( B C ) B3Z - Syk reporter expressing indicated mutant or WT DNGR - 1 receptors ( mouse , long isoform ) were incubated with UV - irradiated 293T ( dead : reporter is indicated ) , or with plate - bound anti - DNGR - 1 antibody or medium alone at 37 ° C overnight . Activation of NFAT reporter was measured at end of incubation period . Data are plotted as mean ± SD of experimental duplicates . One representative of three experiments is shown .

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(B and C) B3Z-Syk reporter cells expressing indicated mutant or WT DNGR-1 receptors (mouse, long isoform) were incubated with UV-irradiated 293T cells (ratio dead:reporter cells is indicated), or with plate-bound anti-DNGR-1 antibody or medium alone at 37°C overnight. Activation of the NFAT reporter was measured at the end of the incubation period. Data are plotted as mean ± SD of experimental duplicates. One representative of three experiments is shown.


( D ) Phoenix expressing indicated mutant or WT DNGR - 1 ( mouse , long isoform ) were treated with F - actin , F - actin buffer , anti - DNGR - 1 antibody or isotype - matched antibody of irrelevant specificity for 45 minutes , fixed in PFA , surface - stained for DNGR - 1 analyzed by flow cytometry . One representative of three experiments is shown .

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(D) Phoenix cells expressing indicated mutant or WT DNGR-1 proteins (mouse, long isoform) were treated with F-actin, F-actin buffer, anti-DNGR-1 antibody or isotype-matched antibody of irrelevant specificity for 45 minutes, fixed in PFA, surface-stained for DNGR-1 and analyzed by flow cytometry. One representative of three experiments is shown.


( A ) DNGR - 1 KO MuTu DCs were transduced to express indicated DNGR - 1 , FACS - sorted expanded . Surface ( left panel ) total ( right panel ) DNGR - 1 expression was determined by flow cytometry .

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(A) DNGR-1 KO MuTu DCs were transduced to express indicated DNGR-1 proteins, FACS-sorted and expanded. Surface (left panel) and total (right panel) DNGR-1 expression was determined by flow cytometry.


( B C ) Ovalbumin - expressing mouse embryonic fibroblasts ( bm1 T OVA MEFs ) were UV - irradiated , left to undergo secondary necrosis incubated overnight with MuTu transduced with indicated WT or mutant DNGR - 1 or an empty - vector control ( dead : MuTu is indicated ) OVA - specific pre - activated OT - I CD8 + T - lymphocytes . amount of IFNγ accumulating in culture medium was assessed using ELISA . One representative of two ( B ) or four ( C ) experiments is shown .

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(B and C) Ovalbumin-expressing mouse embryonic fibroblasts (bm1 T OVA MEFs) were UV-irradiated, left to undergo secondary necrosis and incubated overnight with MuTu cells transduced with indicated WT or mutant DNGR-1 proteins or an empty-vector control (ratio dead cells:MuTu cells is indicated) and OVA-specific pre-activated OT-I CD8+ T-lymphocytes. The amount of IFNγ accumulating in the culture medium was assessed using ELISA. One representative of two (B) or four (C) experiments is shown.


( B ) Immunoblot showing CENP - AEA level at indicated time in RPE - 1 .

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(B) Immunoblot showing CENP-AEA protein level at the indicated time in RPE-1 cells.


( C ) Representative immunofluorescence showing CENP - A reloading at CENP - B - marked centromeres in DLD - 1 . dashed circles contour nuclei . Scale bar , 5 µm .

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(C) Representative immunofluorescence images showing CENP-A reloading at CENP-B-marked centromeres in DLD-1 cells. White dashed circles contour nuclei. Scale bar, 5 µm.


( D ) of of showing centromeric CENP - A 24 hr or 48 hr after IAA WO . Each dot represents one experiment ( ~ 30 - 50 per condition per experiment ) , error bars represent standard deviation ( SD ) of 5 independent experiments .

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(D) Quantification of the percentage of cells showing centromeric CENP-A 24 hr or 48 hr after IAA WO. Each dot represents one experiment (~30-50 cells per condition per experiment), error bars represent standard deviation (SD) of 5 independent experiments.


( E ) of centromeric CENP - A normalized to non - treated level . Each dot represents one experiment , error bars represent SD . Unpaired t : * * * p = 0 . 0005 .

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(E) Quantification of centromeric CENP-A levels normalized to non-treated level. Each dot represents one experiment, error bars represent SD. Unpaired t test: ***p= 0.0005.


( F ) Stills of live imaging to follow de novo CENP - AEA reloading in RPE - 1 harboring endogenously tagged CENP - Bmcherry . were taken every 15 min . dashed circles contour nuclei prior / after mitosis during mitosis on bright field .

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(F) Stills of live cell imaging to follow de novo CENP-AEA reloading in RPE-1 cells harboring endogenously tagged CENP-Bmcherry. Images were taken every 15 min. White dashed circles contour nuclei prior/after mitosis and cells during mitosis based on bright field images.


( G ) Dot plot showing timing of CENP - AEA reloading after anaphase onset in indicated lines . Each dot represents one , error bars represent standard deviation .

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(G) Dot plot showing the timing of CENP-AEA reloading after anaphase onset in the indicated cell lines. Each dot represents one cell, error bars represent standard deviation.


( B ) Full coverage plot of chromosome 17 centromeric array ( chr17 : 22 , 500 , 000 - 26 , 900 , 000 of hg38 assembly ) showing enrichment of CENP - A by CUT & RUN - seq in untreated ( NT ) IAA wash out ( WO ) sample . CENP - A is reloaded to cen17 _ 1 ( D17Z1 ) . A shift of CENP - A occupancy to cen17 _ 2 ( D17Z1B ) is not detected .

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(B) Full coverage plot of chromosome 17 centromeric array (chr17:22,500,000-26,900,000 of the hg38 assembly) showing enrichment of CENP-A by CUT&RUN-seq in the untreated (NT) and IAA wash out (WO) sample. CENP-A is reloaded to cen17_1 (D17Z1). A shift of CENP-A occupancy to cen17_2 (D17Z1B) is not detected.


( F ) Representative showing ectopic CENP - A on a chromosome spread after induction of CENP - A overexpression in DLD - 1 . Centromere position is marked using immunofluorescence for CENP - B . Yellow dashed lines contour representative chromosomes . Scale bar , 2µm . Schematic to right illustrate observed CENP - AmRFP - DHFR localization pattern on a chromosome . Right panel : Comparison of CENP - ADHFR level per pixel at centromere on chromosome arms following IAA DOX / TMP treatment . Each dot represents one centromere or a region on chromosome arms .

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(F) Representative images showing ectopic CENP-A on a chromosome spread after induction of CENP-A overexpression in DLD-1 cells. Centromere position is marked using immunofluorescence staining for CENP-B. Yellow dashed lines contour representative chromosomes. Scale bar, 2µm. Schematic to the right illustrate the observed CENP-AmRFP-DHFR localization pattern on a chromosome. Right panel: Comparison of CENP-ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX/TMP treatment. Each dot represents one centromere or a region on the chromosome arms.


( G ) Schematic illustration for experiments shown in H , I . ( H ) Representative of chromosome spreads after re - expression of endogenous CENP - AEA into a CENP - AmRFP - DHFR overexpression background subsequent removal of overexpression as illustrated in ( G ) . Antibody against CENP - C was used to score for presence of functional centromere . Scale bar , 5µm . ( I ) of indicated events after one CENP - AOFF / ON cycle compared to treatment shown in ( G ) .

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(G) Schematic illustration for the experiments shown in H, I. (H) Representative images of chromosome spreads after re-expression of endogenous CENP-AEA into a CENP-AmRFP-DHFR overexpression background and subsequent removal of the overexpression as illustrated in (G). Antibody against CENP-C was used to score for the presence of the functional centromere. Scale bar, 5µm. (I) Quantification of the indicated events after one CENP-AOFF/ON cycle compared to treatment shown in (G).


( A ) Schematic illustration of CENP - AOFF / ON cycle performed in experiments shown in B , C . ( B ) Representative of de novo CENP - A reloading in CENP - B wild - type ( + / + ) CENP - B knock - out ( - / - ) . with centromeric CENP - A are marked with a red dashed contour while a yellow contour lines mark without centromeric CENP - A . Scale bar , 5 µm . ( C ) of relative number of RPE - 1 with centromeric CENP - A in indicated conditions . Each dot represents one experiment with at least 20 per condition . Error bars represent standard error of mean ( ) from 3 independent experiments .

