Figure 7 . S . typhimurium - infected macrophages exhibited signs of autophagocytosis . Caspase - 1 − / − BMDPM were infected with wild - type S . typhimurium or it isogenic translocation - defective sipD mutant . 6 h after infection , macrophages were either stained with MDC to label autophagosomes ( I ) or processed for EM ( II ) as described in the legend to Fig . 3 . Macrophages infected with wild - type S . typhimurium ( but not those infected with the sipD mutant ) exhibited a large number of vesicular structures that stained with MDC ( I ) Under the electron microscope ( II ) , macrophages infected with wild - type Salmonella exhibited multivesicular structures resembling autophagosomes ( A - D ) Often , electron - dense material , presumably damaged organelles , were readily observed within the multivesicular structures ( B - D ) . In contrast , macrophages infected with type III secretion - defective sipD mutant did not exhibit the large number of multivesicular structures observed in wild - type - infected cells . Bars ( A and E ) 500 nm ; ( B , C , D and F ) 250 nm .

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Figure 7.S. typhimurium-infected macrophages exhibited signs of autophagocytosis. Caspase-1−/− BMDPM were infected with wild-type S. typhimurium or it isogenic translocation-defective sipD mutant. 6 h after infection, macrophages were either stained with MDC to label autophagosomes (I) or processed for EM (II) as described in the legend to Fig. 3. Macrophages infected with wild-type S. typhimurium (but not those infected with the sipD mutant) exhibited a large number of vesicular structures that stained with MDC (I) Under the electron microscope (II), macrophages infected with wild-type Salmonella exhibited multivesicular structures resembling autophagosomes (A-D) Often, electron-dense material, presumably damaged organelles, were readily observed within the multivesicular structures (B-D). In contrast, macrophages infected with type III secretion-defective sipD mutant did not exhibit the large number of multivesicular structures observed in wild-type-infected cells. Bars (A and E) 500 nm; (B, C, D and F) 250 nm.


Figure 8 . The S . typhimurium - induced macrophage death does not require caspase activity . BMDPM were infected with wild - type S . typhimurium or the TTSS - defective invG mutant in the presence or absence of 50 μM of the pan - caspase inhibitor Z - VAD , and cell death was measured as indicated in the legend to Fig . 1 .

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Figure 8. The S. typhimurium-induced macrophage death does not require caspase activity. BMDPM were infected with wild-type S. typhimurium or the TTSS-defective invG mutant in the presence or absence of 50 μM of the pan-caspase inhibitor Z-VAD, and cell death was measured as indicated in the legend to Fig. 1.


Figure 1B Volcano plot showing proteins enrichment ( fold change in log10 scale ) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT ( untagged ) ARHGAP45 proteins prior to ( unstimulated ) and at 120 s after ( stimulated ) TCR stimulation . ARHGAP45 interacting proteins with a greater than 2 - fold enrichment and a p - value < 0 . 01 were selected as specific ARHGAP45 interactors ( Dataset EV1 ) and some of them are specified in red . Red lines represent the thresholds set on P - value and enrichment to identify specific ARHGAP45 interactors .

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Figure 1B Volcano plot showing proteins enrichment (fold change in log10 scale) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT (untagged) ARHGAP45 proteins prior to (unstimulated) and at 120 s after (stimulated) TCR stimulation. ARHGAP45 interacting proteins with a greater than 2-fold enrichment and a p-value < 0.01 were selected as specific ARHGAP45 interactors (Dataset EV1) and some of them are specified in red. Red lines represent the thresholds set on P-value and enrichment to identify specific ARHGAP45 interactors.


Figure 3B The ratio of CTV / CMTPX - labeled cells present in each organ ( Ro ) was determined and normalized by dividing it with Ri , the ratio of CTV / CMTPX - labeled cells present in the cell mixtures prior to injection . Ro / Ri ratio corresponding to each of the specified mice are shown . Data shown for Arhgap45 - / - mice correspond to 4 independent experiments ( E1 to E4 ) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments ( E1 and E2 ) involving a total of 5 individual mice . The Arhgap45 - / - mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments . Mean and SD are shown . ns , non - significant , * * * * P ≤ 0 . 0001 ; multiple unpaired T tests . C Expression of CCR7 , and LFA - 1 on naïve CD4 + and CD8 + T cells found in LNs of WT and Arhgap45 - / - mice , analyzed by flow cytometry . Gray shaded curves , isotype control staining . D Quantification of data shown in ( C ) . The MFI corresponding to each analyzed mouse is shown together with mean and SD . Each dot represents an individual mouse and CD4 + T cells from at least 4 mice have been analyzed for each condition . Mice analyzed on the same day are represented by similar dots ( see key in bottom right corner ) . ns , non - significant , * * P ≤ 0 . 002 ; multiple unpaired T tests .

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Figure 3B The ratio of CTV/CMTPX-labeled cells present in each organ (Ro) was determined and normalized by dividing it with Ri, the ratio of CTV/CMTPX-labeled cells present in the cell mixtures prior to injection. Ro/Ri ratio corresponding to each of the specified mice are shown. Data shown for Arhgap45-/-mice correspond to 4 independent experiments (E1 to E4) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments (E1 and E2) involving a total of 5 individual mice. The Arhgap45-/- mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments. Mean and SD are shown. ns, non-significant, **** P ≤ 0.0001; multiple unpaired T tests.C Expression of CCR7, and LFA-1 on naïve CD4+ and CD8+ T cells found in LNs of WT and Arhgap45-/- mice, analyzed by flow cytometry. Gray shaded curves, isotype control staining. D Quantification of data shown in (C). The MFI corresponding to each analyzed mouse is shown together with mean and SD. Each dot represents an individual mouse and CD4+ T cells from at least 4 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). ns, non-significant, ** P ≤ 0.002; multiple unpaired T tests.


Figure 4The cells specified below were loaded into the upper chamber of a Transwell migration device without ( no ) or with the specified chemokines in the lower chamber . The percentage of cells that migrated to the bottom chamber after 2 h ( T cells ) or 4 h ( B cells ) is shown . A Chemotaxis of naïve CD4 + T cells from WT and Arghap45 − / − mice in response to CCL19 , CCL21 and CXCL12 . B Chemotaxis of naïve CD8 + T cells from WT and Arghap45 − / − mice in response to CCL19 , CCL21 and CXCL12 . C Chemotaxis of naïve B cells from WT and Arghap45 − / − mice in response to CCL19 D Adding CCL19 in both the upper and lower chambers ( CCL19 UL ) prevented migration of WT and Arghap45 − / − naïve T cells whereas the use of Transwell membrane inserts precoated with ICAM - 1 ( CCL19 + ICAM - 1 ) did not change the pattern of chemotaxis observed using ' bare ' membrane inserts ( CCL19 ) . E Chemotaxis of naive CD4 + T cells and B cells from WT , Arghap45 − / − , and Arhgap45 ∆ T / ∆ T mice in response to CCL19 . F Chemotaxis of activated CD4 + T cells from WT and Arghap45 − / − mice in response to CCL19 . Data information : In ( A ) to ( F ) each dot represents an individual mouse and as shown CD4 + T cells from 3 to 9 mice have been analyzed for each condition . Mice analyzed on the same day are represented by similar dots ( see key in bottom right corner ) . Mean and SD are shown . * P ≤ 0 . 033 , * * P ≤ 0 . 002 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ; A , B , C , D , F multiple unpaired T test ; E two - way ANOVA test .

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Figure 4The cells specified below were loaded into the upper chamber of a Transwell migration device without (no) or with the specified chemokines in the lower chamber. The percentage of cells that migrated to the bottom chamber after 2 h (T cells) or 4 h (B cells) is shown. A Chemotaxis of naïve CD4+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12. B Chemotaxis of naïve CD8+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12. C Chemotaxis of naïve B cells from WT and Arghap45−/− mice in response to CCL19 D Adding CCL19 in both the upper and lower chambers (CCL19 UL) prevented migration of WT and Arghap45−/− naïve T cells whereas the use of Transwell membrane inserts precoated with ICAM-1 (CCL19 + ICAM-1) did not change the pattern of chemotaxis observed using 'bare' membrane inserts (CCL19). E Chemotaxis of naive CD4+ T cells and B cells from WT, Arghap45−/−, and Arhgap45∆T/∆T mice in response to CCL19. F Chemotaxis of activated CD4+ T cells from WT and Arghap45−/− mice in response to CCL19. Data information: In (A) to (F) each dot represents an individual mouse and as shown CD4+ T cells from 3 to 9 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). Mean and SD are shown. *P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.001, **** P ≤ 0.0001; A, B, C, D, F multiple unpaired T test; E two-way ANOVA test.


Figure 5Analysis of migration patterns of WT and Arghap45 − / − activated T cells on 2D surface coated with ICAM - 1 . Each track represents the migratory path of individual WT and Arghap45 − / − activated T cells recorded over 466 s in a single field of view , Trajectories were plotted to a common starting point , and > 200 T cells were recorded per plot using time - lapse microscopy at 10 x magnification . Analysis of migration patterns of WT and Arghap45 − / − naive T cells on 2D surface coated with ICAM - 1 and CCL21 . Same conditions as in ( A ) .

