Figure 4 ( A - B ) The indicated cell lines were transfected with either miR - 515 - 5p or a non - targeting miR ( miR NC ) together with a MARK4 expressing or an empty vector ( EV ) plasmid DNA . MARK4 over - expression rescues the inhibition by miR - 515 - 5p of random ( A ) and directed ( B ) migration of the indicated cell lines . ( A - left panel ) Plots show overlays of representative trajectories described for MDA - MB - 231 cells ( n = 30 ) . ( A - right panel ) The migration distance was quantified and represented as the normalised mean ± SEM . ( B - left panel ) Representative field of view from the bottom side of the transwell membrane for MDA - MB - 231 . ( B - right panel ) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field ( n = 5 fields per condition ) . Data are mean of three experiments ± SEM . ( A - C ) P values were calculated by t - test between the transfected and the respective control condition ( EV + miR - NC ) ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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

Figure 4(A-B) The indicated cell lines were transfected with either miR-515-5p or a non-targeting miR (miR NC) together with a MARK4 expressing or an empty vector (EV) plasmid DNA. MARK4 over-expression rescues the inhibition by miR-515-5p of random (A) and directed (B) migration of the indicated cell lines. (A-left panel) Plots show overlays of representative trajectories described for MDA-MB-231 cells (n=30). (A-right panel) The migration distance was quantified and represented as the normalised mean ± SEM. (B-left panel) Representative field of view from the bottom side of the transwell membrane for MDA-MB-231. (B-right panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the transfected and the respective control condition (EV+miR-NC) (*,P<0.05; **,P<0.01; ***,P<0.001).


Figure 5 ( A - B ) Overall survival of ER - negative metastatic breast cancer ( n = 226 ) ( A ) and lung adenocarcinoma ( n = 719 ) ( B ) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier - plotter [ 25 ] . ( C ) A cDNA array for 24 matched lung cancers and surrounding normal tissue was analysed for MARK4 expression . Expression levels from tumors were normalised to their respective normal control and the percentage of cases for the indicated fold expression in the tumor represented as a pie - chart . ( D - F ) MARK4 protein expression was determined by histoimmunochemistry in ( D - E ) a tissue microarray comprising 90 lung tumor and 10 normal lung samples or ( F ) a home - made tissue microarray comprising 100 matched cases of primary and metastatic tumors . ( D - E ) The percentage of positivity for MARK4 staining in lung tumors versus normal lung samples ( D ) or in lung tumors according to their histological subtypes ( E ) is represented . ( F ) The percentage of cases with increased , decreased or unchanged levels of MARK4 in metastatic lesions as compared to the corresponding primary tumors is represented .

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

Figure 5(A-B) Overall survival of ER-negative metastatic breast cancer (n=226) (A) and lung adenocarcinoma (n=719) (B) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier-plotter [25].(C) A cDNA array for 24 matched lung cancers and surrounding normal tissue was analysed for MARK4 expression. Expression levels from tumors were normalised to their respective normal control and the percentage of cases for the indicated fold expression in the tumor represented as a pie-chart.(D-F) MARK4 protein expression was determined by histoimmunochemistry in (D-E) a tissue microarray comprising 90 lung tumor and 10 normal lung samples or (F) a home-made tissue microarray comprising 100 matched cases of primary and metastatic tumors. (D-E) The percentage of positivity for MARK4 staining in lung tumors versus normal lung samples (D) or in lung tumors according to their histological subtypes (E) is represented. (F) The percentage of cases with increased, decreased or unchanged levels of MARK4 in metastatic lesions as compared to the corresponding primary tumors is represented.


Figure 6 ( A - D ) Low expression of miR - 515 - 5p is linked to survival in both Lung and Breast cancer . ( A ) Survival analysis of miR - 515 - 5p from 195 lung adenocarcinoma samples from the TCGA . ( B ) Survival analysis of miR - 515 - p from 34 lung squamous cell carcinoma from the TCGA . ( C ) Survival analysis of miR - 515 - 5p from 60 Breast cancer samples from the Lyng Ditzel Breast Study ( GSE37405 ) . ( D ) Survival analysis of miR - 515 - 5p from 33 Breast cancer from the GSE7405 Breast study . Analysis and plot were obtained with SurvMicro http : / / bioinformatica . mty . itesm . mx : 8080 / Biomatec / Survmicro . jsp for ( A - C ) and with MIRUMIR for ( D ) . ( E ) Comparison of miR - 515 - 5p expression in 18 primary and matched metastatic lesions from breast cancer patients . miR - 515 - 5p levels were quantified by TaqMan qRT - PCR . Absolute values were normalized to u6 snRNA values and displayed as a dot - plot . Bar ; Mean , Whiskers ; SEM . ( F ) MDA - MB - 231 cells were inoculated into the mammary fat pads of nude mice and allowed to form metastatic deposits [ 22 ] . miR - 515 - 5p and MARK4 levels were then determined in the primary and indicated secondary lesions . Data are average ± SEM of technical triplicates . ( G ) Luciferase - expressing A549 cells overexpressing miR - 515 - 5p or a non - targeting control ( miR - NC ) were injected in the tail - vein of SCID mice and the development of tumor burden monitored by whole animal - imaging in response to luciferin administration at week 1 and 4 following injection . ( G - left panel ) Representative imaging of the same animals for the indicated conditions at week 1 and 4 . ( G - Right panel ) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1 . Data represented are representative of three independent experiments . ( E - G ) P values were calculated by t - test ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6(A-D) Low expression of miR-515-5p is linked to survival in both Lung and Breast cancer. (A) Survival analysis of miR-515-5p from 195 lung adenocarcinoma samples from the TCGA. (B) Survival analysis of miR-515-p from 34 lung squamous cell carcinoma from the TCGA. (C) Survival analysis of miR-515-5p from 60 Breast cancer samples from the Lyng Ditzel Breast Study (GSE37405). (D) Survival analysis of miR-515-5p from 33 Breast cancer from the GSE7405 Breast study. Analysis and plot were obtained with SurvMicro http://bioinformatica.mty.itesm.mx:8080/Biomatec/Survmicro.jsp for (A-C) and with MIRUMIR for (D).(E) Comparison of miR-515-5p expression in 18 primary and matched metastatic lesions from breast cancer patients. miR-515-5p levels were quantified by TaqMan qRT-PCR. Absolute values were normalized to u6 snRNA values and displayed as a dot-plot. Bar; Mean, Whiskers; SEM.(F) MDA-MB-231 cells were inoculated into the mammary fat pads of nude mice and allowed to form metastatic deposits [22]. miR-515-5p and MARK4 levels were then determined in the primary and indicated secondary lesions. Data are average ± SEM of technical triplicates.(G) Luciferase-expressing A549 cells overexpressing miR-515-5p or a non-targeting control (miR-NC) were injected in the tail-vein of SCID mice and the development of tumor burden monitored by whole animal-imaging in response to luciferin administration at week 1 and 4 following injection. (G-left panel) Representative imaging of the same animals for the indicated conditions at week 1 and 4. (G-Right panel) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1. Data represented are representative of three independent experiments. (E-G) P values were calculated by t-test (*, P<0.05; **, P<0.01; ***, P<0.001).


