Figure 5HeLa cells co - transfected with H2B - RFP ( a chromosome marker ) , PACT - Ce3 - FRB , and YFP - FKBP - Luciferase were sequentially incubated with thymidine ( 2 mM ) for 16 - 18 hr and RO3306 ( 2 . 5 ng / mL ) for 12 hr to synchronize cells in G2 / M phase ( Interphase cells in left panel ) . The probe trapping experiment in interphase cells was performed right after RO3306 washout . Anaphase cells were chosen for probe trapping experiment 45 - 60 min after RO3306 washout . Transfected cells in interphase or anaphase were treated with 100 nM rapamycin ( Rapa ) . The centrosome regions are shown in lower panels . The normalized fluorescence intensity of YFP - FKBP - Luciferase accumulation at centrosomes in interphase ( Green , n = 12 cells ) or anaphase ( Red , n = 10 cells ) cells upon rapamycin ( 100 nM ) treatment . HeLa cells co - transfected with H2B - RFP ( a chromosome marker ) , PACT - Ce3 - FRB , and YFP - FKBP - Luciferase were synchronized in anaphase with or without CK666 treatment ( 0 . 4 mM ) . Rapamycin ( 100 nM ) was added for 5 min to trigger the YFP - FKBP - Luciferase trapping at centrosomes . The right panel is the enlarged images of centrosome regions and arrows indicated centrosome sites . The percentage of cells in D exhibiting probe translocation . Data are shown as mean ± S . E . M . ( n = 47 and 31 cells in DMSO and CK666 treated groups , respectively ; 3 - 4 independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5HeLa cells co-transfected with H2B-RFP (a chromosome marker), PACT-Ce3-FRB, and YFP-FKBP-Luciferase were sequentially incubated with thymidine (2 mM) for 16-18 hr and RO3306 (2.5 ng/mL) for 12 hr to synchronize cells in G2/M phase (Interphase cells in left panel). The probe trapping experiment in interphase cells was performed right after RO3306 washout. Anaphase cells were chosen for probe trapping experiment 45-60 min after RO3306 washout. Transfected cells in interphase or anaphase were treated with 100 nM rapamycin (Rapa). The centrosome regions are shown in lower panels. The normalized fluorescence intensity of YFP-FKBP-Luciferase accumulation at centrosomes in interphase (Green, n = 12 cells) or anaphase (Red, n =10 cells) cells upon rapamycin (100 nM) treatment.HeLa cells co-transfected with H2B-RFP (a chromosome marker), PACT-Ce3-FRB, and YFP-FKBP-Luciferase were synchronized in anaphase with or without CK666 treatment (0.4 mM). Rapamycin (100 nM) was added for 5 min to trigger the YFP-FKBP-Luciferase trapping at centrosomes. The right panel is the enlarged images of centrosome regions and arrows indicated centrosome sites. The percentage of cells in D exhibiting probe translocation. Data are shown as mean ± S.E.M. (n = 47 and 31 cells in DMSO and CK666 treated groups, respectively; 3-4 independent experiments).


Figure 6U2Os cells were incubated with antibodies against 𝑟 - tubulin ( green ; a marker of 𝑟 - TuRC ) or Pericentrin ( red ; a marker of centrosomes ) in the absence ( 0 . 1 % DMSO ) or presence of CK666 treatment ( 0 . 4 mM ) for 2 hr . The right panel shows magnified images of the indicated region of interest ( ROI ) . S The normalized intensity of 𝑟 - tubulin at centrosome in control ( 0 . 1 % DMSO , 2 hr ) or CK666 ( 0 . 4 mM , 2 hr ) - treated cells . Individual data points and the mean ± SD ( in red ) are shown ( n = 150 and 150 cells in DMSO and CK666 - treated group , respectively ; four independent experiments ) . U2Os cells transfected with GCP4 - GFP were treated with or without CK666 ( 0 . 4 mM , 2 hr ) and then observed for the indicated time after photobleaching . The mobile fraction percentage ( left ) and mobile recovery half - time ( right ) of GCP4 - GFP in FRAP experiments ( c ) . Data ( red ) are shown as meanU2Os cells expressing EB1 - mNeon were incubated with 0 . 1 % DMSO or 0 . 4 mM CK666 for 2 hr . Images were captured over 150 sec and were overlaid to show the overall tracks of the microtubules . The insets show enlarged images of the centrosomal regions . F , G . The number of microtubule tracks that emanate from centrosomes ( F ) and the microtubule elongation rate ( G ) in the absence and presence of CK666 treatment ( 0 . 4 mM ) . Individual data points and the mean ± SD ( in red ) are shown ( n = 78449 and 54283 microtubule tracks from 27 and 22 cells in the DMSO - and CK666 - treated groups , respectively ; three independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6U2Os cells were incubated with antibodies against 𝑟-tubulin (green; a marker of 𝑟-TuRC) or Pericentrin (red; a marker of centrosomes) in the absence (0.1% DMSO) or presence of CK666 treatment (0.4 mM) for 2 hr. The right panel shows magnified images of the indicated region of interest (ROI). S The normalized intensity of 𝑟-tubulin at centrosome in control (0.1% DMSO, 2 hr) or CK666 (0.4 mM, 2 hr)-treated cells. Individual data points and the mean ± SD (in red) are shown (n = 150 and 150 cells in DMSO and CK666-treated group, respectively; four independent experiments).U2Os cells transfected with GCP4-GFP were treated with or without CK666 (0.4 mM, 2 hr) and then observed for the indicated time after photobleaching. The mobile fraction percentage (left) and mobile recovery half-time (right) of GCP4- GFP in FRAP experiments (c). Data (red) are shown as meanU2Os cells expressing EB1-mNeon were incubated with 0.1% DMSO or 0.4 mM CK666 for 2 hr. Images were captured over 150 sec and were overlaid to show the overall tracks of the microtubules. The insets show enlarged images of the centrosomal regions. F, G. The number of microtubule tracks that emanate from centrosomes (F) and the microtubule elongation rate (G) in the absence and presence of CK666 treatment (0.4 mM). Individual data points and the mean ± SD (in red) are shown (n = 78449 and 54283 microtubule tracks from 27 and 22 cells in the DMSO- and CK666-treated groups, respectively; three independent experiments).


Figure 1 . ( A - C ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS ( Earle ' s Balanced Salt Solution ) for 30 min . Total membranes ( mem ) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells . Reactions contained the indicated concentrations of PI3K inhibitor ( PI3KI ) 3 - methyladenine ( 3 - MA ) ( B ) or FYVE protein ( C ) . A diagram of the experimental scheme is shown in ( A ) . RPN1 , Ribophorin 1 ( D , E ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS in the absence or presence of 20 nM wortmannin ( Wtm ) or 10 mM 3 - methyladenine ( 3 - MA ) for 30 min . Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K × g , 25K × g and 100K × g pellet fractions followed by a lipidation assay as above ( E ) . A diagram is shown in ( D ) . ( F , G ) Atg5 KO MEFs were starved for 30 min . Membranes in the 25K × g and 100K × g pellets from a differential centrifugation were collected as described above . A similar lipidation assay was performed in the presence of indicated concentrations ( Conc in G ) of 3 - MA , wortmannin ( F ) and FYVE protein as well as a PI3P binding - deficient FYVE mutant protein ( C / S ) ( G ) . Quantification of lipidation activity is shown as the ratio of LC3 - II to LC3 - I ( II / I ) .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS (Earle's Balanced Salt Solution) for 30 min. Total membranes (mem) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells. Reactions contained the indicated concentrations of PI3K inhibitor (PI3KI) 3-methyladenine (3-MA) (B) or FYVE protein (C). A diagram of the experimental scheme is shown in (A). RPN1, Ribophorin 1(D, E) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS in the absence or presence of 20 nM wortmannin (Wtm) or 10 mM 3-methyladenine (3-MA) for 30 min. Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K ×g, 25K ×g and 100K ×g pellet fractions followed by a lipidation assay as above (E). A diagram is shown in (D).(F, G) Atg5 KO MEFs were starved for 30 min. Membranes in the 25K ×g and 100K ×g pellets from a differential centrifugation were collected as described above. A similar lipidation assay was performed in the presence of indicated concentrations (Conc in G) of 3-MA, wortmannin (F) and FYVE protein as well as a PI3P binding-deficient FYVE mutant protein (C/S) (G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).


