Figure 2 ( B - C ) Catalytically active sACE22 . v2 . 4 - IgG1 ( B ) and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 ( C ) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte ( 0 - 60 s ) and returned to buffer to measure dissociation ( 60 - 360 s ) . RBD concentrations are indicated on the right of the sensorgrams . ( D - E ) Catalytically active sACE22 . v2 . 4 - IgG1 and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 were aerosolized ( 7 . 5 ml protein at 8 . 3 mg / ml in 25 minutes ) and delivered by inhalation to K18 - hACE2 transgenic mice at 12 h , 48 h , and 84 h post - inoculation with SARS - CoV - 2 gamma variant . 10 mice in each group were observed for survival ( D ) and weight loss ( E ) . The P - value of survival curve by Gehan - Breslow - Wilcoxon test is shown . Error bars for mouse weight are centered on the mean and show SEM . Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Catalytically active sACE22.v2.4-IgG1 (B) and catalytically dead sACE22.v2.4(NN)-IgG1 (C) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the sensorgrams.(D-E) Catalytically active sACE22.v2.4-IgG1 and catalytically dead sACE22.v2.4(NN)-IgG1 were aerosolized (7.5 ml protein at 8.3 mg/ml in 25 minutes) and delivered by inhalation to K18-hACE2 transgenic mice at 12 h, 48 h, and 84 h post-inoculation with SARS-CoV-2 gamma variant. 10 mice in each group were observed for survival (D) and weight loss (E). The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown. Error bars for mouse weight are centered on the mean and show SEM. Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1.


Figure 3 ( B - C ) Survival ( B ) and weight loss ( C ) following treatment with an IgG1 isotype control ( red ) versus catalytically active ( blue ) and dead ( green ) sACE22 . v2 . 4 - IgG1 . N = 8 mice for IgG1 group and catalytic active group , N = 9 for catalytic dead group . Error bars for mouse weight are centered on the mean and show SEM . Gehan - Breslow - Wilcoxon test was applied to analyze the survival curves . P = 0 . 0099 for catalytic active group compared to catalytic dead group . P = 0 . 017 for catalytic dead group compared to IgG1 control . ( D - E ) Catalytically active sACE22 . v2 . 4 - IgG1 ( D ) and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 ( E ) were immobilized on BLI biosensors and transferred to solutions of RBD ( original variant ) as the soluble analyte ( 0 - 60 s ) and returned to buffer to measure dissociation ( 60 - 360 s ) . RBD concentrations are indicated on the right of the BLI sensorgrams .

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 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 3(B-C) Survival (B) and weight loss (C) following treatment with an IgG1 isotype control (red) versus catalytically active (blue) and dead (green) sACE22.v2.4-IgG1. N = 8 mice for IgG1 group and catalytic active group, N = 9 for catalytic dead group. Error bars for mouse weight are centered on the mean and show SEM. Gehan-Breslow-Wilcoxon test was applied to analyze the survival curves. P = 0.0099 for catalytic active group compared to catalytic dead group. P = 0.017 for catalytic dead group compared to IgG1 control.(D-E) Catalytically active sACE22.v2.4-IgG1 (D) and catalytically dead sACE22.v2.4(NN)-IgG1 (E) were immobilized on BLI biosensors and transferred to solutions of RBD (original variant) as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the BLI sensorgrams.


Figure 4 ( A ) Binding of monomeric sACE2 - 8his proteins to Expi293F cells expressing S of BA . 1 omicron was measured by flow cytometry . ( B ) Binding of dimeric sACE22 - IgG1 proteins was compared to the binding of antibodies authorized for therapeutic use in COVID - 19 patients . Binding to Expi293F cells expressing BA . 1 omicron S was measured by flow cytometry . ( C ) Neutralization of BA . 1 omicron pseudovirus . sACE22 - IgG1 ( grey ) or sACE22 . v2 . 4 - IgG1 ( blue ) were incubated with pseudovirus for 1 h before adding to HeLa - hACE2 - 11 cells . Infection 48 h later was measured by luciferase reporter gene expression . Data are mean ± SD , N = 3 independent biological replicates . ( D - E ) Authentic BA . 1 omicron virus ( isolate USA / MD - HP20874 / 2021 ) was incubated with sACE22 - IgG1 ( D ) or sACE22 . v2 . 4 - IgG1 ( E ) for 1 h and added to Calu - 3 cells . Infection 48 h later was measured by RT - qPCR for the viral N gene . 3 µM remdesivir ( black columns ) is a positive neutralization control .

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

Figure 4(A) Binding of monomeric sACE2-8his proteins to Expi293F cells expressing S of BA.1 omicron was measured by flow cytometry.(B) Binding of dimeric sACE22-IgG1 proteins was compared to the binding of antibodies authorized for therapeutic use in COVID-19 patients. Binding to Expi293F cells expressing BA.1 omicron S was measured by flow cytometry.(C) Neutralization of BA.1 omicron pseudovirus. sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) were incubated with pseudovirus for 1 h before adding to HeLa-hACE2-11 cells. Infection 48 h later was measured by luciferase reporter gene expression. Data are mean ± SD, N = 3 independent biological replicates.(D-E) Authentic BA.1 omicron virus (isolate USA/MD-HP20874/2021) was incubated with sACE22-IgG1 (D) or sACE22.v2.4-IgG1 (E) for 1 h and added to Calu-3 cells. Infection 48 h later was measured by RT-qPCR for the viral N gene. 3 µM remdesivir (black columns) is a positive neutralization control.


Figure 5 ( B - C ) Survival ( B ) and weight loss ( C ) . N = 8 ( IgG1 control ) or 9 ( sACE22 . v2 . 4 - IgG1 treated ) mice . Error bars for mouse weight are centered on the mean and show SEM . ( E ) Viral Spike , Nsp , and Rdrp mRNA levels in lung tissue were measured by RT - qPCR at Days 8 and 14 , normalized to Ppia transcript levels . Data are presented as mean ± SEM , N = 4 mice per group . ns , not significant ; * * , p < 0 . 01 by two - ways ANOVA , corrected by Tukey .

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 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

Figure 5(B-C) Survival (B) and weight loss (C). N = 8 (IgG1 control) or 9 (sACE22.v2.4-IgG1 treated) mice. Error bars for mouse weight are centered on the mean and show SEM.(E) Viral Spike, Nsp, and Rdrp mRNA levels in lung tissue were measured by RT-qPCR at Days 8 and 14, normalized to Ppia transcript levels. Data are presented as mean ± SEM, N = 4 mice per group. ns, not significant; **, p < 0.01 by two-ways ANOVA, corrected by Tukey.


Figure 6 ( A ) Using flow cytometry , binding to Expi293F cells expressing S of BA . 2 omicron was measured for monomeric sACE2 - 8his proteins . ( B ) Binding of antibodies and dimeric sACE22 - IgG1 proteins to Expi293F cells expressing BA . 2 omicron S measured by flow cytometry . ( C ) BA . 2 omicron pseudovirus was incubated with sACE22 - IgG1 ( grey ) or sACE22 . v2 . 4 - IgG1 ( blue ) for 1 h and added to HeLa - hACE2 - 11 cells . Infection was measured after 48 h . ( D - E ) Binding of monomeric sACE2 - 8his proteins ( D ) and dimeric IgG1 proteins ( E ) to Expi293F cells expressing BA . 4 / BA . 5 omicron S as measured by flow cytometry .

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

Figure 6(A) Using flow cytometry, binding to Expi293F cells expressing S of BA.2 omicron was measured for monomeric sACE2-8his proteins. (B) Binding of antibodies and dimeric sACE22-IgG1 proteins to Expi293F cells expressing BA.2 omicron S measured by flow cytometry.(C) BA.2 omicron pseudovirus was incubated with sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) for 1 h and added to HeLa-hACE2-11 cells. Infection was measured after 48 h.(D-E) Binding of monomeric sACE2-8his proteins (D) and dimeric IgG1 proteins (E) to Expi293F cells expressing BA.4/BA.5 omicron S as measured by flow cytometry.