Figure 7A549 - Luc cells transfected either with miR - 515 - 5p precursor ( miR ) , siRNAs to MARK4 ( siMARK4 ) or the corresponding control sequences ( C and NT , respectively ) were injected in Scid mice through the tail vein . 4 weeks later , the mice were sacrificed , their lungs extracted and fixed prior to staining for Luciferase expression . ( A ) Representative pictures showing the presence of A549 - Luc cells in the lungs of NT mice either as single cells ( upper panel ) or as a tumour mass ( lower panel ) . ( B ) Percentage of the area covered by tumour cells on randomly observed fields of views . Quantification was performed using Photoshop CS4 . Each spot corresponds to quantification of an individual 1 mm x 1 . 5 mm field . Transversal bar ; Median . Statistics : Student t - test . * , p ≤ 0 . 05 .

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Figure 7A549-Luc cells transfected either with miR-515-5p precursor (miR), siRNAs to MARK4 (siMARK4) or the corresponding control sequences (C and NT, respectively) were injected in Scid mice through the tail vein. 4 weeks later, the mice were sacrificed, their lungs extracted and fixed prior to staining for Luciferase expression. (A) Representative pictures showing the presence of A549-Luc cells in the lungs of NT mice either as single cells (upper panel) or as a tumour mass (lower panel). (B) Percentage of the area covered by tumour cells on randomly observed fields of views. Quantification was performed using Photoshop CS4. Each spot corresponds to quantification of an individual 1 mm x 1.5 mm field. Transversal bar; Median. Statistics: Student t-test. *, p≤0.05.


Figure 1B , qRT - PCR of piwi - 1 relative to GAPDH in RNAi animals as indicated , 2dpi . Circles represent individual biological replicates averaged from 3 technical replicates . D , Percentage of X1 cells from control and atm ( RNAi ) animals , 1dpi or unirradiated .

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Figure 1B, qRT-PCR of piwi-1 relative to GAPDH in RNAi animals as indicated, 2dpi. Circles represent individual biological replicates averaged from 3 technical replicates.D, Percentage of X1 cells from control and atm(RNAi) animals, 1dpi or unirradiated.


Figure 2E , qRT - PCR of piwi - 1 relative to GAPDH in RNAi animals as indicated , 2dpi . Data are averages ± SD ; circles represent biological replicates ; statistics represent ordinary one - way ANOVA compared to controls , * * * * P < 0 . 0001 , ns = not significant . F , piwi - 1 fluorescent in situ hybridization ( FISH ) of RNAi animals as indicated , 2 dpi .

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Figure 2E, qRT-PCR of piwi-1 relative to GAPDH in RNAi animals as indicated, 2dpi. Data are averages ± SD; circles represent biological replicates; statistics represent ordinary one-way ANOVA compared to controls, ****P<0.0001, ns=not significant.F, piwi-1 fluorescent in situ hybridization (FISH) of RNAi animals as indicated, 2 dpi.


Figure 3A , Top : Strategy for EdU pulse and animal fixation . Bottom : Confocal images of EdU - stained atm and control RNAi animals . B , Quantification by area of EdU + cells from A . Circles represent individual animals . Inset , expanded scale showing compressed data below . Data includes two independent experiments . C , Graph showing percentage of G2 / M cells in RNAi animals on indicated dpi . D , Confocal images of phosphohistone H3 ( pH3 ) - stained RNAi animals after radiation exposure . Blue , DAPI . E and F , Quantification by area of pH3 cells in radiated ( E ) or unirradiated ( F ) animals after atm ( solid line ) and control ( dashed line ) RNAi .

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Figure 3A, Top: Strategy for EdU pulse and animal fixation. Bottom: Confocal images of EdU-stained atm and control RNAi animals. B, Quantification by area of EdU+ cells from A. Circles represent individual animals. Inset, expanded scale showing compressed data below. Data includes two independent experiments.C, Graph showing percentage of G2/M cells in RNAi animals on indicated dpi.D, Confocal images of phosphohistone H3 (pH3)-stained RNAi animals after radiation exposure. Blue, DAPI. E and F, Quantification by area of pH3 cells in radiated (E) or unirradiated (F) animals after atm (solid line) and control (dashed line) RNAi.


Figure 4A , Confocal images of piwi - 1 FISH in RNAi animals after radiation exposure . Scale bars = 250µm . B and C , Percentage of animal area occupied by piwi - 1 FISH divided by total animal area , in radiated ( B ) or unirradiated ( C ) RNAi animals . D , Total number of piwi - 1 + cells in RNAi animals , 7 dpi . Experiments in ( A ) - ( D ) were repeated twice . E , Schematic of experimental design . Below , Kaplan - Meier survival curves of intact ( solid line ) and decapitated ( dashed line ) animals after RNAi as indicated . n = 45 animals , repeated twice .

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Figure 4A, Confocal images of piwi-1 FISH in RNAi animals after radiation exposure. Scale bars=250µm. B and C, Percentage of animal area occupied by piwi-1 FISH divided by total animal area, in radiated (B) or unirradiated (C) RNAi animals. D, Total number of piwi-1+ cells in RNAi animals, 7 dpi. Experiments in (A) - (D) were repeated twice.E, Schematic of experimental design. Below, Kaplan-Meier survival curves of intact (solid line) and decapitated (dashed line) animals after RNAi as indicated. n = 45 animals, repeated twice.


Figure 5A - C , Kaplan - Meier survival curves of intact RNAi animals after radiation , n = 50 animals , repeated twice . Experiments in A and B were performed simultaneously but split into two graphs for clarity ; control and atm ( RNAi ) values are replicated in both panels . D , Confocal images of piwi - 1 FISH in RNAi animals after exposure to 20Gy , 2dpi . Scale bars = 250µm . E , Quantification by area of piwi - 1 + cells in RNAi animals after 20Gy exposure , 7dpi .

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Figure 5A - C, Kaplan-Meier survival curves of intact RNAi animals after radiation, n = 50 animals, repeated twice. Experiments in A and B were performed simultaneously but split into two graphs for clarity; control and atm(RNAi) values are replicated in both panels.D, Confocal images of piwi-1 FISH in RNAi animals after exposure to 20Gy, 2dpi. Scale bars=250µm. E, Quantification by area of piwi-1+ cells in RNAi animals after 20Gy exposure, 7dpi.


Figure 1Figure 1 . Helix - 7 dynamically interacts with the miRNA seed region . A ) Cartoon representation of the Ago2 - miRNA crystal structure . Ago2 is colored gray except for helix - 7 , which is yellow . miRNA guide colored red . C ) Close - up view of the unpaired complex shows helix - 7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7 . B ) Crystal structure of the Ago2 - miRNA - target RNA ternary complex . Target RNA is colored blue . D ) Close - up view of the Ago2 - miRNA - target complex shows helix - 7 docks into the minor groove of the guide : target duplex , directly contacting base pairs at positions g6 and g7 .

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Figure 1Figure 1. Helix-7 dynamically interacts with the miRNA seed region. A) Cartoon representation of the Ago2-miRNA crystal structure. Ago2 is colored gray except for helix-7, which is yellow. miRNA guide colored red.C) Close-up view of the unpaired complex shows helix-7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7.B) Crystal structure of the Ago2-miRNA-target RNA ternary complex. Target RNA is colored blue. D) Close-up view of the Ago2-miRNA-target complex shows helix-7 docks into the minor groove of the guide:target duplex, directly contacting base pairs at positions g6 and g7.