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

(A) Schematic illustration of the CENP-AOFF/ON cycle performed in the experiments shown in B, C. (B) Representative images of de novo CENP-A reloading in CENP-B wild-type (+/+) and CENP-B knock-out (-/-) cells. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. Scale bar, 5 µm. (C) Quantification of relative number of RPE-1 cells with centromeric CENP-A in the indicated conditions. Each dot represents one experiment with at least 20 cells per condition. Error bars represent standard error of the mean (SEM) from 3 independent experiments.


( E ) Representative of DLD - 1 CENP - B ( + / + ) or ( - / - ) in late M phase following one CENP - AOFF / ON cycle . EdU was used to confirm successful wash - out of Palbociclib progressing through S - phase . Yellow dashed lines contour nucleus of in late M phase . Scale bar 5 µm . ( F ) of indicated events observed in late M phase in indicated lines . Error bars show from 4 independent experiments .

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(E) Representative images of DLD-1 CENP-B (+/+) or (-/-) cells in late M phase following one CENP-AOFF/ON cycle. EdU staining was used to confirm successful wash-out of Palbociclib and cell progressing through S-phase. Yellow dashed lines contour nucleus of cells in late M phase. Scale bar 5 µm. (F) Quantification of indicated events observed in late M phase cells in the indicated cell lines. Error bars show SEM from 4 independent experiments.


( C ) Representative IF - FISH of mitotic spreads in non - treated ( NT ) following a CENP - AOFF / ON cycle ( IAA WO ) . Chromosomes are contoured by a green ( X ) , magenta ( Y ) or ( other autosomes ) dashed . centromeres of chromosome X / Y are highlighted by a green / magenta arrow . Scale bar , 2 µm .

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(C) Representative IF-FISH images of mitotic spreads in non-treated cell (NT) and following a CENP-AOFF/ON cycle (IAA WO). Chromosomes are contoured by a green (X), magenta (Y) or white (other autosomes) dashed line. The centromeres of chromosome X/Y are highlighted by a green/magenta arrow. Scale bar, 2 µm.


( D ) of CENP - C ( as read - out of CENP - A ) presence at X ( green ) or Y ( magenta ) centromere in indicated lines . Each dot represents one experiment . Error bars represent SD from 3 independent experiments . Unpaired t , * * p = 0 . 0042 .

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(D) Quantification of CENP-C (as read-out of CENP-A) presence at the X (green) or Y (magenta) centromere in the indicated cell lines. Each dot represents one experiment. Error bars represent SD from 3 independent experiments. Unpaired t test, **p=0.0042.


( G ) Representative IF - FISH showing CENP - C localization on Y chromosome in growing under selective pressure ( G418 ) following a CENP - AOFF / ON cycle . Chromosomes are highlighted by a dashed contour . CENP - C was found at native centromere position ( top panel , blue ) , Y chromosome fused to another chromosome indicated by increased of chromosome absence of Chr - Y painting probe ( second panel , orange ) or CENP - C was found on a different location on Y chromosome did not overlap with Cen - Y ( lower panels , red ) . Scale bar , 2 µm . ( H ) of indicated events depicted in G . Each dot represents one experiment ( > 20 spreads for experiment ) . Error bars represent from 3 independent experiments . Unpaired t - , * p = 0 . 0137 0 . 039 .

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

(G) Representative IF-FISH images showing CENP-C localization on the Y chromosome in cell growing under selective pressure (G418) following a CENP-AOFF/ON cycle. Chromosomes are highlighted by a white dashed contour line. CENP-C was found at the native centromere position (top panel, blue), the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr-Y painting probe staining (second panel, orange) or CENP-C was found on a different location on the Y chromosome and did not overlap with Cen-Y DNA (lower panels, red). Scale bar, 2 µm. (H) Quantification of indicated events depicted in G. Each dot represents one experiment (>20 spreads for experiment). Error bars represent SEM from 3 independent experiments. Unpaired t-test, *p=0.0137 and 0.039.


( B ) Representative immunofluorescence showing CENP - C recruitment at LacO array in indicated conditions . LacO array is displayed in insets .

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(B) Representative immunofluorescence images showing CENP-C recruitment at the LacO array in the indicated conditions. LacO array is displayed in the insets.


( C ) Bar plot showing frequency of CENP - C ( orange ) or CENP - A ( green ) recruitment to LacO array in indicated conditions . Each dot represents one independent experiment ( > 20 analyzed for experiment ) .

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(C) Bar plot showing frequency of CENP-C (orange) or CENP-A (green) recruitment to LacO array in the indicated conditions. Each dot represents one independent experiment (>20 cell analyzed for experiment).


( D ) of CENP - C ( orange ) or CENP - A ( green ) at LacO array in indicated conditions normalized to intensities in non - treated conditions at mCherry - LacI - CENP - B - LacO array using CENP - A or CENP - C antibody . Each dot represents one independent experiment ( > 20 analyzed for experiment ) . Error bars represent from 3 independent experiments .

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(D) Quantification of CENP-C (orange) or CENP-A (green) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non-treated conditions at the mCherry-LacI-CENP-B-LacO array using CENP-A or CENP-C antibody. Each dot represents one independent experiment (>20 cell analyzed for experiment). Error bars represent SEM from 3 independent experiments.


( E ) Representative immunofluorescence showing CENP - A recruitment to ectopic mCherry - LacI - CENP - B in indicated conditions . LacO array is shown in inset .

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(E) Representative immunofluorescence images showing CENP-A recruitment to ectopic mCherry-LacI-CENP-B in the indicated conditions. LacO array is shown in the inset.


( F ) of frequency of CENP - C recruitment to LacO arrays by different LacI constructs . Error bars represent from 5 independent experiments .

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(F) Quantification of the frequency of CENP-C recruitment to LacO arrays by different LacI constructs. Error bars represent SEM from 5 independent experiments.


( G ) CENP - B immunoblot following GST pulldown experiments using GST tagged CENP - C ( 1 - 727 ) as bait with indicated as preys .

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(G) CENP-B immunoblot following GST pulldown experiments using GST tagged CENP-C (1-727) as bait and with the indicated proteins as preys.


( C ) Representative immunofluorescence showing CENP - A CENP - C reloading in indicated conditions . with centromeric CENP - A / CENP - C are marked with a red dashed contour while a yellow contour lines mark without centromeric CENP - A . centromere position is marked using immunofluorescence for CENP - B . dashed lines contour DAPI stained nucleus of representative .

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(C) Representative immunofluorescence images showing CENP-A and CENP-C reloading in the indicated conditions. Cells with centromeric CENP-A/CENP-C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. The centromere position is marked using immunofluorescence staining for CENP-B. White dashed lines contour DAPI stained nucleus of representative cells.


( D ) Bar plot showing of with CENP - A CENP - C co - localization after a CENP - A / COFF / ON cycle in indicated conditions . Error bars show SD from 5 independent experiments .

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(D) Bar plot showing the percentage of cells with CENP-A and CENP-C co-localization after a CENP-A/COFF/ON cycle in the indicated conditions. Error bars show SD from 5 independent experiments.


( E ) Box plot of CUT & RUN qPCR for CENP - A ( green ) or CENP - C ( orange ) . Box plot shows median , 25th 75th percentiles , whiskers show minimum maximum values . Results from 3 independent experiments including combined data from qPCR reactions using alpha - satellite primers primers binding at centromere of chromosome 4 . Enrichment is measured relative to IgG control normalized to Alu repeats .

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(E) Box plot of CUT&RUN qPCR for CENP-A (green) or CENP-C (orange). Box plot shows median, 25th and 75th percentiles, whiskers show minimum and maximum values. Results from 3 independent experiments including combined data from qPCR reactions using alpha-satellite primers and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.


( C ) Representative immunofluorescence showing exogenous CENP - AmRFP - DHFR reloading in presence ( control ) , but not in absence ( sample ) of endogenous CENP - A CENP - C . with centromeric CENP - A are marked with a red dashed contour while a yellow contour marks without centromeric CENP - A . ( D ) of relative number of showing centromeric eCENP - AmRFP - DHFR in indicated conditions . Error bars represent from 3 independent experiments .

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(C) Representative immunofluorescence images showing exogenous CENP-AmRFP-DHFR reloading in the presence (control), but not in the absence (sample) of endogenous CENP-A and CENP-C. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-A. (D) Quantification of relative number of cells showing centromeric eCENP-AmRFP-DHFR in the indicated conditions. Error bars represent SEM from 3 independent experiments.


( E ) Bar plot of CUT & RUN qPCR using CENP - A antibody primers binding at centromere of chromosome 4 . Enrichment is measured relative to IgG control normalized to Alu repeats .