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Figure 5Analysis of migration patterns of WT and Arghap45−/− activated T cells on 2D surface coated with ICAM-1. Each track represents the migratory path of individual WT and Arghap45−/− activated T cells recorded over 466 s in a single field of view, Trajectories were plotted to a common starting point, and > 200 T cells were recorded per plot using time-lapse microscopy at 10 x magnification. Analysis of migration patterns of WT and Arghap45−/− naive T cells on 2D surface coated with ICAM-1 and CCL21. Same conditions as in (A).


Figure 6A , B Representative kymographs of WT ( A ) and Arghap45 − / − ( B ) naive CD4 + T cells on 2D surface coated with ICAM - 1 and CCL21 .

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Figure 6A, B Representative kymographs of WT (A) and Arghap45−/− (B) naive CD4+ T cells on 2D surface coated with ICAM-1 and CCL21.


Figure 7A Irradiated H - 2 Kb - positive spleen cells isolated from T cell - deficient mice were pulsed for 2 hours with the N4 agonist OVA peptide and cultured with CD8 + T cells purified from WT OT - I ( WT ) or Arhgap45 − / − OT - I ( Arhgap45 − / − ) mice . Cell proliferation was measured by luminescence after 48 h . Data are representative of 2 independent experiments with 3 mice per genotype ( mean and SEM of triplicate are shown ) .

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Figure 7A Irradiated H-2 Kb- positive spleen cells isolated from T cell-deficient mice were pulsed for 2 hours with the N4 agonist OVA peptide and cultured with CD8+ T cells purified from WT OT-I (WT) or Arhgap45−/− OT-I (Arhgap45−/−) mice. Cell proliferation was measured by luminescence after 48 h. Data are representative of 2 independent experiments with 3 mice per genotype (mean and SEM of triplicate are shown).


Figure 1 ( A ) NIH3T3 fibroblasts co - expressing DsRed - CA7 and EGFP - CA2 ( n = 4 independent replicates ) . ( B - E ) Co - localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing ( B ) EGFP , ( C ) EGFP - CA2 , or ( D , E ) EGFP - CA7 and stained with phalloidin - 594 to visualize F - actin ( n = 4 , 10 and 8 independent replicates , respectively ) . A magnification of the area marked with the yellow rectangle in ( C ) and ( D ) shows the localization of EGFP - CA2 and EGFP - CA7 compared to phalloidin - 594 . ( E ) EGFP - CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles ( arrow ) and plasmalemmal protrusions ( arrowhead ) . ( F - H ) The normalized fluorescence emission intensity profiles for F - actin ( red line ) and ( F ) EGFP , ( G ) EGFP - CA2 , or ( H ) EGFP - CA7 ( black line ) .

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Figure 1(A) NIH3T3 fibroblasts co-expressing DsRed-CA7 and EGFP-CA2 (n = 4 independent replicates).(B-E) Co-localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing (B) EGFP, (C) EGFP-CA2, or (D, E) EGFP-CA7 and stained with phalloidin-594 to visualize F-actin (n = 4, 10 and 8 independent replicates, respectively). A magnification of the area marked with the yellow rectangle in (C) and (D) shows the localization of EGFP-CA2 and EGFP-CA7 compared to phalloidin-594. (E) EGFP-CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles (arrow) and plasmalemmal protrusions (arrowhead).(F-H) The normalized fluorescence emission intensity profiles for F-actin (red line) and (F) EGFP, (G) EGFP-CA2, or (H) EGFP-CA7 (black line).


Figure 2 ( A ) Actin co - sedimentation assay shows that CA7 binds to F - actin . The binding is enhanced at more acidic pH ( 6 . 5 vs . 7 . 4 ) n = 3 independent replicates at each actin concentration , two - way ANOVA , P < 0 . 001 . ( B ) Fluorescence time ‐ lapse images of F - actin bundling in an in vitro bundling assay . A mixture of unlabeled and Rhodamine labelled non - muscle actin was polymerized in the absence ( PBS control , upper panel ) or presence of mCA7 ( lower panel ) . Numbers in images indicate the time after the onset of the experiment ( 0 , 5 and 23 min ) . Intensity based Fire - coloring ( Fiji ) was used to visualize intensity changes . Scale bar 10 µm . ( C ) Quantification of the mean increase in filament length ( n = 10 filaments at each time point ) and the mean relative fluorescence intensity values of cross - sections for individual filaments / bundles ( n = 30 - 31 ) in the absence and presence of mCA7 ( 1 . 12 µM ) . The data were analyzed using a general mixed model with time as a within - unit factor and the presence of CA7 as a between - unit factor . n = 3 independent repetitions , experiment repeats were included as a covariate and were non - significant . ( D ) Kymographs showing different time points in the line of interest ( line width 1 µm ) from the experiments analyzed in ( C ) . Kymographs were generated with Fiji Multi Kymograph function . Total time is 159 frames = 26 . 5 minutes . Scale bar 5 μm ( full height is 20 μm ) . F - actin bundles can be detected as clear lines in kymographs . ( E ) Fluorescence time ‐ lapse images of F - actin bundling in an in vitro bundling assay . A mixture of unlabeled and Rhodamine labelled non - muscle actin was polymerized in the presence of mCA7 ( 0 . 11 µM ) . Numbers above images indicate the time after the onset of the experiment ( 22 min - 23 min 20 sec ) . Intensity based Fire - coloring ( Fiji ) was used to visualize intensity changes . White arrows highlight the bundling filaments . Scale bar 5 μm . This is a representative video from 8 similar experiments .

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Figure 2(A) Actin co-sedimentation assay shows that CA7 binds to F-actin. The binding is enhanced at more acidic pH (6.5 vs. 7.4) n = 3 independent replicates at each actin concentration, two-way ANOVA, P < 0.001.(B) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the absence (PBS control, upper panel) or presence of mCA7 (lower panel). Numbers in images indicate the time after the onset of the experiment (0, 5 and 23 min). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. Scale bar 10 µm. (C) Quantification of the mean increase in filament length (n = 10 filaments at each time point) and the mean relative fluorescence intensity values of cross-sections for individual filaments /bundles (n = 30 - 31) in the absence and presence of mCA7 (1.12 µM). The data were analyzed using a general mixed model with time as a within-unit factor and the presence of CA7 as a between-unit factor. n = 3 independent repetitions, experiment repeats were included as a covariate and were non-significant. (D) Kymographs showing different time points in the line of interest (line width 1 µm) from the experiments analyzed in (C). Kymographs were generated with Fiji Multi Kymograph function. Total time is 159 frames = 26.5 minutes. Scale bar 5 μm (full height is 20 μm). F-actin bundles can be detected as clear lines in kymographs.(E) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the presence of mCA7 (0.11 µM). Numbers above images indicate the time after the onset of the experiment (22 min -23 min 20 sec). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. White arrows highlight the bundling filaments. Scale bar 5 μm. This is a representative video from 8 similar experiments.


Figure 3 ( A , B ) NIH3T3 cells transfected with DsRed ( A ) or DsRed - CA7 ( B ) were incubated in growth medium with 5 µM Latrunculin B for 0 , 2 , 5 , 10 , or 30 minutes , or in an equal amount of DMSO for 60 minutes . Analyses of experiments show that in cells transfected with DsRed - CA7 F - actin structures collapse more slowly ( 0 min : 83 % " normal " ; 2 min : 80 % , p = 0 . 44 ; 5 min : 74 % , p = 0 . 39 ; 10 min : 27 % , p = 0 . 08 ; 30 min : 16 % , p = 0 . 02 ; DMSO : 82 % , p = 0 . 99 ; tested against 0 min with two - way ANOVA , Dunnett ' s multiple comparison test ) than in the DsRed - transfected ones ( 0 min : 89 % " normal " ; 2 min : 48 % , p = 0 . 14 ; 5 min : 31 % , p = 0 . 04 ; 10 min : 2 % , p = 0 . 001 ; 30 min : 0 . 7 % , p = 0 . 001 ; DMSO : 88 % , p = 0 . 8 ; tested against 0 min with two - way ANOVA , Dunnett ' s multiple comparison test ) . For the analysis , cells were categorized to three groups as " normal " , " some shape / F - actin left " and " round " . The upper panel shows example images of the cells in all three categories for ( A ) DsRed - or ( B ) DsRed - CA7 transfected cells ( actin visualized with Phalloidin - 488 ) .