Figure 1Mouse bone marrow - derived macrophages ( BMDMs ) infected with Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - or 2 - hour intervals for 132 hours BMDMs infected with aggregates of Mtb Erd - tdTomato from axenic culture and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours ( A - B ) Percentage of macrophages surviving after interaction with an extracellular Mtb aggregate originating from the debris of dead macrophages ( A ) or from axanic culture ( B ) . Each line represents an independent experimental replicate ( n = 3 replicates with ≥ 100 cells per replicate ) . BMDMs infected with aggregates of Mtb Erd - tdTomato from axenic culture and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours E , F ( E ) Example of a macrophage that interacts with an Mtb aggregate ( 0h00 ) , fragments it ( 3h00 ) , redistributes the bacteria in a " bullseye " pattern around the nucleus ( 12h00 ) , and dies ( 16h00 ) . Scale bar , 20 μm . ( F ) Example of a macrophage that stably interacts with an Mtb aggregate without fragmenting it ( 0h00 - 09h00 ) and ultimately dies ( 12h00 ) . In ( E and F ) , Draq7 staining of the nucleus ( in blue ) is used as a marker for cell death . Scale bar , 20 μm . BMDMs infected with aggregates of Mtb Erd - tdTomato from axenic culture and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours ( G - H ) BMDMs infected with aggregates of Mtb were imaged by time - lapse microscopy ( every 30 min for up to 13 . 5h ) followed by fixation , immunostaining ( nuclei stained with Hoechst , membrane staining with anti - CD45 antibody ) and imaging by confocal microscopy . White arrows indicate intracellular bacteria , cyan arrows indicate extracellular bacteria . All scale bars , 10 μM . ( G . I , H . I ) Time - lapse microscopy image - series of macrophages that interacts with Mtb aggregates and fragment ( G ) or do not fragment ( H ) them . ( G . II , H . II ) Max intensity projection of confocal microscopy images of the same macrophages shown in panels I . ( G . III , H . III ) 3 - D reconstruction of the cells imaged in panels II , images are cropped in x or y in the position indicated by the white dotted lines in panels II to show the inside of the cell . BMDMs infected with aggregates of Mtb Erd - tdTomato from axenic culture and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours ( I - M ) BMDMs infected with aggregates of Mtb were imaged by time - lapse fluorescence microscopy followed by SEM . ( I ) Example of a macrophage showing the typical " bullseye " pattern of bacterial redistribution around the cell nucleus . Scale bar , 10 μm . ( J ) Correlative SEM image of ( I ) . ( K ) Example of a macrophage interacting with an extracellular Mtb aggregate without complete fragmentation and redistribution of bacteria . White arrows indicate intracellular bacteria that have been detached from the main aggregate and internalized . Scale bar , 10 μm . Time - lapse microscopy image - series of these macrophages are shown in Appendix Fig S4A . ( L - M ) Correlative SEM image of ( K ) . Scale bar , 2 μm ( M ) . Mouse bone marrow - derived macrophages ( BMDMs ) infected with Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - or 2 - hour intervals for 132 hours ( O ) Percentage of macrophages surviving over time after uptake and fragmentation of Mtb aggregates or after contact without fragmentation . Each line represents an independent biological replicate . The vertical dotted line marks the 12h post - interaction time - point . BMDMs were treated with cytochalasin D to prevent bacterial uptake , infected with aggregates of Mtb and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . ( P ) Percentage survival over time for macrophages in contact with Mtb aggregates . Time 0 represents the time when stable contact with an aggregate begins . Each line represents an independent biological replicate ( n = 3 replicates with ≥ 100 cells per replicate ) .

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 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 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 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 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

Figure 1Mouse bone marrow-derived macrophages (BMDMs) infected with Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1- or 2-hour intervals for 132 hours BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (A-B) Percentage of macrophages surviving after interaction with an extracellular Mtb aggregate originating from the debris of dead macrophages (A) or from axanic culture (B). Each line represents an independent experimental replicate (n = 3 replicates with ≥ 100 cells per replicate).BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours E, F (E) Example of a macrophage that interacts with an Mtb aggregate (0h00), fragments it (3h00), redistributes the bacteria in a "bullseye" pattern around the nucleus (12h00), and dies (16h00). Scale bar, 20 μm. (F) Example of a macrophage that stably interacts with an Mtb aggregate without fragmenting it (0h00-09h00) and ultimately dies (12h00). In (E and F), Draq7 staining of the nucleus (in blue) is used as a marker for cell death. Scale bar, 20 μm.BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (G-H) BMDMs infected with aggregates of Mtb were imaged by time-lapse microscopy (every 30 min for up to 13.5h) followed by fixation, immunostaining (nuclei stained with Hoechst, membrane staining with anti-CD45 antibody) and imaging by confocal microscopy. White arrows indicate intracellular bacteria, cyan arrows indicate extracellular bacteria. All scale bars, 10 μM. (G.I,H.I) Time-lapse microscopy image-series of macrophages that interacts with Mtb aggregates and fragment (G) or do not fragment (H) them. (G.II,H.II) Max intensity projection of confocal microscopy images of the same macrophages shown in panels I. (G.III,H.III) 3-D reconstruction of the cells imaged in panels II, images are cropped in x or y in the position indicated by the white dotted lines in panels II to show the inside of the cell.BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (I-M) BMDMs infected with aggregates of Mtb were imaged by time-lapse fluorescence microscopy followed by SEM. (I) Example of a macrophage showing the typical "bullseye" pattern of bacterial redistribution around the cell nucleus. Scale bar, 10 μm. (J) Correlative SEM image of (I). (K) Example of a macrophage interacting with an extracellular Mtb aggregate without complete fragmentation and redistribution of bacteria. White arrows indicate intracellular bacteria that have been detached from the main aggregate and internalized. Scale bar, 10 μm. Time-lapse microscopy image-series of these macrophages are shown in Appendix Fig S4A. (L-M) Correlative SEM image of (K). Scale bar, 2 μm (M).Mouse bone marrow-derived macrophages (BMDMs) infected with Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1- or 2-hour intervals for 132 hours (O) Percentage of macrophages surviving over time after uptake and fragmentation of Mtb aggregates or after contact without fragmentation. Each line represents an independent biological replicate. The vertical dotted line marks the 12h post-interaction time-point. BMDMs were treated with cytochalasin D to prevent bacterial uptake, infected with aggregates of Mtb and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (P) Percentage survival over time for macrophages in contact with Mtb aggregates. Time 0 represents the time when stable contact with an aggregate begins. Each line represents an independent biological replicate (n = 3 replicates with ≥ 100 cells per replicate).


Figure 2BMDMs were treated with cytochalasin D and infected with aggregated or non - aggregated Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . ( B - E ) Examples of macrophages infected with low ( B ) , medium ( C ) , or high ( D ) doses of non - aggregated bacteria or with bacterial aggregates ( E ) . Scale bars , 20 μm . BMDMs were treated with cytochalasin D and infected with aggregated or non - aggregated Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . ( F ) Percentage survival over time for macrophages in contact with increasing doses of non - aggregated bacteria or with bacterial aggregates . Time 0 for cells " in contact " represents the time when stable contact with the bacteria begins . Each line represents a biological replicate ( n ≥ 100 cells per condition ) .

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

Figure 2BMDMs were treated with cytochalasin D and infected with aggregated or non-aggregated Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (B-E) Examples of macrophages infected with low (B), medium (C), or high (D) doses of non-aggregated bacteria or with bacterial aggregates (E). Scale bars, 20 μm.BMDMs were treated with cytochalasin D and infected with aggregated or non-aggregated Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (F) Percentage survival over time for macrophages in contact with increasing doses of non-aggregated bacteria or with bacterial aggregates. Time 0 for cells "in contact" represents the time when stable contact with the bacteria begins. Each line represents a biological replicate (n ≥ 100 cells per condition).


Figure 3 ( A ) BMDMs expressing a membrane - targeted tdTomato were treated with cytochalasin D , infected with aggregates of Mtb expressing GFP , and imaged by time - lapse microscopy at 20 - minute intervals for 24 hours . Example of a BMDM interacting with an extracellular Mtb aggregate . Top panels show the macrophage plasma membrane and the bacterial aggregate . Intimate interaction between the macrophage plasma membrane and the bacterial aggregate begins at 4h00 ( indicated by the white arrow ) . Bottom panels show the Annexin V - 647 positive plasma membrane domain that appears at 5 hours 20 minutes and co - localizes with the bacterial aggregate . At 8h00 , plasma membrane integrity is lost ( top panel ) and Annexin V accumulates throughout the dead cell ( bottom panel ) . ( E - G ) Cytochalasin D - treated BMDMs were infected with aggregates of Mtb Erd - tdTomato in the presence of Annexin V - FITC . Selected cells were imaged by time - lapse fluorescence microscopy followed by SEM . Arrows in top panels indicate the cell imaged by SEM . Top left : brightfield image of macrophage . Top left and right : fluorescence image of the Mtb aggregate . Top right : fluorescence image of Annexin V staining . Bottom : correlative SEM image . Scale bars , 20 μm in top panels and 1 μm in bottom panels . ( E ) Example of a bystander BMDM that is not in contact with a bacterial aggregate . ( F ) Example of an Annexin V - negative BMDM interacting with a Mtb aggregate . Arrows in bottom panel indicate areas of intact plasma membrane interacting with bacterial aggregates . ( G ) Example of a BMDM with blebbed Annexin V - positive membrane domains interacting with an Mtb aggregate .