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(E) Bar plot of CUT&RUN qPCR quantification using CENP-A antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.


( F ) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP - CmRFP - DHFR in presence ( control ) in absence ( sample ) of endogenous CENP - A CENP - C . were arrested with Colcemid for 3 h prior to spread . with centromeric CENP - C are marked with a red dashed contour while a yellow contour marks without centromeric CENP - C . Chromosomes in inset are highlighted using a dashed . ( G ) of relative number of with eCENP - CmRFP - DHFR in indicated conditions . Each dot represents one independent experiment with at least 30 per condition .

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(F) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP-CmRFP-DHFR in the presence (control) and in the absence (sample) of endogenous CENP-A and CENP-C. Cells were arrested with Colcemid for 3 h prior to spread. Cells with centromeric CENP-C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-C. Chromosomes in inset are highlighted using a white dashed line. (G) Quantification of relative number of cells with eCENP-CmRFP-DHFR in the indicated conditions. Each dot represents one independent experiment with at least 30 cells per condition.


( H ) Bar plot of CUT & RUN qPCR results using CENP - C antibody primers binding at centromere of chromosome 4 . Enrichment is measured relative to IgG control normalized to Alu repeats . Each dot represents one independent experiment .

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

(H) Bar plot of CUT&RUN qPCR results using CENP-C antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats. Each dot represents one independent experiment.


( A ) Gating strategy for sorting of freshly purified resting human CD4 + T on CENP - A expression . Dead were excluded ( first plot ) using Fixable Viability Dye eFluor 780 , CENP - Ahigh CENP - Alow ( both CENP - B + ) were sorted ( second plot ) . Post - sort of each population is then shown ( third fourth plots ) .

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

(A) Gating strategy for the sorting of freshly purified resting human CD4+ T cells based on CENP-A expression. Dead cells were excluded (first plot) using Fixable Viability Dye eFluor 780, CENP-Ahigh and CENP-Alow cells (both CENP-B+) were sorted (second plot). Post-sort analysis of each population is then shown (third and fourth plots).


( B ) Representative of sorted CD4 + T with high or low CENP - A level . nucleus is contoured by a dashed . Scale bar , 5 µm .

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(B) Representative images of sorted CD4+ T cells with high or low CENP-A level. The nucleus is contoured by a white dashed line. Scale bar, 5 µm.


( D ) Tracking of division by CFSE dilution . black separates that have not divided ( nº of div = 0 , CFSE - high ) from those that have undergone at least one division ( nº of div ≥ 1 , CFSE - low ) . ( E ) CENP - A expression CFSE dilution . Black lines were set on CENP - A isotype control CFSE maximal at day 0 ( in shaded quadrants are CFSE - high / CENP - A - low ) . One representative donor is depicted . ( F ) Absolute number of CFSE - high / CENP - A - low CD4 + T ( shaded gate in E ) . Symbols represent donors . Error bars represent from 6 independent donors . One - way ANOVA , multiple comparisons , n = 6 . * p < 0 . 05 ; * * p < 0 . 01 . ( G ) Frequency of without centromeric CENP - A determined by immunofluorescence microscopy before post activation . Symbols represent donors , n = 4 . Error bars represent from 6 independent donors .

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

(D) Tracking of cell division by CFSE dilution. The black line separates cells that have not divided (nº of div=0, CFSE-high) from those that have undergone at least one division (nº of div≥1, CFSE-low). (E) CENP-A expression and CFSE dilution. Black lines were set based on CENP-A isotype control and CFSE maximal staining at day 0 (cells in shaded quadrants are CFSE-high/CENP-A-low). One representative donor is depicted. (F) Absolute number of CFSE-high/CENP-A-low CD4+ T cells (shaded gate in E). Symbols represent individual donors. Error bars represent SEM from 6 independent donors. One-way ANOVA, multiple comparisons, n=6. *p < 0.05; **p < 0.01. (G) Frequency of cells without centromeric CENP-A determined by immunofluorescence microscopy before and post activation. Symbols represent individual donors, n=4. Error bars represent SEM from 6 independent donors.


A A bright field image of a wild - type adult C . elegans . position of AIY interneurons in head is indicated with green , gut is outlined with dashed lines . A ' A cartoon diagram of C . elegans head modified from wormatlas with permission . AIY processes soma are indicated in grey , presynaptic sites are marked with green ( Altun et al . , 2002 - 2019 ) .

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

A A bright field image of a wild-type adult C. elegans. The position of the AIY interneurons in the head is indicated with green, and the gut is outlined with dashed lines. A' A cartoon diagram of the C. elegans head modified from wormatlas with permission. The AIY processes and soma are indicated in grey, and the presynaptic sites are marked with green (Altun et al., 2002-2019).


B - G ' Confocal of AIY labeled with synaptic vesicle marker GFP : : RAB - 3 ( B - G , green ) active zone marker GFP : : SYD - 1 ( B ' - G ' , pseudo - red ) in wild - type ( B , B ' ) , cwn - 2 ( ok895 ) ( C , C ' ) , cfz - 2 ( ok1201 ) ( D , D ' ) , pop - 1 ( hu9 ) ( E , E ' ) , sys - 1 RNAi ( G , G ' ) . H - H ' of of AIY RAB - 3 ( H ) SYD - 1 ( H ' ) for indicated genotypes .

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

B-G' Confocal images of the AIY labeled with synaptic vesicle marker GFP::RAB-3 (B-G, green) and active zone marker GFP::SYD-1 (B'-G', pseudo-red) in wild-type (B, B'), cwn-2(ok895) (C, C'), cfz-2(ok1201) (D, D'), pop-1(hu9) (E, E'), and sys-1 RNAi (G, G') animals. H-H' Quantification of the fluorescence intensity of AIY RAB-3 (H) and SYD-1 (H') for the indicated genotypes.


I - M ' Confocal of AIY labeled with mCherry : : RAB - 3 ( pseudo - GFP ) ( I - M ) , or GFP : : RAB - 3 ( I ' - M ' ) of tissue - specific RNAi treatment for control ( I - I ' ) , cwn - 2 ( J - J ' ) , cfz - 2 ( K - K ' ) , sys - 1 ( L - L ' ) pop - 1 ( M - M ' ) . N - N ' of of AIY mCherry : : RAB - 3 or GFP : : RAB - 3 for indicated RNAi treatment .

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

I-M' Confocal images of AIY labeled with mCherry::RAB-3 (pseudo-GFP) (I-M), or GFP::RAB-3 (I'-M') of tissue-specific RNAi treatment for control (I-I'), cwn-2(J-J'), cfz-2(K-K'), sys-1(L-L') and pop-1(M-M'). N-N' Quantification of the fluorescence intensity of AIY mCherry::RAB-3 or GFP::RAB-3 for the indicated RNAi treatment.


A of of AIY GFP : : RAB - 3 in wild - type , egl - 3 ( n589 ) aex - 5 ( sa23 ) mutants .

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A Quantification of the fluorescence intensity of AIY GFP::RAB-3 in wild-type , egl-3(n589) and aex-5(sa23) mutants.


C of of AIY GFP : : RAB - 3 for indicated RNAi treatment . Candidates are intestinal expressed nlp or flp .

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C Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated RNAi treatment. Candidates are intestinal expressed nlp or flp.


Confocal of AIY labeled with GFP : : RAB - 3 in wild - type ( E ) with a wild type nlp - 40 transgene .

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Confocal images of AIY labeled with GFP::RAB-3 in wild-type (E) with a wild type nlp-40 transgene.


Confocal of AIY labeled with nlp - 40 ( tm4085 ) ( F ) , nlp - 40 ( vj3 ) ( G ) , nlp - 40 ( tm4085 ) ( H ) or nlp - 40 ( vj3 ) ( I ) with a wild type nlp - 40 transgene . J of of AIY GFP : : RAB - 3 for indicated genotypes . Transgenic data are averaged from at least two independent lines .

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Confocal images of AIY labeled with nlp-40(tm4085) (F), nlp-40(vj3) (G), nlp-40(tm4085) (H) or nlp-40(vj3) (I) with a wild type nlp-40 transgene. J Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated genotypes. Transgenic data are averaged from at least two independent lines.


K - L . Confocal of AIY synaptic marker GFP : : RAB - 3 for indicated intestinal - specific RNAi treatment . M for ( K ) ( L ) .

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K-L. Confocal images of AIY synaptic marker GFP::RAB-3 for the indicated intestinal-specific RNAi treatment. M Quantification for (K) and (L).