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Figure 3(A,B) NIH3T3 cells transfected with DsRed (A) or DsRed-CA7 (B) were incubated in growth medium with 5 µM Latrunculin B for 0, 2, 5, 10, or 30 minutes, or in an equal amount of DMSO for 60 minutes. Analyses of experiments show that in cells transfected with DsRed-CA7 F-actin structures collapse more slowly (0 min: 83% "normal"; 2 min: 80%, p = 0.44; 5 min: 74%, p = 0.39; 10 min: 27%, p = 0.08; 30 min: 16%, p = 0.02; DMSO: 82%, p = 0.99; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) than in the DsRed-transfected ones (0 min: 89% "normal"; 2 min: 48%, p = 0.14; 5 min: 31%, p = 0.04; 10 min: 2%, p = 0.001; 30 min: 0.7%, p = 0.001; DMSO: 88%, p = 0.8; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) . For the analysis, cells were categorized to three groups as "normal", "some shape/F-actin left" and "round". The upper panel shows example images of the cells in all three categories for (A) DsRed- or (B) DsRed-CA7 transfected cells (actin visualized with Phalloidin-488).


Figure 5 ( A - C ) NIH3T3 fibroblasts expressing EGFP - CA7 - mutant1 ( A ) , EGFP - CA7 - mutant2 ( B ) , and EGFP - CA7 - mutant3 ( C ) . F - actin is visualized with Phalloidin - 594 . In the right - most panel of ( A - C ) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal ( black ) and actin ( red ) and the yellow line in left - most panels indicates the cross - section from which the pixel intensities were measured . Scale bars 20 µm . ( D ) Analysis of the mutated CA7 and F - actin co - localization in cultured fibroblasts . Scatterplots of fluorescent intensities per pixel ( EGFP vs . Phalloidin - 594 ) along a cross section through a representative cell . Pearson ' s correlation coefficient ( r ) for the analyzed cell is given in each panel . ( E ) Pearson ' s correlation coefficient values calculated for the depicted constructs and compared to CA7 . F - actin had a strong positive correlation coefficient with EGFP - CA7 ( r = 0 . 91 ± 0 . 02 , n = 26 cells ) . Neither EGFP alone ( r = 0 . 01 ± 0 . 03 , n = 56 ) nor EGFP - CA2 ( r = 0 . 09 ± 0 . 03 , n = 53 ) co - localized with F - actin ( P < 0 . 0001 for both constructs , when compared to CA7 ) . From the five mutated CA7 constructs , EGFP - CA7 - mutant1 ( r = 0 . 51 ± 0 . 04 , P < 0 . 0001 , n = 25 ) and EGFP - CA7 - mutant3 ( r = 0 . 72 ± 0 . 03 , P = 0 . 05 , n = 21 ) co - localized less with F - actin actin when compared to CA7 . The co - localization of the other four mutated CA7 constructs , EGFP - CA7 - mutant2 ( r = 0 . 92 ± 0 . 02 , P = 0 . 99 , n = 18 ) , EGFP - CA7 - R223E ( r = 0 . 86 ± 0 . 02 , P = 0 . 99 , n = 24 ) and EGFP - CA7 - H96 / 98C ( r = 0 . 79 ± 0 . 04 , P = 0 . 14 , n = 18 ) did not differ significantly from that of CA7 . Data are shown as mean ± SEM . Data did not pass Shapiro - Wilk test for normality , statistical comparison against CA7 was done with Kruskall - Wallis test corrected for multiple comparisons .

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

Figure 5(A-C) NIH3T3 fibroblasts expressing EGFP-CA7-mutant1 (A), EGFP-CA7-mutant2 (B), and EGFP-CA7-mutant3 (C). F-actin is visualized with Phalloidin-594. In the right-most panel of (A-C) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal (black) and actin (red) and the yellow line in left-most panels indicates the cross-section from which the pixel intensities were measured. Scale bars 20 µm.(D) Analysis of the mutated CA7 and F-actin co-localization in cultured fibroblasts. Scatterplots of fluorescent intensities per pixel (EGFP vs. Phalloidin-594) along a cross section through a representative cell. Pearson's correlation coefficient (r) for the analyzed cell is given in each panel.(E) Pearson's correlation coefficient values calculated for the depicted constructs and compared to CA7. F-actin had a strong positive correlation coefficient with EGFP-CA7 (r = 0.91 ± 0.02, n = 26 cells). Neither EGFP alone (r = 0.01 ± 0.03, n = 56) nor EGFP-CA2 (r = 0.09 ± 0.03, n = 53) co-localized with F-actin (P < 0.0001 for both constructs, when compared to CA7). From the five mutated CA7 constructs, EGFP-CA7-mutant1 (r= 0.51 ± 0.04, P < 0.0001, n = 25) and EGFP-CA7-mutant3 (r = 0.72 ± 0.03, P = 0.05, n = 21) co-localized less with F-actin actin when compared to CA7. The co-localization of the other four mutated CA7 constructs, EGFP-CA7-mutant2 (r = 0.92 ± 0.02, P = 0.99, n = 18), EGFP-CA7-R223E (r = 0.86 ± 0.02, P = 0.99, n = 24) and EGFP-CA7-H96/98C (r = 0.79 ± 0.04, P = 0.14, n = 18) did not differ significantly from that of CA7. Data are shown as mean ± SEM. Data did not pass Shapiro-Wilk test for normality, statistical comparison against CA7 was done with Kruskall-Wallis test corrected for multiple comparisons.


Figure 6 ( A ) Isoform - specific subcellular localization shown in cultured hippocampal neurons ( DIV14 ) co - expressing DsRed - CA7 ( left ) and EGFP - CA2 ( middle ) . ( B , C ) Representative confocal images of precocious in vivo expression of ( B ) EGFP - CA2 and ( C ) EGFP - CA7 in P40 mouse cortical layer 2 / 3 pyramidal neurons . Neurons were transfected at E14 . 5 with EGFP - CA2 or EGFP - CA7 using in utero electroporation and images were taken from fixed slices . Right panels in ( B ) and ( C ) show higher magnification of the primary apical dendrite marked with a box . ( D ) The expression of EGFP - CA7 disrupted the normal spine morphology and induced the formation of thick , filopodia - like protrusions .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Isoform-specific subcellular localization shown in cultured hippocampal neurons (DIV14) co-expressing DsRed-CA7 (left) and EGFP-CA2 (middle).(B,C) Representative confocal images of precocious in vivo expression of (B) EGFP-CA2 and (C) EGFP-CA7 in P40 mouse cortical layer 2/3 pyramidal neurons. Neurons were transfected at E14.5 with EGFP-CA2 or EGFP-CA7 using in utero electroporation and images were taken from fixed slices. Right panels in (B) and (C) show higher magnification of the primary apical dendrite marked with a box. (D) The expression of EGFP-CA7 disrupted the normal spine morphology and induced the formation of thick, filopodia-like protrusions.


Figure 7 ( A ) Comparison of mEPSCs in cortical layer 2 / 3 pyramidal neurons from P30 - P40 WT and CA7 KO mice . Sample traces of mEPSC recordings from WT and CA7 KO neurons , low - pas filtered at 1 kHz ( left ) . The data are summarized in the bar diagrams ( right ) . mEPSC frequency ( P = 0 . 63 ) and amplitude ( P = 0 . 90 ) were not significantly different between WT and CA7 KO and neurons ( n = 7 and 5 neurons , respectively , Student ' s independent samples t - test ) . ( B ) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2 / 3 pyramidal neurons from WT and CA7 KO mice . The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice . Scale bar 2 µm . ( C ) Summary of the spine density analysis done from the Lucifer Yellow injected neurons . Spine density was a higher in CA7 KO neurons both in apical and basal dendrites ( n = 28 neurons for both ) compared to WT ( n = 29 neurons for apical and n = 30 for basal dendrite analysis ) ( P = 0 . 000002 for apical dendrites , analyzed with Mann - Whitney test , and P = 6 , 8 x 10 - 8 for basal dendrites , Student ' s t - test with Welch - correction . ) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice . ( D ) The spine head width distribution7differed significantly between the genotypes ( n = 467 spines from WT and n = 421 spines from CA7 KO animals , 15 neurons analyzed from both genotypes , Wilcoxon rank sum test with continuity correction , W = 134540 , P < 0 . 001 ) .

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

Figure 7(A) Comparison of mEPSCs in cortical layer 2/3 pyramidal neurons from P30 - P40 WT and CA7 KO mice. Sample traces of mEPSC recordings from WT and CA7 KO neurons, low-pas filtered at 1 kHz (left). The data are summarized in the bar diagrams (right). mEPSC frequency (P = 0.63) and amplitude (P = 0.90) were not significantly different between WT and CA7 KO and neurons (n = 7 and 5 neurons, respectively, Student's independent samples t-test).(B) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2/3 pyramidal neurons from WT and CA7 KO mice. The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice. Scale bar 2 µm.(C) Summary of the spine density analysis done from the Lucifer Yellow injected neurons. Spine density was a higher in CA7 KO neurons both in apical and basal dendrites (n = 28 neurons for both) compared to WT (n= 29 neurons for apical and n=30 for basal dendrite analysis) (P = 0.000002 for apical dendrites, analyzed with Mann-Whitney test, and P = 6,8 x 10-8 for basal dendrites, Student's t-test with Welch-correction.) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice.(D) The spine head width distribution7differed significantly between the genotypes (n= 467 spines from WT and n = 421 spines from CA7 KO animals, 15 neurons analyzed from both genotypes, Wilcoxon rank sum test with continuity correction, W = 134540, P < 0.001).