Figure 2 . ( B ) A small vesicle generation assay as above was performed with the indicated conditions . Slowly - sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction . 3 - MA , 5 mM ; Wortmannin , 20 nM ; S , starved Atg5 KO MEF cytosol ; N , untreated Atg5 KO MEF cytosol ; − / AP , in the absence of ATP regeneration and GTP and in the presence of apyrase ( AP ) . ( D ) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K , P and F1 - 4 fractions ( upper four panels : Small vesicle ) . SDS - PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction ( lower five panels : Fraction input ) . TFR , transferrin receptor ( E ) A small vesicle generation assay as shown in ( A ) was performed in the presence of indicated proteins or drugs . Slowly - sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST , H79G ( Sar1A ( H79G ) , 0 . 7 μM ) , wortmannin ( 20 nM ) , or BFA ( brefeldin A , 0 . 5 μg / ml ) . ( F ) A small vesicle generation assay as shown in ( A ) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A ( H79G ) or 3 - MA followed by a lipidation reaction . ( G ) Atg5 KO MEFs were starved for 30 min . Differential centrifugation as shown in Figure 1D was performed to collect the 25K × g and 100K × g pellet fractions . The LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 ( H79G ) . Quantification of lipidation activity is shown as the ratio of LC3 - II to LC3 - I ( II / I ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.(B) A small vesicle generation assay as above was performed with the indicated conditions. Slowly-sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction. 3-MA, 5 mM; Wortmannin, 20 nM; S, starved Atg5 KO MEF cytosol; N, untreated Atg5 KO MEF cytosol; −/AP, in the absence of ATP regeneration and GTP and in the presence of apyrase (AP).(D) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K, P and F1-4 fractions (upper four panels: Small vesicle). SDS-PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction (lower five panels: Fraction input). TFR, transferrin receptor(E) A small vesicle generation assay as shown in (A) was performed in the presence of indicated proteins or drugs. Slowly-sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST, H79G (Sar1A (H79G), 0.7 μM), wortmannin (20 nM), or BFA (brefeldin A, 0.5 μg/ml).(F) A small vesicle generation assay as shown in (A) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A (H79G) or 3-MA followed by a lipidation reaction.(G) Atg5 KO MEFs were starved for 30 min. Differential centrifugation as shown in Figure 1D was performed to collect the 25K ×g and 100K ×g pellet fractions. The LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 (H79G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).


Figure 3 . ( A ) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min . Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 ( F2 in Figure 2C , D ) of the OptiPrep gradient in the three - step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes . ( B ) Atg5 KO MEFs were transfected with a plasmid encoding an HA - tagged SEC31A . After transfection ( 24 hr ) , the cells were treated as shown in ( A ) . Immunofluorescence was performed with anti - ERGIC53 and anti - HA antibodies and the cells were examined by confocal microscopy . Bar : 10 μM ( C ) Quantification of SEC31A overlapped with ERGIC53 shown in ( B ) using the Pearson ' s correlation coefficient ( PCC ) . Error bars represent standard deviations from ∼ 20 cells in three independent experiments . p values were calculated by T - test .

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

Figure 3.(A) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min. Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 (F2 in Figure 2C,D) of the OptiPrep gradient in the three-step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes.(B) Atg5 KO MEFs were transfected with a plasmid encoding an HA-tagged SEC31A. After transfection (24 hr), the cells were treated as shown in (A). Immunofluorescence was performed with anti-ERGIC53 and anti-HA antibodies and the cells were examined by confocal microscopy. Bar: 10 μM (C) Quantification of SEC31A overlapped with ERGIC53 shown in (B) using the Pearson's correlation coefficient (PCC). Error bars represent standard deviations from ∼20 cells in three independent experiments. p values were calculated by T-test.


Figure 1 ( A ) Super - resolved average intensity ( bar : high 8 to low 0 density ) , average diffusion coefficient ( bar : Log10D high 1 to low - 5 mobility ) and trajectory maps ( 16000 frames ) of BoNT / Aiwt - At647N on the plasma membrane ( uPAINT ) and following endocytosis ( sdTIM ) . B , C Number of BoNT / Aiwt - At647N and mutant trajectories ( B ) on the plasma membrane ( uPAINT ) and ( C ) following endocytosis ( sdTIM ) . N = 17 technical replicates / condition from 5 - 6 independent biological replicates ( E ) EM analysis of endocytosed HRP - tagged BoNT / Ai . Representative images show cytochemically stained HRP precipitate ( arrowheads ) . Endo / lysosomes , multivesicular bodies ( MVB ) , structures with the morphology of autophagosomes ( AP ) , and ( i ) presynaptic ( Ps ) and ( ii ) axonal ( Ax ) plasma membrane ( PM ) . Postsynaptic density ( asterisks ) , non - stained endocytic compartments ( arrows ) and background binding on the PM ( arrows in ii ) are indicated . Scale bars 100 nm . F , G ( F ) Percentage of total BoNT / Aiwt - HRP and indicated mutants localized on the PM or in the indicated endocytic structures , and ( G ) the average sectional area of the HRP - stained endocytic compartments . ( F , G ) n = 60 electron micrographs / condition from 3 independent biological replicates .

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 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 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 1(A) Super-resolved average intensity (bar: high 8 to low 0 density), average diffusion coefficient (bar: Log10D high 1 to low -5 mobility) and trajectory maps (16000 frames) of BoNT/Aiwt-At647N on the plasma membrane (uPAINT) and following endocytosis (sdTIM).B, C Number of BoNT/Aiwt-At647N and mutant trajectories (B) on the plasma membrane (uPAINT) and (C) following endocytosis (sdTIM). N = 17 technical replicates/ condition from 5-6 independent biological replicates(E) EM analysis of endocytosed HRP-tagged BoNT/Ai. Representative images show cytochemically stained HRP precipitate (arrowheads). Endo/lysosomes, multivesicular bodies (MVB), structures with the morphology of autophagosomes (AP), and (i) presynaptic (Ps) and (ii) axonal (Ax) plasma membrane (PM). Postsynaptic density (asterisks), non-stained endocytic compartments (arrows) and background binding on the PM (arrows in ii) are indicated. Scale bars 100 nm.F, G (F) Percentage of total BoNT/Aiwt-HRP and indicated mutants localized on the PM or in the indicated endocytic structures, and (G) the average sectional area of the HRP-stained endocytic compartments. (F,G) n = 60 electron micrographs/ condition from 3 independent biological replicates.