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Figure 3(A) BMDMs expressing a membrane-targeted tdTomato were treated with cytochalasin D, infected with aggregates of Mtb expressing GFP, and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Example of a BMDM interacting with an extracellular Mtb aggregate. Top panels show the macrophage plasma membrane and the bacterial aggregate. Intimate interaction between the macrophage plasma membrane and the bacterial aggregate begins at 4h00 (indicated by the white arrow). Bottom panels show the Annexin V-647 positive plasma membrane domain that appears at 5 hours 20 minutes and co-localizes with the bacterial aggregate. At 8h00, plasma membrane integrity is lost (top panel) and Annexin V accumulates throughout the dead cell (bottom panel).(E-G) Cytochalasin D-treated BMDMs were infected with aggregates of Mtb Erd-tdTomato in the presence of Annexin V-FITC. Selected cells were imaged by time-lapse fluorescence microscopy followed by SEM. Arrows in top panels indicate the cell imaged by SEM. Top left: brightfield image of macrophage. Top left and right: fluorescence image of the Mtb aggregate. Top right: fluorescence image of Annexin V staining. Bottom: correlative SEM image. Scale bars, 20 μm in top panels and 1 μm in bottom panels. (E) Example of a bystander BMDM that is not in contact with a bacterial aggregate. (F) Example of an Annexin V-negative BMDM interacting with a Mtb aggregate. Arrows in bottom panel indicate areas of intact plasma membrane interacting with bacterial aggregates. (G) Example of a BMDM with blebbed Annexin V-positive membrane domains interacting with an Mtb aggregate.


Figure 4Cytochalasin D - treated BMDMs were stained with the membrane - permeable dye Oregon Green 488 Bapta - 1 AM to visualize intracellular Ca2 + , infected with aggregates of Mtb Erdman WT and imaged by time - lapse microscopy at 20 - minute intervals for 24 hours . Oregon Green 488 Bapta - 1 AM fluorescence values at each time point were normalized to time 0 for uninfected bystander cells and to the time of first contact with an Mtb aggregate for infected cells . ( A ) Examples of BMDMs dying with ( yellow outline ) or without ( white outline ) interacting with extracellular Mtb aggregates . Cell outlines are based on brightfield images Cell death is indicated by Draq7 nuclear staining . Scale bar , 10 μm . ( B ) Oregon Green 488 Bapta - 1 AM fluorescence over time in dying bystander macrophages ( n = 15 ) and in dying macrophages in contact with an Mtb aggregates ( n = 63 ) . Lines represent median fluorescence values for all cells , error bars represent interquartile ranges . The distributions of the fluorescence values at time of death in bystander and dying macrophages are significantly different , p value < 0 . 0001 , calculated using a Welch ' s t test . ( E ) BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd - tdTomato and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . Percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate without ( untr . ) or with supplementation of BAPTA - AM . Each symbol represents the percentage of dead macrophages for a single biological replicate ( n ≥ 60 cells per replicate ) . Bars represent means and standard deviations , P value calculated using a 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Cytochalasin D-treated BMDMs were stained with the membrane-permeable dye Oregon Green 488 Bapta-1 AM to visualize intracellular Ca2+, infected with aggregates of Mtb Erdman WT and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Oregon Green 488 Bapta-1 AM fluorescence values at each time point were normalized to time 0 for uninfected bystander cells and to the time of first contact with an Mtb aggregate for infected cells. (A) Examples of BMDMs dying with (yellow outline) or without (white outline) interacting with extracellular Mtb aggregates. Cell outlines are based on brightfield images Cell death is indicated by Draq7 nuclear staining. Scale bar, 10 μm. (B) Oregon Green 488 Bapta-1 AM fluorescence over time in dying bystander macrophages (n=15) and in dying macrophages in contact with an Mtb aggregates (n=63). Lines represent median fluorescence values for all cells, error bars represent interquartile ranges. The distributions of the fluorescence values at time of death in bystander and dying macrophages are significantly different, p value <0.0001, calculated using a Welch's t test.(E) BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. Percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate without (untr.) or with supplementation of BAPTA-AM. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 60 cells per replicate). Bars represent means and standard deviations, P value calculated using a t test.


Figure 5BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd - tdTomato , fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways . Controls and representative microscopy images for all the antibodies used are provided in Macrophages are defined as " in contact " when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel ( A ) Percentage of dead uninfected bystander macrophages ( byst . ) or dead macrophages in contact ( cont . ) with an extracellular Mtb aggregate that stain positive for cleaved Caspase - 8 ( cCasp8 ) . Each symbol represents the percentage of positive dead macrophages for a single biological replicate ( n ≥ 50 cells per replicate ) . Bars represent means and standard deviations . P values were calculated using a t test . ( B ) Percentage of dead uninfected bystander macrophages ( byst . ) or dead macrophages in contact with an extracellular Mtb aggregate ( cont . ) that display ASC - specks . Each symbol represents the percentage of positive dead macrophages for a single biological replicate ( n ≥ 80 cells per replicate ) . Bars represent means and standard deviations . P values were calculated using a t test . BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd - tdTomato , fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways . Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as " in contact " when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel ( C ) Representative example of a dead macrophage in contact with an extracellular Mtb aggregate that display a ASC - speck ( white arrow ) . Nuclei stained with Hoechst . Scale bar , 10 μm . BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd - tdTomato , fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways . Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as " in contact " when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel Median fluorescence values for bystander macrophages ( byst . ) or macrophages in contact with an extracellular Mtb aggregate ( cont . ) stained with anti - cleaved Caspase - 1 antibody ( cCasp1 ) , an ( E ) anti - phosphorylated RIP3 antibody ( pRIP3 ) or ( F ) anti - phosphorylated MLKL antibody ( pMLKL ) . In ( G ) BMDMs were treated with MCC950 ( NLRP3 inhibitor ) or with VX765 ( Caspase - 1 inhibitor ) during the course of the infection . Each symbol represents a single macrophage . Black bars represent median and interquartile range . P - value were calculated using an unpaired Mann - Whitney test ; ns , P values > 0 . 05 . E : n = 182 , 112 respectively ; F : n = 186 , 84 respectively ; G : n = 117 , 121 , 111 , 129 respectively ; n represents the number of individual cells analyzed per condition ) .

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

Figure 5BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided in Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel (A) Percentage of dead uninfected bystander macrophages (byst.) or dead macrophages in contact (cont.) with an extracellular Mtb aggregate that stain positive for cleaved Caspase-8 (cCasp8). Each symbol represents the percentage of positive dead macrophages for a single biological replicate (n ≥ 50 cells per replicate). Bars represent means and standard deviations. P values were calculated using a t test. (B) Percentage of dead uninfected bystander macrophages (byst.) or dead macrophages in contact with an extracellular Mtb aggregate (cont.) that display ASC-specks. Each symbol represents the percentage of positive dead macrophages for a single biological replicate (n ≥ 80 cells per replicate). Bars represent means and standard deviations. P values were calculated using a t test.BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel (C) Representative example of a dead macrophage in contact with an extracellular Mtb aggregate that display a ASC-speck (white arrow). Nuclei stained with Hoechst. Scale bar, 10 μm.BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel Median fluorescence values for bystander macrophages (byst.) or macrophages in contact with an extracellular Mtb aggregate (cont.) stained with anti-cleaved Caspase-1 antibody (cCasp1), an (E) anti-phosphorylated RIP3 antibody (pRIP3) or (F) anti-phosphorylated MLKL antibody (pMLKL). In (G) BMDMs were treated with MCC950 (NLRP3 inhibitor) or with VX765 (Caspase-1 inhibitor) during the course of the infection. Each symbol represents a single macrophage. Black bars represent median and interquartile range. P-value were calculated using an unpaired Mann-Whitney test; ns, P values > 0.05. E: n=182, 112 respectively; F: n=186, 84 respectively; G: n=117, 121, 111, 129 respectively; n represents the number of individual cells analyzed per condition).