Figure 4 ( F - G ) Thermal shift assay traces of human Teneurin4WT ( F ) and human Teneurin4Mut ( G ) in the presence of physiological calcium concentrations ( + Ca2 + ; black ) , low calcium concentration ( + / - Ca2 + ; blue and red , respectively ) and no calcium ( - Ca2 + ; grey ) .

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

Figure 4(F-G) Thermal shift assay traces of human Teneurin4WT (F) and human Teneurin4Mut (G) in the presence of physiological calcium concentrations (+ Ca2+; black), low calcium concentration (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey).


Figure 5 ( A ) Dimensionless Kratky plots for Teneurin4WT ( left ) and Teneurin4Mut ( right ) in physiological calcium concentrations ( + Ca2 + ; black ) , low calcium concentrations ( + / - Ca2 + ; blue and red , respectively ) and no calcium ( - Ca2 + ; grey ) . I ( 0 ) and Rg were obtained from Guinier analysis ( Fig EV5A , B ) . Crosshairs indicate the maximum position for a fully folded globular protein . ( B ) Pair - distance distribution plots for Teneurin4 proteins colored as in A .

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

Figure 5(A) Dimensionless Kratky plots for Teneurin4WT (left) and Teneurin4Mut (right) in physiological calcium concentrations (+ Ca2+; black), low calcium concentrations (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey). I(0) and Rg were obtained from Guinier analysis (Fig EV5A, B). Crosshairs indicate the maximum position for a fully folded globular protein.(B) Pair-distance distribution plots for Teneurin4 proteins colored as in A.


Figure 6 ( A ) Representative images of HEK293 - T cells transfected with GFP ( top ) , GFP - Teneurin4WT ( GFP - Ten4WT ; middle ) or GFP - Teneurin4Mut ( GFP - Ten4Mut ; bottom ) for 24h and stained with Alexa 568 Phalloidin ( magenta ) and Dapi ( blue ) . Images are maximum intensity projections of 14 - 16 stacks . Scale bar , 10 µm . ( B ) Zoom shows the regions indicated in A . White arrows indicate the tip of the filopodium from the transfected cell . Scale bar , 2 µm . ( ( C ) Average number of filopodia per cell in cells transfected with GFP , GFP - Teneurin4WT ( Ten4 WT ; red ) and GFP - Teneurin4Mut ( Ten4Mut ; blue ) . ( D ) Fraction of filopodia enriched with GFP per cell in GFP - , GFP - Teneurin4WT ( Ten4WT ; blue ) - and GFP - Teneurin4Mut ( Ten4Mut ; red ) - transfected cells . (

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

Figure 6(A) Representative images of HEK293-T cells transfected with GFP (top), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP- Teneurin4Mut (GFP-Ten4Mut; bottom) for 24h and stained with Alexa 568 Phalloidin (magenta) and Dapi (blue). Images are maximum intensity projections of 14-16 stacks. Scale bar, 10 µm. (B) Zoom shows the regions indicated in A. White arrows indicate the tip of the filopodium from the transfected cell. Scale bar, 2 µm. ((C) Average number of filopodia per cell in cells transfected with GFP, GFP-Teneurin4WT (Ten4 WT; red) and GFP- Teneurin4Mut (Ten4Mut; blue). (D) Fraction of filopodia enriched with GFP per cell in GFP-, GFP-Teneurin4WT (Ten4WT; blue)- and GFP-Teneurin4Mut (Ten4Mut; red)-transfected cells. (


Figure 7 ( A ) Representative images of non - adherent K562 cells electroporated with GFP ( left ) , GFP - Teneurin4WT ( GFP - Ten4WT ; middle ) or GFP - Teneurin4Mut ( GFP - Ten4Mut ; right ) . Zoomed images show the formation of cellular clusters in cells expressing GFP - Ten4WT and GFP - Ten4Mut , but not in GFP - expressing cells . Scale bar top panel , 50 µm . Scale bar bottom panel , 20 µm . ( C ) Fraction of cell clusters detected per image in GFP - ( black ) , GFP - Teneurin4WT ( blue ) - and GFP - Teneurin4Mut ( red ) - electroporated cells .

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

Figure 7(A) Representative images of non-adherent K562 cells electroporated with GFP (left), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP-Teneurin4Mut (GFP-Ten4Mut; right). Zoomed images show the formation of cellular clusters in cells expressing GFP-Ten4WT and GFP-Ten4Mut, but not in GFP-expressing cells. Scale bar top panel, 50 µm. Scale bar bottom panel, 20 µm.(C) Fraction of cell clusters detected per image in GFP- (black), GFP-Teneurin4WT (blue)- and GFP-Teneurin4Mut (red)-electroporated cells.


Figure 1WB analysis of smc5 - AID clones in the absence or presence of Auxin using FLAG . Tubulin is used as loading control and wt cells is used as a negative control for the FLAG tag . Proliferation curve of wt and smc5 - AID cells in the presence or absence of Auxin . The data represents means ± SD of 3 independent experiments . Proliferation curves of wt and 2 independent smc5 clones grown at 39 . 5ºC , with the estimated doubling time on the right . Data represents means ± SD of 4 experiments . Asterisks indicate p value ≤ 0 . 05 , as derived from unpaired t - test type 3 , two - sample unequal variance , carried out to check statistical significance between smc5 clones and wt . smc5 clone 1 versus wt , p = 0 . 016 , smc5 clone 2 versus wt , p = 0 . 04 . Bidimensional FACS analysis shows significant accumulation in G2 / M and decrease in the S phase in smc5 mutants . The data represents means ± SD of 3 independent experiments for smc5 clones 2 and 3 , and 4 independent experiments for wt and smc5 clone 1 . Asterisks indicate p value ≤ 0 . 05 , detailed below , using paired t - test . S - phase ( smc5 clones 1 , 2 , 3 versus wt : p = 0 . 0031 ; p = 0 . 0002 ; p = 0 . 004 , respectively ) . G2 / M ( smc5 clones 1 , 2 , 3 versus wt : p = 0 . 029 ; p = 0 . 0014 ; p = 0 . 02 , respectively ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1WB analysis of smc5-AID clones in the absence or presence of Auxin using FLAG. Tubulin is used as loading control and wt cells is used as a negative control for the FLAG tag.Proliferation curve of wt and smc5-AID cells in the presence or absence of Auxin. The data represents means ± SD of 3 independent experiments.Proliferation curves of wt and 2 independent smc5 clones grown at 39.5ºC, with the estimated doubling time on the right. Data represents means ± SD of 4 experiments. Asterisks indicate p value ≤ 0.05, as derived from unpaired t-test type 3, two-sample unequal variance, carried out to check statistical significance between smc5 clones and wt. smc5 clone 1 versus wt, p=0.016, smc5 clone 2 versus wt, p=0.04.Bidimensional FACS analysis shows significant accumulation in G2/M and decrease in the S phase in smc5 mutants. The data represents means ± SD of 3 independent experiments for smc5 clones 2 and 3, and 4 independent experiments for wt and smc5 clone 1. Asterisks indicate p value ≤ 0.05, detailed below, using paired t-test. S-phase (smc5 clones 1, 2, 3 versus wt: p= 0.0031; p=0.0002; p=0.004, respectively). G2/M (smc5 clones 1, 2, 3 versus wt: p= 0.029; p=0.0014; p=0.02, respectively).


Figure 2Survival curve of smc5 cells treated with different concentrations of cisplatin ( CDDP ) . Data represents means ± SD of 3 experiments . Survival curve of smc5 cells complemented or not with chicken SMC5 cDNA and treated with different concentrations of cisplatin . The experiment is carried out at 39 . 5ºC . Data represents means ± SD of at least 3 experiments . Survival curve of smc5 ku70 cells treated with different concentrations of cisplatin . Data represents means ± SD of the 3 experiments . Survival curve of smc5 fancc cells treated with different concentrations of cisplatin . Data represents means ± SD of 3 experiments . WB analysis of CHK1 - P levels at steady state or after treatment with 1 μM cisplatin for 3 hours . CHK1 total protein is used as loading control . Quantitative analysis of CHK1 - P levels at steady state versus total CHK1 is shown in the bottom panel . Data represents means ± SD of 4 experiments . WB analysis of FANCD2 ubiquitination levels at steady state after treatment with 1 μM cisplatin for 3 hours . Analysis of the ratio between FANCD2 ubiquitinated / non - ubiquitinated forms is shown on the bottom panel . Data represents means ± SD of 3 experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Survival curve of smc5 cells treated with different concentrations of cisplatin (CDDP). Data represents means ± SD of 3 experiments.Survival curve of smc5 cells complemented or not with chicken SMC5 cDNA and treated with different concentrations of cisplatin. The experiment is carried out at 39.5ºC. Data represents means ± SD of at least 3 experiments.Survival curve of smc5 ku70 cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.Survival curve of smc5 fancc cells treated with different concentrations of cisplatin. Data represents means ± SD of 3 experiments.WB analysis of CHK1-P levels at steady state or after treatment with 1 μM cisplatin for 3 hours. CHK1 total protein is used as loading control. Quantitative analysis of CHK1-P levels at steady state versus total CHK1 is shown in the bottom panel. Data represents means ± SD of 4 experiments.WB analysis of FANCD2 ubiquitination levels at steady state after treatment with 1 μM cisplatin for 3 hours. Analysis of the ratio between FANCD2 ubiquitinated/non-ubiquitinated forms is shown on the bottom panel. Data represents means ± SD of 3 experiments.