Figure 2A , B Low resolution images and the corresponding super - resolved uPAINT average intensity ( bar : high 8 to low 0 density ) , average diffusion coefficient ( bar : Log10D high 1 to low - 5 mobility ) and trajectory maps of SV2Awt - pH / At565nb in ( A ) control conditions ( high K + ; Ctrl ) , and ( B ) following treatment with 100 pM BoNT / Aiwt - At647N ( supplemented in high K + ) . ( C ) Scatter plots of the average diffusion coefficient ( µm2 s - 1 ) single - molecule mobility of SV2Awt - pH / At565nb and SV2AT84A - pH / 565nb in the control condition ( high K + ; Ctrl ) and following treatment with 100 pM BoNT / Aiwt - At647N or BoNT / AiSV2 - At647N ( supplemented in high K + ) . ( D ) Scatter plots of the average diffusion coefficient ( µm2 s - 1 ) single - molecule mobility of Syt1wt - pH / At565nb , Syt1K362A , K328A - pH / 565nb and Syt1K52A - pH / At565nb in the control condition ( high K + ; Ctrl ) and following treatment with 100 pM BoNT / Aiwt - At647N ( supplemented in high K + ) .

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 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

Figure 2A, B Low resolution images and the corresponding super-resolved uPAINT average intensity (bar: high 8 to low 0 density), average diffusion coefficient (bar: Log10D high 1 to low -5 mobility) and trajectory maps of SV2Awt-pH/At565nb in (A) control conditions (high K+; Ctrl), and (B) following treatment with 100 pM BoNT/Aiwt-At647N (supplemented in high K+).(C) Scatter plots of the average diffusion coefficient (µm2 s-1) single-molecule mobility of SV2Awt-pH/At565nb and SV2AT84A-pH/565nb in the control condition (high K+; Ctrl) and following treatment with 100 pM BoNT/Aiwt-At647N or BoNT/AiSV2-At647N (supplemented in high K+). (D) Scatter plots of the average diffusion coefficient (µm2 s-1) single-molecule mobility of Syt1wt-pH/At565nb, Syt1K362A,K328A-pH/565nb and Syt1K52A-pH/At565nb in the control condition (high K+; Ctrl) and following treatment with 100 pM BoNT/Aiwt-At647N (supplemented in high K+).


Figure 3A - C ( A ) Representative low - resolution image of SV2Awt - pH ( green ) , and corresponding dual - colour super - resolved uPAINT trajectories of SV2Awt - pH / At565nb ( cyan ) imaged simultaneously with BoNT / Aiwt - At647N ( magenta ) . Regions ( i - ii ) are magnified below , and ( iii ) is magnified in ( B ) showing a representative time - lapse sequence of the single molecules of SV2Awt - pH / At565nb ( cyan ) and BoNT / Aiwt - At647N ( magenta ) detected coincidentally in a neuronal projection . The 10 x 10 pixel boxed region ( iv ) is magnified in ( C ) , showing frame - by - frame coincidental detection of BoNT / Aiwt - At647N and SV2Awt - pH / At565nb obtained using a 50 Hz ( 20 ms exposure time ) 16 , 000 frame acquisition as indicated . H , I Representative images of NASTIC nanocluster analysis of SV2Awt - pH / At565nb in ( H ) the control condition ( high K + ; Ctrl ) and ( I ) following high K + stimulation in the presence of 100 pM BoNT / Aiwt - At647N , with 2D kernel density estimation ( KDE , high density in red ) , instantaneous Log10 diffusion coefficient map ( low mobility in red ) , and nanoclusters with their respective trajectories ( white ) and centroids ( orange ) shown . Boxed areas are magnified on the right , with arrowheads indicating nanoclusters . J - N Quantification of the ( J ) SV2Awt - pH / At565nb , ( K ) SV2AT84A - pH / At565nb , ( L ) Syt1wt - pH / At565nb , ( M ) Syt1K326A , K328A - pH / At565nb and ( N ) Syt1K52A - pH / At565nb nanoclustering in control conditions ( high K + ; Ctrl ) and following BoNT / Aiwt - At647N treatment ( diluted in high K + ) . ( J - N ) N = 11 - 19 technical replicates from 3 - 5 independent biological replicates ,

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Figure 3A-C (A) Representative low-resolution image of SV2Awt-pH (green), and corresponding dual-colour super-resolved uPAINT trajectories of SV2Awt-pH/At565nb (cyan) imaged simultaneously with BoNT/Aiwt-At647N (magenta). Regions (i-ii) are magnified below, and (iii) is magnified in (B) showing a representative time-lapse sequence of the single molecules of SV2Awt-pH/At565nb (cyan) and BoNT/Aiwt-At647N (magenta) detected coincidentally in a neuronal projection. The 10 x 10 pixel boxed region (iv) is magnified in (C), showing frame-by-frame coincidental detection of BoNT/Aiwt-At647N and SV2Awt-pH/At565nb obtained using a 50 Hz (20 ms exposure time) 16,000 frame acquisition as indicated.H, I Representative images of NASTIC nanocluster analysis of SV2Awt-pH/At565nb in (H) the control condition (high K+; Ctrl) and (I) following high K+ stimulation in the presence of 100 pM BoNT/Aiwt-At647N, with 2D kernel density estimation (KDE, high density in red), instantaneous Log10 diffusion coefficient map (low mobility in red), and nanoclusters with their respective trajectories (white) and centroids (orange) shown. Boxed areas are magnified on the right, with arrowheads indicating nanoclusters.J-N Quantification of the (J) SV2Awt-pH/At565nb, (K) SV2AT84A-pH/At565nb, (L) Syt1wt-pH/At565nb, (M) Syt1K326A,K328A-pH/At565nb and (N) Syt1K52A-pH/At565nb nanoclustering in control conditions (high K+; Ctrl) and following BoNT/Aiwt-At647N treatment (diluted in high K+). (J-N) N = 11-19 technical replicates from 3-5 independent biological replicates,


Figure 4 ( A ) Representative confocal Z - stack sum projections of neurons transduced with TagBFP2 - expressing ( blue ) control ( ctrl ; non - targeting sgRNA ) and CRISPRi Syt1 KD ( Syt1 sgRNA1 - 3 ) lentiviruses . Neurons were treated for 30 min with 10 units of AbobotulinumtoxinA , and immunostained for endogenous Syt1 ( yellow ) and BoNT / A - cleaved SNAP - 25 ( SNAP - 25 / A ; magenta ) . Boxed regions ( i - iv ) are magnified on the right , with neuronal somas outlined by dashed lines for clarity . B , C Quantification of mean fluorescence intensity ( MFI ) of endogenous ( B ) Syt1 , and ( C ) cleaved SNAP - 25 ( SNAP - 25 / A ) , in control and Syt1 sgRNA1 - 3 KD neurons following AbobotulinumtoxinA treatment . ( B - C ) n = 21 technical replicates / condition from 2 independent biological replicates . Statistical significance was assessed using a non - parametric Kruskal - Wallis multiple comparison testH , I Quantification of ( H ) Syt1 and ( I ) SNAP - 25 / A MFI in non - transduced neurons ( neurons negative for TagBFP2 ) with and without pLenti6 . 3 - Syt1wt - pH expression following AbobotulinumtoxinA treatment ( 10 units , 30 min . ) n = 3 technical replicates / condition from one biological experiment .