Figure 6 ( A - D ) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains and imaged by time - lapse microscopy at 1 - hour intervals for 60 hours . The plots represent the percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate . Each symbol represents the percentage of dead macrophages for a single biological replicate ( n ≥ 3 replicates with ≥ 70 cells per replicate ) . Bars represent means and standard deviations . P - values were calculated using a one - way ANOVA test ( A , C , D ) or t test ( B ) comparing each strain to the wild - type reference strain ( A - D ) ; ns , P values > 0 . 05 . ( A ) Macrophages in contact with aggregates of Mtb H37Rv wild - type ( WT ) , ∆ RD1 mutant and complemented ∆ RD1 mutant ( ∆ RD1 : : 2F9 ) . ( B ) Macrophages in contact with aggregates of Mtb Erdman wild - type ( WT ) or PDIM - deficient ( fadD26 ) strains . ( C ) Macrophages in contact with aggregates of Mtb Erdman wild - type ( WT ) or mutant strains ( espI eccD1 , esxA , espA ) and the complemented strains ( esxA : : esxBA , espA : : espA ) . ( D ) Macrophages in contact with aggregates of Mtb Erdman wild - type ( WT ) or mutant strains ( espB , espA espB ) and the espB - complemented strains ( espB : : espB , espA espB : : espB ) . ( F ) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains , incubated with Annexin V and imaged by time - lapse microscopy at 1 - hour intervals for 48 hours . Percentage of macrophages that show Annexin V - positive membrane domains within the first 12 hours after entering in contact with Mtb aggregates . Each symbol represents a single biological replicate ( n ≥ 3 replicates with ≥ 90 cells per replicate ) . Bars represent means and standard deviations . ( G ) Cytochalasin D - treated BMDMs were infected with aggregates of Mtb Erdman WT , stained with the membrane - permeable dye Oregon Green 488 Bapta - 1 AM to visualize cytosolic Ca2 + and imaged by time - lapse microscopy at 20 - minute intervals for 24 hours . Oregon Green 488 Bapta - 1 AM fluorescence values at each time point were normalized to the time of first contact with an Mtb aggregate for infected cells . Values in the plot correspond to the time of death after first contact or 16 hours post - contact for cells that survive . Each symbol represents a single macrophage . ( n = 123 , 64 , 48 , 71 , 78 , 55 respectively ) . Black bars represent median and interquartile range . P - value were calculated using a one sample Wilcoxon test ; ns , P values > 0 . 05 . ( H ) BMDMs were treated as in ( A - D ) . Percentage of macrophages that die within the first 12 hours after interaction with an Mtb aggregate . Infected cells are incubated with different concentration of BTP15 ( 0 , 2 , 10 , 50 μM ) during the course of the experiment . Each symbol represents the percentage of dead macrophages for a single biological replicate ( n ≥ 3 replicates with ≥ 70 cells per replicate ) . Bars represent means and standard deviations . P - values were calculated using a one - way ANOVA test comparing the treated vs . untreated samples .

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

Figure 6(A-D) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. The plots represent the percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P-values were calculated using a one-way ANOVA test (A,C,D) or t test (B) comparing each strain to the wild-type reference strain (A-D); ns, P values > 0.05. (A) Macrophages in contact with aggregates of Mtb H37Rv wild-type (WT), ∆RD1 mutant and complemented ∆RD1 mutant (∆RD1::2F9). (B) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or PDIM-deficient (fadD26) strains. (C) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or mutant strains (espI eccD1, esxA, espA) and the complemented strains (esxA::esxBA, espA::espA). (D) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or mutant strains (espB, espA espB) and the espB-complemented strains (espB::espB, espA espB::espB).(F) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains, incubated with Annexin V and imaged by time-lapse microscopy at 1-hour intervals for 48 hours. Percentage of macrophages that show Annexin V - positive membrane domains within the first 12 hours after entering in contact with Mtb aggregates. Each symbol represents a single biological replicate (n ≥ 3 replicates with ≥ 90 cells per replicate). Bars represent means and standard deviations.(G) Cytochalasin D-treated BMDMs were infected with aggregates of Mtb Erdman WT, stained with the membrane-permeable dye Oregon Green 488 Bapta-1 AM to visualize cytosolic Ca2+ and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Oregon Green 488 Bapta-1 AM fluorescence values at each time point were normalized to the time of first contact with an Mtb aggregate for infected cells. Values in the plot correspond to the time of death after first contact or 16 hours post-contact for cells that survive. Each symbol represents a single macrophage. (n=123, 64, 48, 71, 78, 55 respectively). Black bars represent median and interquartile range. P-value were calculated using a one sample Wilcoxon test; ns, P values > 0.05.(H) BMDMs were treated as in (A-D). Percentage of macrophages that die within the first 12 hours after interaction with an Mtb aggregate. Infected cells are incubated with different concentration of BTP15 (0, 2, 10, 50 μM) during the course of the experiment. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P-values were calculated using a one-way ANOVA test comparing the treated vs. untreated samples.


Figure 2 ( A ) Hybridization Chain Reaction ( HCR ) detection of CGG repeat RNA along with immunocytochemistry ( ICC ) to detect SRSF1 - FLAG in U2OS cells . Expanded view of merged channels shows the CGG RNA foci ( arrowhead ) and an example of SRSF1 - CGG RNA foci co - localization ( arrow ) . DAPI marks the nucleus . Scale bars are 10 µm . ( B ) Co - localization analysis of CGG RNA ( red traces ) and SRSF1 ( green traces ) showing overlaps of CGG RNA and SRSF1 . ( a . u . = arbitrary unit ) ( C ) Co - immunoprecipitation of indicated SRSF proteins with PP7 - tagged control sequence ( partial nLUC sequence ) to correct for non - specific binding , AUG and CGG reporter RNAs . SRSF1 and 2 specifically immunoprecipitated with PP7 - tagged CGG reporter .

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 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 2(A) Hybridization Chain Reaction (HCR) detection of CGG repeat RNA along with immunocytochemistry (ICC) to detect SRSF1-FLAG in U2OS cells. Expanded view of merged channels shows the CGG RNA foci (arrowhead) and an example of SRSF1-CGG RNA foci co-localization (arrow). DAPI marks the nucleus. Scale bars are 10 µm.(B) Co-localization analysis of CGG RNA (red traces) and SRSF1 (green traces) showing overlaps of CGG RNA and SRSF1. (a.u. = arbitrary unit)(C) Co-immunoprecipitation of indicated SRSF proteins with PP7-tagged control sequence (partial nLUC sequence) to correct for non-specific binding, AUG and CGG reporter RNAs. SRSF1 and 2 specifically immunoprecipitated with PP7-tagged CGG reporter.


Figure 3 ( D ) Representative photographs of fly eyes and quantitation ( below ) of rough eye scores with fly SRSF1 overexpression ( dSF2 OE ) ; n = 20 - 32 / genotype . Error bars represent mean + / - SD . Data information : For eye scoring , target siRNA lines were compared to non - targeting control siRNA lines using a two - tailed student ' s t test with Welch ' s correction for multiple comparisons . ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . Human orthologs of fly genes are used for labeling . ( F ) Quantitation of ( GGGGCC ) 28 - EGFP rough eye phenotype with SRSF modifiers ( n > 30 flies / genotype ) . Error bars represent mean + / - SD . Data information : For eye scoring , target siRNA lines were compared to non - targeting control siRNA lines using a two - tailed student ' s t test with Welch ' s correction for multiple comparisons . ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . Human orthologs of fly genes are used for labeling . ( G ) Representative photographs of fly eyes expressing GMR - GAL4 driven ( GGGGCC ) 28 - EGFP at 29 ˚ C along with the quantifications of necrosis and eye width . n = 28 - 30 / genotype . Error bars represent mean + / - SD . ( H - I ) Survival assays of flies expressing ( CGG ) 90 - EGFP under Tub5 - GS ( H ) and ELAV - GS ( I ) drivers with control or SRSF1 siRNAs . Expression of ( CGG ) 90 - EGFP was initiated with addition of drug starting 1 day post eclosion and continued through experiment ( Log - rank Mantel - Cox test ; n = 98 - 101 / genotype for Tub - GS and n = 120 - 141 / genotype for ELAV - GS flies ) ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 . ( J ) Survival assays of ( GGGGCC ) 28 - EGFP expressing fly under Tub5 - GS driver ( Log - rank Mantel - Cox test ; n = 71 - 93 / genotype ) with control or SRSF1 siRNAs . ∗ ∗ p < 0 . 01 .

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 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 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

Figure 3(D) Representative photographs of fly eyes and quantitation (below) of rough eye scores with fly SRSF1 overexpression (dSF2 OE); n=20-32/genotype. Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling.(F) Quantitation of (GGGGCC)28-EGFP rough eye phenotype with SRSF modifiers (n > 30 flies/ genotype). Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling.(G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP at 29˚C along with the quantifications of necrosis and eye width. n=28-30/genotype. Error bars represent mean +/- SD.(H-I) Survival assays of flies expressing (CGG)90-EGFP under Tub5-GS (H) and ELAV-GS (I) drivers with control or SRSF1 siRNAs. Expression of (CGG)90-EGFP was initiated with addition of drug starting 1 day post eclosion and continued through experiment (Log-rank Mantel-Cox test; n=98-101/genotype for Tub-GS and n=120-141/genotype for ELAV-GS flies) ∗p < 0.05, ∗∗p < 0.01. (J) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel-Cox test; n=71-93/genotype) with control or SRSF1 siRNAs. ∗∗p < 0.01.