A - C of of AIY presynaptic marker GFP : : RAB - 3 ( A , B ) nlp - 40 mRNA level ( C ) for indicated genotypes . AIY synaptic region was quantified for synaptic .

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A-C Quantification of the fluorescence intensity of AIY presynaptic marker GFP::RAB-3 (A, B) and nlp-40 mRNA level (C) for the indicated genotypes. The AIY synaptic region was quantified for the synaptic intensity analysis.


D - I Confocal of transcriptional nlp - 40 reporter ( Pnlp - 40 : : GFP ) in wild - type ( D ) , cwn - 2 ( ok895 ) ( E ) , cfz - 2 ( ok1201 ) ( F ) , pop - 1 ( hu9 ) ( G ) , control or sys - 1 RNAi ( H I ) at adult Day 1 stage . J of Pnlp - 40 : : GFP for indicated genotypes .

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D-I Confocal images of the transcriptional nlp-40 reporter (Pnlp-40::GFP) in wild-type (D), cwn-2(ok895) (E), cfz-2(ok1201) (F), pop-1(hu9) (G), and control or sys-1 RNAi (H and I) animals at adult Day 1 stage. J Quantification of the Pnlp-40::GFP fluorescence intensity for the indicated genotypes.


K - P Confocal of an intestinal - specific reporter ERM - 1 : : GFP in wild type ( K ) , cwn - 2 ( ok895 ) ( L ) , cfz - 2 ( ok1201 ) ( M ) , pop - 1 ( hu9 ) ( N ) , control or sys - 1 RNAi ( O P ) at adult Day1 stage . Q of ERM - 1 : : GFP for indicated genotype .

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K-P Confocal images of an intestinal-specific reporter ERM-1::GFP in wild type (K), cwn-2(ok895) (L), cfz-2(ok1201) (M), pop-1(hu9) (N), and control or sys-1 RNAi (O and P) animals at the adult Day1 stage. Q Quantification of the ERM-1::GFP fluorescence intensity for the indicated genotype.


A Diagram of aex - 2 genomic structure . boxes lines represent exons introns . Black gray indicate coding UTRs . asterisk marks location of R232Q mutation site of sa3 allele . B - K Confocal of AIY presynaptic site labeled with GFP : : RAB - 3 in wild type ( B ) , nlp - 40 ( tm4085 ) ( C ) , aex - 2 ( sa3 ) ( D ) , nlp - 40 ( tm4085 ) ; aex - 2 ( sa3 ) ( E ) over - expression NLP - 40 in aex - 2 ( sa3 ) mutants ( F ) aex - 2 ( sa3 ) with a wild - type aex - 2 transgene driven by endogenous promoter ( Paex - 2 ) ( G ) , neuron - specific promoter ( Prab - 3 ) ( H ) , AIY - specific promoter ( Pttx - 3 ) ( I ) intestinal - specific promoter ( Pges - 1 ) ( J ) or muscle - specific promoter ( Pmyo - 3 ) ( K ) . L - M of of AIY GFP : : RAB - 3 for indicated genotypes . Each dot represents one . Data were collected from at least three independent experiments . Transgenic data are averaged from at least two independent lines . tg : Paex - 2 : : aex - 2 , Pttx - 3 : : aex - 2 or Pges - 1 : : aex - 2 transgene , + : wild type ; - : mutant or without tg . AIY synaptic region was quantified for synaptic .

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

A Diagram of aex-2 genomic structure. The boxes and lines represent exons and introns. Black and gray indicate coding sequence and UTRs. The white asterisk marks the location of the R232Q mutation site of sa3 allele. B-K Confocal images of AIY presynaptic site labeled with GFP::RAB-3 in wild type(B), nlp-40(tm4085)(C), aex-2(sa3)(D), nlp-40(tm4085); aex-2(sa3) (E) over-expression NLP-40 in aex-2(sa3) mutants(F) and aex-2(sa3) with a wild-type aex-2 transgene driven by the endogenous promoter (Paex-2) (G), neuron-specific promoter (Prab-3) (H) , AIY-specific promoter (Pttx-3) (I) intestinal-specific promoter (Pges-1) (J) or muscle-specific promoter (Pmyo-3)(K). L-M Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated genotypes. Each dot represents one animal. Data were collected from at least three independent experiments. Transgenic data are averaged from at least two independent lines. tg: Paex-2::aex-2, Pttx-3::aex-2 or Pges-1::aex-2 transgene, +: wild type ; -: mutant or without tg. The AIY synaptic region was quantified for the synaptic intensity analysis.


N - Q ' Confocal of Paex - 2 : : mNeonGreen of whole with bright field ( N ) , head region labeled with Paex - 2 : : mNeonGreen Pttx - 3 : : mCherry with green channel ( O ) , red channel ( P ) merged channels ( Q ) . ( O ' - Q ' ) are cross sections corresponding to dashed sites .

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N-Q' Confocal images of Paex-2::mNeonGreen of whole animal with bright field (N), the head region labeled with Paex-2::mNeonGreen and Pttx-3::mCherry with green channel (O), red channel (P) and the merged channels (Q). (O'-Q') are the cross sections corresponding to the dashed line sites.


A - A ' Representative confocal showing inactive ( A ) active ( A ' ) state of AIY : : GCaMP6s ( with mod - 1 promoter ) colabeled with mCherry ( with ttx - 3 promoter ) ( red ) . Dashed ovals mark AIY zone 2 region where GCaMP was quantified .

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A-A' Representative confocal images showing inactive (A) and active(A') state of AIY::GCaMP6s (with mod-1 promoter) colabeled with mCherry (with ttx-3 promoter) (red). Dashed ovals mark the AIY zone 2 region where GCaMP was quantified.


B Representative AIY calcium activity over 60 seconds for indicated genotypes . C - D of peak frequency amplitude of AIY : : GCaMP6s in for indicated genotypes .

O O O O O 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 Representative AIY calcium activity over 60 seconds for the indicated genotypes. C-D Quantification of the peak frequency and amplitude of AIY::GCaMP6s in for the indicated genotypes.


B - G Confocal of Prab - 3 : : GFP : : RAB - 3 in nerve region corresponding to dashed box in ( A ) of wild type ( B ) , cwn - 2 ( ok895 ) ( C ) , cfz - 2 ( ok1201 ) ( D ) , pop - 1 ( hu9 ) ( E ) , nlp - 40 ( tm4085 ) ( F ) aex - 2 ( sa3 ) ( G ) mutants . H - I of GFP : : RAB - 3 in nerve ring synaptic region for indicated genotypes . Each dot represents one . Data were collected from at least three independent experiments . Transgenic data are averaged from at least two independent lines .

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

B-G Confocal images of Prab-3::GFP::RAB-3 in the nerve region corresponding to the dashed box in (A) of wild type (B), cwn-2(ok895) (C), cfz-2(ok1201) (D), pop-1(hu9) (E), nlp-40(tm4085) (F) and aex-2(sa3) (G) mutants. H-I Quantification of the GFP::RAB-3 intensity in the nerve ring synaptic region for the indicated genotypes. Each dot represents one animal. Data were collected from at least three independent experiments. Transgenic data are averaged from at least two independent lines.


A APB Northern blot using a tRNAHis probe . m5C38 of tRNAAspGUC were measured by 454 bisulfite sequencing at indicated time points . Both queuosinylation methylation could be restored by addition of queuine . slower migration of tRNAHis is eliminated by oxidizing ribose with periodate , producing a single faster - migrating band ( ox ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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A APB Northern blot using a tRNAHis probe. m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points. Both queuosinylation and methylation levels could be restored by the addition of queuine. The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate, producing a single faster-migrating band (ox). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B addition of queuine to HCT116 cultured in serum free medium resulted in an increase of both queuosinylation m5C38 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C addition of 20 nM queuine to SF medium is sufficient to restore tRNAHisQUG in HeLa , whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa, whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B Corresponding queuosinylation methylation , as determined by Northern blot using a tRNAHisGUG probe bisulfite sequencing of tRNAAspGUC . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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B Corresponding queuosinylation and methylation levels, as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C Representative metaplot of SF . 25 - 32 mer ribosome footprint reads were summed across annotated reading frames with AUG staring codon at position zero . In - frame 27 - 30 mer ribosome footprints showing correct periodicity .

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C Representative metaplot of SF cells. 25-32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero. In-frame 27-30 mer ribosome footprints showing the correct periodicity.