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Figure 4(A) Representative confocal Z-stack sum projections of neurons transduced with TagBFP2-expressing (blue) control (ctrl; non-targeting sgRNA) and CRISPRi Syt1 KD (Syt1 sgRNA1-3) lentiviruses. Neurons were treated for 30 min with 10 units of AbobotulinumtoxinA, and immunostained for endogenous Syt1 (yellow) and BoNT/A-cleaved SNAP-25 (SNAP-25/A; magenta). Boxed regions (i-iv) are magnified on the right, with neuronal somas outlined by dashed lines for clarity. B, C Quantification of mean fluorescence intensity (MFI) of endogenous (B) Syt1, and (C) cleaved SNAP-25 (SNAP-25/A), in control and Syt1 sgRNA1-3 KD neurons following AbobotulinumtoxinA treatment. (B-C) n = 21 technical replicates/ condition from 2 independent biological replicates. Statistical significance was assessed using a non-parametric Kruskal-Wallis multiple comparison testH, I Quantification of (H) Syt1 and (I) SNAP-25/A MFI in non-transduced neurons (neurons negative for TagBFP2) with and without pLenti6.3-Syt1wt-pH expression following AbobotulinumtoxinA treatment (10 units, 30 min.) n = 3 technical replicates/ condition from one biological experiment.


Figure 5 ( A ) Representative patch - clamp spontaneous miniature inhibitory postsynaptic current ( mPSC ) recordings in naïve neurons , and in control ( non - targeting ) and Syt1 KD ( CRISPRi sgRNA1 ) neurons following a 30 min treatment with AbobotulinumtoxinA ( 10 units ) . B , C Quantification of mPSC ( B ) peak amplitudes and ( C ) frequencies for indicated conditions . Error bars are shown as standard error of the mean ( ± SEM ) . N = 5 - 12 technical replicates / condition from 2 independent biological replicates in ( B , C ) statistical significance was assessed using a non - parametric Kruskal - Wallis multiple comparison test ( B ) , an ordinary one - way ANOVA multiple comparison test ( C ) ( D ) EM analysis of endocytosed BoNT / Aiwt - HRP following lentiviral control ( ctrl ; non - targeting sgRNA ) and Syt1 sgRNA1 CRISPRi KD transduction . HRP precipitate in synaptic vesicles ( SVs ; arrowheads ) and tubular structures ( arrows ) in presynapses ( Ps ) are indicated . The postsynaptic density is marked with an asterisk . ( E ) Percentage of total BoNT / Aiwt - HRP localized in SVs , endo / lysosomes ( E ) , tubular structures ( T ) , multivesicular bodies ( MVB ) , structures resembling autophagosomes ( AP ) and on the plasma membrane ( PM ) in the indicated conditions . n = 56 regions of interest / condition from 2 independent biological replicates ( E ) two - way ANOVA multiple comparison test ( E ) .

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Figure 5(A) Representative patch-clamp spontaneous miniature inhibitory postsynaptic current (mPSC) recordings in naïve neurons, and in control (non-targeting) and Syt1 KD (CRISPRi sgRNA1) neurons following a 30 min treatment with AbobotulinumtoxinA (10 units). B, C Quantification of mPSC (B) peak amplitudes and (C) frequencies for indicated conditions. Error bars are shown as standard error of the mean (±SEM). N = 5-12 technical replicates/ condition from 2 independent biological replicates in (B, C) statistical significance was assessed using a non-parametric Kruskal-Wallis multiple comparison test (B), an ordinary one-way ANOVA multiple comparison test (C)(D) EM analysis of endocytosed BoNT/Aiwt-HRP following lentiviral control (ctrl; non-targeting sgRNA) and Syt1 sgRNA1 CRISPRi KD transduction. HRP precipitate in synaptic vesicles (SVs; arrowheads) and tubular structures (arrows) in presynapses (Ps) are indicated. The postsynaptic density is marked with an asterisk. (E) Percentage of total BoNT/Aiwt-HRP localized in SVs, endo/lysosomes (E), tubular structures (T), multivesicular bodies (MVB), structures resembling autophagosomes (AP) and on the plasma membrane (PM) in the indicated conditions. n = 56 regions of interest/ condition from 2 independent biological replicates (E) two-way ANOVA multiple comparison test (E).


Figure 1d , 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 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 .

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Figure 1d, 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 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.


Figure 2b , 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 .

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Figure 2b, 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.


Figure 3a , 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 ) and SARS - CoV - 2 ( NP ) in the lung . Scale bars = 50 μm . 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

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Figure 3a, 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) and SARS-CoV-2 (NP) in the lung. Scale bars = 50 μm.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


Figure 1C . Dissociation constant values for wild - type AtAGO10 , HsAGO2 , and the AtAGO10 + Hs - loop HsAGO2 + At - loop mutants binding to a target RNA with base pairing complementarity to the miRNA seed region ( guide nucleotides 2 - 7 ) . Guide - target base pairing schematic is shown above . Plotted data represent the best - fit KD values from n = 3 technical experimental trials , each containing 12 data points . Error bars indicate s . e . m . ANOVA with Dunnett ' s posthoc test P values : * * * * < 0 . 0001 . No data points were excluded from the analyses .

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Figure 1C. Dissociation constant values for wild-type AtAGO10, HsAGO2, and the AtAGO10+Hs-loop HsAGO2+At-loop mutants binding to a target RNA with base pairing complementarity to the miRNA seed region (guide nucleotides 2-7). Guide-target base pairing schematic is shown above. Plotted data represent the best-fit KD values from n=3 technical experimental trials, each containing 12 data points. Error bars indicate s.e.m. ANOVA with Dunnett's posthoc test P values: **** < 0.0001. No data points were excluded from the analyses.


Figure 2C . Dissociation constants derived from data in panel B . Data points in represent best - fit KDE . Fraction of target RNA released in the fast phase of curve fits in panel D . Data points in E represent best - fit

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Figure 2C. Dissociation constants derived from data in panel B. Data points in represent best-fit KDE. Fraction of target RNA released in the fast phase of curve fits in panel D. Data points in E represent best-fit KD


Figure 5B . Dissociation constants derived from data in panel A . Data in panels B represent best - fit values of data in panels No data points were excluded from the analyses . D . Concentration of cleaved product generated by HsAGO2 or HsAGO2 + At - loop at 22 ˚ C or 36 ˚ C versus time . were obtained in n = 3 independent technical trials . No data points were excluded from the analyses . E . k2 and k3 values derived from fitting data in D to the burst - and - steady - state equation Data in panels E represent best - fit values of data in panels No data points were excluded from the analyses .

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Figure 5B. Dissociation constants derived from data in panel A. Data in panels B represent best-fit values of data in panels No data points were excluded from the analyses.D. Concentration of cleaved product generated by HsAGO2 or HsAGO2+At-loop at 22 ˚C or 36 ˚C versus time. were obtained in n=3 independent technical trials. No data points were excluded from the analyses.E. k2 and k3 values derived from fitting data in D to the burst-and-steady-state equation Data in panels E represent best-fit values of data in panels No data points were excluded from the analyses.


Figure 6B . Fold repression of Renilla expression ( compared to reporter plasmid transfection alone ) versus the amount of transfected siRNA duplex , wild - type HsAGO2 - siRNA complex , or HsAGO2 + At - loop - siRNA complex . The 2 - 8 target ( left panel ) indicates miRNA - like targeting . The 2 - 19 target ( right panel ) indicates siRNA - like targeting . Dotted line indicates the normalized expression level of the positive control ( no repression ) . Graphed data in B represent the individual and the ratio of mean values of n = 3 independent biological trials , respectively . Error bars indicate s . e . m . ANOVA with Dunnett ' s posthoc test P values : * * = 0 . 0062 , * = 0 . 0363 . No data points were excluded from the analyses .

