAPI to visualize cells surrounding thyroid follicles . ( E ) Boxplot depicting the proportion of thyrocytes present in transverse sections obtained from three Tg ( tg : nls - EGFP ) animals at 3 mpf . Each dot represents a transverse section . The Tukey boxplot marks the 25th percentile , median and 75th percentile with whiskers extending from smallest to largest values . ( F , G ) Representative FACS plot of single cells from Tg ( tg : nls - mVenus - T2A - NTR ) animals at 2 mpf ( F ) and 8 mpf ( G ) . Calcein ( Pacific Blue ) labels live cells , while green fluorescence ( FITC ) labels thyrocytes . Percentage values represent proportion of calcein + thyrocytes within total calcein + cells .

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

Figure 1(A , B) A brightfield image showing the zebrafish thyroid gland along with surrounding tissue. The thyroid follicles reside in the soft tissue surrounding the ventral aorta, which extends from the outflow tract of the heart into the gills towards the basibranchial cartilage in the lower jaw. The thyroid follicular cells, or thyrocytes, are labeled in green in the Tg(tg:nls-mVenus-NTR) transgenic line (B'). Scale bars: 500 µm(C) Maximum intensity projection of 3D confocal stack obtained from the dissected thyroid gland of Tg(tg:nls-EGFP) animal and labeled with DAPI. (D) Confocal scan of a transverse section across the dissected thyroid gland from Tg(tg:nls-EGFP) animal at 3 mpf. Sections were stained with DAPI to visualize cells surrounding thyroid follicles. (E) Boxplot depicting the proportion of thyrocytes present in transverse sections obtained from three Tg(tg:nls-EGFP) animals at 3 mpf. Each dot represents a transverse section. The Tukey boxplot marks the 25th percentile, median and 75th percentile with whiskers extending from smallest to largest values. (F , G) Representative FACS plot of single cells from Tg(tg:nls-mVenus-T2A-NTR) animals at 2 mpf (F) and 8 mpf (G). Calcein (Pacific Blue) labels live cells, while green fluorescence (FITC) labels thyrocytes. Percentage values represent proportion of calcein+ thyrocytes within total calcein+ cells.


Figure 2 ( A ) A t - SNE plot displaying the 6249 single - cells profiled in the zebrafish thyroid gland atlas . The colors represent cell clusters denoting a specific cell - type . ( B - D ) Cluster # 1 represents the thyrocytes that express tg , slc5a5 ( NIS ) and tpo . The color scale represents the normalized expression counts for each gene ranging from lowest ( grey ) to highest ( red ) .

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

Figure 2(A) A t-SNE plot displaying the 6249 single-cells profiled in the zebrafish thyroid gland atlas. The colors represent cell clusters denoting a specific cell-type. (B-D) Cluster #1 represents the thyrocytes that express tg, slc5a5 (NIS) and tpo. The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (red).


Figure 3 ( A ) Heatmap depicting five genes specifically expressed in each one of the seven clusters of the zebrafish thyroid gland atlas . The color scale represents expression as a z - score ranging from - 2 ( purple ) to 2 ( yellow ) . NFE : Non - Follicular Epithelium . ( B - G ) t - SNE plots overlaid with the expression of a gene specific to each of the cluster . The endothelium cluster ( cluster # 2 ) is a mix of blood vessels and lymphatic vessels while the immune cell cluster ( cluster # 4 ) is a mix of macrophages , neutrophils and lymphocytes The color scale represents the normalized expression counts for each gene ranging from lowest ( grey ) to highest ( blue ) .

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

Figure 3(A) Heatmap depicting five genes specifically expressed in each one of the seven clusters of the zebrafish thyroid gland atlas. The color scale represents expression as a z-score ranging from -2 (purple) to 2 (yellow). NFE: Non-Follicular Epithelium.(B - G) t-SNE plots overlaid with the expression of a gene specific to each of the cluster. The endothelium cluster (cluster #2) is a mix of blood vessels and lymphatic vessels while the immune cell cluster (cluster #4) is a mix of macrophages, neutrophils and lymphocytes The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (blue).


Figure 4 ( A - D ) Images show immunofluorescence labeling of thyroid gland from adult zebrafish . Transverse sections were utilized for imaging . The organ was isolated from tissue - specific transgenic lines to allow marking of a particular cell - type adjacent to the thyroid follicle . Blood vessels were marked using Tg ( kdrl : EGFP ) ( A ) , macrophages using Tg ( mpeg1 . 1 : mCherry ) ( B ) and stroma using Tg ( col1a2 : mCherry ) ( C ) . Thyrocytes were labeled with pax2amKO2 expression in ( A ) and Tg ( tg : nls - EGFP ) expression in ( B - C ) . ( D ) NFE was labeled using antibody against TP63 in sections of the thyroid gland isolated from Tg ( tg : nls - EGFP ) animals . Gills are marked based on their morphological appearance . DAPI labels nuclei . Scale bars : 10 µm ( A - B ) , 50 µm ( C - D ) .

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

Figure 4(A-D) Images show immunofluorescence labeling of thyroid gland from adult zebrafish. Transverse sections were utilized for imaging. The organ was isolated from tissue-specific transgenic lines to allow marking of a particular cell-type adjacent to the thyroid follicle. Blood vessels were marked using Tg(kdrl:EGFP) (A), macrophages using Tg(mpeg1.1:mCherry) (B) and stroma using Tg(col1a2:mCherry) (C). Thyrocytes were labeled with pax2amKO2 expression in (A) and Tg(tg:nls-EGFP) expression in (B - C). (D) NFE was labeled using antibody against TP63 in sections of the thyroid gland isolated from Tg(tg:nls-EGFP) animals. Gills are marked based on their morphological appearance. DAPI labels nuclei. Scale bars: 10 µm (A - B), 50 µm (C - D).