D Representative metagene plot of 27 - 30 mer ribosome footprints in S SF culture conditions for two biological replicates ( rep1 rep2 ) . Data information : SF ( serum free medium ) , q ( queuine )

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D Representative metagene plot of 27-30 mer ribosome footprints in S and SF culture conditions for two biological replicates (rep1 and rep2). Data information: SF (serum free medium), q (queuine)


A showing codon occupancy according to color scale . Green bars indicate Q - decoded codons . Asp - GAC codon translated by Q - m5C38 - tRNAAspGUC is shown in yellow Lys Glu codons are in orange . Lack of Q reduces ribosome translation speed at Q dependent codons at near - cognate decoded codons . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

A Heatmap showing codon occupancy according to the color scale. Green bars indicate Q-decoded codons. The Asp-GAC codon translated by Q- and m5C38-tRNAAspGUC is shown in yellow and Lys and Glu codons are in orange. Lack of Q reduces ribosome translation speed at all Q dependent codons and at near-cognate decoded codons. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B Translational speed of C ending codons relative to U ending codons . Error bars : ± SE of permutated ratios of C - ending codon occupancy relative to U for indicated conditions ; ( n = 4 ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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B Translational speed of C ending codons relative to U ending codons. Error bars: ±SE of the permutated ratios of C-ending codon occupancy relative to U for the indicated conditions; (n=4). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C displaying increase or decrease of codon frequencies ( ‰ ) in SILAC down - regulated relative to average frequency of that codon in unchanged .

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C Heatmap displaying increase or decrease of codon frequencies (‰) in SILAC down-regulated proteins relative to the average frequency of that codon in unchanged proteins.


D Codons translated by Q - tRNA are depleted in down - regulated increased in up - regulated in both forward reverse SILAC .

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D Codons translated by Q-tRNA are depleted in down-regulated proteins and increased in up-regulated proteins in both forward and reverse SILAC.


A volcano plot shows differentially translated transcripts against adjusted p - values for SF compared to SF + q . Red dots indicate padj < 0 . 1 in multiple . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine )

A The volcano plot shows differentially translated transcripts against adjusted p-values for SF compared to SF+q. Red dots indicate padj< 0.1 in multiple tests. Data information: S (standard medium), SF (serum free medium), q (queuine)


C Representative polysome profiles of HeLa grown in S SF medium in absence or presence of q . As a measurement for global translation rate fraction of polysomal ribosomes was quantified ; * p < 0 . 05 ( t - ) ; n = 5 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , RO ( ribosome occupancy ) .

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C Representative polysome profiles of HeLa cells grown in S and SF medium in the absence or presence of q. As a measurement for the global translation rate the fraction of polysomal ribosomes was quantified; *p <0.05 (t-test); n=5. Data information: S (standard medium), SF (serum free medium), q (queuine), RO (ribosome occupancy).


D Proliferation of HeLa grown under indicated culture conditions . Population doubling of three biological replicates were calculated for each time point . Error bars : ± SD ; n = 3 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine )

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D Proliferation analysis of HeLa cells grown under the indicated culture conditions. Population doubling levels of three biological replicates were calculated for each time point. Error bars: ±SD; n = 3. Data information: S (standard medium), SF (serum free medium), q (queuine)


A Representative confocal of live HeLa expressing Fluc - EGFP FlucR188Q - EGFP maintained in SF compared to SF + q . Scale bars , 10 μm . Data information : SF ( serum free medium ) , q ( queuine )

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A Representative fluorescence confocal images of live HeLa cells expressing Fluc-EGFP and FlucR188Q-EGFP maintained in SF compared to SF+q. Scale bars, 10 μm. Data information: SF (serum free medium), q (queuine)


B of was quantified as a fraction of of entire . number of showing at least one aggregate is indicated . 10 were imaged for Fluc - EGFP 20 for FlucR188Q - EGFP from 3 independent transfections ; * p < 0 . 05 ( t - ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , AU ( arbitrary units ) .

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B Fluorescence intensity of aggregates was quantified as a fraction of fluorescence of the entire cell. The number of cells showing at least one aggregate is indicated. 10 cells were imaged for Fluc-EGFP and 20 cells for FlucR188Q-EGFP from 3 independent transfections; *p < 0.05 (t-test). Data information: S (standard medium), SF (serum free medium), q (queuine), AU (arbitrary units).


C Specific luminescence activity of EGFP - tagged sensor maintained in SF compared to SF + q . Luci ferase activity was normalized to luciferase content in sample . Error bars : ± SD ; * p < 0 . 05 ( t - ) ; n = 8 . Data information : SF ( serum free medium ) , q ( queuine )

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C Specific luminescence activity of EGFP-tagged sensor proteins maintained in SF compared to SF+q. Luciferase activity was normalized to the luciferase protein content in the sample. Error bars: ±SD; *p < 0.05 (t-test); n = 8. Data information: SF (serum free medium), q (queuine)


A Transmission electron microscopy showing increase of cystic ER vacuolae upon q depletion . Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks . Scale bar 250 nm . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) .

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A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion. Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks. Scale bar 250 nm. Data information: S (standard medium), SF (serum free medium), q (queuine).


B Representative showing immunofluorescence of HeLa grown in S SF medium in absence or presence of Q . Anti - KDEL ( ER stress marker ) ; anti - eIF2 - pS51 ( ER stress - induced inhibitor of translation ) ; anti - ATF4 ( transcription factor that activates ER stress - responsive genes ) ; anti - Hps70 ( chaperone ) . Nuclei were stained with Hoechst . Scale bar 10 µm . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) .

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B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q. Anti-KDEL (ER stress marker); anti-eIF2-pS51 (ER stress-induced inhibitor of translation); anti-ATF4 (transcription factor that activates ER stress-responsive genes); anti-Hps70 (chaperone). Nuclei were stained with Hoechst. Scale bar 10 µm. Data information: S (standard medium), SF (serum free medium), q (queuine).


A APB Northern blot using a tRNAHis probe . m5C38 were determined by 454 bisulfite sequencing of from same liver tissue . Both queuosinylation methylation could be restored by addition of q to feed . results are shown for three biological replicates . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , + q ( n = 3 ) mice were fed with same synthetic diet supplemented with 40 nM queuine for 60 days .

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A APB Northern blot using a tRNAHis probe. m5C38 levels were determined by 454 bisulfite sequencing of RNA from the same liver tissue. Both queuosinylation and methylation levels could be restored by the addition of q to the feed. The results are shown for three biological replicates. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


B Representative polysome profiles showing a reduction of translation rate in liver of axenic mice fed with a q - free synthetic diet . Addition of q to q - free diet significantly rescued translation rate . n = 3 . Asterisks indicate statistically significant ( p < 0 . 05 , t - ) differences . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , + q ( n = 3 ) mice were fed with same synthetic diet supplemented with 40 nM queuine for 60 days .

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B Representative polysome profiles showing a reduction of the protein translation rate in the liver of axenic mice fed with a q-free synthetic diet. Addition of q to the q-free diet significantly rescued the translation rate. n=3. Asterisks indicate statistically significant (p<0.05, t-test) differences. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


C Box plot showing of mice fed a q - free synthetic diet for 60 days , center lines show medians ; box limits indicate 25th 75th percentiles , whiskers extend 1 . 5 times interquartile range ; n = 3 . Addition of q to q - free diet significantly rescued weights . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , + q ( n = 3 ) mice were fed with same synthetic diet supplemented with 40 nM queuine for 60 days .

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C Box plot showing the body weight of mice fed a q-free synthetic diet for 60 days, center lines show the medians; box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range; n=3. Addition of q to the q-free diet significantly rescued body weights. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


D Dimethyl - labeling of - q versus + q liver tissue . Top 10 % of deregulated are indicated in red blue . UPR effectors : HSPA5 / BiP , HSP90b1 CALR are in orange . Data information : - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , + q ( n = 3 ) mice were fed with same synthetic diet supplemented with 40 nM queuine for 60 days .

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D Dimethyl-labeling analysis of -q versus +q liver tissue. Top 10% of deregulated proteins are indicated in red and blue. UPR effectors: HSPA5/BiP, HSP90b1 and CALR are in orange. Data information: -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


F Liver sections from indicated mice were stained with anti - KDEL antibody . Arrowheads indicate ER stressed hepatocytes . Scale bar 25 µm . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , + q ( n = 3 ) mice were fed with same synthetic diet supplemented with 40 nM queuine for 60 days .

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F Liver sections from indicated mice were stained with anti-KDEL antibody. Arrowheads indicate ER stressed hepatocytes. Scale bar 25 µm. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


( A ) Dot plot depicting expression of mRNA for neurogenic transcription factor MEIS2 in prospectively isolated populations from SEZ .

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(A) Dot plot depicting the expression of mRNA for neurogenic transcription factor MEIS2 in prospectively isolated cellular populations from the SEZ.