Figure 4 ( A , B ) Representative ( A ) photographs of fly eyes and ( B ) quantitation with siRNA mediated knockdown of SRPK1 ( dSRPK1 ) ; n = 30 - 34 / genotype . Error bars represent mean + / - SD . Data information : Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . ( C ) Representative photographs of fly eyes expressing GMR - GAL4 driven ( GGGGCC ) 28 - EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion . ( D ) Quantitation of rough eye phenotypes . t - test with Welch corrections for comparisons with the control ; n = 31 - 34 flies / genotype . Error bars represent mean + / - SD . Data information : Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . ( E ) Representative external eye imaging for detection of GFP aggregates caused by ( CGG ) 90 - EGFP transgene expression ( top ) . Converted images used to quantify total intensity of GFP puncta ( bottom ) . ( F ) Depletion of SRSF1 or SRPK1 by RNAi results in reduced ( CGG ) 90 - EGFP puncta compared to control siRNA as quantified by total intensity ( a . u . = arbitrary unit ) . n = 13 - 15 flies / genotype . Error bars represent mean + / - SD . Data information : Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction , ∗ ∗ ∗ 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 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

Figure 4(A, B) Representative (A) photographs of fly eyes and (B) quantitation with siRNA mediated knockdown of SRPK1 (dSRPK1); n=30-34/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.(C) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (D) Quantitation of rough eye phenotypes. t-test with Welch corrections for comparisons with the control; n= 31-34 flies/ genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.(E) Representative external eye imaging for detection of GFP aggregates caused by (CGG)90-EGFP transgene expression (top). Converted images used to quantify total intensity of GFP puncta (bottom). (F) Depletion of SRSF1 or SRPK1 by RNAi results in reduced (CGG)90-EGFP puncta compared to control siRNA as quantified by total intensity (a.u. = arbitrary unit). n=13-15 flies/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗


Figure 5 ( B ) Anti - FLAG immunoblot of DMSO and SRPIN340 pre - treated HEK293T cells expressing AUG - nLuc - 3xFLAG control or CGG - nLuc - 3xFLAG RAN translation reporters . β - Actin is used as a loading control . To prevent signal saturation , AUG - nLuc lysate was diluted 1 : 3 in sample buffer prior to loading ( n = 3 biological replicates ) . Schematics of the AUG - nLUC - 3xFLAG and + 1CGG ( 100 ) - nLuc - 3xFLAG reporters presented on top . Data information : Error bars represent mean + / - SD . Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 . To prevent over - exposure , the AUG - nLuc lysate was diluted 1 : 3 in sample buffer . ( D ) Anti - FLAG immunoblot of DMSO and SRPIN340 pre - treated HEK293T cells expressing GGGGCC - nLuc - 3xFLAG ( GA70 ) and + 2CGG - nLuc - 3xFLAG ( FMRpolyA ) RAN translation reporters ( n = 3 biological replicates ) . Schematics of the GA70 ( GGGGCCx70 ) and + 2CGG reporters presented on top . Data information : Error bars represent mean + / - SD . Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 . ( E ) Immunoblot of DMSO and SPHINX31 pre - treated HEK293T cells expressing AUG - nLuc - 3xFLAG control or CGG - nLuc - 3xFLAG RAN translation reporters . ( n = 3 biological replicates ) . ( F ) Anti - FLAG of DMSO and SPHINX31 pre - treated HEK293T cells expressing GGGGCC - nLuc - 3xFLAG ( GA70 ) and + 2CGG - nLuc - 3xFLAG ( FMRpolyA ) RAN translation reporters ( n = 3 biological replicates ) . Data information : Error bars represent mean + / - SD . Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 5(B) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3 biological replicates). Schematics of the AUG-nLUC-3xFLAG and +1CGG(100)-nLuc-3xFLAG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in sample buffer.(D) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.(E) Immunoblot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3 biological replicates). (F) Anti-FLAG of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


Figure 6 ( C , D ) Expression of GGGGCC - nLuc - 3xFLAG ( C ) and + 2CGG - nLuc - 3xFLAG ( D ) RAN translation reporters in HEK293T cells treated with 2 μM TG ( for stress induction ) analyzed by immunoblot ( n = 3 biological replicates ) . To evaluate effects of SRPK1 inhibition cells were pre - treated with DMSO or SRPIN340 before reporter transfection . Data information : Error bars represent mean + / - SD . Two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 . ( E ) SRPK1 inhibition by SRPIN340 did not alter eIF2α phosphorylation in response to 2 μM TG ( for stress induction ) in HEK293T cells by immunoblot ( n = 3 biological replicates ) . ( F ) SPRK1 inhibition suppressed eIF2α phosphorylation in HEK293T cells treated with 2 μM TG ( for stress induction ) in the presence of the + 1CGG reporter . Cells were pre - treated with DMSO or SRPIN340 before reporter transfection ( n = 3 biological replicates ) . Data information : Error bars represent mean + / - SD . Two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

Figure 6(C, D) Expression of GGGGCC-nLuc-3xFLAG (C) and +2CGG-nLuc-3xFLAG (D) RAN translation reporters in HEK293T cells treated with 2 μM TG (for stress induction) analyzed by immunoblot (n=3 biological replicates). To evaluate effects of SRPK1 inhibition cells were pre-treated with DMSO or SRPIN340 before reporter transfection. Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.(E) SRPK1 inhibition by SRPIN340 did not alter eIF2α phosphorylation in response to 2 μM TG (for stress induction) in HEK293T cells by immunoblot (n=3 biological replicates). (F) SPRK1 inhibition suppressed eIF2α phosphorylation in HEK293T cells treated with 2 μM TG (for stress induction) in the presence of the +1CGG reporter. Cells were pre-treated with DMSO or SRPIN340 before reporter transfection (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


Figure 7 ( B ) ICC images of FLAG - tagged SRSF1 after SRPIN340 treatment compared to vehicle ( DMSO ) . Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal Error bars indicate mean + / - 95 % CI ( n = 85 - 126 cells / condition ) . Statistical analysis was performed using t test with Welch ' s correction , ∗ ∗ ∗ ∗ p < 0 . 0001 . ( C ) Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle ( DMSO ) as detected by HCR . Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal as parts of whole . Error bars indicate mean + / - 95 % CI ( n = 124 - 151 cells / condition ) . Statistical analysis was performed using t test with Welch ' s correction , ∗ ∗ ∗ ∗ p < 0 . 0001 . ( D ) Anti - FLAG immunoblot blot of DMSO and SRPIN340 pre - treated HEK293T cells transfected with in vitro transcribed CGG - nLuc - 3xFLAG reporter RNA . β - Actin is used as a loading control . Error bars represent mean + / - SD ( n = 6 biological replicates ) . Statistical analysis was performed using Student ' s t test with Welch ' s correction . ∗ p < 0 .

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 O O O O O O O O O O O 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

Figure 7(B) ICC images of FLAG-tagged SRSF1 after SRPIN340 treatment compared to vehicle (DMSO). Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal Error bars indicate mean +/- 95% CI (n= 85-126 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.(C) Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle (DMSO) as detected by HCR. Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal as parts of whole. Error bars indicate mean +/- 95% CI (n= 124-151 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.(D) Anti-FLAG immunoblot blot of DMSO and SRPIN340 pre-treated HEK293T cells transfected with in vitro transcribed CGG-nLuc-3xFLAG reporter RNA. β-Actin is used as a loading control. Error bars represent mean +/- SD (n=6 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05


Figure 8 ( A , B ) Pharmacological targeting of SPRK1 with ( A ) 40 μM SRPIN340 or ( B ) 8 μM SPHINX31 reduced the cumulative risk of death in + 1 ( CGG ) 100 - EGFP ( encoding FMRpolyG ) expressing neurons . n = # of neurons quantified for each condition ; Cox proportional hazard analysis , ∗ ∗ ∗ 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

Figure 8(A, B) Pharmacological targeting of SPRK1 with (A) 40 μM SRPIN340 or (B) 8 μM SPHINX31 reduced the cumulative risk of death in +1(CGG)100-EGFP (encoding FMRpolyG) expressing neurons. n= # of neurons quantified for each condition; Cox proportional hazard analysis, ∗∗∗p < 0.001.


figf1a , Sections of the thymus and muscle of fed and starved GFP - LC3 mice were stained with anti - GFP Alexa 488 antibody ( green ) and 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Images are representative of n = 6 . b , Thymic stromal cells from GFP - LC3 mice were fixed onto glass slides and stained with anti - GFP Alexa 488 and DAPI . Typical autophagy - positive cTECs and mTEChi are shown . The average frequencies ± s . d . of cTECs and mTEChi scoring autophagy positive ( that is , ≥ 5 autophagosomes ) in two independent preparations are indicated . Less than 1 % of dendritic cells ( DCs ) and mTEClo were positive . c , Relative distribution of the number of autophagosomes in autophagy - positive cTECs and mTEChi ; a . u . , arbitrary units .