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Figure 6B. Fold repression of Renilla expression (compared to reporter plasmid transfection alone) versus the amount of transfected siRNA duplex, wild-type HsAGO2-siRNA complex, or HsAGO2+At-loop-siRNA complex. The 2-8 target (left panel) indicates miRNA-like targeting. The 2-19 target (right panel) indicates siRNA-like targeting. Dotted line indicates the normalized expression level of the positive control (no repression). Graphed data in B represent the individual and the ratio of mean values of n=3 independent biological trials, respectively. Error bars indicate s.e.m. ANOVA with Dunnett's posthoc test P values: ** = 0.0062, * = 0.0363. No data points were excluded from the analyses.


Figure 1B . Numbers of CD4 + and CD8 + in 1 × 106 uterine cells on gd 18 . 5 after preconceptual infection of the mother in comparison with non - infected mothers ( n = 7 , n = 9 , n : biological replicates ) . T cells were identified as CD45 + CD3 + and either CD4 + or CD8 + cells . Data information : In data are presented as mean ± SEM . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 B , Mann - Whitney - U test ) . Percentage of EM T cells ( CD44 + , CD62L - ) among CD8 + T cells ; ( D ) peripheral blood ( n = 6 , n = 9 ) , ( E ) uterus - draining lymph nodes ( n = 6 , n = 9 ) n : biological replicates . Data information : In data are presented as mean ± SEM . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 ( E , Student ' s t - test Mann - Whitney - U test ) . I - L . Percentage of ovalbumin - specific CD8 + EM T cells ; ( I ) peripheral blood ( n = 7 , n = 9 ) , ( J ) uterus - draining lymph nodes ( n = 7 , n = 9 ) , ( K ) spleen ( n = 8 each ) , ( L ) uterus ( n = 6 , n = 9 ) ; n : biological replicates . Data information : I - L , data are presented as mean ± SEM . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 L : Student ' s t - test I : Mann - Whitney - U test ) .

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Figure 1B. Numbers of CD4+ and CD8+ in 1×106 uterine cells on gd 18.5 after preconceptual infection of the mother in comparison with non-infected mothers (n=7, n=9, n: biological replicates). T cells were identified as CD45+CD3+ and either CD4+ or CD8+ cells. Data information: In data are presented as mean ± SEM. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001 B, Mann-Whitney-U test).Percentage of EM T cells (CD44+, CD62L-) among CD8+ T cells; (D) peripheral blood (n=6, n=9), (E) uterus-draining lymph nodes (n=6, n=9) n: biological replicates. Data information: In data are presented as mean ± SEM. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001 (E, Student's t-test Mann-Whitney-U test).I-L. Percentage of ovalbumin-specific CD8+ EM T cells; (I) peripheral blood (n=7, n=9), (J) uterus-draining lymph nodes (n=7, n=9), (K) spleen (n=8 each), (L) uterus (n=6, n=9); n: biological replicates. Data information: I-L, data are presented as mean ± SEM. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001 L: Student's t-test I: Mann-Whitney-U test).


Figure 2C - E . Percentage of CD3 + T cells of total MMc ; ( C ) fetal bone marrow ( n = 13 , n = 14 ) , ( D ) fetal spleen ( n = 12 , n = 13 ) , ( E ) fetal liver ( n = 13 , n = 14 ) ; n : biological replicates . Data information : In C - E data are presented as mean ± SEM . * P ≤ 0 . 05 ( C , D , E Student ' s t - testG - I . percentage of OVA - specific MMc stained with OVA tetramers among CD3 + microchimeric T cells ; ( G ) bone marrow ( n = 13 each ) , ( H ) spleen ( n = 12 each ) , ( I ) liver ( n = 11 , n = 12 ) ; n : biological replicates . Data information : In data are presented as mean ± SEM . * P ≤ 0 . 05 H : Student ' s t - test ; G , I : Mann - Whitney - U ) . J . Serum anti - Lm IgG titer of mothers and fetuses at gd 18 . 5 after preconceptual infection of the mother with LmOVA ; n = 3 biological replicates , each averaged from n = 2 technical replicates ) . Data information data are presented as mean ± SEM . * P ≤ 0 .

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

Figure 2C-E. Percentage of CD3+ T cells of total MMc; (C) fetal bone marrow (n=13, n=14), (D) fetal spleen (n=12, n=13), (E) fetal liver (n=13, n=14); n: biological replicates. Data information: In C-E data are presented as mean ± SEM. *P≤0.05 (C, D, E Student's t-testG-I. percentage of OVA-specific MMc stained with OVA tetramers among CD3+ microchimeric T cells; (G) bone marrow (n=13 each), (H) spleen (n=12 each), (I) liver (n=11, n=12); n: biological replicates. Data information: In data are presented as mean ± SEM. *P≤0.05 H: Student's t-test; G, I: Mann-Whitney-U).J. Serum anti-Lm IgG titer of mothers and fetuses at gd 18.5 after preconceptual infection of the mother with LmOVA; n=3 biological replicates, each averaged from n=2 technical replicates). Data information data are presented as mean ± SEM. *P≤0.05


Figure 3Percentages of innate immune cell subsets in the fetal spleen ; ( B ) CD45 + leukocytes ( n = 20 , n = 11 ) , n : biological replicates . Data information : In data are presented as mean ± SEM . * P ≤ 0 . 05 ( B Ordinary one - way ANOVAPercentages of innate immune cell subsets in the fetal spleen ( C ) CD45 + CD11b + myeloid cells ( n = 19 , n = 11 ) , ( D ) CD45 + CD11b + Gr - 1 - monocytes ( n = 19 , n = 11 ) , ( E ) CD45 + CD11b + Gr - 1 + neutrophils ( n = 19 , n = 11 ) and ( F ) CD45 + CD11b + F4 / 80 + macrophages ( n = 20 , n = 11 ) ; n : biological replicates . Data information : In data are presented as mean ± SEM . * P ≤ 0 . 05 Ordinary one - way ANOVA ; D - F , : Kruskal - Wallis test ) . G - K . Percentages of innate immune cell subsets in the fetal liver ; ( G ) CD45 + leukocytes ( n = 20 , n = 10 ) , ( H ) CD45 + CD11b + myeloid cells ( n = 20 , n = 10 ) , ( I ) CD45 + CD11b + Gr - 1 - monocytes ( n = 20 , n = 10 ) , ( J ) CD45 + CD11b + Gr - 1 + neutrophils ( n = 20 , n = 10 ) and ( K ) CD45 + CD11b + F4 / 80 + macrophages ( n = 20 , n = 10 ) ; n : biological replicates . Data information : In data are presented as mean ± SEM . * P ≤ 0 . 05 G - I : Ordinary one - way ANOVA K , Kruskal - Wallis test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Percentages of innate immune cell subsets in the fetal spleen; (B) CD45+ leukocytes (n=20, n=11), n: biological replicates. Data information: In data are presented as mean ± SEM. *P≤0.05 (B Ordinary one-way ANOVAPercentages of innate immune cell subsets in the fetal spleen (C) CD45+CD11b+ myeloid cells (n=19, n=11), (D) CD45+CD11b+Gr-1- monocytes (n=19, n=11), (E) CD45+CD11b+Gr-1+ neutrophils (n=19, n=11) and (F) CD45+CD11b+F4/80+ macrophages (n=20, n=11); n: biological replicates. Data information: In data are presented as mean ± SEM. *P≤0.05 Ordinary one-way ANOVA; D-F, : Kruskal-Wallis test).G-K. Percentages of innate immune cell subsets in the fetal liver; (G) CD45+ leukocytes (n=20, n=10), (H) CD45+CD11b+ myeloid cells (n=20, n=10), (I) CD45+CD11b+Gr-1- monocytes (n=20, n=10), (J) CD45+CD11b+Gr-1+ neutrophils (n=20, n=10) and (K) CD45+CD11b+F4/80+ macrophages (n=20, n=10); n: biological replicates. Data information: In data are presented as mean ± SEM. *P≤0.05 G-I: Ordinary one-way ANOVA K, Kruskal-Wallis test).