( B ) depicting expression of neurogenic fate determinant Meis2 mRNA MEIS2 in acutely dissociated SEZ . Note that Meis2 mRNA positive LRCs have low ( no ) MEIS2 .

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(B) Micrographs depicting the expression of neurogenic fate determinant Meis2 mRNA and MEIS2 protein in acutely dissociated SEZ cells. Note that Meis2 mRNA positive LRCs have low (no) MEIS2 protein.


( C labeling for MEIS2 ( C ) of LRCs positive for BrdU only neuroblasts marked by DCX .

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(C IHC labeling for MEIS2 (C) of LRCs positive for BrdU only and neuroblasts marked by DCX.


D ) labeling for MCM6 ( D ) of LRCs positive for BrdU only neuroblasts marked by DCX .

D) IHC labeling for MCM6 (D) of LRCs positive for BrdU only and neuroblasts marked by DCX.


( E ) Dot plot showing proportion of BrdU + LRCs negative for MEIS2 or MCM6 .

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(E) Dot plot showing the proportion of BrdU+ LRCs negative for MEIS2 or MCM6 protein.


( F ) Dot plot depicting expression of miR - 204 in prospectively isolated of neural lineage .

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(F) Dot plot depicting the expression of miR-204 in prospectively isolated cells of neural lineage.


( B ) Dot plot depicting immunoreactivity of LRCs for MEIS2 in control antagomirs treated 7 dpi .

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(B) Dot plot depicting the immunoreactivity of LRCs for MEIS2 in control and antagomirs treated animals 7 dpi.


( C , D ) showing for neurogenic priming factor MEIS2 in SEZ 7 days after injection of artificial CSF ( control , C ) or AntimiR204 ( D ) . arrows indicate LRCs without detectable MEIS2 . arrowheads are pointing out loss of neurogenic priming ( LRCs positive for MEIS2 ) upon antagomir treatments .

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(C, D) Micrographs showing IHC for the neurogenic priming factor MEIS2 in the SEZ 7 days after injection of artificial CSF (control, C) or AntimiR204 (D). White arrows indicate LRCs without detectable MEIS2 protein. The white arrowheads are pointing out the loss of neurogenic priming (LRCs positive for MEIS2 protein) upon the antagomir treatments.


( A , B ) depicting composition in SEZ 7 days after injection of aCSF ( control , A ) or AntimiR204 ( B ) . Boxed areas correspond to higher magnifications in adjacent panels ( A ' , B ' ) .

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(A, B) Micrographs depicting the cellular composition in the SEZ 7 days after injection of aCSF (control, A) or AntimiR204 (B). Boxed areas correspond to higher magnifications in adjacent panels (A', B').


Dot plots depicting abundance of LRCs ( BrdU + , Ki67 - , DCX - ) in SEZ 7 days after aCSF or AntimiR204 injection .

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Dot plots depicting the abundance of LRCs (BrdU+, Ki67-, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.


Dot plots depicting abundance of aNSCs TAPs ( BrdU - , Ki67 + , DCX - ) in SEZ 7 days after aCSF or AntimiR204 injection .

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Dot plots depicting the abundance of aNSCs and TAPs (BrdU-, Ki67+, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.


Dot plots depicting abundance of neuroblasts ( DCX + Ki67 + or DCX + Ki67 - ) in SEZ 7 days after aCSF or AntimiR204 injection .

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Dot plots depicting the abundance of neuroblasts (DCX+ Ki67+ or DCX+ Ki67-) in the SEZ 7 days after aCSF or AntimiR204 injection.


( F , G ) depicting elbow - most ventral region of RMS ( F ) OB neuronal layers ( G ) upon injection of artificial CSF ( control ) or AntimiR204 . ( H ) Dot plot showing density of BrdU + ( progeny of NSCs ) in RMS in OB 7 days after artificial CSF or AntimiR204 injection .

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(F, G) Micrographs depicting the elbow - the most ventral region of the RMS (F) and OB neuronal layers (G) upon injection of artificial CSF (control) or AntimiR204. (H) Dot plot showing the density of BrdU+ cells (progeny of NSCs) in the RMS and in the OB 7 days after artificial CSF or AntimiR204 injection.


( A ) of in situ hybridization for miR - 204 in an adult mouse brain section . ( A ' A ' ' ) are magnifications of boxed areas in ( A or A ' ) , respectively .

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(A) Micrographs of in situ hybridization for miR-204 in an adult mouse brain section. (A' and A'') are magnifications of boxed areas in (A or A'), respectively.


( B ) Dot plot showing miR - 204 in ChP SEZ in adult mouse brain measured by RT - qPCR . ( C ) Agarose gel of RT - qPCR product loaded after saturation phase showing presence of miR - 204 U6 in mouse ChP , SEZ CSF .

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(B) Dot plot showing miR-204 levels in ChP and SEZ in the adult mouse brain measured by RT-qPCR. (C) Agarose gel of RT-qPCR product loaded after the saturation phase showing presence of miR-204 and U6 in mouse ChP, SEZ and CSF.


Plots depicting number of EVs isolated from human CSF ( D )

Plots depicting the number and size of EVs isolated from human CSF (D)


Plots depicting miR - 204 ( E ) in EVs EV - free CSF ( n = 2 ) . ( F ) Agarose gel of RT - qPCR depicting of miR - 204 in EV - free supernatant EV containing fraction of human CSF loaded at saturation phase .

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Plots depicting the miR-204 levels (E) in EVs and EV-free CSF (n=2). (F) Agarose gel of RT-qPCR analysis depicting levels of miR-204 in the EV-free supernatant and EV containing fraction of human CSF loaded at the saturation phase.


( G ) of transmission electron microscopy imaged extracellular vesicles ( EVs ) isolated from sham treated ( upper row ) GW4869 treated ( lower row ) . ( G ' ' G ' ' ' ) are magnifications of boxed areas in overview to left . ( H ) Dot plot depicting miR - 204 in CSF isolated 2 h after ventricular injection of GW4869 inhibitor .

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(G) Micrographs of transmission electron microscopy imaged extracellular vesicles (EVs) isolated from sham treated (upper row) and GW4869 treated animals (lower row). (G'' and G''') are magnifications of boxed areas in overview images to the left. (H) Dot plot depicting miR-204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor.


( A ) Gel pictures of saturation phase of RT - qPCR for miR - 204 in SEZ culture , ChP co - culture ChP explants 7 day post preparation .

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(A) Gel pictures of the saturation phase of RT-qPCR analysis for miR-204 in SEZ primary culture cells, ChP co-culture and ChP explants 7 day post preparation.


( B ) Gel electrophoresis of RT - qPCR loaded at saturation phase for miR - 204 in SEZ culture medium SEZ medium from ChP co - culture .

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(B) Gel electrophoresis of RT-qPCR analysis loaded at the saturation phase for miR-204 in SEZ culture medium and SEZ medium from the ChP co-culture.


( C ) Dot plot showing proportion of neural ( GFAP + ) out of DAPI + in SEZ / ChP co - culture with or without treatment with GW4869 inhibitor or AntimiR204 .

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(C) Dot plot showing the proportion of neural stem cells (GFAP+) out of DAPI+ cells in primary SEZ/ChP co-culture with or without treatment with GW4869 inhibitor or AntimiR204.


( D - G ) depicting compositions of SEZ cultures after co - culturing with control treated , GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating . Boxed areas correspond to higher magnification in adjacent panels ( D ' , E ' , F ' , G ' ) .

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(D-G) Micrographs depicting the cellular compositions of SEZ cultures after co-culturing with control treated, GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating. Boxed areas correspond to the higher magnification images in adjacent panels (D', E', F', G').


( B , C ) depicting specific expression of AAV5 encoded GFP in ChP 7 days after viral delivery in lateral ventricle . ( B ' C ' ) are magnifications of SEZ ( B ' ' C ' ' ) of ChP . ( D ) Dot plot depicting expression of miR - 204 in ChP 7 days after second ventricular injection of AAV5 encoding for miR - 204 - specific TuD compared to scrambled control .

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(B, C) Micrographs depicting specific expression of AAV5 encoded GFP in the ChP 7 days after viral delivery in the lateral ventricle. (B' and C') are magnifications of the SEZ and (B'' and C'') of the ChP. (D) Dot plot depicting expression of miR-204 in ChP 7 days after second ventricular injection of AAV5 encoding for miR-204-specific TuD compared to scrambled control.


depicting immunoreactivity for MEIS2 ( E , F ) priming factors in LRCs ( BrdU + only ) 7 days after ChP - specific miR - 204 inhibition scrambled control arrows point out primed LRCs , arrowheads label MEIS2 Dot plots depicting proportion of LRCs immunoreactive for priming MEIS2 after ChP specific miR - 204 interference .