Figure 2Figure 2 . Helix - 7 is not required for target recognition . A ) Plot of g2 - g8 target RNA bound ( 0 . 1 nM ) versus Ago2 - miR122 concentration for wild type ( WT ) , helix - 7 double point mutant ( MI - AA ) , and Ηhelix - 7 Ago2 . Average values from at least three independent experiments ± standard deviation ( SD ) are plotted . Top panel shows target RNA paired to seed region of the guide RNA ( miRNA - 122 ) . B ) Dissociation of a 32P - labeled target RNA ( 0 . 1 nM ) from the Ago2 - miR122 complex ( 1 nM ) was monitored in the presence of unlabeled target RNA ( 100 nM ) . Fraction of the target RNA bound to Ago2 - miR122 is plotted as a function of time for WT and Ηhelix - 7 Ago2 . Average values from at least three independent experiments ± SD were fit to single exponential decays .

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Figure 2Figure 2. Helix-7 is not required for target recognition. A) Plot of g2-g8 target RNA bound (0.1 nM) versus Ago2-miR122 concentration for wild type (WT), helix-7 double point mutant (MI-AA), and Ηhelix-7 Ago2. Average values from at least three independent experiments ± standard deviation (SD) are plotted. Top panel shows target RNA paired to seed region of the guide RNA (miRNA-122).B) Dissociation of a 32P-labeled target RNA (0.1 nM) from the Ago2-miR122 complex (1 nM) was monitored in the presence of unlabeled target RNA (100 nM). Fraction of the target RNA bound to Ago2-miR122 is plotted as a function of time for WT and Ηhelix-7 Ago2. Average values from at least three independent experiments ± SD were fit to single exponential decays.


Figure 3C ) Representative fluorescence time traces ( with a resolution of 100 ms for wt , 300 ms for MI - AA and Ηhelix - 7 ) show docking and dissociation of wild type ( top ) , MI - AA ( middle ) , and Ηhelix - 7 ( bottom ) Ago2 - miRNA at single spots in the microfluidic chamber . D ) Dwell time histograms obtained for N = 4 , 5 and 6 , with wild type Ago2 ; histograms were fit with single exponential decay . The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution . The bin size is 0 . 5 sec for N = 4 and 1 sec for N = 5 and 6 . E ) Bar plot showing the dependence of dwell time on N for wild type Ago2 . The dashed red line indicates the observation time limit ( 300 s ) , which is constrained by photobleaching . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days . F ) Dwell time histogram for different N ' s with the MI - AA helix - 7 mutant . The dwell time histogram for N = 6 fit with double exponential decay ( green ; R2 = 0 . 98 ) ; The dwell time for N = 6 with single exponential fit was 15 . 2 ± 3 . 41 sec , R2 = 0 . 96 ) . G ) Bar plot for dwell times of MI - AA Ago2 as a function of N . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days . H ) Dwell time histogram for different N ' s with the ∆ helix - 7 mutant . The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays ( green ; R2 = 0 . 97 for N = 5 and R2 = 0 . 96 for N = 6 ) ; The dwell time for N = 5 and N = 6 with single exponential fit was 9 . 1 ± 0 . 91 sec ( R2 = 0 . 86 ) and 22 . 3 ± 5 . 08 sec ( R2 = 0 . 90 ) respectively ) . I ) Bar plot for dwell times of ∆ helix - 7 Ago2 as a function of N . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

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

Figure 3C) Representative fluorescence time traces (with a resolution of 100 ms for wt, 300 ms for MI-AA and Ηhelix-7) show docking and dissociation of wild type (top), MI-AA (middle), and Ηhelix-7 (bottom) Ago2-miRNA at single spots in the microfluidic chamber.D) Dwell time histograms obtained for N = 4, 5 and 6, with wild type Ago2; histograms were fit with single exponential decay. The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution. The bin size is 0.5 sec for N =4 and 1 sec for N =5 and 6.E) Bar plot showing the dependence of dwell time on N for wild type Ago2. The dashed red line indicates the observation time limit (300 s), which is constrained by photobleaching.For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.F) Dwell time histogram for different N's with the MI-AA helix-7 mutant. The dwell time histogram for N = 6 fit with double exponential decay (green; R2 = 0.98); The dwell time for N = 6 with single exponential fit was 15.2 ± 3.41 sec, R2 = 0.96).G) Bar plot for dwell times of MI-AA Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.H) Dwell time histogram for different N's with the ∆helix-7 mutant. The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays (green; R2 = 0.97 for N = 5 and R2 = 0.96 for N = 6); The dwell time for N = 5 and N = 6 with single exponential fit was 9.1 ± 0.91 sec (R2 = 0.86) and 22.3 ± 5.08 sec (R2 = 0.90) respectively).I) Bar plot for dwell times of ∆helix-7 Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


Figure 4Figure 4 . Helix - 7 discourages non - Watson Crick pairing at g6 and g7 . A - C ) Plots of target RNA ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 ( wild type and Ηhelix - 7 ) concentration for targets with an increasing number of G : U wobble pairs in the seed 3 ' end . D ) Plot of target RNA ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 concentration for a target with mismatches at positions g6 - g8 . For A - E , average values from at least three independent experiments ± SD are plotted . E ) Bar plots of the Kd of wild type ( left ) Ηhelix - 7 ( right ) Ago2 - miR122 for indicated target RNAs . For A - E , average values from at least three independent experiments ± SD are plotted .

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Figure 4Figure 4. Helix-7 discourages non-Watson Crick pairing at g6 and g7. A-C) Plots of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 (wild type and Ηhelix-7) concentration for targets with an increasing number of G:U wobble pairs in the seed 3' end. D) Plot of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 concentration for a target with mismatches at positions g6-g8. For A-E, average values from at least three independent experiments ± SD are plotted.E) Bar plots of the Kd of wild type (left) Ηhelix-7 (right) Ago2-miR122 for indicated target RNAs. For A-E, average values from at least three independent experiments ± SD are plotted.


Figure 5Figure 5 . ΗHelix - 7 Ago2 is sensitive to inhibition by off - target RNAs . A ) Ago2 - miRNA122 ( 1 nM ) was incubated with a 32P - labeled , seed - matched target RNA ( 0 . 1 nM ) in the presence of increasing concentrations of unlabeled a competitor RNA ( top panel ) . Fraction target RNA bound to Ago2 is plotted as a function of off - target competitor RNA concentration . B ) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA . For A - B , average values from at least three independent experiments ± SD are plotted .

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

Figure 5Figure 5. ΗHelix-7 Ago2 is sensitive to inhibition by off-target RNAs. A) Ago2-miRNA122 (1 nM) was incubated with a 32P-labeled, seed-matched target RNA (0.1 nM) in the presence of increasing concentrations of unlabeled a competitor RNA (top panel). Fraction target RNA bound to Ago2 is plotted as a function of off-target competitor RNA concentration. B) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA. For A-B, average values from at least three independent experiments ± SD are plotted.


Figure 6Figure 6 . Helix - 7 mutants display reduced target - binding rates . A ) Ago2 - miR122 complexes were mixed with a 32P - labeled seed matched target RNA ; bound and free RNAs were then separated using a filter - binding apparatus at various times . Representative time course for target RNA ( 0 . 1 nM ) binding to wild type , MI - AA , and Δhelix - 7 Ago2 ( 1 nM ) . Average values from at least three independent experiments ± SD are plotted . B ) Observed binding rates ( kon , obs ) plotted as a function of Ago2 - miR122 concentration . Average values ± SD are plotted . C ) Arrival time distribution of single Ago2 - miRNA complexes binding immobilized targets in a microfluidic chamber . The value is the average of three independent measurements .