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 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

figf1a, Sections of the thymus and muscle of fed and starved GFP-LC3 mice were stained with anti-GFP Alexa 488 antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). Images are representative of n = 6.b, Thymic stromal cells from GFP-LC3 mice were fixed onto glass slides and stained with anti-GFP Alexa 488 and DAPI. Typical autophagy-positive cTECs and mTEChi are shown. The average frequencies ± s.d. of cTECs and mTEChi scoring autophagy positive (that is, ≥5 autophagosomes) in two independent preparations are indicated. Less than 1% of dendritic cells (DCs) and mTEClo were positive.c, Relative distribution of the number of autophagosomes in autophagy-positive cTECs and mTEChi; a.u., arbitrary units.


figf2a , Thymi from wild - type ( WT ) or Atg5 - / - embryos were transplanted under the kidney capsule of wild - type recipients and analysed 6 weeks later . The average cellularities ± s . d . are shown ; n = 5 . b , Normal compartmentalization of Atg5 - / - grafts in cytokeratin - 8 - positive ( red ; K8 ) cortical and cytokeratin - 5 - positive ( green ; K5 ) medullary regions ; n = 8 . c , Expression of MHC class I and II on cTECs or mTECs from Atg5 - / - to wild - type ( WT ) grafts ( red ) , or wild - type to wild - type grafts ( blue ) . Staining controls are depicted in grey ; data are representative of three experiments . dd , RT - PCR analysis of autoimmune regulator ( Aire ) and TRA expression in mTECs isolated from Atg5 - / - to wild - type , or wild - type to wild - type grafts . Expression of β - actin ( Actb ) , intestinal trefoil factor ( Tff3 ) , casein 2 ( Csn2 ) and salivary protein 1 ( Spt1 ) are also shown ( 5 - fold serial dilutions ) . Data are representative of two experiments .

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 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

figf2a, Thymi from wild-type (WT) or Atg5-/- embryos were transplanted under the kidney capsule of wild-type recipients and analysed 6 weeks later. The average cellularities ± s.d. are shown; n = 5.b, Normal compartmentalization of Atg5-/- grafts in cytokeratin-8-positive (red; K8) cortical and cytokeratin-5-positive (green; K5) medullary regions; n = 8.c, Expression of MHC class I and II on cTECs or mTECs from Atg5-/- to wild-type (WT) grafts (red), or wild-type to wild-type grafts (blue). Staining controls are depicted in grey; data are representative of three experiments. dd, RT-PCR analysis of autoimmune regulator (Aire) and TRA expression in mTECs isolated from Atg5-/- to wild-type, or wild-type to wild-type grafts. Expression of β-actin (Actb), intestinal trefoil factor (Tff3), casein 2 (Csn2) and salivary protein 1 (Spt1) are also shown (5-fold serial dilutions). Data are representative of two experiments.


figf3a , Polyclonal T - cell development in Atg5 - / - to wild - type ( WT ) , or wild - type to wild - type grafts . The top dot plots show CD4 and CD8 staining ; the bottom dot plots show expression of CD25 and Foxp3 by CD4 SP thymocytes ; n ≥ 5 . b , Atg5 - / - or wild - type lobes were grafted into TCR - HA transgenic recipients . Graft cellularity ± s . d . and the expression of the transgenic TCR on CD4 SP cells were analysed after 6 weeks . The frequency ± s . d . of TCR - HA + cells among CD4 SP cells was 32 . 1 % ± 3 . 2 % in wild - type to TCR - HA , and 19 . 6 % ± 4 . 3 % in Atg5 - / - to TCR - HAlobes ; P = 1 . 8 × 10 - 8 ( n ≥ 14 ) . c , Atg5 - / - or wild - type lobes were grafted into AND transgenic mice . Graft cellularity ± s . d . and the expression of the transgenic TCR ( Vb3 - Va11 ) on CD4 SP cells were analysed after 6 weeks . The frequency ± s . d . of Vb3 - Va11 + CD4 SP cells was 93 . 5 % ± 0 . 2 % in wild - type to ANDlobes ( n = 3 ) and 97 . 9 % ± 1 . 1 % ( n = 6 ) in Atg5 - / - to ANDlobes ( P = 9 × 10 - 4 ) . d , Thymi from wild - type or Atg5 - / - embryos were grafted into P14 , OT - I or HY TCR - transgenic recipients . The dot plots show the expression of the transgenic TCR V - segments on CD8 SP cells . Thymus cellularities ± s . d . are shown in the respective bar diagrams . Data are representative of n = 4 ( P14 ) , n ≥ 4 ( OT - I ) and n ≥ 2 ( HY ) . e , Thymi from wild - type or ( BALB / c3C57BL / 6 ) F1 Atg5 - / - embryos were transplanted into ( BALB / c3C57BL / 6 ) F1 recipients . Six weeks after grafting , expression of the I - Ea52 - 68 - I - Ab complex on cTECs was analysed using the monoclonal antibody Y - Ae . Atg5 - / - cTECs are shown in red ; wild - type cTECs are shown in blue . Control stainings are shown in orange ( Atg5 - / - ) and green ( wild type ) . Data are representative of two independent experiments .

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

figf3a, Polyclonal T-cell development in Atg5-/- to wild-type (WT), or wild-type to wild-type grafts. The top dot plots show CD4 and CD8 staining; the bottom dot plots show expression of CD25 and Foxp3 by CD4 SP thymocytes; n ≥ 5.b, Atg5-/- or wild-type lobes were grafted into TCR-HA transgenic recipients. Graft cellularity ± s.d. and the expression of the transgenic TCR on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of TCR-HA+ cells among CD4 SP cells was 32.1% ± 3.2% in wild-type to TCR-HA, and 19.6% ± 4.3% in Atg5-/- to TCR-HAlobes; P = 1.8 × 10-8 (n ≥ 14).c, Atg5-/- or wild-type lobes were grafted into AND transgenic mice. Graft cellularity ± s.d. and the expression of the transgenic TCR (Vb3-Va11) on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of Vb3-Va11+ CD4 SP cells was 93.5% ± 0.2% in wild-type to ANDlobes (n = 3) and 97.9% ± 1.1% (n = 6) in Atg5-/- to ANDlobes (P = 9 × 10-4).d, Thymi from wild-type or Atg5-/- embryos were grafted into P14, OT-I or HY TCR-transgenic recipients. The dot plots show the expression of the transgenic TCR V-segments on CD8 SP cells. Thymus cellularities ± s.d. are shown in the respective bar diagrams. Data are representative of n = 4 (P14), n ≥ 4 (OT-I) and n ≥ 2 (HY).e, Thymi from wild-type or (BALB/c3C57BL/6)F1 Atg5-/- embryos were transplanted into (BALB/c3C57BL/6)F1 recipients. Six weeks after grafting, expression of the I-Ea52-68-I-Ab complex on cTECs was analysed using the monoclonal antibody Y-Ae. Atg5-/- cTECs are shown in red; wild-type cTECs are shown in blue. Control stainings are shown in orange (Atg5-/-) and green (wild type). Data are representative of two independent experiments.


figf4Atg5 - / - or wild - type ( WT ) lobes were grafted into athymic ( nu / nu ) mice ( nu / nuAtg5 - / - or nu / nuWT , respectively ) . Data are representative of three experiments with n ≥ 4 . a , Expression of activation markers on lymph node CD4 T cells in nu / nuAtg5 - / - or nu / nuWT chimaeras . b , The changes in body weight over time are shown . c , The appearance of nu / nuAtg5 - / - or nu / nuWT chimaeras . The colon ( arrow ) and uterus ( arrowhead ) are highlighted ; note the absence of fat pads ( asterisk ) in nu / nuAtg5 - / - chimaeras . d , Haematoxylin and eosin staining of organs from nu / nuAtg5 - / - or nu / nuWT chimaeras . Arrows indicate infiltrates .

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figf4Atg5-/- or wild-type (WT) lobes were grafted into athymic (nu/nu) mice (nu/nuAtg5-/- or nu/nuWT, respectively). Data are representative of three experiments with n ≥ 4. a, Expression of activation markers on lymph node CD4 T cells in nu/nuAtg5-/- or nu/nuWT chimaeras.b, The changes in body weight over time are shown.c, The appearance of nu/nuAtg5-/- or nu/nuWT chimaeras. The colon (arrow) and uterus (arrowhead) are highlighted; note the absence of fat pads (asterisk) in nu/nuAtg5-/- chimaeras.d, Haematoxylin and eosin staining of organs from nu/nuAtg5-/- or nu/nuWT chimaeras. Arrows indicate infiltrates.