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Micrographs depicting immunoreactivity for MEIS2 (E, F) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MEIS2 Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MEIS2 after ChP specific miR-204 interference.


depicting immunoreactivity for MCM6 ( H , I ) priming factors in LRCs ( BrdU + only ) 7 days after ChP - specific miR - 204 inhibition scrambled control arrows point out primed LRCs , arrowheads label MCM6 + LRCs . Dot plots depicting proportion of LRCs immunoreactive for priming MCM6 ( J ) after ChP specific miR - 204 interference .

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Micrographs depicting immunoreactivity for MCM6 (H, I) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MCM6+ LRCs. Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MCM6 (J) after ChP specific miR-204 interference.


( K , L ) showing composition in SEZ 7 days after injection of AVV5 encoding for scrambled control ( K ) miR - 204 specific TuD ( L ) . Note reduction of primed LRCs ( BrdU + only , arrows in K L ) upon TuD - 204 injection . ( M ) Dot plot depicting number of LRCs ( BrdU + only ) in SEZ 7 days after miR - 204 inhibition .

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(K, L) Micrographs showing the cellular composition in the SEZ 7 days after injection of AVV5 encoding for scrambled control (K) and miR-204 specific TuD (L). Note the reduction of primed LRCs (BrdU+ only, white arrows in K and L) upon TuD-204 injection. (M) Dot plot depicting the number of LRCs (BrdU+ only) in the SEZ 7 days after miR-204 inhibition.


D Heatmaps showing pairwise comparison of MPRA activity for constructs ( mRNA / ) between donors - left panel : resting CD4 T ; right panel : stimulated CD4 T .

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D Heatmaps showing pairwise comparison of MPRA activity for all constructs (mRNA/DNA) between donors - left panel: resting CD4 T cells; right panel: stimulated CD4 T cells.


B Activity of each allele at 10 positive control SNPs 1 negative control SNP in stimulated T . GATA1 , NF - κB , RUNX1 constructs were designed to include a binding site for indicated transcription factors ( + ) or with that site disrupted ( - ) . Fixed effects meta - P value is shown : * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 . Box whisker plots represent median IQR ( box ) min - to - max ( whiskers ) .

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B Activity of each allele at 10 positive control SNPs and 1 negative control SNP in stimulated T cells. GATA1, NF-κB, and RUNX1 constructs were designed to include a binding site for the indicated transcription factors (+) or with that site disrupted (-). Fixed effects meta-analysis P value is shown: *<0.05; **<0.01, ***<0.001, ****<0.0001. Box and whisker plots represent median and IQR (box) and min-to-max (whiskers).


D Expression - modulating effect of each SNP [ log2 ( OR ) ] as measured in MPRA validation experiments . OR were calculated using median activity of allelic constructs are presented with respect to risk allele ( luciferase : n = 5 , MPRA : n = 12 ) .

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D Expression-modulating effect of each SNP [log2(OR)] as measured in MPRA and validation experiments. OR were calculated using the median activity of allelic constructs and are presented with respect to the risk allele (luciferase: n=5, MPRA: n=12).


A Pie chart depicting fine - mapping results ( Huang et al , 2017 ) ( posterior probabilities ) for an IBD - associated locus on 21q21 ( left panel ) . MPRA results in resting ( centre panel ) stimulated CD4 T ( right panel ) showing that putative causal variant has significant expression - modulating effect .

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A Pie chart depicting fine-mapping results (Huang et al, 2017) (posterior probabilities) for an IBD-associated locus on 21q21 (left panel). MPRA results in resting (centre panel) and stimulated CD4 T cells (right panel) showing that the putative causal variant has significant expression-modulating effect.


B Pie chart depicting Bayesian fine - mapping results for an AS - associated locus on 2p15 ( left panel ) . MPRA results in stimulated T ( right panel ) showing that rs6759298 has a significant expression - modulating effect ( strongest of any variant at this locus ) while other candidate SNPs have negligible effects .

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B Pie chart depicting Bayesian fine-mapping results for an AS-associated locus on 2p15 (left panel). MPRA results in stimulated T cells (right panel) showing that rs6759298 has a significant expression-modulating effect (the strongest of any variant at this locus) while the other candidate SNPs have negligible effects.


C GWAS results ( Liu et al , 2015 ) at a Crohn ' s disease multiple sclerosis - associated locus on 6p23 ( left panel ) . MPRA for candidate SNPs in stimulated T ( right panel ) identifying a SNP ( rs34421390 ) with by far greatest expression - modulating effect at this locus ( blue , risk allele reduces expression ; red , risk allele increases expression ) . Dotted horizontal represents significance threshold ( corrected for multiple - testing ) .

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C GWAS results (Liu et al, 2015) at a Crohn's disease and multiple sclerosis-associated locus on 6p23 (left panel). MPRA for candidate SNPs in stimulated T cells (right panel) identifying a SNP (rs34421390) with by far the greatest expression-modulating effect at this locus (blue, risk allele reduces expression; red, risk allele increases expression). Dotted horizontal line represents significance threshold (corrected for multiple-testing).


D GWAS results at a Type 1 Diabetes - associated locus on 14q32 ( Onengut - Gumuscu et al , 2015 ; left panel ) . MPRA for candidate SNPs in stimulated T ( right panel ) . construct with largest most significant effect contains risk alleles for 2 SNPs ( rs1988588 rs3902659 ) , each of which has a smaller effect when tested individually ( position indicated by vertical dotted ) . Dotted horizontal represents significance threshold ( corrected for multiple - testing ) .

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D GWAS results at a Type 1 Diabetes-associated locus on 14q32 (Onengut-Gumuscu et al, 2015; left panel). MPRA for the candidate SNPs in stimulated T cells (right panel). The construct with the largest and most significant effect contains the risk alleles for 2 SNPs (rs1988588 and rs3902659), each of which has a smaller effect when tested individually (position indicated by vertical dotted line). Dotted horizontal line represents significance threshold (corrected for multiple-testing).


A IBD GWAS results ( Liu et al , 2015 ) at a multi - disease - associated locus on chromosome 6q23 .

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A IBD GWAS results (Liu et al, 2015) at a multi-disease-associated locus on chromosome 6q23.


C A single variant ( rs6927172 ) has largest most significant expression - modulating activity in resting ( left panel ) stimulated CD4 T ( right panel ) with risk allele reducing transcription . Plots represent median IQR ( box ) min - to - max ( whiskers ) . FDR - corrected meta - P value shown .

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C A single variant (rs6927172) has the largest and most significant expression-modulating activity in resting (left panel) and stimulated CD4 T cells (right panel) with the risk allele reducing transcription. Plots represent median and IQR (box) and min-to-max (whiskers). FDR-corrected meta-analysis P value shown.


E Allele - specific NF - κB binding in CD4 T from rs6927172 heterozygotes , demonstrating reduced NF - κB binding to risk allele following stimulation ( n = 8 ; one - sample t - , two - ) .

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E Allele-specific NF-κB binding in CD4 T cells from rs6927172 heterozygotes, demonstrating reduced NF-κB binding to the risk allele following stimulation (n=8; one-sample t-test, two-tailed).


F Allele - specific expression of enhancer in heterozygous CD4 T . used for technical control ( n = 6 ; paired t - ; two - ) .

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F Allele-specific expression of enhancer RNA in heterozygous CD4 T cells. DNA used for technical control (n=6; paired t-test; two-tailed).


G Genome - wide H3K27ac ChIP - seq in stimulated CD4 T from major - minor - allele homozygotes at rs6927172 ( n = 6 ) . Upper panels show input - normalised H3K27ac signals plotted against enhancer rank . Super - enhancers are defined above inflection point of curve . Lower panels show H3K27ac reads from a major ( left ) a minor ( risk ) allele homozygote ( right ) in a 9kb window around rs6927172 .

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G Genome-wide H3K27ac ChIP-seq in stimulated CD4 T cells from major- and minor-allele homozygotes at rs6927172 (n=6). Upper panels show input-normalised H3K27ac signals plotted against enhancer rank. Super-enhancers are defined above the inflection point of the curve. Lower panels show H3K27ac reads from a major (left) and a minor (risk) allele homozygote (right) in a 9kb window around rs6927172.


I Expression of genes on 6q23 in CD4 T from 131 with active IBD , stratified by rs6927172 genotype ( qPCR ; one - way ANOVA ) . Error bars represent SD . Expression of IL20RA IL22RAR2 not detected .