Figure 3Left panel : Growth curve was carried out to test smc5 fancm cell proliferation . Data represents means ± SD of 3 experiments . Right panel : Survival curve of smc5 fancm cells treated with different concentrations of cisplatin . Data represents means ± SD of the 3 experiments . Clonogenic assay to test cisplatin sensitivity of smc5 fancm cells in long term assays . Data represents means ± SD of 2 independent experiments with two independent clones analyzed for smc5 fancm . Left panel : Growth curve was carried out to test smc5 rad17 ( C ) cell proliferation . Data represents means ± SD of 3 experiments . Right panel : Survival curve of smc5 rad17 ( C ) cells treated with different concentrations of cisplatin . Data represents means ± SD of the 3 experiments . Left panel : Growth curve was carried out to test smc5 ddx11 ( D ) cell proliferation . Data represents means ± SD of 3 experiments . Right panel : Survival curve of smc5 ddx11 ( D ) cells treated with different concentrations of cisplatin . Data represents means ± SD of the 3 experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Left panel: Growth curve was carried out to test smc5 fancm cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 fancm cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.Clonogenic assay to test cisplatin sensitivity of smc5 fancm cells in long term assays. Data represents means ± SD of 2 independent experiments with two independent clones analyzed for smc5 fancm.Left panel: Growth curve was carried out to test smc5 rad17 (C) cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 rad17 (C) cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.Left panel: Growth curve was carried out to test smc5 ddx11 (D) cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 ddx11 (D) cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.


Figure 4Left panel : Representative Western blots illustrating the expression of SMC6 , SMC5 and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs . Right panel : Relative expression of SMC5 , SMC6 and FANCD2 . We performed individual experiments for each cell line with technical replicates . In each individual experiment , we first normalized SMC5 , SMC6 or FANCD2 expression to that of Vinculin ( internal control ) and calculated its relative level in each cell line in relation to the target protein / Vinculin ratio of the siLacZ transfected cells , which was set as one in each experiment . Data present the mean + / - S . E . M . and were analyzed by unpaired t - test . SMC5 in siSMC6 : n = 7 and p = 0 . 0002 ; SMC5 in siFANCD2 : n = 7 and p = 0 . 0016 ; SMC6 in siSMC5 : n = 8 and p < 0 . 0001 ; SMC6 in siFANCD2 : n = 24 and p < 0 . 0001 ; FANCD2 in siSMC6 : n = 30 and p < 0 . 0001 ; FANCD2 in siSMC5 : n = 10 and p = 0 . 0008 . Top . Representative Western blots illustrating the expression of SMC6 , FANCJ and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs . Bottom . Survival curves of cells transfected with indicated siRNA and treated with different concentrations of mitomycin C ( MMC ) . Data represents the means and SEM ( standard error of the mean ) of 3 independent experiments .

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

Figure 4Left panel: Representative Western blots illustrating the expression of SMC6, SMC5 and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs. Right panel: Relative expression of SMC5, SMC6 and FANCD2. We performed individual experiments for each cell line with technical replicates. In each individual experiment, we first normalized SMC5, SMC6 or FANCD2 expression to that of Vinculin (internal control) and calculated its relative level in each cell line in relation to the target protein/Vinculin ratio of the siLacZ transfected cells, which was set as one in each experiment. Data present the mean+/-S.E.M. and were analyzed by unpaired t-test. SMC5 in siSMC6: n=7 and p=0.0002; SMC5 in siFANCD2: n=7 and p=0.0016; SMC6 in siSMC5: n=8 and p<0.0001; SMC6 in siFANCD2: n=24 and p<0.0001; FANCD2 in siSMC6: n=30 and p<0.0001; FANCD2 in siSMC5: n=10 and p=0.0008.Top. Representative Western blots illustrating the expression of SMC6, FANCJ and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs. Bottom. Survival curves of cells transfected with indicated siRNA and treated with different concentrations of mitomycin C (MMC). Data represents the means and SEM (standard error of the mean) of 3 independent experiments.


Figure 5Representative Western blots illustrating the expression of SMC6 and of monoubiquitylated FANCD2 in protein extracts from HeLa cells transfected with the indicated siRNAs and treated 48 hours after transfection with HU ( 5mM , 18 hours ) or Mitomycin C ( 200 ng / ml , 18 hours ) . Vinculin is used as loading control . Top . Representative images of nuclei ( DAPI stained , blue ) with RAD51 ( green ) foci in HeLa cells transfected with the indicated siRNAs and treated with HU or MMC , as indicated in panel A . The scale bar represents 10 μm . Bottom . Histograms presenting the percentage of RAD51 foci - positive cells as evaluated in the different conditions of above . Data represent the means + / - S . E . M . and were analyzed by unpaired t - test . siSMC6 vs siLacZ : n = 3 , NT , MMC or HU p = n . s . ; siFANCD2 vs siLacZ : n = 4 , NT p = n . s . , MMC p = 0 . 0003 , HU p = 0 . 0479 ; siSMC6 + siFANCD2 vs siLacZ : n = 3 , NT p = n . s . , MMC p = 0 . 0439 , HU p = 0 . 0458 .

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

Figure 5Representative Western blots illustrating the expression of SMC6 and of monoubiquitylated FANCD2 in protein extracts from HeLa cells transfected with the indicated siRNAs and treated 48 hours after transfection with HU (5mM, 18 hours) or Mitomycin C (200 ng/ml, 18 hours). Vinculin is used as loading control.Top. Representative images of nuclei (DAPI stained, blue) with RAD51 (green) foci in HeLa cells transfected with the indicated siRNAs and treated with HU or MMC, as indicated in panel A. The scale bar represents 10 μm. Bottom. Histograms presenting the percentage of RAD51 foci-positive cells as evaluated in the different conditions of above. Data represent the means+/-S.E.M. and were analyzed by unpaired t-test. siSMC6 vs siLacZ: n=3, NT, MMC or HU p=n.s.; siFANCD2 vs siLacZ: n=4, NT p=n.s., MMC p=0.0003, HU p=0.0479; siSMC6+siFANCD2 vs siLacZ: n=3, NT p=n.s., MMC p=0.0439, HU p=0.0458.


Figure 6Left panel : Representative images of anaphase HeLa cells with micronuclei ( white arrows ) . 48 hours after transfection with indicated siRNAs cells were incubated 18 hours with 2 μg / ml cytochalasyn B to block cytokinesis . The scale bar represents 20 μm . Right panel : Histograms presenting the percentage of binucleate cells with micronuclei . Data represent the mean + / - SEM of 3 independent experiments , t - test , unpaired . siSMC6 vs siLacZ , p = 0 . 0085 ; siFANCD2 vs siLacZ , p = 0 . 0057 ; siSMC6 + siFANCD2 vs siLacZ , p = 0 . 0019 . Top : Examples of HeLa mitotic catastrophes and anaphase bridges as observed 48 hours after siRNA transfection . The scale bar represents 20 μm . Bottom : Histograms presenting the percentage of cells with mitotic abnormalities . Data represent the mean + / - S . E . M . of 3 independent experiments . t - test , unpaired . siSMC6 vs siLacZ : Aberrant mitosis p = 0 . 0125 , Nuclear bridges p = 0 . 0198 , Mitotic catastrophes p = 0 . 0145 ; siFANCD2 vs siLacZ : Aberrant mitosis p = 0 . 0005 , Nuclear bridges p = 0 . 0002 , Mitotic catastrophes p = 0 . 0011 ; siSMC6 + siFANCD2 vs siLacZ : Aberrant mitosis p = 0 . 0012 , Nuclear bridges p = 0 . 0116 , Mitotic catastrophes p = 0 . 0033 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6Left panel: Representative images of anaphase HeLa cells with micronuclei (white arrows). 48 hours after transfection with indicated siRNAs cells were incubated 18 hours with 2 μg/ml cytochalasyn B to block cytokinesis. The scale bar represents 20 μm. Right panel: Histograms presenting the percentage of binucleate cells with micronuclei. Data represent the mean +/- SEM of 3 independent experiments, t-test, unpaired. siSMC6 vs siLacZ, p=0.0085; siFANCD2 vs siLacZ, p=0.0057; siSMC6+siFANCD2 vs siLacZ, p=0.0019.Top: Examples of HeLa mitotic catastrophes and anaphase bridges as observed 48 hours after siRNA transfection. The scale bar represents 20 μm. Bottom: Histograms presenting the percentage of cells with mitotic abnormalities. Data represent the mean +/- S.E.M. of 3 independent experiments. t-test, unpaired. siSMC6 vs siLacZ: Aberrant mitosis p=0.0125, Nuclear bridges p=0.0198, Mitotic catastrophes p=0.0145; siFANCD2 vs siLacZ: Aberrant mitosis p=0.0005, Nuclear bridges p=0.0002, Mitotic catastrophes p=0.0011; siSMC6+siFANCD2 vs siLacZ: Aberrant mitosis p=0.0012, Nuclear bridges p=0.0116, Mitotic catastrophes p=0.0033.