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

Figure 6Figure 6. Helix-7 mutants display reduced target-binding rates. A) Ago2-miR122 complexes were mixed with a 32P-labeled seed matched target RNA; bound and free RNAs were then separated using a filter-binding apparatus at various times. Representative time course for target RNA (0.1 nM) binding to wild type, MI-AA, and Δhelix-7 Ago2 (1 nM). Average values from at least three independent experiments ± SD are plotted.B) Observed binding rates (kon, obs) plotted as a function of Ago2-miR122 concentration. Average values ± SD are plotted.C) Arrival time distribution of single Ago2-miRNA complexes binding immobilized targets in a microfluidic chamber. The value is the average of three independent measurements.


Figure 7Figure 7 . Structure of a helix - 7 Ago2 mutant . A ) Superposition of wild type ( white ) and MI - AA ( gray ) Ago2 . Helix - 7 colored yellow ; ordered region of the guide RNAs colored in red . B ) Close - up view shows helix - 7 tilts away from the seed region in the MI - AA mutant . C ) Side - by - side comparison of the seed region in wild type ( left ) and MI - AA ( right ) Ago2 crystal structures .

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Figure 7Figure 7. Structure of a helix-7 Ago2 mutant. A) Superposition of wild type (white) and MI-AA (gray) Ago2. Helix-7 colored yellow; ordered region of the guide RNAs colored in red.B) Close-up view shows helix-7 tilts away from the seed region in the MI-AA mutant.C) Side-by-side comparison of the seed region in wild type (left) and MI-AA (right) Ago2 crystal structures.


Figure 1A . Cryo - sections of human organoids were analyzed for the presence of enterocytes ( E - cad ) , Goblet cells ( Muc - 2 ) and tight junctions ( ZO - 1 ) by indirect immunofluorescence . Nuclei are stained with DAPI . Scale bar 25 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test . B . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . 16hpi organoids were frozen , cryo - sectioned , and HAstV1 infected cells were visualized by indirect immunofluorescence ( HAstV1 ( red ) , nuclei ( DAPI , blue ) . Scale bar 25 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test . C . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells ( green ) were visualized by indirect immunofluorescence . Apical and basolateral membranes were immunostained for actin ( magenta ) and Laminin ( red ) respectively . Nuclei are stained with DAPI ( blue ) . Scale bar is 20 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test . E . Human intestinal organoids were infected with HAstV1 . At indicated time post - infection the increase in viral copy number was determined by q - RT - PCR . F . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . At 24 hpi the presence of IFNλ in the media was tested by ELISA . Dotted line indicates detection limit of the assay . G . Same as E but for the induced levels of either type I IFN ( IFNβ1 ) or type III IFN ( IFNλ ) . H . Human intestinal organoids were pre - treated for 24h with 2000IU / mL of IFNβ1 or 300ng / mL of IFNλ1 - 3 . Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q - RT - PCR .

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

Figure 1A. Cryo-sections of human organoids were analyzed for the presence of enterocytes (E-cad), Goblet cells (Muc-2) and tight junctions (ZO-1) by indirect immunofluorescence. Nuclei are stained with DAPI. Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.B. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. 16hpi organoids were frozen, cryo-sectioned, and HAstV1 infected cells were visualized by indirect immunofluorescence (HAstV1 (red), nuclei (DAPI, blue). Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.C. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells (green) were visualized by indirect immunofluorescence. Apical and basolateral membranes were immunostained for actin (magenta) and Laminin (red) respectively. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.E. Human intestinal organoids were infected with HAstV1. At indicated time post-infection the increase in viral copy number was determined by q-RT-PCR.F. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. At 24 hpi the presence of IFNλ in the media was tested by ELISA. Dotted line indicates detection limit of the assay.G. Same as E but for the induced levels of either type I IFN (IFNβ1) or type III IFN (IFNλ).H. Human intestinal organoids were pre-treated for 24h with 2000IU/mL of IFNβ1 or 300ng/mL of IFNλ1-3. Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q-RT-PCR.


Figure 3J - M . Percentage of stem cells ( OLFM4 ) goblet cell ( FGCBP ) , mature enterocytes ( APOA4 ) and cells of the enterocyte lineage ( FGCBP ) from the total organoid cells population ( black bar ) in mock - treated organoids and after HAstV1 infection . The percentage of infected cells ( red bars ) and bystander cells ( blue bars ) within each cell type is shown . The data was obtained from multiplex in situ RNA hybridization in 2D organoids . n = 10 , mean ± SEM . Statistics show comparison of infected cells between mock - treated , 4h pi and 16h pi organoids . An Ordinary one - way ANOVA and Tukey ' s multiple comparisons test was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Figure 3J-M. Percentage of stem cells (OLFM4) goblet cell (FGCBP), mature enterocytes (APOA4) and cells of the enterocyte lineage (FGCBP) from the total organoid cells population (black bar) in mock-treated organoids and after HAstV1 infection. The percentage of infected cells (red bars) and bystander cells (blue bars) within each cell type is shown. The data was obtained from multiplex in situ RNA hybridization in 2D organoids. n=10, mean ± SEM. Statistics show comparison of infected cells between mock-treated, 4h pi and 16h pi organoids. An Ordinary one-way ANOVA and Tukey's multiple comparisons test was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001.


Figure 5A . One image per condition was used to show the multiplex in situ RNA FISH . A representative region of the images was chosen and cropped in order to have a zoom - in of a specific area for better visualization . Shown are the enterocyte lineage marker FABP6 ( green ) , HAstV1 infection ( white ) , and CCL2 gene expression ( red ) of mock - treated and infected organoids . DAPI is in blue . White arrows show co - localization of FABP6 and CCL2 with HAstV1 signal . Scale bar 100µm . B - D . Fluorescence intensity ( arbitrary units ) of innate immune marker expression in stem cells ( OLFM4 positive ) , goblet cells ( FCGBP positive ) , enterocyte lineage cells ( FABP6 positive ) , and mature enterocytes ( APOA4 positive ) . Bystander cells are in blue and infected cells in red . 10 - 90 percentile box plots , each dot represents one cell ( mock N = 2274 , 4h N = 3682 , 16h N = 1510 . C . mock N = 2693 , 4h N = 3706 , 16h N = 1921 . D . mock N = 2274 , 4h N = 3706 , 16h N = 1514 . ) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages . Ordinary one - way ANOVA and Tukey ' s multiple comparisons test were used . Grey statistics show the comparison between infected and bystander cells within each cell lineage . Unpaired t - test with Welch ' s correction was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. One image per condition was used to show the multiplex in situ RNA FISH. A representative region of the images was chosen and cropped in order to have a zoom-in of a specific area for better visualization. Shown are the enterocyte lineage marker FABP6 (green), HAstV1 infection (white), and CCL2 gene expression (red) of mock-treated and infected organoids. DAPI is in blue. White arrows show co-localization of FABP6 and CCL2 with HAstV1 signal. Scale bar 100µm.B-D. Fluorescence intensity (arbitrary units) of innate immune marker expression in stem cells (OLFM4 positive), goblet cells (FCGBP positive), enterocyte lineage cells (FABP6 positive), and mature enterocytes (APOA4 positive). Bystander cells are in blue and infected cells in red. 10-90 percentile box plots, each dot represents one cell (mock N=2274, 4h N=3682, 16h N=1510. C. mock N=2693, 4h N=3706, 16h N=1921. D. mock N=2274, 4h N=3706, 16h N=1514.) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages. Ordinary one-way ANOVA and Tukey's multiple comparisons test were used. Grey statistics show the comparison between infected and bystander cells within each cell lineage. Unpaired t-test with Welch's correction was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001


Figure 1C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography . C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Total leukocyte and red blood cell count in PB . CoPP increases the number of all WBC populations . Granulocyte and monocyte percentage are increasing , whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP . C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay . CoPP increases the concentrations of a group of cytokines ( red box ) . C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Selected cytokine and growth factor concentrations in plasma measured by Luminex assay . CoPP increases concentrations of G - CSF , MCP - 1 , IP - 10 , IL - 6 and IL - 5 . Results are shown as mean + SEM , one - way Anova with Bonferroni post - test , n = 5 mice per group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O 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 1C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography.C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Total leukocyte and red blood cell count in PB. CoPP increases the number of all WBC populations. Granulocyte and monocyte percentage are increasing, whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP.C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases the concentrations of a group of cytokines (red box).C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Selected cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases concentrations of G-CSF, MCP-1, IP-10, IL-6 and IL-5. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 5 mice per group.