Figure 4C . Neonatal body weight in mg ( n = 20 - 55 ) . ( C ) day 14 after birth ( 7 days after infection ) ; n : biological replicates . D , E . Percentages of OVA tetramer + cells among CD3 + MMc ( percentages of OVA tetramer + among CD3 + MMc at gd 18 . 5 ( Fig 2h , i ) are included for direct comparison ) ; ( D ) spleen ( n = 5 - 10 ) , ( E ) liver ( n = 5 - 10 ) ; n : biological replicates . Data information : data are presented as mean ± SEM . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 Ordinary one - way ANOVA ; : Kruskal - Wallis test ) . F - I . Percentage of ( F ) CD3 + T cells of CD45 + spleen cells ( n = 6 - 14 ) , ( G ) OVA - specific cells of CD3 + T cells from spleen cells ( n = 6 - 14 ) , ( H ) CD11b + cells of CD3 + T cells from spleen cells ( n = 6 - 14 ) , and ( I ) CD11c + cells of CD3 + T cells from spleen cells ( n = 6 - 14 ) ; n : biological replicates . Data information : In data are presented as mean ± SEM . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 G : Ordinary one - way ANOVA , H , I : Kruskal - Wallis test ) .

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

Figure 4C. Neonatal body weight in mg (n=20-55). (C) day 14 after birth (7 days after infection); n: biological replicates. D, E. Percentages of OVA tetramer+ cells among CD3+ MMc (percentages of OVA tetramer+ among CD3+ MMc at gd 18.5 (Fig 2h, i) are included for direct comparison); (D) spleen (n=5-10), (E) liver (n=5-10); n: biological replicates. Data information: data are presented as mean ± SEM. *P≤0.05; **P≤0.01; ****P≤0.0001 Ordinary one-way ANOVA; : Kruskal-Wallis test).F-I. Percentage of (F) CD3+ T cells of CD45+ spleen cells (n=6-14), (G) OVA-specific cells of CD3+ T cells from spleen cells (n=6-14), (H) CD11b+ cells of CD3+ T cells from spleen cells (n=6-14), and (I) CD11c+ cells of CD3+ T cells from spleen cells (n=6-14); n: biological replicates. Data information: In data are presented as mean ± SEM. *P≤0.05; **P≤0.01; ****P≤0.0001 G: Ordinary one-way ANOVA , H, I: Kruskal-Wallis test).


Figure 1B . The 5 ' UTR of rne is bound by RNase E and non - genomically encoded A - tails are maximally recovered - 9nt from the rne start codon . Known stem loop strucutres ( HP1 - 3 ) and the ribosomal binding site ( RBS ) are shaded grey . C . RNase E binding and oligoadenylation of the pdlB - yigL dicistronic transcript . The reported RNase E cleavage site ( red dashed line ) and SgrS binding site ( grey shading ) are indicated . D . Length of non - genomically encoded oligo ( A ) tails recovered from RNase E bound reads . E . Position of Hfq binding and oliogadenylation relative to RNase E binding peaks . The cumulative position of Hfq and oligoadenylation peaks were determined relative to RNase E binding peaks for 672 RNase E binding sites that were within 1kb of an Hfq binding peak . A detailed description of the data processing is presented in the Appended Supplementary Methods .

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 B-PANEL_START 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 1B. The 5'UTR of rne is bound by RNase E and non-genomically encoded A-tails are maximally recovered -9nt from the rne start codon. Known stem loop strucutres (HP1-3) and the ribosomal binding site (RBS) are shaded grey.C. RNase E binding and oligoadenylation of the pdlB-yigL dicistronic transcript. The reported RNase E cleavage site (red dashed line) and SgrS binding site (grey shading) are indicated.D. Length of non-genomically encoded oligo(A) tails recovered from RNase E bound reads.E. Position of Hfq binding and oliogadenylation relative to RNase E binding peaks. The cumulative position of Hfq and oligoadenylation peaks were determined relative to RNase E binding peaks for 672 RNase E binding sites that were within 1kb of an Hfq binding peak. A detailed description of the data processing is presented in the Appended Supplementary Methods.


Figure 2B . Hfq binding ( blue ) , RNase E binding ( grey ) , and oligoadenylation of the MicA binding site in the ompA mRNA . The MicA seed sequence is shaded blue . C . Interaction strength ( Gibbs free energy ) of all RNA - RNA interactions recovered by RNase E - CLASH ( solid line ) and for randomly paired hybrid read halves ( dashed line ) . D . Interaction strength of sRNA - mRNA hybrid read halves ( solid line ) and randomly paired sRNA and mRNA read halves ( dashed line ) . E . Position of sRNA - mRNA interactions relative to the mRNA start codon .

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

Figure 2B. Hfq binding (blue), RNase E binding (grey), and oligoadenylation of the MicA binding site in the ompA mRNA. The MicA seed sequence is shaded blue.C. Interaction strength (Gibbs free energy) of all RNA-RNA interactions recovered by RNase E-CLASH (solid line) and for randomly paired hybrid read halves (dashed line).D. Interaction strength of sRNA-mRNA hybrid read halves (solid line) and randomly paired sRNA and mRNA read halves (dashed line).E. Position of sRNA-mRNA interactions relative to the mRNA start codon.


Figure 3RNA classes are labeled on the outer ring ( rRNA has been omitted to clarify interactions between other RNA classes ) . RNA - RNA interactions with more than two unique hybrid sequences are presented . The thickness of the link represents the number of unique hybrid sequences recovered ( up to a maximum of 50 sequences ) . RNA - RNA interactions where both hybrid halves overlap an Hfq binding site are colored blue and non - overlapping interactions are colored green .

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

Figure 3RNA classes are labeled on the outer ring (rRNA has been omitted to clarify interactions between other RNA classes). RNA-RNA interactions with more than two unique hybrid sequences are presented. The thickness of the link represents the number of unique hybrid sequences recovered (up to a maximum of 50 sequences). RNA-RNA interactions where both hybrid halves overlap an Hfq binding site are colored blue and non-overlapping interactions are colored green.