Figure 7Immunoprecipitation of FLAG - SMC6 and FLAG - NSMCE2 with FANCI in HEK293T cells overexpressing the SMC6 or NSMCE2 , respectively , in the presence or absence of Ethidium bromide ( EtBr ) . The experiment was performed 3 times , and the blot shows a representative experiment . The band under the one indicated as FANCI may represent a degradation product . Co - immunoprecipitation of endogenous SMC5 - mAID - EGFP and FANCD2 in TK6 cells in the presence of benzonase . Both experiments were performed twice independently , with the note that the cisplatin condition was performed only in one of the experiments , and the FANCD2 pull down in the experiment not shown was performed without benzonase .

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

Figure 7Immunoprecipitation of FLAG-SMC6 and FLAG-NSMCE2 with FANCI in HEK293T cells overexpressing the SMC6 or NSMCE2, respectively, in the presence or absence of Ethidium bromide (EtBr). The experiment was performed 3 times, and the blot shows a representative experiment. The band under the one indicated as FANCI may represent a degradation product.Co-immunoprecipitation of endogenous SMC5-mAID-EGFP and FANCD2 in TK6 cells in the presence of benzonase. Both experiments were performed twice independently, with the note that the cisplatin condition was performed only in one of the experiments, and the FANCD2 pull down in the experiment not shown was performed without benzonase.


Figure 1A . Confocal images show Iba1 - immunoreactive microglia and macrophages in RPE / choroid whole mount preparations 48 hours after laser coagulation and intravitreal application of low ( 0 . 2 µg ) and high ( 3 µg ) dose polySia avDP20 or PBS - vehicle . Laser spots of vehicle - injected SIGLEC11 transgenic ( tg ) and wild type control animals revealed strong accumulation of activated microglia and macrophages , which was effectively decreased in polySia avDP20 - injected SIGLEC11 tg animals and to a lower extent in wild type controls . Representative images out of at least three independent experiments are shown . Scale bar : 50µm . B . Quantification of the average pixel intensity of Iba1 - positive area in laser spots reflects accumulation of reactive microglia / macrophages on RPE / choroid whole mounts . In comparison to the accumulation of reactive microglia / macrophages in control animals , there is a reduced pixel intensity found in polySia avDP20 - treated SIGLEC11 tg animals . Notably , treatment with a high polySia avDP20 dose ( 3 µg ) also reduced accumulation of microglia / macrophages in laser spots of wild type animals . Data show mean + / - SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 24 spots ) , WT 0 . 2 µg Sia ( n = 37 spots ) , WT 3 µg Sia ( n = 20 spots ) , SIGLEC11 tg PBS ( n = 25 spots ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 35 spots ) , SIGLEC11 tg 3 µg Sia ( n = 31 spots ) , WT PBS vs WT 3 µg Sia p = 0 . 0147 , WT 0 . 2 µg Sia vs SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0046 , SIGLEC11 tg PBS vs SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0071 , SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p < 0 . 0001 . C . Confocal images of retinal whole mounts show Iba1 - immunoreactive microglial cells . Retinal microglia of polySia avDP20 - treated SIGLEC11 tg mice had a more ramified microglial morphology in the laser spot compared to PBS - vehicle treated mice . Interestingly , high polySia avDP20 dose ( 3 µg ) also exerted a weak therapeutic effect in wild type animals . Representative images out of at least three independent experiments are shown . Scale bar : 20 µm . D . Percentage of retina showing activated microglial cells within the laser spots was quantified . PolySia avDP20 reduced the percentage of laser spots with activated microglia in SIGLEC11 tg animals and at high dose ( 3 µg ) also in wild type controls . Data show mean + / - SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 6 retinas ) , WT 0 . 2 µg Sia ( n = 8 retinas ) , WT 3 µg Sia ( n = 6 retinas ) , SIGLEC11 tg PBS ( n = 8 retinas ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 7 retinas ) , SIGLEC11 tg 3 µg Sia ( n = 6 retinas ) , WT PBS vs WT 3 µg Sia p = 0 . 0052 , WT 0 . 2 µg Sia vs SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0005 , SIGLEC11 tg PBS vs SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0159 , SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0 . 001 .

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

Figure 1A. Confocal images show Iba1-immunoreactive microglia and macrophages in RPE/choroid whole mount preparations 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Laser spots of vehicle-injected SIGLEC11 transgenic (tg) and wild type control animals revealed strong accumulation of activated microglia and macrophages, which was effectively decreased in polySia avDP20-injected SIGLEC11 tg animals and to a lower extent in wild type controls. Representative images out of at least three independent experiments are shown. Scale bar: 50µm.B. Quantification of the average pixel intensity of Iba1-positive area in laser spots reflects accumulation of reactive microglia/macrophages on RPE/choroid whole mounts. In comparison to the accumulation of reactive microglia/macrophages in control animals, there is a reduced pixel intensity found in polySia avDP20-treated SIGLEC11 tg animals. Notably, treatment with a high polySia avDP20 dose (3 µg) also reduced accumulation of microglia/macrophages in laser spots of wild type animals. Data show mean +/- SD. * p< 0.05, ** p< 0.01, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 24 spots), WT 0.2 µg Sia (n = 37 spots), WT 3 µg Sia (n = 20 spots), SIGLEC11 tg PBS (n = 25 spots), SIGLEC11 tg 0.2 µg Sia (n = 35 spots), SIGLEC11 tg 3 µg Sia (n = 31 spots), WT PBS vs WT 3 µg Sia p = 0.0147, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0046, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0071, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p < 0.0001.C. Confocal images of retinal whole mounts show Iba1-immunoreactive microglial cells. Retinal microglia of polySia avDP20-treated SIGLEC11 tg mice had a more ramified microglial morphology in the laser spot compared to PBS-vehicle treated mice. Interestingly, high polySia avDP20 dose (3 µg) also exerted a weak therapeutic effect in wild type animals. Representative images out of at least three independent experiments are shown. Scale bar: 20 µm.D. Percentage of retina showing activated microglial cells within the laser spots was quantified. PolySia avDP20 reduced the percentage of laser spots with activated microglia in SIGLEC11 tg animals and at high dose (3 µg) also in wild type controls. Data show mean +/- SEM. * p< 0.05, ** p< 0.01, ***p<0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 6 retinas), WT 0.2 µg Sia (n = 8 retinas), WT 3 µg Sia (n = 6 retinas), SIGLEC11 tg PBS (n = 8 retinas), SIGLEC11 tg 0.2 µg Sia (n = 7 retinas), SIGLEC11 tg 3 µg Sia (n = 6 retinas),WT PBS vs WT 3 µg Sia p = 0.0052, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0005, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0159, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.001.


Figure 2A . Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low ( 0 . 2 µg ) and high ( 3 µg ) dose polySia avDP20 or PBS - vehicle . Late stage ( 10 - 11 minutes after fluorescein injection ) fundus fluorescein angiography revealed that PBS - treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20 - treated SIGLEC11 transgenic ( tg ) mice had lower levels of vessel leakage compared to PBS - injected wild type or SIGLEC11 tg mice . High polySia avDP20 dose reduced vascular leakage also in wild type animals . Representative images out of at least eight independent experiments are shown . B . Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software ( NIH ) . Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted . PolySia avDP20 - treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS - injected wild type control and SIGLEC11 tg mice . A reduction in vascular leakage was also observed in high dose polySia avDP20 - injected wild type control mice . Data show mean + / - SD . * p < 0 . 05 , * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 20 eyes ) , WT 0 . 2 µg Sia ( n = 17 eyes ) , WT 3 µg Sia ( n = 9 eyes ) , SIGLEC11 tg PBS ( n = 16 eyes ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 20 eyes ) , SIGLEC11 tg 3 µg Sia ( n = 15 eyes ) , WT PBS vs WT 3 µg Sia p = 0 . 0193 , WT 0 . 2 µg Sia vs SIGLEC11 tg 0 . 2 µg Sia p < 0 . 0001 , SIGLEC11 tg PBS vs SIGLEC11 tg 0 . 2 µg Sia p < 0 . 0001 , SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0 . 0002 . C . Anti - C5b - 9 immunostaining of RPE / choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle - injected controls . PolySia avDP20 treatment reduced MAC formation in a dose - dependent fashion independent of SIGLEC11 presence . Scale bar : 100 µm . D . Quantification of C5b - 9 fluorescence signal intensity in the laser lesions . In comparison to the high amount of MAC deposition in vehicle - injected controls , the pixel intensity is reduced in polySia avDP20 - treated eyes in a dose - dependent fashion and regardless of SIGLEC11 presence . Data show mean + / - SD . * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 12 laser spots ) , WT 0 . 2 µg Sia ( n = 12 laser spots ) , WT 3 µg Sia ( n = 7 laser spots ) , SIGLEC11 tg PBS ( n = 6 laser spots ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 10 laser spots ) , SIGLEC11 tg 3 µg Sia ( n = 12 laser spots ) ; all statistical comparisons p < 0 . 0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0004 .