Figure 2C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . ( A ) Cell numbers of main leukocyte populations in PB measured by flow cytometry . Both G - CSF and CoPP increase numbers of CD45 + cells in blood . G - CSF and CoPP similarly increase monocyte numbers , but the increase of granulocytes is higher after G - CSF . Lymphocytes , including T cells , are increased only by G - CSF and not by CoPP . C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Cytokine and growth factor concentrations in plasma measured by Luminex assay . CoPP induces a group of cytokines ( red box ) which are not induced by G - CSF . C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Plasma concentrations of selected cytokines and growth factors . CoPP highly increases concentration of endogenous G - CSF and IL - 6 that are not increased by G - CSF . C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Treatment with G - CSF and CoPP decreases percentage of Ly6G + macrophages in the BMC57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Treatment with G - CSF and CoPP increases relative spleen weight . C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . viSNE maps of CD11b + CD11c - blood cells , colored by SSC value . Three independent experiments were performed and the results were pooled . Results are shown as mean + SEM , one - way Anova with Bonferroni post - test , n = 7 mice per group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. (A) Cell numbers of main leukocyte populations in PB measured by flow cytometry. Both G-CSF and CoPP increase numbers of CD45+ cells in blood. G-CSF and CoPP similarly increase monocyte numbers, but the increase of granulocytes is higher after G-CSF. Lymphocytes, including T cells, are increased only by G-CSF and not by CoPP.C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP induces a group of cytokines (red box) which are not induced by G-CSF.C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Plasma concentrations of selected cytokines and growth factors. CoPP highly increases concentration of endogenous G-CSF and IL-6 that are not increased by G-CSF.C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP decreases percentage of Ly6G+ macrophages in the BMC57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP increases relative spleen weight.C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. viSNE maps of CD11b+ CD11c- blood cells, colored by SSC value. Three independent experiments were performed and the results were pooled. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 7 mice per group.


Figure 3C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after the last injection . ( A ) Representative flow cytometry plots show higher granularity ( SSC ) and Ly6G expression in CoPP - mobilized granulocytes than in G - CSF mobilized granulocytes . C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after the last injection . Relative abundance of different granulocyte phenotypes in blood of mice treated with G - CSF and CoPP . Mice treated with CoPP have higher proportion of granulocytes with mature phenotype , than mice treated with G - CSF . C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after the last injection . Relative abundance of different granulocyte phenotypes in BM ( C ) of mice treated with G - CSF and CoPP . Mice treated with CoPP have higher proportion of granulocytes with mature phenotype , than mice treated with G - CSF . C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after the last injection . The percentage of cells isolated from C57BL / 6xFVB mice treated with G - CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli .

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

Figure 3C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. (A) Representative flow cytometry plots show higher granularity (SSC) and Ly6G expression in CoPP-mobilized granulocytes than in G-CSF mobilized granulocytes.C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. Relative abundance of different granulocyte phenotypes in blood of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF.C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. Relative abundance of different granulocyte phenotypes in BM (C) of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF.C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. The percentage of cells isolated from C57BL/6xFVB mice treated with G-CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli.


Figure 4Gating strategy of KLS ( c - Kit + Lin - Sca - 1 + ) cells in blood , using CD48 and CD150 to distinguish HSC ( hematopoietic stem cells ) , MPP ( multipotent progenitors ) and HPC ( hematopoietic progenitors ) . CoPP mobilizes higher numbers of HSC ( KLS CD48 - CD150 + ) , MPP ( KLS CD48 - CD150 - ) , HPC - 1 ( KLS CD48 + CD150 - ) and HPC - 2 ( KLS CD48 + CD150 + ) cells than G - CSF to the PB . Treatment with CoPP increases percentage of KLS cells in BM , what is related to increase in HPC populations , but not in LT - HSC fraction . The number of KLS - ( c - Kit + Lin - Sca - 1 - ) is higher in mice treated with CoPP than in mice treated with G - CSF . Numbers of lineage committed progenitors : GMP ( granulocyte - macrophage progenitors , KLS - CD48 + CD150 - CD34 + ) and MEP ( megakaryocyte - erythroid progenitors , KLS - CD48 + CD150 + CD34 - ) were higher after CoPP administration than after G - CSF . G - CSF and CoPP similarly increase EP numbers ( erythrocyte progenitors , KLS - CD48 - CD150 - CD34 - ) . Both G - CSF and CoPP treatments decrease the KLS - percentage in the BM , however the decrease after CoPP is smaller . G - CSF and CoPP decrease percentages of MEP and EP , but only G - CSF decreases the percentage of GMP . Results are shown as mean + individual values , one - way Anova with Bonferroni post - test , n = 7 mice per group .

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

Figure 4Gating strategy of KLS (c-Kit+Lin-Sca-1+) cells in blood, using CD48 and CD150 to distinguish HSC (hematopoietic stem cells), MPP (multipotent progenitors) and HPC (hematopoietic progenitors).CoPP mobilizes higher numbers of HSC (KLS CD48-CD150+), MPP (KLS CD48-CD150-), HPC-1 (KLS CD48+CD150-) and HPC-2 (KLS CD48+CD150+) cells than G-CSF to the PB.Treatment with CoPP increases percentage of KLS cells in BM, what is related to increase in HPC populations, but not in LT-HSC fraction.The number of KLS- (c-Kit+Lin-Sca-1-) is higher in mice treated with CoPP than in mice treated with G-CSF. Numbers of lineage committed progenitors: GMP (granulocyte-macrophage progenitors, KLS- CD48+CD150-CD34+) and MEP (megakaryocyte-erythroid progenitors, KLS- CD48+CD150+CD34-) were higher after CoPP administration than after G-CSF. G-CSF and CoPP similarly increase EP numbers (erythrocyte progenitors, KLS- CD48-CD150-CD34-).Both G-CSF and CoPP treatments decrease the KLS- percentage in the BM, however the decrease after CoPP is smaller. G-CSF and CoPP decrease percentages of MEP and EP, but only G-CSF decreases the percentage of GMP. Results are shown as mean + individual values, one-way Anova with Bonferroni post-test, n = 7 mice per group.