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I Expression of genes on 6q23 in CD4 T cells from 131 patients with active IBD, stratified by rs6927172 genotype (qPCR; one-way ANOVA). Error bars represent SD. Expression of IL20RA and IL22RAR2 not detected.


B CD4 T editing efficiency for indicated combinations of 5 ' 3 ' gRNAs ( n = 6 for DB , DH , FH , n = 2 for FB ) . Distribution of indels assessed using ICE .

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B Primary CD4 T cell editing efficiency for indicated combinations of 5' and 3' gRNAs (n=6 for DB, DH, FH, and n=2 for FB). Distribution of indels assessed using ICE.


D Expression of genes on 6q23 in EU - containing mRNA ( EU added at time of stimulation ) showing that deletion of NF - κB binding site specifically reduces transcription of TNFAIP3 , but not other genes at this locus ( n = 6 ; one sample t - ) . Representative data from DH gRNA combination .

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D Expression of genes on 6q23 in EU-containing mRNA (EU added at time of stimulation) showing that deletion of the NF-κB binding site specifically reduces transcription of TNFAIP3, but not other genes at this locus (n=6; one sample t-test). Representative data from the DH gRNA combination.


E Expression of CD69 , an activation marker , following CRISPR editing with indicated gRNA combinations or non - targeting ( negative ) control ( NTC ) - data shown for ATTO - 550 positive ( RNP - containing ) negative ( n = 6 ; paired t - , one - ) . Inset flow cytometry plot depicting representative gating of ATTO - 550 positive negative .

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E Expression of CD69, an activation marker, following CRISPR editing with indicated gRNA combinations or the non-targeting (negative) control (NTC) - data shown for ATTO-550 positive (RNP-containing) and negative cells (n=6; paired t-test, one-tailed). Inset flow cytometry plot depicting representative gating of ATTO-550 positive and negative cells.


F Correlation between editing efficiency ( total indel rate ) of phosphorylated IκBα in CD4 T ( normalised to mean in NTC ; n = 9 , linear regression ) .

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F Correlation between editing efficiency (total indel rate) and levels of phosphorylated IκBα in CD4 T cells (normalised to the mean fluorescence intensity in NTC; n=9, linear regression).


G Secretion of effector cytokines following deletion of NF - κB binding site - Th1 ( left panel , IFNγ ) , Th17 ( centre panel , IL - 17A ) Th2 subsets ( right panel , IL - 4 ) ( n = 6 , paired t - , one - ) .

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G Secretion of effector cytokines following deletion of the NF-κB binding site - Th1 (left panel, IFNγ), Th17 (centre panel, IL-17A) and Th2 subsets (right panel, IL-4) (n=6, paired t-test, one-tailed).


d , between CPD ( copy per droplets , normalized to γ − actin Hprt reference genes relative expression ) of structural [ N , nucleocapsid ] non - structural [ IP4 : RdRp , - dependent polymerase ] viral gene expression determined by digital droplet PCR ( ddPCR ) in nasal turbinates in lungs at 4 dpi . Data information : Horizontal lines indicate medians . p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney M : hamsters ; F : hamsters . Data were obtained from two independent experiments for each sex .

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d, ratio between the CPD (copy per droplets, normalized to γ−actin and Hprt reference genes relative expression) of structural [N, nucleocapsid] and non-structural [IP4: RdRp, RNA-dependent RNA polymerase] viral gene expression determined by digital droplet PCR (ddPCR) in the nasal turbinates and in the lungs at 4 dpi. Data information: Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test M: male hamsters; F: female hamsters. Data were obtained from two independent experiments for each sex.


f , cytokines chemokines transcripts in nasal turbinates at 4dpi in SARS - CoV - 2 infected hamsters , treated with saline or with 400 µg / kg ivermectin . Data information : Horizontal lines indicate medians . p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney M : hamsters ; F : hamsters . Data were obtained from two independent experiments for each sex .

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f, cytokines and chemokines transcripts in the nasal turbinates at 4dpi in male and female SARS-CoV-2 infected hamsters, treated with saline or with 400 µg/kg ivermectin. Data information: Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test M: male hamsters; F: female hamsters. Data were obtained from two independent experiments for each sex.


b , Validation targets in lung at 4 dpi . Horizontal lines indicate medians . p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney . Data information : M : hamsters ; F : hamsters . Data were obtained from two independent experiments for each sex .

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b, Validation targets in the lung at 4 dpi. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test. Data information: M: male hamsters; F: female hamsters. Data were obtained from two independent experiments for each sex.


a , representative histopathology photomicrographies of lungs according to different groups : mock _ saline , CoV _ saline CoV _ ivermectin . Top panels : whole lung sections . Bottom panels : high magnification . CoV _ saline section exhibits important congestion ( * ) , edema associated with few mononuclear ( arrowheads ) . Note thickening of alveolar walls . CoV _ ivermectin section exhibits important amounts of mononuclear ( black arrowheads ) less marked signs of congestion or edema . Hematoxylin Eosin . Scale bars = 1 mm ( top panels ) 20 μm ( bottom panels ) .

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a, representative histopathology photomicrographies of lungs according to the different groups: mock_saline, CoV_saline and CoV_ivermectin. Top panels: whole lung sections. Bottom panels: high magnification. CoV_saline section exhibits important congestion (*), edema associated with few mononuclear cells (white arrowheads). Note the thickening of the alveolar walls. CoV_ivermectin section exhibits important amounts of mononuclear cells (black arrowheads) and less marked signs of congestion or edema. Hematoxylin and Eosin. Scale bars = 1 mm (top panels) and 20 μm (bottom panels).


b , representative immunofluorescence photomicrographies of neutrophils ( Ly - 6G ) , monocytes / macrophages ( Iba1 ) , M2 macrophages ( Arg1 ) SARS - CoV - 2 ( NP ) in lung . Scale bars = 50 μm .

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b, representative immunofluorescence photomicrographies of neutrophils (Ly-6G), monocytes/macrophages (Iba1), M2 macrophages (Arg1) and SARS-CoV-2 (NP) in the lung. Scale bars = 50 μm.


c , of Iba1 + , Arg1 + Iba1 + Arg1 + in lungs . mock _ saline n = 3 ( ) , mock _ ivermectin n = 4 ( ) , CoV _ saline n = 9 ( 6 3 ) , CoV _ ivermectin n = 6 ( 4 2 ) . Data information : M : hamsters ; F : hamsters . Horizontal lines indicate medians . p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney

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c, quantification of Iba1+ cells, Arg1+ cells and Iba1+Arg1+ cells in the lungs. mock_saline n=3 (males), mock_ivermectin n=4 (males), CoV_saline n=9 (6 males and 3 females), CoV_ivermectin n=6 (4 males and 2 females). Data information: M: male hamsters; F: female hamsters. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test


B . qRT - PCR expression of lncMN2 isoforms ( left panel ) embedded miRNAs ( right panel ) in mESC EBs differentiated for 6 days ( EB6 ) . Data , that are expressed as mean ( error bars represent SD ) , were normalized over Atp50 mRNA ( left ) snoRNA U25 ( right ) reported as fold over control ( mESC ) , set as 1 . N = 3 biological replicates . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

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B. qRT-PCR expression analysis of lncMN2 isoforms (left panel) and embedded miRNAs (right panel) in mESC and EBs differentiated for 6 days (EB6). Data, that are expressed as mean (error bars represent SD), were normalized over Atp50 mRNA (left) and snoRNA U25 (right) levels and reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


C . qRT - PCR expression of lncMN2 - 202 / 203 / 204 isoforms ( left panel ) , miR - 325 - 3p ( middle panel ) miR - 384 - 5p ( right panel ) in WT , KO1 KO2 EB6 . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

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C. qRT-PCR expression analysis of lncMN2-202/203/204 isoforms (left panel), miR-325-3p (middle panel) and miR-384-5p (right panel) in WT, KO1 and KO2 EB6. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


D . Cytofluorimetric of from dissociated EB6 . left diagram reports distribution of ( of peak ) according to GFP , from WT ( gray ) , KO1 ( orange ) KO2 ( blue ) EB . In histogram aside , of GFP + in WT , KO1 KO2 is reported . Data are expressed as mean error bars represent SD . N = 3 biological replicates . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

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D. Cytofluorimetric analysis of cells from dissociated EB6. The left diagram reports the distribution of cells (percentage of the peak) according to GFP levels, from WT (gray), KO1 (orange) and KO2 (blue) EB. In the histogram aside, the percentage of GFP+ cells in WT, KO1 and KO2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).