Figure 1Heat map showing absolute counts of unique peptides for S . indica apoplastic proteins present at all three time points and in culture filtrate . S1 / S2 / S3 = biologically independent sample 1 , 2 and 3 ; d = deglycosylated .

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Figure 1Heat map showing absolute counts of unique peptides for S. indica apoplastic proteins present at all three time points and in culture filtrate. S1/S2/S3 = biologically independent sample 1, 2 and 3; d = deglycosylated.


Figure 2eATP levels in APF from S . indica inoculated and mock - treated barley root samples collected at the predominantly biotrophic phase ( 3 and 5 dpi ) as well as the predominantly cell death associated phase ( 7 and 10 dpi ) . Error bars show the standard error of the mean obtained from three independent biological replicates at p < 0 . 05 ( * ) , 0 . 01 ( * * ) analyzed by Student ' s t - test . eATP levels measured from culture medium collected from mock treated and S . indica colonized Arabidopsis seedlings at 3 , 5 , 7 and 10 dpi . Error bars represent ± SE of the mean from three independent biological replicates . RLU : relative light units . Asterisks indicate significance at p < 0 . 05 ( * ) , 0 . 01 ( * * ) analyzed by Student ' s t - test . S . indica colonization of Arabidopsis dorn1 - 3 mutant and the parental Col - 0aq lines quantified by qPCR at 3 , 5 , 7 and 10 dpi . The ratio of fungal ( SiTEF ) to plant ( AtUBI ) amplicons representing fungal colonization levels in plant root tissue was calculated using gDNA as template and the 2 - ΔCT method . Error bars represent standard error of the mean of three technical replicates . The experiment was repeated 3 times for 3 , 5 and 7 dpi with similar outcomes . Transcript levels of S . indica E5 ′ NT during colonization of barley and Arabidopsis at different symbiotic stages and in axenic culture . Error bars represent standard error of the mean of three independent biological replicates . CM = complex medium .

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Figure 2eATP levels in APF from S. indica inoculated and mock-treated barley root samples collected at the predominantly biotrophic phase (3 and 5 dpi) as well as the predominantly cell death associated phase (7 and 10 dpi). Error bars show the standard error of the mean obtained from three independent biological replicates at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.eATP levels measured from culture medium collected from mock treated and S. indica colonized Arabidopsis seedlings at 3, 5, 7 and 10 dpi. Error bars represent ±SE of the mean from three independent biological replicates. RLU: relative light units. Asterisks indicate significance at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.S. indica colonization of Arabidopsis dorn1-3 mutant and the parental Col-0aq lines quantified by qPCR at 3, 5, 7 and 10 dpi. The ratio of fungal (SiTEF) to plant (AtUBI) amplicons representing fungal colonization levels in plant root tissue was calculated using gDNA as template and the 2-ΔCT method. Error bars represent standard error of the mean of three technical replicates. The experiment was repeated 3 times for 3, 5 and 7 dpi with similar outcomes.Transcript levels of S. indica E5′NT during colonization of barley and Arabidopsis at different symbiotic stages and in axenic culture. Error bars represent standard error of the mean of three independent biological replicates. CM = complex medium.


Figure 4Ecto - 5 ′ - nucleotidase activity measured in membrane protein preparations of Arabidopsis plants expressing Pro35S : : E5 ′ NT ( # 303 ) , Pro35S : : SPE5 ′ NT : mCherry : E5 ′ NTwoSP ( # 304 ) or Pro35S : : mCherry ( # 305 ) . E5 ′ NT activity was measured after incubation with 100 µM of either ATP , ADP or AMP . In the membrane protein preparations from Pro35S : : E5 ′ NT ( # 303 ) lines phosphate release was specifically increased upon incubation with purines . Error bars represent the standard error of the mean from three technical repetitions . The coomassie stained SDS - PAGE shows the protein pattern of the membrane fractions for the individual transgenic lines . Equal volumes were loaded . The experiment was repeated two times with similar results . Confocal microscopy images of Arabidopsis roots expressing either cytosolic mCherry ( # 305 ) or Pro35S : : SPE5 ′ NT : mCherry : E5 ′ NTwoSP ( # 304 ) showing secretion of the E5 ′ NT fusion protein . mCherry images show z - stacks of 14 image planes of 1 µm each . Scale bar = 20 µm . The transgenic Arabidopsis line Pro35S : : E5 ′ NT ( # 303 ) expressing untagged full length SiE5 ′ NT was better colonized by S . indica . Error bars of the qPCR data represent ± SE of the mean from three independent biological replicates . Asterisks indicate significance ( Student ' s t - test , * p < 0 . 05 ) . S . indica induced eATP release in different Arabidopsis transgenic lines . Culture medium was collected from mock treated or S . indica inoculated seedlings growing in liquid medium at 5 dpi and released eATP was measured . RLU : relative light units . Error bars represent ± SE of the mean from three biological replicates . Asterisks indicate significance to all other samples within the same treatment group ( ANOVA , p < 0 . 05 ) . S . indica colonization of transgenic lines at 5 dpi . Error bars represent ± SE of the mean from three biological replicates . Asterisks indicate significance to all other samples within the same treatment group ( ANOVA , p < 0 . 05 ) . Expression analysis of the eATP responsive gene At1g58420 measured by qRT - PCR . Error bars represent ± SE of the mean from three independent biological replicates ( independent from those shown in Fig . 2C ) . Letters indicate significant groups ( ANOVA , p < 0 . 01 , for the line 305 p < 0 . 05 ) .

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Figure 4Ecto-5′-nucleotidase activity measured in membrane protein preparations of Arabidopsis plants expressing Pro35S::E5′NT (#303), Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) or Pro35S::mCherry (#305). E5′NT activity was measured after incubation with 100 µM of either ATP, ADP or AMP. In the membrane protein preparations from Pro35S::E5′NT (#303) lines phosphate release was specifically increased upon incubation with purines. Error bars represent the standard error of the mean from three technical repetitions. The coomassie stained SDS-PAGE shows the protein pattern of the membrane fractions for the individual transgenic lines. Equal volumes were loaded. The experiment was repeated two times with similar results.Confocal microscopy images of Arabidopsis roots expressing either cytosolic mCherry (#305) or Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) showing secretion of the E5′NT fusion protein. mCherry images show z-stacks of 14 image planes of 1 µm each. Scale bar=20 µm.The transgenic Arabidopsis line Pro35S::E5′NT (#303) expressing untagged full length SiE5′NT was better colonized by S. indica. Error bars of the qPCR data represent ±SE of the mean from three independent biological replicates. Asterisks indicate significance (Student's t-test, * p< 0.05).S. indica induced eATP release in different Arabidopsis transgenic lines. Culture medium was collected from mock treated or S. indica inoculated seedlings growing in liquid medium at 5 dpi and released eATP was measured. RLU: relative light units. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).S. indica colonization of transgenic lines at 5 dpi. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).Expression analysis of the eATP responsive gene At1g58420 measured by qRT-PCR. Error bars represent ±SE of the mean from three independent biological replicates (independent from those shown in Fig. 2C). Letters indicate significant groups (ANOVA, p<0.01, for the line 305 p<0.05).


Figure 5Relative fluxes of phosphates ( Pi ) and sugars ( C ) across the fungal plasma membrane . Without additional Pi - source in the apoplast ( value 0 at the x - axis , black dots ) there is a constant flux of phosphate via the proton - coupled phosphate ( H / P ) transporter from the fungus to the apoplast and a constant flux of sugar from the apoplast to the fungus . For better comparison these fluxes were normalized to 1 and - 1 , respectively , and all other fluxes were calculated relative to these control values . With increasing ATP - release and Pi - production the Pi - efflux gets smaller and is zero at a relative Pi - production rate of 1 ( grey triangles ) . At higher ATP - release and Pi - production rates the fungus imports Pi , i . e . the transport direction of phosphate has been inverted in comparison to the control condition ( white square ) . The C - flux via the proton - coupled sugar ( H / C ) transporter is not affected by the additional Pi - source ( horizontal grey line ) . Relative fluxes of Pi and C across the plant plasma membrane . Without additional Pi - source in the apoplast ( value 0 at the x - axis , black dots ) there is a constant flux of sugar via the H / C transporter from the plant to the apoplast ( and thereafter to the fungus ) and a constant flux of phosphate ( coming from the fungus ) via the H / P transporter from the apoplast to the plant . With increasing ATP - release and Pi - production the Pi - influx via the H / P transporter increases while the C - flux via the H / C transporter is unaffected by the additional Pi - source ( horizontal grey line ) . Phosphate fluxes across the plant plasma membrane . Besides the Pi - uptake via the H / P transporter ( B , black line , for clarity not shown in C ) , the plant loses Pi due to the ATP - release ( light grey line ) . The difference between Pi - uptake and Pi - loss is the net Pi - balance of the plant ( dark grey line ) . Without ATP - release ( value 0 at the x - axis , black dots ) the plant gains Pi . At a relative ATP - release and Pi - production of 1 ( grey triangles ) the plant release as much phosphate as it takes up , while at higher ATP - release values , the plant loses Pi .