Figure 5Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor cells . After 18 or 20 weeks we performed secondary transplantation of primary recipients ' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks . ( A ) Effect of G - CSF and CoPP - induced mobilization in C57BL / 6 - Tg ( UBC - GFP ) 30Sch / J donor mice ( mean + individual values , one - way Anova with Bonferroni post - test , n = 16 mice per group ) : Donor mice treated with CoPP had the highest number of mobilized granulocytes ( i ) and KLS cells ( ii ) , compared to control or G - CSF - treated donor miceDonor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor cells . After 18 or 20 weeks we performed secondary transplantation of primary recipients ' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks . Recipient mice which were transplanted with CoPP - mobilized PBMC had the highest number of WBC , PLT and the highest hematocrit values 2 weeks after PBMC transplantation , compared to G - CSF - mobilized and control PBMC recipients ( mean + individual values , one - way Anova with Bonferroni post - test , NaCl : n = 8 , G - CSF : n = 7 , CoPP : n = 9 mice per group ) . Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor cells . After 18 or 20 weeks we performed secondary transplantation of primary recipients ' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks . Recipient mice which were transplanted with CoPP - mobilized PBMC had the highest chimerism among CD45 + cells , granulocytes and B cells 2 , 4 and 18 weeks after transplantation . Chimerism among T cells was the highest in recipients of G - CSF - mobilized PBMC 2 weeks after transplantation , but 18 weeks after transplantation it was the highest in CoPP - mobilized PBMC recipients ( mean + SEM , two - way Anova with Bonferroni post - test , NaCl : n = 8 , G - CSF : n = 7 , CoPP : n = 9 mice per group ) . Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor cells . After 18 or 20 weeks we performed secondary transplantation of primary recipients ' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks . The majority of primary recipients transplanted with G - CSF and CoPP - mobilized PBMC have detectable GFP chimerism among BM KLS cells . Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor cells . After 18 or 20 weeks we performed secondary transplantation of primary recipients ' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks . Fraction of secondary recipient mice with chimerism in PB CD45 + cells exceeding 1 % tends to be higher after CoPP - mobilized PBMC transplant than G - CSF - mobilized PBMC transplant . Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor cells . After 18 or 20 weeks we performed secondary transplantation of primary recipients ' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks . PB chimerism in secondary recipients is higher in CoPP group than in the control group ( mean + individual values , Kruskal - Wallis test with Dunn ' s post - test , NaCl : n = 7 , G - CSF : n = 6 , CoPP : n = 8 mice per group ) . Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor cells . After 18 or 20 weeks we performed secondary transplantation of primary recipients ' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks . Percentage of secondary recipient mice with GFP chimerism in BM KLS ( G ) and KLS - ( H ) cells higher that 1 % is similar in G - CSF and CoPP groups . Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor cells . After 18 or 20 weeks we performed secondary transplantation of primary recipients ' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks . GFP chimerism in KLS ( I ) and KLS - ( J ) cells in the BM of secondary recipients ( mean + individual values , Kruskal - Wallis test with Dunn ' s post - test , NaCl : n = 7 , G - CSF : n = 6 , CoPP : n = 8 mice per group ) .

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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 5Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. (A) Effect of G-CSF and CoPP-induced mobilization in C57BL/6-Tg(UBC-GFP)30Sch/J donor mice (mean + individual values, one-way Anova with Bonferroni post-test, n = 16 mice per group): Donor mice treated with CoPP had the highest number of mobilized granulocytes (i) and KLS cells (ii), compared to control or G-CSF-treated donor miceDonor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest number of WBC, PLT and the highest hematocrit values 2 weeks after PBMC transplantation, compared to G-CSF-mobilized and control PBMC recipients (mean + individual values, one-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group).Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest chimerism among CD45+ cells, granulocytes and B cells 2, 4 and 18 weeks after transplantation. Chimerism among T cells was the highest in recipients of G-CSF-mobilized PBMC 2 weeks after transplantation, but 18 weeks after transplantation it was the highest in CoPP-mobilized PBMC recipients (mean + SEM, two-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group).Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. The majority of primary recipients transplanted with G-CSF and CoPP-mobilized PBMC have detectable GFP chimerism among BM KLS cells.Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Fraction of secondary recipient mice with chimerism in PB CD45+ cells exceeding 1% tends to be higher after CoPP-mobilized PBMC transplant than G-CSF-mobilized PBMC transplant.Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. PB chimerism in secondary recipients is higher in CoPP group than in the control group (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Percentage of secondary recipient mice with GFP chimerism in BM KLS (G) and KLS-(H) cells higher that 1% is similar in G-CSF and CoPP groups.Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. GFP chimerism in KLS (I) and KLS- (J) cells in the BM of secondary recipients (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).


Figure 6Wild type C57BL / 6 mice were injected with anti - G - CSF antibody alone or in combination with anti - IL - 6 antibody one hour prior to CoPP treatment ; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection . G - CSF and IL - 6 concentration in plasmaWild type C57BL / 6 mice were injected with anti - G - CSF antibody alone or in combination with anti - IL - 6 antibody one hour prior to CoPP treatment ; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection . CD45 + cells , granulocytes and HSC numbers in PB measured by flow cytometry . HO - 1 deficient and control C57BL / 6xFVB mice were injected with CoPP three times , every second day and sacrificed 24 hours after last injection . Total leukocyte counts in PB ( HO - 1 + / + : n = 5 , HO - 1 - / - DMSO : n = 6 , HO - 1 - / - CoPP : n = 4 mice per group ) . Granulocyte percentage among PB leukocytes . HO - 1 deficient and control C57BL / 6xFVB mice were injected with CoPP three times , every second day and sacrificed 24 hours after last injection . Cytokine and growth factor concentrations in plasma ( E - HO - 1 + / + n = 5 , HO - 1 - / - DMSO : n = 5 , HO - 1 - / - CoPP : n = 3 mice per group ) . Results are shown as mean + SEM , two - way Anova with Bonferroni post - test . Nrf2 - deficient and control C57BL / 6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection ( DMSO : n = 4 , CoPP : n = 5 mice per group ) . CD45 + cell numbers in PB measured by flow cytometry . Granulocyte percentage among PB leukocytes . Nrf2 - deficient and control C57BL / 6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection ( DMSO : n = 4 , CoPP : n = 5 mice per group ) . Cytokine and growth factor concentrations in

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 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 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 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

Figure 6Wild type C57BL/6 mice were injected with anti-G-CSF antibody alone or in combination with anti-IL-6 antibody one hour prior to CoPP treatment; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection. G-CSF and IL-6 concentration in plasmaWild type C57BL/6 mice were injected with anti-G-CSF antibody alone or in combination with anti-IL-6 antibody one hour prior to CoPP treatment; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection. CD45+ cells, granulocytes and HSC numbers in PB measured by flow cytometry.HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. Total leukocyte counts in PB (HO-1+/+: n = 5, HO-1-/- DMSO: n = 6, HO-1-/- CoPP: n = 4 mice per group). Granulocyte percentage among PB leukocytes.HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. Cytokine and growth factor concentrations in plasma (E - HO-1+/+ n = 5, HO-1-/- DMSO: n = 5, HO-1-/- CoPP: n = 3 mice per group). Results are shown as mean + SEM, two-way Anova with Bonferroni post-test.Nrf2-deficient and control C57BL/6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection (DMSO: n = 4, CoPP: n = 5 mice per group). CD45+ cell numbers in PB measured by flow cytometry. Granulocyte percentage among PB leukocytes.Nrf2-deficient and control C57BL/6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection (DMSO: n = 4, CoPP: n = 5 mice per group). Cytokine and growth factor concentrations in plasma


Figure 1IL - 1β ELISA of supernatants from LPS - primed ( 1 μg / mL , 4hrs . ) MDMs pre - incubated with 0 . 1 % DMSO or P22077 ( 2 . 5 µM ) 15 mins . before treatment with either nigericin ( 10 μM , 45 mins . ) or CPPD crystals ( 250 μg / mL , 2 hrs . ) . Bars represent the mean , ± S . D . n = 13 and 11 independent blood donors for nigericin and CPPD , respectively . IL - 18 ELISA of supernatants from MDMs treated as in A . Bars represent the mean , ± S . D . n = 11 independent blood donors . LDH - release from MDMs treated as in A . Bars represent the mean percentage of LDH release relative to the total cells lysed , ± S . D . n = 10 and 11 independent blood donors for nigericin and CPPD , respectively . Western blots of supernatants and cell lysates from MDMs treated as in A . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) ; pro - IL - 18 ( 24 kDa ) , mature IL - 18 ( mIL - 18 , 18 kDa ) , pro - caspase - 1 ( pro - Casp - 1 ; 45 kDa ) and mature caspase - 1 ( mCasp - 1 ; 20 kDa ) . β - actin is shown as a loading control . Blots are representative of at least 3 independent blood donors .