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

Figure 2A. Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Late stage (10-11 minutes after fluorescein injection) fundus fluorescein angiography revealed that PBS-treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20-treated SIGLEC11 transgenic (tg) mice had lower levels of vessel leakage compared to PBS-injected wild type or SIGLEC11 tg mice. High polySia avDP20 dose reduced vascular leakage also in wild type animals. Representative images out of at least eight independent experiments are shown.B. Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software (NIH). Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted. PolySia avDP20-treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS-injected wild type control and SIGLEC11 tg mice. A reduction in vascular leakage was also observed in high dose polySia avDP20-injected wild type control mice. Data show mean +/- SD. * p< 0.05, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 20 eyes), WT 0.2 µg Sia (n = 17 eyes), WT 3 µg Sia (n = 9 eyes), SIGLEC11 tg PBS (n = 16 eyes), SIGLEC11 tg 0.2 µg Sia (n = 20 eyes), SIGLEC11 tg 3 µg Sia (n = 15 eyes), WT PBS vs WT 3 µg Sia p = 0.0193, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.0002.C. Anti-C5b-9 immunostaining of RPE/choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle-injected controls. PolySia avDP20 treatment reduced MAC formation in a dose-dependent fashion independent of SIGLEC11 presence. Scale bar: 100 µm.D. Quantification of C5b-9 fluorescence signal intensity in the laser lesions. In comparison to the high amount of MAC deposition in vehicle-injected controls, the pixel intensity is reduced in polySia avDP20-treated eyes in a dose-dependent fashion and regardless of SIGLEC11 presence. Data show mean +/- SD. *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 12 laser spots), WT 0.2 µg Sia (n = 12 laser spots), WT 3 µg Sia (n = 7 laser spots), SIGLEC11 tg PBS (n = 6 laser spots), SIGLEC11 tg 0.2 µg Sia (n = 10 laser spots), SIGLEC11 tg 3 µg Sia (n = 12 laser spots); all statistical comparisons p < 0.0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0.2 µg Sia p = 0.0004.


Figure 3A . Analysis of relative TNFSF2 gene transcription in human control and SIGLEC11 / 16 knockout THP1 - macrophages . The levels of gene transcripts were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and concentrations of 0 . 15 µM and 1 . 5 µM of polySia avDP20 in the human wild type macrophages . No response to polySia avDP20 was detectable in the knockout line . Data show mean + / - SEM . * * p < 0 . 01 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 7 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 4 and p = 0 . 0002 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 3 and p = 0 . 009 , LPS / PolySia avDP20 1 . 5µM n = 5 and p = 0 . 002 . Siglec11 / 16 KO : no treatment n = 5 and p = 0 . 01 , PolySia avDP20 n = 4 and p = 0 . 122 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 3 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 4 and p = 1 . 0 . B . Analysis of relative TNFSF2 protein release in human control and SIGLEC11 / 16 knockout THP1 - macrophages . The released protein levels were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and concentrations of 0 . 15 µM and 1 . 5 µM of polySia avDP20 in the human wild type macrophages . No response to polySia avDP20 was detectable in the knockout line . Data show mean + / - SEM . * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 8 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 5 and p < 0 . 001 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 5 and p = 0 . 002 , LPS / PolySia avDP20 1 . 5µM n = 4 and p = 0 . 0003 . Siglec11 / 16 KO : no treatment n = 6 and p < 0 . 001 , PolySia avDP20 1 . 5 µM n = 5 and p < 0 . 001 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 5 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 5 and p = 1 . 0 . C . Analysis of relative VEGFA gene transcription in human control and SIGLEC11 / 16 knockout THP1 - macrophages . Gene transcripts were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and polySia avDP20 ( 0 . 15 µM and 1 . 5 µM ) in the human wild type macrophages . No response to polySia avDP20 was detectable in the knockout macrophages . Data show mean + / - SEM . * * * p < 0 . 001 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 6 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 5 and p < 0 . 0001 , LPS n = 5 , LPS / PolySia avDP20 0 . 15µM n = 5 and p = 0 . 0002 , LPS / PolySia avDP20 1 . 5µM n = 5 and p < 0 . 0001 . Siglec11 / 16 KO : no treatment n = 5 and p = 0 . 022 , PolySia avDP20 n = 3 and p = 0 . 063 , LPS n = 4 , LPS / PolySia avDP20 0 . 15µM n = 3 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 4 and p = 1 . 0 . D . Analysis of relative VEGFA protein release in human control and SIGLEC11 / 16 knockout THP1 - macrophages . Released protein levels were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and polySia avDP20 ( 0 . 15 µM and 1 . 5 µM ) in the human macrophages . Data show mean + / - SEM . * p < 0 . 05 , * * * p < 0 . 001 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 9 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 6 and p = 0 . 009 , LPS n = 9 , LPS / PolySia avDP20 0 . 15µM n = 6 and p = 0 . 043 , LPS / PolySia avDP20 1 . 5µM n = 7 and p = 0 . 0001 . Siglec11 / 16 KO : no treatment n = 9 and p = 0 . 349 , PolySia avDP20 1 . 5 µM n = 6 and p = 0 . 249 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 6 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 7 and p = 1 . 0 . E . Quantification of human control and SIGLEC11 / 16 knockout THP1 - macrophages having ingested cellular debris . PolySia avDP20 ( 1 . 5 µM ) reduced the percentage of phagocytic cells having ingested drusen - like debris . No response to polySia avDP20 was detectable in the knockout macrophages . Data are presented as mean + / - SEM . n = 6 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 , ANOVA followed by Bonferroni . Debris treated WT macrophages vs debris plus polySia avDP20 treated WT macrophages p = 0 . 028 , debris plus polySia avDP20 treated WT macrophages vs KO macrophages p = 0 . 00012 . F . Prevention of superoxide release in activated human control and SIGLEC11 / 16 knockout THP1 - macrophages by polySia avDP20 . Cultured human THP1 - macrophages were stimulated with RPE cell debris or co - stimulated with debris and polySia avDP20 . Addition of debris stimulated the production of superoxide . 1 . 5 µM polySia avDP20 completely prevented the release of superoxide induced by debris challenge . No response to polySia avDP20 was detectable in the knockout macrophages . Data are presented as mean + / - SEM . n = 6 . p * ≤ 0 . 05 , * * * p ≤ 0 . 001 , ANOVA followed by Bonferroni . Untreated WT macrophages vs debris treated WT macrophages p = 0 . 037 , debris treated WT macrophage vs debris plus polySia avDP20 1 . 5 µM treated WT macrophages p < 0 . 001 , debris plus polySia avDP20 WT macrophages vs KO macrophages p = 0 . 001 , G . PolySia avDP20 ( 0 . 15 to 50 µM ) was added to normal human serum ( NHS ) to evaluate any interference with activation of the classical complement pathway by IgM . Complement activation was determined by C3b deposition . PolySia avDP20 had no effect on C3b deposition induced by activation of the classical complement pathway . Data show mean + / - SEM . n = 3 , n . s . = not significant . H . PolySia avDP20 inhibits the alternative complement pathway . PolySia avDP20 ( 0 . 15 to 50 µM ) was added to NHS and activation of the alternative pathway was induced by LPS . C3b deposition was monitored by ELISA . Data show mean + / - SEM ( n = 4 . * p < 0 . 05 , * * p < 0 . 01 , p * * * < 0 . 001 , ANOVA followed by Bonferroni ) .