Figure 4A B ( top ) . Sequences interacting with ChiX ( A ) or ompA ( B ) were analyzed for conserved motifs using MEME . Motifs that were enriched in the target RNAs and are shown above the heatmap with proportion of target RNAs carrying the motif ( N ) and the expected value for the motif ( e - val ) . The complementary sequence motifs within ChiX or ompA are shown below the logo . ( Middle ) Heatmaps showing the position of predicted basepairing for each interaction within ChiX or ompA . Grey bars boxed below indicate the position of basepairing with experimentally verified mRNAs ( known interactions ) . ( Bottom ) Hfq ( blue ) and RNase E ( grey ) binding sites within ChiX and ompA . Hfq and RNase E - bound sequence , and non - genomically encoded oligo ( A ) tails within ChiX and ompA are shown as line plots where the x - axis position correlates with heatmaps above . C , D E . Cumulative plots of oligoadenylation ( C ) , Hfq ( D ) and RNase E ( E ) binding at predicted seed motifs .

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 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

Figure 4A B (top). Sequences interacting with ChiX (A) or ompA (B) were analyzed for conserved motifs using MEME. Motifs that were enriched in the target RNAs and are shown above the heatmap with proportion of target RNAs carrying the motif (N) and the expected value for the motif (e-val). The complementary sequence motifs within ChiX or ompA are shown below the logo. (Middle) Heatmaps showing the position of predicted basepairing for each interaction within ChiX or ompA. Grey bars boxed below indicate the position of basepairing with experimentally verified mRNAs (known interactions). (Bottom) Hfq (blue) and RNase E (grey) binding sites within ChiX and ompA. Hfq and RNase E-bound sequence, and non-genomically encoded oligo(A) tails within ChiX and ompA are shown as line plots where the x-axis position correlates with heatmaps above.C, D E. Cumulative plots of oligoadenylation (C), Hfq (D) and RNase E (E) binding at predicted seed motifs.


Figure 5A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for frdA - RyhB ( A ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 . A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for zapB - RyhB ( B ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 . A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for hdeA - RyhB ( C ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 . A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) hdeA - GadY ( D ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 . A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for rssA - RyeB ( E ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 . A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for chuA - Esr41 ( F ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 . A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for cirA - Esr41 ( G ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 . A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for bfr - Esr41 ( H ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 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 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 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 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 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 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 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 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

Figure 5A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for frdA-RyhB (A). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for zapB-RyhB (B). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for hdeA-RyhB (C). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) hdeA-GadY (D). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for rssA-RyeB (E). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for chuA-Esr41 (F). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for cirA-Esr41 (G). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for bfr-Esr41 (H). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


Figure 6A - C . FACS analysis of constitutively expressed sfGFP fusions to chuA ( A ) , cirA ( B ) , or bfr ( C ) in the presence of Esr41 ( orange ) , RyhB ( blue ) , or a scrambled RNA control ( red ) . The histogram shows GFP fluorescence for each sRNA - mRNA fusion . D . Esr41 confers resistance to Colicin 1A and 1B in the sensitive background , E . coli DH5alpha . Top agar lawns of DH5alpha expressing a control scrambled RNA ( pJV300 ) , Esr41 ( pZE12 : : esr41 ) , or RyhB ( pZE12 : : ryhB ) were spotted with colicins indicated ( top ) . Zones of clearing indicate sensitivity to the tested colicin . E . Esr41 confers a competitive disadvantage on EHEC under iron - limiting conditions . Wild type EHEC and isogenic ∆ esr41 strain ( black ) , or EHEC ∆ esr41and the chromosomally repaired strain EHEC ∆ esr41 : : esr41 were inoculated at equal densities and cultured in MEM - HEPES media for 3 days . The proportion of each strain was determined at each time point ( days ) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness ( see Appended Supplementary Methods ) .

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

Figure 6A-C. FACS analysis of constitutively expressed sfGFP fusions to chuA (A), cirA (B), or bfr (C) in the presence of Esr41 (orange), RyhB (blue), or a scrambled RNA control (red). The histogram shows GFP fluorescence for each sRNA-mRNA fusion.D. Esr41 confers resistance to Colicin 1A and 1B in the sensitive background, E. coli DH5alpha. Top agar lawns of DH5alpha expressing a control scrambled RNA (pJV300), Esr41 (pZE12::esr41), or RyhB (pZE12::ryhB) were spotted with colicins indicated (top). Zones of clearing indicate sensitivity to the tested colicin.E. Esr41 confers a competitive disadvantage on EHEC under iron-limiting conditions. Wild type EHEC and isogenic ∆esr41 strain (black), or EHEC ∆esr41and the chromosomally repaired strain EHEC ∆esr41::esr41 were inoculated at equal densities and cultured in MEM-HEPES media for 3 days. The proportion of each strain was determined at each time point (days) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness (see Appended Supplementary Methods).


Figure 1A . qPCR analysis of Ahr expression in B cell subsets purified from C57Bl / 6 mice . Ahr expression was normalized to Hprt1 . n = 3 - 5 independent experiments ; mean ± SD . B . qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 4h as indicated . Ahr expression was normalized to Hprt1 . Ahr expression among groups was normalized to Medium . n = 4 independent experiments ; mean ± SEM ; one - way ANOVA , Dunnett ' s multiple comparison test . C . qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 4h with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Ahr expression was normalized to Hprt1 . Ahr expression among groups was normalized to Medium . n = 3 independent experiments ; mean ± SEM ; one - way ANOVA , Tukey ' s multiple comparison test . D . Western blot of protein extracts from splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 4h with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Values above the blots indicate AhR protein quantification obtained by densitometry , normalized to β - actin and compared to Medium sample . Sample A and B indicate 2 independent replicates . E . Quantification by densitometric analysis of the results shown in C . n = 2 independent experiments ; mean ± range . F . qPCR analysis of Ahr expression in purified splenic B cell subsets isolated from C57Bl / 6 mice and cultured as indicated for 4h . Ahr expression was normalized to Hprt1 . n = 2 independent experiments ; mean ± range . G . qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for the indicated time points with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Ahr expression was normalized to Hprt1 . Ahr expression among groups was normalized to Medium . n = 5 independent experiments ; mean ± SEM ; one - way ANOVA , Dunnett ' s multiple comparison test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O 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. qPCR analysis of Ahr expression in B cell subsets purified from C57Bl/6 mice. Ahr expression was normalized to Hprt1. n = 3-5 independent experiments; mean ± SD.B. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h as indicated. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 4 independent experiments; mean ± SEM; one-way ANOVA, Dunnett's multiple comparison test.C. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.D. Western blot of protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to β-actin and compared to Medium sample. Sample A and B indicate 2 independent replicates.E. Quantification by densitometric analysis of the results shown in C. n = 2 independent experiments; mean ± range.F. qPCR analysis of Ahr expression in purified splenic B cell subsets isolated from C57Bl/6 mice and cultured as indicated for 4h. Ahr expression was normalized to Hprt1. n = 2 independent experiments; mean ± range.G. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 5 independent experiments; mean ± SEM; one-way ANOVA, Dunnett's multiple comparison test.


Figure 2A . Western blot of nuclear and cytoplasmic protein extracts from splenic CD19 + cells isolated from C57Bl / 6 mice and cultured with anti - IgM ( α - IgM ) for 4h , followed by the indicated treatment for 2h . Values above the blots indicate AhR protein quantification obtained by densitometry , normalized to Sam68 or Tubulin and compared to the DMSO - treated sample . Representative data of n = 3 independent experiments . B . qPCR analysis of Cyp1a1 expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 24h as indicated . Cyp1a1 expression was normalized to Hprt1 . Cyp1a1 expression among groups was normalized to Medium DMSO . n = 2 independent experiments ; mean ± range . C . Flow cytometry analysis of Cyp1a1 expression , reported by eYFP , in splenic CD19 + cells isolated from Cyp1a1Cre R26R eYFP mice and cultured for 72h as indicated . Representative data of n = 3 independent experiments . D . qPCR analysis of Cyp1a1 expression in purified splenic B cell subsets isolated from C57Bl / 6 mice and cultured as indicated for 24h . Cyp1a1 expression was normalized to Hprt1 . n = 2 independent experiments ; mean ± range .