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Figure 1IL-1β ELISA of supernatants from LPS-primed (1 μg/mL, 4hrs.) MDMs pre-incubated with 0.1% DMSO or P22077 (2.5 µM) 15 mins. before treatment with either nigericin (10 μM, 45 mins.) or CPPD crystals (250 μg/mL, 2 hrs.). Bars represent the mean, ± S.D. n = 13 and 11 independent blood donors for nigericin and CPPD, respectively. IL-18 ELISA of supernatants from MDMs treated as in A. Bars represent the mean, ± S.D. n = 11 independent blood donors.LDH-release from MDMs treated as in A. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 10 and 11 independent blood donors for nigericin and CPPD, respectively.Western blots of supernatants and cell lysates from MDMs treated as in A. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa), pro-caspase-1 (pro-Casp-1; 45 kDa) and mature caspase-1 (mCasp-1; 20 kDa). β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.


Figure 2Quantitative RT - PCR showing the mRNA expression levels of IL - 1β , IL - 18 , ASC , CASP1 and NLRP3 in MDMs treated with P22077 ( 2 . 5 µM ) and / or LPS ( 1 μg / mL ; 4 hrs . ) , as indicated . Bars represent the mean fold increase from an untreated ( UT ) controlWestern blots showing the relative abundance of NLRP3 , ASC , pro - caspase - 1 , pro - IL - 1β , pro - IL - 18 , USP7 and USP47 in cell lysates of MDMs treated with P22077 ( 2 . 5 µM ) and / or LPS ( 1 μg / mL ) for 4 hrs . , as indicated . β - actin is shown as a loading control . Blots are representative of at least 3 independent blood donors . IL - 18 ELISA of supernatants from LPS - primed ( 1 μg / mL , 10 mins . ) MDMs pre - incubated with either vehicle ( 0 . 1 % DMSO ) or P22077 ( 2 . 5 µM ) for 15 mins . before treatment with nigericin ( 10 μM , 45 mins . ) . Bars represent the mean , ± S . D . n = 9 independent blood donors . Western blots of supernatants and cell lysates from MDMs as treated in C . Bands in the figure represent : Pro - IL - 18 ( 24 kDa ) , mature IL - 18 ( mIL - 18 , 18 kDa ) and β - actin as a loading control . Blots are representative of at least 3 independent blood donors . LDH - release from MDMs as treated in C . Bars represent the mean percentage of LDH release relative to the total cells lysed , ± S . D . n = 9 independent blood donors .

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Figure 2Quantitative RT-PCR showing the mRNA expression levels of IL-1β, IL-18, ASC, CASP1 and NLRP3 in MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL; 4 hrs.), as indicated. Bars represent the mean fold increase from an untreated (UT) controlWestern blots showing the relative abundance of NLRP3, ASC, pro-caspase-1, pro- IL-1β, pro-IL-18, USP7 and USP47 in cell lysates of MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL) for 4 hrs., as indicated. β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.IL-18 ELISA of supernatants from LPS-primed (1 μg/mL, 10 mins.) MDMs pre-incubated with either vehicle (0.1% DMSO) or P22077 (2.5 µM) for 15 mins. before treatment with nigericin (10 μM, 45 mins.). Bars represent the mean, ± S.D. n = 9 independent blood donors.Western blots of supernatants and cell lysates from MDMs as treated in C. Bands in the figure represent: Pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa) and β-actin as a loading control. Blots are representative of at least 3 independent blood donors.LDH-release from MDMs as treated in C. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 9 independent blood donors.


Figure 3Western blots showing ASC - oligomerisation after DSS - mediated crosslinking in insoluble fractions from THP1 cell lysates . PMA differentiated THP1 cells were LPS - primed ( 1 μg / mL , 4 hrs . ) and pre - incubated for 15 min with either 0 . 1 % DMSO or P22077 ( 2 . 5 µM ) . followed by treatment with nigericin ( 10 μM , 45 mins . ) . The ' input ' represents the abundance of ASC in cell lysates prior to isolation of the insoluble fraction . Blots show membranes that were probed with an anti - ASC antibody . Data is representative of at least three independent experiments . Western blots showing ASC - oligomerisation after DSS - mediated crosslinking in supernatants from THP1s treated as in A . Blots show membranes that were probed with an anti - ASC antibody . The input represents non - crosslinked supernatant . Data is representative of at least three independent experiments . Images from an immunofluorescence experiment showing ASC - speck formation in THP1 cells treated as in A . All images show cells that were stained with an anti - ASC antibody . Images are representative of 3 independent experiments . Scale bar 20µm . Quantification of ASC - speck formation in images depicted in C . The number of ASC specks was quantified and expressed as the percentage of cells containing specks .

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Figure 3Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in insoluble fractions from THP1 cell lysates. PMA differentiated THP1 cells were LPS-primed (1 μg/mL, 4 hrs.) and pre-incubated for 15 min with either 0.1% DMSO or P22077 (2.5 µM). followed by treatment with nigericin (10 μM, 45 mins.). The 'input' represents the abundance of ASC in cell lysates prior to isolation of the insoluble fraction. Blots show membranes that were probed with an anti-ASC antibody. Data is representative of at least three independent experiments.Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in supernatants from THP1s treated as in A. Blots show membranes that were probed with an anti-ASC antibody. The input represents non-crosslinked supernatant. Data is representative of at least three independent experiments.Images from an immunofluorescence experiment showing ASC-speck formation in THP1 cells treated as in A. All images show cells that were stained with an anti-ASC antibody. Images are representative of 3 independent experiments. Scale bar 20µm. Quantification of ASC-speck formation in images depicted in C. The number of ASC specks was quantified and expressed as the percentage of cells containing specks.


Figure 4NLRP3 oligomerisation analysed by native PAGE . LPS - primed ( 1 μg / mL , 4 hrs . ) murine BMDMs pre - incubated with either 0 . 1 % DMSO or P22077 ( at the indicated concentrations ) for 15 mins . before treatment with ATP ( 5 mM , 30 min . ) . Digitonin - solubilised cell lysates were analysed by native - PAGE or SDS - PAGE and NLRP3 western - blotting . Western blots of supernatants and cell lysates for IL - 1β are also shown as a control for P22077 inflammasome inhibition . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) and NLRP3 ( 120 kDa ) . β - actin is shown as a loading control . Blots are representative of 4 independent murine donors . NLRP3 ubiquitination analysed by western blot . LPS - primed ( 1 μg / mL , 4 hrs . ) murine BMDMs pre - incubated with either 0 . 1 % DMSO or P22077 ( at the indicated concentrations ) for 15 mins . before treatment with ATP ( 5 mM , 30 mins . ) . Endogenous NLRP3 was immunoprecipitated ( IP ) with anti - NLRP3 and probed with either anti - ubiquitin ( Poly - Ub ) , anti - K63 - ubiquitin ( Poly - K63 - Ub ) or anti - NLRP3 . As a negative control , cell lysate made from LPS - primed and 10 μM P22077 treated cells was immunoprecipitated with a nonspecific IgG . Total levels of NLRP3 in cell lysates ( input ) were also probed . Western blots of supernatants and cell lysates for IL - 1β are also shown as a control for P22077 inhibition . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) and NLRP3 ( 120kDa ) . β - actin is shown as a loading control . Blots are representative of 2 independent murine donors for 2 . 5µM P22077 and 4 independent murine donors for 10 µM .

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Figure 4NLRP3 oligomerisation analysed by native PAGE. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 min.). Digitonin-solubilised cell lysates were analysed by native-PAGE or SDS-PAGE and NLRP3 western-blotting. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inflammasome inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120 kDa). β-actin is shown as a loading control. Blots are representative of 4 independent murine donors.NLRP3 ubiquitination analysed by western blot. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 mins.). Endogenous NLRP3 was immunoprecipitated (IP) with anti-NLRP3 and probed with either anti-ubiquitin (Poly-Ub), anti-K63-ubiquitin (Poly-K63-Ub) or anti-NLRP3. As a negative control, cell lysate made from LPS-primed and 10 μM P22077 treated cells was immunoprecipitated with a nonspecific IgG. Total levels of NLRP3 in cell lysates (input) were also probed. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120kDa). β-actin is shown as a loading control. Blots are representative of 2 independent murine donors for 2.5µM P22077 and 4 independent murine donors for 10 µM.


Figure 5Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin . MDMs were either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) and then treated with nigericin ( 10 μM ) for differing lengths of time , as indicated . Relative , activity - based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs . Higher panels show lysates incubated with ABPs ( labelled ) , with the active DUB bound to ABP indicated by the suffix ' - VME ' . Lower panels , without probe labelling , show total USP7 or USP47 levels . ( Unlabelled ) . Western blots are representative of 3 independent MDM donors . Western blots showing activity changes in both USP7 and USP47 upon treatment with CPPD . MDMs were either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) and then pre - incubated with either 0 . 1 % DMSO or P22077 ( 2 . 5 μM ) prior to incubation with CPPD crystals ( 250 µg / mL , 3 hrs . ) . Blots are labelled as in B and are representative of 3 independent MDM donors . Western blots showing activity changes in both USP7 and USP47 in LPS - primed ( 1 μg / mL , 4 hrs . ) MDMs pre - incubated with different P22077 concentrations , as indicated , prior to activation by nigericin treatment ( 10 μM ; 45 mins . ) . Blots are labelled as in B and are representative of 3 independent MDM donors . Western blots showing activity changes in both USP7 and USP47 upon activation in high extracellular potassium ( High K + ) conditions . MDMs were LPS - primed ( 1 μg / mL , 4 hrs . ) and then changed to media that either had normal or high concentrations of K + , and subsequently treated with nigericin ( 10 μM ; 45 mins . ) . Blots are labelled as in B and are representative of at least 2 independent MDM donors . Pro - IL - 1β ( 31 kDa ) and mature IL - 1β ( mIL - 1β , 17 kDa ) in the supernatants of these cells are shown as a control . Non - specific bands are highlighted with an asterisk .

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Figure 5Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then treated with nigericin (10 μM) for differing lengths of time, as indicated. Relative, activity-based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs. Higher panels show lysates incubated with ABPs (labelled), with the active DUB bound to ABP indicated by the suffix '-VME'. Lower panels, without probe labelling, show total USP7 or USP47 levels. (Unlabelled). Western blots are representative of 3 independent MDM donors.Western blots showing activity changes in both USP7 and USP47 upon treatment with CPPD. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then pre-incubated with either 0.1% DMSO or P22077 (2.5 μM) prior to incubation with CPPD crystals (250 µg/mL, 3 hrs.). Blots are labelled as in B and are representative of 3 independent MDM donors.Western blots showing activity changes in both USP7 and USP47 in LPS-primed (1 μg/mL, 4 hrs.) MDMs pre-incubated with different P22077 concentrations, as indicated, prior to activation by nigericin treatment (10 μM; 45 mins.). Blots are labelled as in B and are representative of 3 independent MDM donors.Western blots showing activity changes in both USP7 and USP47 upon activation in high extracellular potassium (High K+) conditions. MDMs were LPS-primed (1 μg/mL, 4 hrs.) and then changed to media that either had normal or high concentrations of K+, and subsequently treated with nigericin (10 μM; 45 mins.). Blots are labelled as in B and are representative of at least 2 independent MDM donors. Pro-IL-1β (31 kDa) and mature IL-1β (mIL-1β, 17 kDa) in the supernatants of these cells are shown as a control. Non-specific bands are highlighted with an asterisk.


Figure 6Western blots of supernatants and cell lysates from THP - 1 cells engineered by CRISPR / Cas9 to induce USP7 and / or USP47 KOs . Deficiency of both USP7 and USP47 was induced by doxycycline ( Doxy ) treatment ( 1 µg / mL ; 3 days ) , as indicated . All cells were differentiated with PMA and either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) and treated with nigericin ( 10 μM , 45 mins . ) , as indicated . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) ; pro - caspase - 1 ( pro - Casp - 1 ; 45 kDa ) ; mature caspase - 1 ( mCasp - 1 ; 20 kDa ) ; USP7 and USP47 . β - actin is shown as a loading control . Blots are representative of at least 3 independent experiments . IL - 1β release measured from doxycycline treated ( 1 µg / mL ; 3 days ) PMA - differentiated and LPS - primed ( 1 μg / mL , 4 hrs . ) , nigericin activated ( 10 μM , 45 mins . ) THP - 1 cells , which contain the indicated sgRNA . Bars represent the mean percentage of IL - 1β release relative to their respective non - doxycycline treated control cells , ± S . D . LDH - release measured from cells as treated in B . Bars represent the mean percentage of LDH release relative to their respective non - doxycycline treated control cells , ± S . D .

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Figure 6Western blots of supernatants and cell lysates from THP-1 cells engineered by CRISPR/Cas9 to induce USP7 and/or USP47 KOs. Deficiency of both USP7 and USP47 was induced by doxycycline (Doxy) treatment (1 µg/mL; 3 days), as indicated. All cells were differentiated with PMA and either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and treated with nigericin (10 μM, 45 mins.), as indicated. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-caspase-1 (pro-Casp-1; 45 kDa); mature caspase-1 (mCasp-1; 20 kDa); USP7 and USP47. β-actin is shown as a loading control. Blots are representative of at least 3 independent experiments.IL-1β release measured from doxycycline treated (1 µg/mL; 3 days) PMA-differentiated and LPS-primed (1 μg/mL, 4 hrs.), nigericin activated (10 μM, 45 mins.) THP-1 cells, which contain the indicated sgRNA. Bars represent the mean percentage of IL-1β release relative to their respective non-doxycycline treated control cells, ± S.D. LDH-release measured from cells as treated in B. Bars represent the mean percentage of LDH release relative to their respective non-doxycycline treated control cells, ± S.D.