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

Figure 3A. Analysis of relative TNFSF2 gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. The levels of gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=7 and p< 0.0001, PolySia avDP20 1.5 µM n=4 and p=0.0002, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=0.009, LPS/PolySia avDP20 1.5µM n=5 and p=0.002. Siglec11/16 KO: no treatment n=5 and p=0.01, PolySia avDP20 n=4 and p=0.122, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.B. Analysis of relative TNFSF2 protein release in human control and SIGLEC11/16 knockout THP1-macrophages. The released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=8 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=0.002, LPS/PolySia avDP20 1.5µM n=4 and p=0.0003. Siglec11/16 KO: no treatment n=6 and p<0.001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=1.0, LPS/PolySia avDP20 1.5µM n=5 and p=1.0.C. Analysis of relative VEGFA gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. Gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout macrophages. Data show mean +/- SEM. *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=6 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.0001, LPS n=5, LPS/PolySia avDP20 0.15µM n=5 and p=0.0002, LPS/PolySia avDP20 1.5µM n=5 and p<0.0001. Siglec11/16 KO: no treatment n=5 and p=0.022, PolySia avDP20 n=3 and p=0.063, LPS n=4, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.D. Analysis of relative VEGFA protein release in human control and SIGLEC11/16 knockout THP1-macrophages. Released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human macrophages. Data show mean +/- SEM. * p < 0.05, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=9 and p<0.0001, PolySia avDP20 1.5 µM n=6 and p=0.009, LPS n=9, LPS/PolySia avDP20 0.15µM n=6 and p=0.043, LPS/PolySia avDP20 1.5µM n=7 and p=0.0001. Siglec11/16 KO: no treatment n=9 and p=0.349, PolySia avDP20 1.5 µM n= 6 and p=0.249, LPS n=7, LPS/PolySia avDP20 0.15µM n=6 and p=1.0, LPS/PolySia avDP20 1.5µM n=7 and p=1.0.E. Quantification of human control and SIGLEC11/16 knockout THP1-macrophages having ingested cellular debris. PolySia avDP20 (1.5 µM) reduced the percentage of phagocytic cells having ingested drusen-like debris. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. *p ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Debris treated WT macrophages vs debris plus polySia avDP20 treated WT macrophages p = 0.028, debris plus polySia avDP20 treated WT macrophages vs KO macrophages p = 0.00012.F. Prevention of superoxide release in activated human control and SIGLEC11/16 knockout THP1-macrophages by polySia avDP20. Cultured human THP1-macrophages were stimulated with RPE cell debris or co-stimulated with debris and polySia avDP20. Addition of debris stimulated the production of superoxide. 1.5 µM polySia avDP20 completely prevented the release of superoxide induced by debris challenge. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. p* ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Untreated WT macrophages vs debris treated WT macrophages p = 0.037, debris treated WT macrophage vs debris plus polySia avDP20 1.5 µM treated WT macrophages p < 0.001, debris plus polySia avDP20 WT macrophages vs KO macrophages p = 0.001,G. PolySia avDP20 (0.15 to 50 µM) was added to normal human serum (NHS) to evaluate any interference with activation of the classical complement pathway by IgM. Complement activation was determined by C3b deposition. PolySia avDP20 had no effect on C3b deposition induced by activation of the classical complement pathway. Data show mean +/- SEM. n = 3, n.s.= not significant.H. PolySia avDP20 inhibits the alternative complement pathway. PolySia avDP20 (0.15 to 50 µM) was added to NHS and activation of the alternative pathway was induced by LPS. C3b deposition was monitored by ELISA. Data show mean +/- SEM (n = 4. * p < 0.05, ** p < 0.01, p***<0.001, ANOVA followed by Bonferroni).


Figure 1Fluorescent images ( left panels ) and tracings ( right panels ) of PVD neurons of the indicated genotypes . PVD is visualized by the wdIs52 transgene . Primary ( 1º ) , secondary ( 2º ) , tertiary ( 3º ) , and quaternary ( 4º ) dendrites are indicated , and the cell body is marked with an asterisk . Anterior is to the left and dorsal is up in all panels . Scale bars represent 20μm . Quantification of the number of quaternary branches ( indicated in schematic ) in different aman - 2 alleles and in wild type control animals . All loss of function alleles of aman - 2 display a significant decrease in PVD quaternary branch number . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and is indicated ( * * * * p ≤ 0 . 0001 ) . n = 15 for all genotypes and are biological replicates . Quantification of the number of full tertiaries ( marked by secondary and tertiary branches as shown in schematic ) in the genotypes indicated . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and is indicated ( * * * * p ≤ 0 . 0001 ; ns = not significant ) . n = 15 for all genotypes and are biological replicates . Fluorescent images and tracings of PVD in mnr - 1 ( dz213 ) hypomorphic animals alone and in combination with an aman - 2 ( gk248486 ) null mutant . The dz213 introduces a L135F missense mutation in the DUF2181 domain of MNR - 1 / Menorin . PVD is visualized by the dzIs53 transgene . The cell body is marked with an asterisk . Scale bars represent 20μm . Quantification of the number of full tertiaries in genotypes indicated and traced in ( F ) . The loss of aman - 2 severely enhances the mnr - 1 ( dz213 ) phenotype . Control indicates wild type control animals as shown in ( A ) . Data are represented as mean ± SEM . Statistical significance was calculated using the Mann - Whitney test and is indicated ( * * * * p ≤ 0 . 0001 ; ns = not significant ) . n > 23 for all genotypes and are biological replicates . Fluorescent images and tracings of PVD in kpc - 1 ( dz254 ) hypomorph animals alone and in combination with an aman - 2 ( gk248486 ) null mutant . PVD is visualized by the dzIs53 transgene . White arrows indicate self - avoidance defects in tertiary branches . Red arrows show gaps between tertiary branches ( no self - avoidance defects ) . The cell body is marked with an asterisk . Scale bars represent 20μm . Quantification of the percent of self - avoidance defects in genotypes indicated and traced in ( G ) . Control indicates wild type control animals as shown in ( A ) . The loss of aman - 2 suppresses defects in the kpc - 1 ( dz254 ) phenotype . Data are represented as mean . Statistical significance was calculated using the Z - test and is indicated ( * * * * p ≤ 0 . 0001 ) . n > 15 for all genotypes and are biological replicates .

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

Figure 1Fluorescent images (left panels) and tracings (right panels) of PVD neurons of the indicated genotypes. PVD is visualized by the wdIs52 transgene. Primary (1º), secondary (2º), tertiary (3º), and quaternary (4º) dendrites are indicated, and the cell body is marked with an asterisk. Anterior is to the left and dorsal is up in all panels. Scale bars represent 20μm.Quantification of the number of quaternary branches (indicated in schematic) in different aman-2 alleles and in wild type control animals. All loss of function alleles of aman-2 display a significant decrease in PVD quaternary branch number. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (****p ≤ 0.0001). n = 15 for all genotypes and are biological replicates.Quantification of the number of full tertiaries (marked by secondary and tertiary branches as shown in schematic) in the genotypes indicated. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (****p ≤ 0.0001; ns = not significant). n = 15 for all genotypes and are biological replicates.Fluorescent images and tracings of PVD in mnr-1(dz213) hypomorphic animals alone and in combination with an aman-2(gk248486) null mutant. The dz213 introduces a L135F missense mutation in the DUF2181 domain of MNR-1/Menorin. PVD is visualized by the dzIs53 transgene. The cell body is marked with an asterisk. Scale bars represent 20μm. Quantification of the number of full tertiaries in genotypes indicated and traced in (F). The loss of aman-2 severely enhances the mnr-1(dz213) phenotype. Control indicates wild type control animals as shown in (A). Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n >23 for all genotypes and are biological replicates.Fluorescent images and tracings of PVD in kpc-1(dz254) hypomorph animals alone and in combination with an aman-2(gk248486) null mutant. PVD is visualized by the dzIs53 transgene. White arrows indicate self-avoidance defects in tertiary branches. Red arrows show gaps between tertiary branches (no self-avoidance defects). The cell body is marked with an asterisk. Scale bars represent 20μm. Quantification of the percent of self-avoidance defects in genotypes indicated and traced in (G). Control indicates wild type control animals as shown in (A). The loss of aman-2 suppresses defects in the kpc-1(dz254) phenotype. Data are represented as mean. Statistical significance was calculated using the Z-test and is indicated (****p ≤ 0.0001). n >15 for all genotypes and are biological replicates.


Figure 2Fluorescent images and tracings of wild type control ( top ) and lect - 2 ( gk846764 ) hypomorphic animals ( bottom ) fed on plates with 300µm swainsonine vs a DMSO control . PVD is visualized by the wyIs581 transgene . The cell body is denoted with an asterisk . Anterior is to the left and dorsal is up in all panels . Vesicular gut autofluorescence is visible as white circular staining . Scale bars represent 20μm . Quantification of the number of full tertiaries in denoted genetic backgrounds ( aman - 2 null is gk248486 ) . Black data points indicate DMSO and orange data points show swainsonine treated animals . Data are represented as mean ± SEM . Statistical significance was calculated using the Mann - Whitney test and is indicated ( * * * * p ≤ 0 . 0001 ; ns = not significant ) . n = 12 for all genotypes and are biological replicates . Quantification of the number of quaternary dendrites in wild type control animals fed on plates with and without 300µm swainsonine . Animals treated with swainsonine show a significant decrease in quaternary branch number , Data are represented as mean ± SEM . Statistical significance was calculated using the Mann - Whitney test and is indicated ( * * * * p ≤ 0 . 0001 ) . n = 12 for each experiment and are biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Fluorescent images and tracings of wild type control (top) and lect-2(gk846764) hypomorphic animals (bottom) fed on plates with 300µm swainsonine vs a DMSO control. PVD is visualized by the wyIs581 transgene. The cell body is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Vesicular gut autofluorescence is visible as white circular staining. Scale bars represent 20μm. Quantification of the number of full tertiaries in denoted genetic backgrounds (aman-2 null is gk248486). Black data points indicate DMSO and orange data points show swainsonine treated animals. Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n = 12 for all genotypes and are biological replicates. Quantification of the number of quaternary dendrites in wild type control animals fed on plates with and without 300µm swainsonine. Animals treated with swainsonine show a significant decrease in quaternary branch number, Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001). n = 12 for each experiment and are biological replicates.