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 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 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

Figure 2A. Western blot of nuclear and cytoplasmic protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured with anti-IgM (α-IgM) for 4h, followed by the indicated treatment for 2h. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to Sam68 or Tubulin and compared to the DMSO-treated sample. Representative data of n = 3 independent experiments.B. qPCR analysis of Cyp1a1 expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 24h as indicated. Cyp1a1 expression was normalized to Hprt1. Cyp1a1 expression among groups was normalized to Medium DMSO. n = 2 independent experiments; mean ± range.C. Flow cytometry analysis of Cyp1a1 expression, reported by eYFP, in splenic CD19+ cells isolated from Cyp1a1Cre R26R eYFP mice and cultured for 72h as indicated. Representative data of n = 3 independent experiments.D. qPCR analysis of Cyp1a1 expression in purified splenic B cell subsets isolated from C57Bl/6 mice and cultured as indicated for 24h. Cyp1a1 expression was normalized to Hprt1. n = 2 independent experiments; mean ± range.


Figure 3B . Representative flow cytometry analysis of Cyp1a1 expression , reported by eYFP , in CD45 . 1 + cells harvested from mice challenged as in A . Gating is indicated above the plots . Representative data of n = 3 independent experiments . GC cells : CD95 + GL - 7 + ; non - GC cells : CD95 - GL - 7 - . C , D . Summary of percentage of GC ( C ) and non - GC eYFP + ( D ) cells from flow cytometry analysis . n = 3 independent experiments ; lines indicate mean ; one - way ANOVA , Tukey ' s multiple comparison test .

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Figure 3B. Representative flow cytometry analysis of Cyp1a1 expression, reported by eYFP, in CD45.1+ cells harvested from mice challenged as in A. Gating is indicated above the plots. Representative data of n = 3 independent experiments. GC cells: CD95+ GL-7+; non-GC cells: CD95- GL-7-.C, D. Summary of percentage of GC (C) and non-GC eYFP+ (D) cells from flow cytometry analysis. n = 3 independent experiments; lines indicate mean; one-way ANOVA, Tukey's multiple comparison test.


Figure 4A . Flow cytometry analysis of CTV dilution in splenic CD19 + cells sorted from non - immune Ahrfl / + mb1Cre + ( Ahr + / - B cells , solid grey ) and Ahrfl / - mb1Cre + ( Ahr - / - B cells , black ) mice stimulated for 72h as indicated . Treatment of Ahr + / - CD19 + cells with CH223191 is indicated in red . Representative data of n = 4 - 5 independent experiments . B - D . Flow cytometry analysis of expansion index ( B ) , % of divided cells ( C ) and replication index ( D ) of splenic CD19 + cells sorted from non - immune Ahrfl / + mb1Cre + ( black ) and Ahrfl / - mb1Cre + ( white ) mice stimulated for 72h as indicated . Treatment of Ahr + / - CD19 + cells with CH223191 is indicated in red . n = 4 - 5 independent experiments ; mean ± SEM ; 2 - way ANOVA , Sidak ' s multiple comparison test . E . Flow cytometry analysis of cell cycle distribution of splenic CD19 + cells purified from non - immune Ahrfl / + mb1Cre + ( black ) and Ahrfl / - mb1Cre + ( white ) mice stimulated for 48h with 5 µg / ml α - IgM . Data representative of n = 2 independent experiments ; lines indicate mean ; 2 - way ANOVA , Sidak ' s multiple comparison test .

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

Figure 4A. Flow cytometry analysis of CTV dilution in splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (Ahr+/- B cells, solid grey) and Ahrfl/-mb1Cre+ (Ahr-/- B cells, black) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. Representative data of n = 4-5 independent experiments.B-D. Flow cytometry analysis of expansion index (B), % of divided cells (C) and replication index (D) of splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (black) and Ahrfl/-mb1Cre+ (white) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. n = 4-5 independent experiments; mean ± SEM; 2-way ANOVA, Sidak's multiple comparison test.E. Flow cytometry analysis of cell cycle distribution of splenic CD19+ cells purified from non-immune Ahrfl/+mb1Cre+ (black) and Ahrfl/-mb1Cre+ (white) mice stimulated for 48h with 5 µg/ml α-IgM. Data representative of n = 2 independent experiments; lines indicate mean; 2-way ANOVA, Sidak's multiple comparison test.


Figure 5A , B . Flow cytometry analysis of distribution of CD45 . 1 + and CD45 . 2 + cells in indicated cell subsets purified from bone marrow ( A ) and lymph node ( B ) of sub lethally - irradiated Rag1 - / - mice 8 weeks after reconstitution with equal numbers of bone marrow cells from Ahr + / + ( CD45 . 1 + ) and Ahr - / - ( CD45 . 2 + ) mice . Dashed lines indicate 50 % level . Representative data of n = 2 independent experiments ; two - tailed paired t test . D . Flow cytometry analysis of distribution of CD45 . 1 + CD45 . 2 + and CD45 . 2 + cells harvested from host mice challenged as indicated above the dot plots . Representative data of n = 3 independent experiments . E . Flow cytometry analysis of distribution of Ahr + / + CD45 . 1 + CD45 . 2 + ( black ) and Ahr - / - CD45 . 2 + ( white ) cells harvested from host mice . Indicated distribution is quantified relative to transferred cells . X symbols indicate SRBC - mock - treated control . Dashed line indicates 50 % threshold . Representative data of n = 3 independent experiments ; two - tailed paired t test . F , G . Flow cytometry analysis of distribution ( F ) and cell numbers ( G ) of IgG1 + and IgG1 - HEL3x - binding Ahr + / + CD45 . 1 + CD45 . 2 + ( black ) and Ahr - / - CD45 . 2 + ( white ) cells harvested from host mice . Representative data of n = 2 independent experiments ; two - tailed paired t test .

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

Figure 5A, B. Flow cytometry analysis of distribution of CD45.1+ and CD45.2+ cells in indicated cell subsets purified from bone marrow (A) and lymph node (B) of sub lethally-irradiated Rag1-/- mice 8 weeks after reconstitution with equal numbers of bone marrow cells from Ahr+/+ (CD45.1+) and Ahr-/- (CD45.2+) mice. Dashed lines indicate 50% level. Representative data of n = 2 independent experiments; two-tailed paired t test.D. Flow cytometry analysis of distribution of CD45.1+CD45.2+ and CD45.2+ cells harvested from host mice challenged as indicated above the dot plots. Representative data of n = 3 independent experiments.E. Flow cytometry analysis of distribution of Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Indicated distribution is quantified relative to transferred cells. X symbols indicate SRBC-mock-treated control. Dashed line indicates 50% threshold. Representative data of n = 3 independent experiments; two-tailed paired t test.F, G. Flow cytometry analysis of distribution (F) and cell numbers (G) of IgG1+ and IgG1- HEL3x-binding Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Representative data of n = 2 independent experiments; two-tailed paired t test.