Figure 5 ( C ) A dotplot depicting examples of paracrine and autocrine signaling in the thyroid gland . The size of the dots represent percentage of cells expressing the gene in a particular cluster , while the color scale represents average expression of the gene in the cluster after scaling .

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

Figure 5(C) A dotplot depicting examples of paracrine and autocrine signaling in the thyroid gland. The size of the dots represent percentage of cells expressing the gene in a particular cluster, while the color scale represents average expression of the gene in the cluster after scaling.


Figure 6 ( A ) Dot plot depicting expression entropy on Y - axis against average gene expression on X - axis for the thyrocyte population . Each dot depicts a gene , with red dots depicting genes that show statistically significant ( p - value < 0 . 05 ) difference in entropy from expected value . Expected value is represented by black regression line . pax2a and ctsba are marked on the graph . ( B ) t - SNE plot of the thyrocyte cluster with expression of tg , pax2a and ctsba . The color scale represents the normalized expression counts for each gene ranging from lowest ( yellow ) to highest ( red ) . ( B ' ) Histogram overlaid with density plots depicting the distribution of normalized gene expression for tg , pax2a and ctsba .

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

Figure 6(A) Dot plot depicting expression entropy on Y-axis against average gene expression on X-axis for the thyrocyte population. Each dot depicts a gene, with red dots depicting genes that show statistically significant (p-value < 0.05) difference in entropy from expected value. Expected value is represented by black regression line. pax2a and ctsba are marked on the graph.(B) t-SNE plot of the thyrocyte cluster with expression of tg, pax2a and ctsba. The color scale represents the normalized expression counts for each gene ranging from lowest (yellow) to highest (red). (B') Histogram overlaid with density plots depicting the distribution of normalized gene expression for tg, pax2a and ctsba.


Figure 7 ( B ) Whole mount immunofluorescence of 9 . 5hpf pax2amKO2 embryos stained with anti - mKO2 antibody ( red ) and anti - PAX2A antibody ( green ) . Anterior is to the left , and dorsal side is to the top . Scale bar : 0 . 1 mm . ( C ) Whole mount immunofluorescence of 55 hpf pax2amKO2 ; Tg ( tg : nls - EGFP ) stained with PAX2A antibody ( PAX2A - Ab ) displays an overlap of mKO2 and PAX2A - Ab signal . The otic vesicle ( OV ) , mid - hindbrain barrier ( MHB ) , interneurons ( IN ) and thyroid gland ( THY ) is labelled . White dashed line represents the outline of the zebrafish larvae . Scale bar : 100 µm . Anterior to the right . ( D - F ) Confocal microscopy imaging of a sagittal section of a 55 hpf pax2amKO2 ( red ) ; Tg ( tg : nls - EGFP ) ( green ) embryos immunostained with PAX2A antibody ( cyan ) showing co - localization of mKO2 and pax2a in the pronephros ( D ) , thyroid gland ( E ) and mid - hindbrain barrier ( F ) . In the thyroid gland , mKO2 ( red ) , PAX2A - Ab ( cyan ) and thyrocyte - specific GFP ( green ) show co - localization . Scale bar : 50 µm . ( G , H ) Snapshots from live imaging of 55 hpf pax2amKO2 ; Tg ( tg : nls - EGFP ) embryos injected with sgRNA targeting pax2a coding sequence . The anterior part of a representative control embryo ( G ) is shown alongside a representative crispant ( H ) . Crispants display a strong reduction of mKO2 fluorescence , as well as an absence of GFP signal suggesting absence of thyroid ( THY ) tissue . White dashed line represents the outline of the zebrafish larvae . Scale bar : 100 µm . Anterior to the right .

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

Figure 7(B) Whole mount immunofluorescence of 9.5hpf pax2amKO2 embryos stained with anti-mKO2 antibody (red) and anti-PAX2A antibody (green). Anterior is to the left, and dorsal side is to the top. Scale bar: 0.1 mm.(C) Whole mount immunofluorescence of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) stained with PAX2A antibody (PAX2A-Ab) displays an overlap of mKO2 and PAX2A-Ab signal. The otic vesicle (OV), mid-hindbrain barrier (MHB), interneurons (IN) and thyroid gland (THY) is labelled. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.(D - F) Confocal microscopy imaging of a sagittal section of a 55 hpf pax2amKO2 (red); Tg(tg:nls-EGFP) (green) embryos immunostained with PAX2A antibody (cyan) showing co-localization of mKO2 and pax2a in the pronephros (D), thyroid gland (E) and mid-hindbrain barrier (F). In the thyroid gland, mKO2 (red), PAX2A-Ab (cyan) and thyrocyte-specific GFP (green) show co-localization. Scale bar: 50 µm.(G , H) Snapshots from live imaging of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) embryos injected with sgRNA targeting pax2a coding sequence. The anterior part of a representative control embryo (G) is shown alongside a representative crispant (H). Crispants display a strong reduction of mKO2 fluorescence, as well as an absence of GFP signal suggesting absence of thyroid (THY) tissue. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.