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Figure 5Relative fluxes of phosphates (Pi) and sugars (C) across the fungal plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of phosphate via the proton-coupled phosphate (H/P) transporter from the fungus to the apoplast and a constant flux of sugar from the apoplast to the fungus. For better comparison these fluxes were normalized to 1 and -1, respectively, and all other fluxes were calculated relative to these control values. With increasing ATP-release and Pi-production the Pi-efflux gets smaller and is zero at a relative Pi-production rate of 1 (grey triangles). At higher ATP-release and Pi-production rates the fungus imports Pi, i.e. the transport direction of phosphate has been inverted in comparison to the control condition (white square). The C-flux via the proton-coupled sugar (H/C) transporter is not affected by the additional Pi-source (horizontal grey line).Relative fluxes of Pi and C across the plant plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of sugar via the H/C transporter from the plant to the apoplast (and thereafter to the fungus) and a constant flux of phosphate (coming from the fungus) via the H/P transporter from the apoplast to the plant. With increasing ATP-release and Pi-production the Pi-influx via the H/P transporter increases while the C-flux via the H/C transporter is unaffected by the additional Pi-source (horizontal grey line).Phosphate fluxes across the plant plasma membrane. Besides the Pi-uptake via the H/P transporter (B, black line, for clarity not shown in C), the plant loses Pi due to the ATP-release (light grey line). The difference between Pi-uptake and Pi-loss is the net Pi-balance of the plant (dark grey line). Without ATP-release (value 0 at the x-axis, black dots) the plant gains Pi. At a relative ATP-release and Pi-production of 1 (grey triangles) the plant release as much phosphate as it takes up, while at higher ATP-release values, the plant loses Pi.


Figure 1 ( A ) Dot plot depicting the expression of mRNA for neurogenic transcription factor MEIS2 in prospectively isolated cellular populations from the SEZ . ( B ) Micrographs depicting the expression of neurogenic fate determinant Meis2 mRNA and MEIS2 protein in acutely dissociated SEZ cells . Note that Meis2 mRNA positive LRCs have low ( no ) MEIS2 protein . ( C IHC labeling for MEIS2 ( C ) of LRCs positive for BrdU only and neuroblasts marked by DCX . D ) IHC labeling for MCM6 ( D ) of LRCs positive for BrdU only and neuroblasts marked by DCX . ( E ) Dot plot showing the proportion of BrdU + LRCs negative for MEIS2 or MCM6 protein . ( F ) Dot plot depicting the expression of miR - 204 in prospectively isolated cells of neural lineage .

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Figure 1(A) Dot plot depicting the expression of mRNA for neurogenic transcription factor MEIS2 in prospectively isolated cellular populations from the SEZ.(B) Micrographs depicting the expression of neurogenic fate determinant Meis2 mRNA and MEIS2 protein in acutely dissociated SEZ cells. Note that Meis2 mRNA positive LRCs have low (no) MEIS2 protein.(C IHC labeling for MEIS2 (C) of LRCs positive for BrdU only and neuroblasts marked by DCX.D) IHC labeling for MCM6 (D) of LRCs positive for BrdU only and neuroblasts marked by DCX.(E) Dot plot showing the proportion of BrdU+ LRCs negative for MEIS2 or MCM6 protein.(F) Dot plot depicting the expression of miR-204 in prospectively isolated cells of neural lineage.


Figure 2 ( B ) Dot plot depicting the immunoreactivity of LRCs for MEIS2 in control and antagomirs treated animals 7 dpi . ( C , D ) Micrographs showing IHC for the neurogenic priming factor MEIS2 in the SEZ 7 days after injection of artificial CSF ( control , C ) or AntimiR204 ( D ) . White arrows indicate LRCs without detectable MEIS2 protein . The white arrowheads are pointing out the loss of neurogenic priming ( LRCs positive for MEIS2 protein ) upon the antagomir treatments .

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Figure 2(B) Dot plot depicting the immunoreactivity of LRCs for MEIS2 in control and antagomirs treated animals 7 dpi.(C, D) Micrographs showing IHC for the neurogenic priming factor MEIS2 in the SEZ 7 days after injection of artificial CSF (control, C) or AntimiR204 (D). White arrows indicate LRCs without detectable MEIS2 protein. The white arrowheads are pointing out the loss of neurogenic priming (LRCs positive for MEIS2 protein) upon the antagomir treatments.


Figure 3 ( A , B ) Micrographs depicting the cellular composition in the SEZ 7 days after injection of aCSF ( control , A ) or AntimiR204 ( B ) . Boxed areas correspond to higher magnifications in adjacent panels ( A ' , B ' ) . Dot plots depicting the abundance of LRCs ( BrdU + , Ki67 - , DCX - ) in the SEZ 7 days after aCSF or AntimiR204 injection . Dot plots depicting the abundance of aNSCs and TAPs ( BrdU - , Ki67 + , DCX - ) in the SEZ 7 days after aCSF or AntimiR204 injection . Dot plots depicting the abundance of neuroblasts ( DCX + Ki67 + or DCX + Ki67 - ) in the SEZ 7 days after aCSF or AntimiR204 injection . ( F , G ) Micrographs depicting the elbow - the most ventral region of the RMS ( F ) and OB neuronal layers ( G ) upon injection of artificial CSF ( control ) or AntimiR204 . ( H ) Dot plot showing the density of BrdU + cells ( progeny of NSCs ) in the RMS and in the OB 7 days after artificial CSF or AntimiR204 injection .

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Figure 3(A, B) Micrographs depicting the cellular composition in the SEZ 7 days after injection of aCSF (control, A) or AntimiR204 (B). Boxed areas correspond to higher magnifications in adjacent panels (A', B').Dot plots depicting the abundance of LRCs (BrdU+, Ki67-, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.Dot plots depicting the abundance of aNSCs and TAPs (BrdU-, Ki67+, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.Dot plots depicting the abundance of neuroblasts (DCX+ Ki67+ or DCX+ Ki67-) in the SEZ 7 days after aCSF or AntimiR204 injection.(F, G) Micrographs depicting the elbow - the most ventral region of the RMS (F) and OB neuronal layers (G) upon injection of artificial CSF (control) or AntimiR204. (H) Dot plot showing the density of BrdU+ cells (progeny of NSCs) in the RMS and in the OB 7 days after artificial CSF or AntimiR204 injection.


Figure 4 ( A ) Micrographs of in situ hybridization for miR - 204 in an adult mouse brain section . ( A ' and A ' ' ) are magnifications of boxed areas in ( A or A ' ) , respectively . ( B ) Dot plot showing miR - 204 levels in ChP and SEZ in the adult mouse brain measured by RT - qPCR . ( C ) Agarose gel of RT - qPCR product loaded after the saturation phase showing presence of miR - 204 and U6 in mouse ChP , SEZ and CSF . Plots depicting the number and size of EVs isolated from human CSF ( D ) Plots depicting the miR - 204 levels ( E ) in EVs and EV - free CSF ( n = 2 ) . ( F ) Agarose gel of RT - qPCR analysis depicting levels of miR - 204 in the EV - free supernatant and EV containing fraction of human CSF loaded at the saturation phase . ( G ) Micrographs of transmission electron microscopy imaged extracellular vesicles ( EVs ) isolated from sham treated ( upper row ) and GW4869 treated animals ( lower row ) . ( G ' ' and G ' ' ' ) are magnifications of boxed areas in overview images to the left . ( H ) Dot plot depicting miR - 204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor .

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Figure 4(A) Micrographs of in situ hybridization for miR-204 in an adult mouse brain section. (A' and A'') are magnifications of boxed areas in (A or A'), respectively.(B) Dot plot showing miR-204 levels in ChP and SEZ in the adult mouse brain measured by RT-qPCR. (C) Agarose gel of RT-qPCR product loaded after the saturation phase showing presence of miR-204 and U6 in mouse ChP, SEZ and CSF.Plots depicting the number and size of EVs isolated from human CSF (D)Plots depicting the miR-204 levels (E) in EVs and EV-free CSF (n=2). (F) Agarose gel of RT-qPCR analysis depicting levels of miR-204 in the EV-free supernatant and EV containing fraction of human CSF loaded at the saturation phase.(G) Micrographs of transmission electron microscopy imaged extracellular vesicles (EVs) isolated from sham treated (upper row) and GW4869 treated animals (lower row). (G'' and G''') are magnifications of boxed areas in overview images to the left. (H) Dot plot depicting miR-204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor.