Figure 1A ) Differential interference contrast ( DIC ) image of cortical neurons in culture . The arrowhead points to a neuron expressing Ub - R - GFP . B ) GFP fluorescence before and after exposure to lactacystin ( 10µM ) . C ) A neuron coexpressing Ub - R - GFP and CFP before ( left ) and 10 hours after exposure to lactacystin ( right ) . Top row - GFP fluorescence ; bottom row - CFP fluorescence . Bars : 50µm ( A , B ) , 20 µm ( C ) . D ) Changes in GFP and CFP fluorescence following exposure to lactacystin at t = 1 hour ( vertical dashed line ) . Changes normalized to initial fluorescence in same cells . 9 neurons from 5 experiments ; averages and SEM . E ) Same as in D , except that here neurons were exposed only to carrier solution . 11 neurons from 5 experiments ; averages and SEM ( barely observable ) . F ) Comparison of GFP fluorescence accumulation rates . Linear fits shown as black lines ; fit parameters shown next to fits . Same data as in D , and E .

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Figure 1A) Differential interference contrast (DIC) image of cortical neurons in culture. The arrowhead points to a neuron expressing Ub-R-GFP. B) GFP fluorescence before and after exposure to lactacystin (10µM).C) A neuron coexpressing Ub-R-GFP and CFP before (left) and 10 hours after exposure to lactacystin (right). Top row - GFP fluorescence; bottom row - CFP fluorescence. Bars: 50µm (A,B), 20 µm (C). D) Changes in GFP and CFP fluorescence following exposure to lactacystin at t=1 hour (vertical dashed line). Changes normalized to initial fluorescence in same cells. 9 neurons from 5 experiments; averages and SEM. E) Same as in D, except that here neurons were exposed only to carrier solution. 11 neurons from 5 experiments; averages and SEM (barely observable). F) Comparison of GFP fluorescence accumulation rates. Linear fits shown as black lines; fit parameters shown next to fits. Same data as in D, and E.


Figure 2A ) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins ( the postsynaptic scaffold molecule Dlg4 / PSD - 95 , the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A ) following 10 hour incubations in carrier solution ( control , upper panels ) or Lactacystin ( 10 μM ; bottom panels ) . Scale bar , 10μmB ) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution . Numbers within bars represent the number of fields of view analyzed for each condition ( ~ 170 synapses / field of view , on average ) . Error bars , SEM ; P values - two tailed unpaired t - test . Note that a statistically significant accumulation was observed only for RIM , whereas immunolabeling of all other proteins was either unchanged or reduced . C ) Hippocampal neurons in primary culture were grown in the presence of Lactacystin ( 10 μM ) , fixed at two hour intervals and immunolabeled against Shank3 / ProSAP2 . Two representative fields of view at t = 0 and t = 24h . Scale bar , 10μm . D ) Average synaptic Shank3 / ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin . Fluorescence was normalized to mean fluorescence at t = 0 . Numbers under data points reflect the numbers of fields of view analyzed at each time point . Error bars , SEM .

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

Figure 2A) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins (the postsynaptic scaffold molecule Dlg4/PSD-95, the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A) following 10 hour incubations in carrier solution (control, upper panels) or Lactacystin (10 μM; bottom panels). Scale bar, 10μmB) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution. Numbers within bars represent the number of fields of view analyzed for each condition (~170 synapses/field of view, on average). Error bars, SEM; P values - two tailed unpaired t-test. Note that a statistically significant accumulation was observed only for RIM, whereas immunolabeling of all other proteins was either unchanged or reduced.C) Hippocampal neurons in primary culture were grown in the presence of Lactacystin (10 μM), fixed at two hour intervals and immunolabeled against Shank3/ProSAP2. Two representative fields of view at t=0 and t=24h. Scale bar, 10μm. D) Average synaptic Shank3/ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin. Fluorescence was normalized to mean fluorescence at t=0. Numbers under data points reflect the numbers of fields of view analyzed at each time point. Error bars, SEM.


Figure 3B ) Comparisons of H / L ratios obtained for 1409 proteins in lactacystin and untreated cells . H / L ratios are averages of 2 experiments .

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Figure 3B) Comparisons of H/L ratios obtained for 1409 proteins in lactacystin and untreated cells. H/L ratios are averages of 2 experiments.


Figure 4A ) Fold changes in the H / L ratios ( ) measured for each protein in the experiments described in Fig . 3 ( expressed as log2 ( H / L ) Lactacystin - log2 ( H / L ) control ) . H / L ratios used here are the average of ratios obtained in two experiments for each protein and each condition . Proteins were sorted according to the fold change in H / L ratios - from the proteins whose ratios were most strongly reduced ( ~ 8 - fold reduction ) to proteins whose H / L ratios increased to the greatest extent ( > 8 - fold increase ) . Red circles indicate the location of synaptic proteins along this sorted list . The names of 34 synaptic proteins located at the left most region of the sorted list are provided in the order of their appearance . B ) The apparent fold - change in protein half - lives under certain assumptions described in Results . See Materials and Methods for further details on the calculation of the apparent fold change in half - lives .

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Figure 4A) Fold changes in the H/L ratios ( ) measured for each protein in the experiments described in Fig. 3 (expressed as log2(H/L)Lactacystin - log2(H/L)control). H/L ratios used here are the average of ratios obtained in two experiments for each protein and each condition. Proteins were sorted according to the fold change in H/L ratios - from the proteins whose ratios were most strongly reduced (~8-fold reduction) to proteins whose H/L ratios increased to the greatest extent (>8-fold increase). Red circles indicate the location of synaptic proteins along this sorted list. The names of 34 synaptic proteins located at the left most region of the sorted list are provided in the order of their appearance. B) The apparent fold-change in protein half-lives under certain assumptions described in Results. See Materials and Methods for further details on the calculation of the apparent fold change in half-lives.


Figure 6A ) Average log2 ( H / M ) ratios for 1 , 750 proteins at four time points in three separate experiments . Each time point shows the average and standard deviation of three mean log2 ( H / M ) values obtained in three separate experiments . Each mean , in turn , represents the mean log2 ( H / M ) value measured in the population of 1 , 750 proteins in that experiment and time point . B ) Distribution of log2 ( H / M ) values for 1 , 750 proteins at t = 0 . The log2 ( H / M ) value of each protein represents the average value for that protein in the three experiments . In principle , log2 ( H / M ) for all proteins at t = 0 should be 0 , but in practice they are distributed around 0 as shown here . The H / M ratios for 3 standard deviation limits are shown beneath the X axis . C , D ) The same as in A and B but for two experiments in which Epoxomicin was used ( 1 , 823 proteins ) . E , F ) The same as in A and B but for the combined data from all experiments ( 3 experiments in which lactacystin was used and 2 experiments in which epoxomicin was used ; 1 , 469 proteins ) .

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

Figure 6A) Average log2(H/M) ratios for 1,750 proteins at four time points in three separate experiments. Each time point shows the average and standard deviation of three mean log2(H/M) values obtained in three separate experiments. Each mean, in turn, represents the mean log2(H/M) value measured in the population of 1,750 proteins in that experiment and time point. B) Distribution of log2(H/M) values for 1,750 proteins at t=0. The log2(H/M) value of each protein represents the average value for that protein in the three experiments. In principle, log2(H/M) for all proteins at t=0 should be 0, but in practice they are distributed around 0 as shown here. The H/M ratios for 3 standard deviation limits are shown beneath the X axis. C, D) The same as in A and B but for two experiments in which Epoxomicin was used (1,823 proteins). E, F) The same as in A and B but for the combined data from all experiments (3 experiments in which lactacystin was used and 2 experiments in which epoxomicin was used; 1,469 proteins).


Figure 7A ) log2 ( H / M ) values for 5 synaptic proteins for which degradation rates were significantly slowed down in the presence of lactacystin . Each point represents average log2 ( H / M ) from 3 experiments . B ) log2 ( H / M ) values for the same 5 synaptic proteins in experiments in which epoxomicin was used . Each point represents average log2 ( H / M ) from 2 experiments . C ) Average ( ± SEM ) log2 ( H / M ) ratios for the same 5 synaptic proteins , based on all five experiments ( 3 lactacystin , 2 epoxomicin ) . P values - two - tailed Welch ' s t - test , comparing the five log2 ( H / M ) measurements at t = 24 to those measured for the same proteins at t = 0 . D , E , F ) Average ( ± SEM ) Log2 ( H / M ) values for three groups of synaptic proteins ( synaptic vesicle , presynaptic , postsynaptic ) , based on all five experiments . None of the changes observed for these proteins were statistically significant . The temporal changes common to all proteins and all groups most likely represent slight imperfections in the H / M normalization process ( Compare with Fig . 6E ; see Results for further details ) . G ) log2 ( H / M ) values at t = 24 hours for a group of well - studied synaptic proteins , sorted from highest to lowest . P values ( two - tailed Welch ' s t - test ) of comparisons with log2 ( H / M ) values for the same proteins at t = 0 are shown in parentheses . Proteins that exhibited statistically significant changes ( P ≤ 0 . 05 ) at t = 24h are shown in red . Proteins for which ( 0 . 05 < P ≤ 0 . 10 ) at t = 24h are shown in blue / red . The gray region depicts three standard deviations of log2 ( H / M ) values at t = 0 ( Fig . 6F ) , to provide a measure of the method ' s sensitivity .

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 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 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

Figure 7A) log2(H/M) values for 5 synaptic proteins for which degradation rates were significantly slowed down in the presence of lactacystin. Each point represents average log2(H/M) from 3 experiments. B) log2(H/M) values for the same 5 synaptic proteins in experiments in which epoxomicin was used. Each point represents average log2(H/M) from 2 experiments. C) Average (± SEM) log2(H/M) ratios for the same 5 synaptic proteins, based on all five experiments (3 lactacystin, 2 epoxomicin). P values - two-tailed Welch's t-test, comparing the five log2(H/M) measurements at t=24 to those measured for the same proteins at t=0.D,E,F) Average (± SEM) Log2(H/M) values for three groups of synaptic proteins (synaptic vesicle, presynaptic, postsynaptic), based on all five experiments. None of the changes observed for these proteins were statistically significant. The temporal changes common to all proteins and all groups most likely represent slight imperfections in the H/M normalization process (Compare with Fig. 6E; see Results for further details).G) log2(H/M) values at t=24 hours for a group of well-studied synaptic proteins, sorted from highest to lowest. P values (two-tailed Welch's t-test) of comparisons with log2(H/M) values for the same proteins at t=0 are shown in parentheses. Proteins that exhibited statistically significant changes (P≤0.05) at t=24h are shown in red. Proteins for which (0.05 < P ≤ 0.10) at t=24h are shown in blue/red. The gray region depicts three standard deviations of log2(H/M) values at t=0 (Fig. 6F), to provide a measure of the method's sensitivity.


Figure 9Measured log2 ( H / M ) values at four time points as a function of their half - lives estimated here and elsewhere ( Cohen et al , 2013 ) . All log2 ( H / M ) values shown here are averages of values obtained in 5 experiments ( 3 using lactacystin , 2 using epoxomicin ) . Proteins for which statistically significant differences at t = 24 hours were observed ( P < 0 . 05 , two - tailed Welch ' s t - test ) are shown in red . Expected log2 ( H / M ) values based on equation 4 are plotted as light blue lines . To minimize the masking of potential dependencies by the slight imperfections in the H / M normalization process ( which introduces small offsets along the Y scale ) , average population log2 ( H / M ) values of each time point ( Fig . 6E ) were subtracted from all measured Log2 ( H / M ) values . Data for 1 , 416 proteins for which half - life estimates were available .

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Figure 9Measured log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 1,416 proteins for which half-life estimates were available.


Figure 10Measured Log2 ( H / M ) values at four time points as a function of their half - lives estimated here and elsewhere ( Cohen et al , 2013 ) . All log2 ( H / M ) values shown here are averages of values obtained in 5 experiments ( 3 using lactacystin , 2 using epoxomicin ) . Proteins for which statistically significant differences at t = 24 hours were observed ( P < 0 . 05 , two - tailed Welch ' s t - test ) are shown in red . Expected log2 ( H / M ) values based on equation 4 are plotted as light blue lines . To minimize the masking of potential dependencies by the slight imperfections in the H / M normalization process ( which introduces small offsets along the Y scale ) , average population log2 ( H / M ) values of each time point ( Fig . 6E ) were subtracted from all measured Log2 ( H / M ) values . Data for 174 synaptic proteins for which half - life estimates were available .

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

Figure 10Measured Log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 174 synaptic proteins for which half-life estimates were available.


Figure 1B ) Kaplan - Meier curve showing % of mice surviving S . pneumoniae infection . Data was analyzed using Kaplan - Meier statistical analysis . N = 5 - 10 mice / group . Data information : Data presented show mean ± SEM of biological replicates . F ) Bar graphs comparing bacterial burden ( CFU ) in the lung and spleen at 48h , 72h and 7d post - S . pneumoniae infection in unvaccinated and BCG vaccinated mice . N = 3 - 5 mice / group . Data information : Data presented in F ) show mean ± SEM of biological replicates . Statistical analysis for F ) were two - way ANOVA with Sidak ' s multiple comparisons test * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 .

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Figure 1B) Kaplan-Meier curve showing % of mice surviving S. pneumoniae infection. Data was analyzed using Kaplan-Meier statistical analysis. N = 5 - 10 mice/group. Data information: Data presented show mean ± SEM of biological replicates.F) Bar graphs comparing bacterial burden (CFU) in the lung and spleen at 48h, 72h and 7d post-S. pneumoniae infection in unvaccinated and BCG vaccinated mice. N = 3 - 5 mice/group. Data information: Data presented in F) show mean ± SEM of biological replicates. Statistical analysis for F) were two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01; ****p < 0.0001.


Figure 2B ) Dot plots of gated mononuclear cells from the BAL of control and BCG vaccinated mice . Analysis was performed utilizing default FlowJo V . 10 software settings . C ) Line graphs comparing absolute neutrophil cell counts in the airways at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis for ( C , were two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 ; * * * p < 0 . 001 . D ) Dot plots of gated mononuclear cells from the airways of control and BCG vaccinated hosts . Analysis was performed utilizing default FlowJo V . 10 software settings . Line graphs comparing absolute AM cell counts in the airways at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis were two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 ; * * * p < 0 . 001 . G ) Line graphs comparing the levels of neutrophilic chemokines in the BAL fluid at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis were two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 ; * * * 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 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

Figure 2B) Dot plots of gated mononuclear cells from the BAL of control and BCG vaccinated mice. Analysis was performed utilizing default FlowJo V.10 software settings. C) Line graphs comparing absolute neutrophil cell counts in the airways at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (C, were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.D) Dot plots of gated mononuclear cells from the airways of control and BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. Line graphs comparing absolute AM cell counts in the airways at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.G) Line graphs comparing the levels of neutrophilic chemokines in the BAL fluid at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.


Figure 3A ) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts . Analysis was performed utilizing default FlowJo V . 10 software settings . B ) Line graphs comparing absolute neutrophil cell counts in the lung at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis for ( B , were two - way ANOVA with Sidak ' s multiple comparisons test . C ) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts . Analysis was performed utilizing default FlowJo V . 10 software settings . D - Line graphs comparing absolute AM , cell counts in the lung at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis were two - way ANOVA with Sidak ' s multiple comparisons test . Line graphs comparing absolute IM and MDM cell counts in the lung at 18 and 36h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 - 4 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis were two - way ANOVA with Sidak ' s multiple comparisons test .

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

Figure 3A) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. B) Line graphs comparing absolute neutrophil cell counts in the lung at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (B, were two-way ANOVA with Sidak's multiple comparisons test.C) Dot plots of gated mononuclear cells from the lung of control and BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. D - Line graphs comparing absolute AM, cell counts in the lung at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test.Line graphs comparing absolute IM and MDM cell counts in the lung at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test.


Figure 4C ) Bar graphs comparing the killing rates of phagocytosed S . pneumoniae by AMs at 1 and 2h post - ex vivo S . pneumoniae infection . N = 3 replicates / group . Data information : Data presented in C , represent mean ± SEM of technical replicates . Statistical analysis for C , were two - tailed unpaired t - tests * p < 0 . 05 . E ) Line graphs comparing changes in body weight following S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 4 mice / group . Data information : Data presented represent mean ± SEM of technical replicates . Statistical analysis for ( E ) was two - way ANOVA with Sidak ' s multiple comparisons test . * p < 0 . 05 . G ) Bar graphs comparing bacterial counts ( CFU ) in the lung and spleen at 72h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 4 - 5 mice / group . Data information : Data presented represent mean ± SEM of technical replicates . Statistical analysis for G ) were two - tailed unpaired t - tests , * p < 0 . 05 .

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

Figure 4C) Bar graphs comparing the killing rates of phagocytosed S. pneumoniae by AMs at 1 and 2h post-ex vivo S. pneumoniae infection. N = 3 replicates/group. Data information: Data presented in C, represent mean ± SEM of technical replicates. Statistical analysis for C, were two-tailed unpaired t-tests *p < 0.05.E) Line graphs comparing changes in body weight following S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 4 mice/group. Data information: Data presented represent mean ± SEM of technical replicates. Statistical analysis for (E) was two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05.G) Bar graphs comparing bacterial counts (CFU) in the lung and spleen at 72h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 4 - 5 mice/group. Data information: Data presented represent mean ± SEM of technical replicates. Statistical analysis for G) were two-tailed unpaired t-tests, *p < 0.05.


Figure 5B ) Line graphs comparing changes in body weight following S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 4 - 5 mice / group . Data information : Data presented in ( B represent mean ± SEM of biological replicates . Statistical analysis for ( B were two - way ANOVA with Sidak ' s multiple comparisons test * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . C ) Bar graphs comparing bacterial counts ( CFU ) in the lung and spleen at 72h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 4 - 5 mice / group . Data information : Data presented in C ) represent mean ± SEM of biological replicates . Statistical analysis for C ) were two - way ANOVA with Sidak ' s multiple comparisons test , ( C ) was two - tailed unpaired t - test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 .

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

Figure 5B) Line graphs comparing changes in body weight following S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 4 - 5 mice/group. Data information: Data presented in (B represent mean ± SEM of biological replicates. Statistical analysis for (B were two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01; ****p < 0.0001.C) Bar graphs comparing bacterial counts (CFU) in the lung and spleen at 72h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 4 - 5 mice/group. Data information: Data presented in C) represent mean ± SEM of biological replicates. Statistical analysis for C) were two-way ANOVA with Sidak's multiple comparisons test, (C) was two-tailed unpaired t-test. *p < 0.05; **p < 0.01; ****p < 0.0001.


Figure 6B ) Line graphs comparing changes in body weight following S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis for ( B was two - way ANOVA with Sidak ' s multiple comparisons test * p < 0 . 05 ; * * p < 0 . 01 . D ) Bar graphs comparing bacterial counts ( CFU ) in the lung and spleen at 48h post - S . pneumoniae infection in BCG vaccinated and unvaccinated hosts . N = 3 mice / group . Data information : Data presented represent mean ± SEM of biological replicates . Statistical analysis for ( D ) was one - way ANOVA with Tukey ' s multiple comparisons test . * p < 0 . 05 ; * * p < 0 . 01 .

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

Figure 6B) Line graphs comparing changes in body weight following S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (B was two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01.D) Bar graphs comparing bacterial counts (CFU) in the lung and spleen at 48h post- S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (D) was one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05; **p < 0.01.


Figure 1 ( A ) Mean clinical scores ( ± SEM ) of C57BL / 6 → C57BL / 6 controls treated with DTX and CD11c - DTR / GFP → C57BL / 6 treated with DTX or PBS during the whole observation period starting from day - 1 . All animals received in vitro - generated 2d2 . tdRFP Th17 intravenously on day 0 . DC depletion in CD11c - DTR / GFP → C57BL / 6 reduces the encephalitogenicity of adoptively transferred 2d2 . tdRFP Th17 . Mann - Whitney - U - test was performed on mean clinical scores ( n = 11PBS / 13DTX ) ; * p < 0 . 05 , * * p < 0 . 01 ; see also Appendix Table S1 . Pooled data from two independent experiments . ( B ) Mononuclear cells were isolated on day 30 of animals shown in ( A ) from the CNS of PBS - and DTX - treated CD11c - DTR / GFP → C57BL / 6 that had been transferred with 2d2 . tdRFP Th17 . Flow cytometry was performed on surface stained cell samples . Cells were pre - gated on lymphocyte cells ( FSC SSC gate ) and PI - negative ; pooled data of three animals ; representative of two independent experiments . ( C ) Mononuclear cells were isolated from the CNS of PBS - and DTX - treated 2d2 . tdRFPTh17 - transferred CD11c - DTR / GFP → C57BL / 6mice 2 - 3 d after onset of the disease . Upper panels : cells were stimulated with plate - bound anti - CD3 / anti - CD28 , stained for CD4 and cytokines IFN - y , IL - 17 ( pre - gated in addition to FSC / SSC on CD45 + CD4 + ) . Lower panels : expression of transcription factor FoxP3 ; pooled data from three animals , representative for two independent experiments . ( D ) , ( E ) Quantification of cytokine expression data as shown in ( C ) for pooled data from 2 independent experiments ; Mann - Whitney - U - Test * p < 0 . 05 . ( F ) 2d2Th17 were transferred into lymphopenic Rag2 - / - c - gamma - / - . Mononuclear cells were isolated from the spleen and brain at the peak of the disease and analyzed for IL - 17 and GM - CSF production after stimulation with anti - CD3 / anti - CD28 . ( G ) GM - CSF production of IL - 17 producers versus IL - 17 non - producers of CNS - isolated 2d2Th17 cells ( see also ( F ) ) . Each dot / square represents a single animal ; Kruskal - Wallis - test ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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

Figure 1(A) Mean clinical scores (±SEM) of C57BL/6→C57BL/6 controls treated with DTX and CD11c-DTR/GFP→C57BL/6 treated with DTX or PBS during the whole observation period starting from day -1. All animals received in vitro-generated 2d2.tdRFP Th17 intravenously on day 0. DC depletion in CD11c-DTR/GFP→C57BL/6 reduces the encephalitogenicity of adoptively transferred 2d2.tdRFP Th17. Mann-Whitney-U-test was performed on mean clinical scores (n=11PBS/13DTX); * p < 0.05, ** p < 0.01; see also Appendix Table S1. Pooled data from two independent experiments.(B) Mononuclear cells were isolated on day 30 of animals shown in (A) from the CNS of PBS- and DTX-treated CD11c-DTR/GFP→C57BL/6 that had been transferred with 2d2.tdRFP Th17. Flow cytometry was performed on surface stained cell samples. Cells were pre-gated on lymphocyte cells (FSC SSC gate) and PI-negative; pooled data of three animals; representative of two independent experiments.(C) Mononuclear cells were isolated from the CNS of PBS- and DTX-treated 2d2.tdRFPTh17-transferred CD11c-DTR/GFP→C57BL/6mice 2-3 d after onset of the disease. Upper panels: cells were stimulated with plate-bound anti-CD3/anti-CD28, stained for CD4 and cytokines IFN-y, IL-17 (pre-gated in addition to FSC/SSC on CD45+CD4+). Lower panels: expression of transcription factor FoxP3; pooled data from three animals, representative for two independent experiments.(D), (E) Quantification of cytokine expression data as shown in (C) for pooled data from 2 independent experiments; Mann-Whitney-U-Test * p < 0.05.(F) 2d2Th17 were transferred into lymphopenic Rag2-/-c-gamma-/-. Mononuclear cells were isolated from the spleen and brain at the peak of the disease and analyzed for IL-17 and GM-CSF production after stimulation with anti-CD3/anti-CD28.(G) GM-CSF production of IL-17 producers versus IL-17 non-producers of CNS-isolated 2d2Th17 cells (see also (F)). Each dot/square represents a single animal; Kruskal-Wallis-test; * p < 0.05, ** p < 0.01, **** p < 0.0001.


Figure 2 ( B ) Microarray signal intensities of strongest regulated genes from ( A ) are shown in detailed statistical analysis for the different T cell subgroups . Values are depicted as signal intensity mean ± SEM from three independent experiments . Statistical significance was determined using one - way ANOVA with post - hoc Tukey Test for multiple comparisons . P - values < 0 . 05 were regarded as statistically significant . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

Figure 2(B) Microarray signal intensities of strongest regulated genes from (A) are shown in detailed statistical analysis for the different T cell subgroups. Values are depicted as signal intensity mean ± SEM from three independent experiments. Statistical significance was determined using one-way ANOVA with post-hoc Tukey Test for multiple comparisons. P-values < 0.05 were regarded as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.


Figure 3TPLSM of EAE lesions in the brainstem of adoptive transfer EAE at the onset of the disease . EAE was induced by transfer of in vitro differentiated 2d2Th17 cells ( tdRFP - red , IL - 17 - EGFP - green ) into CD11c - DTR / GFPmice ( CD11c - GFP - green ) ; imaging area 300 × 300 µm . ( A ) Intravascular IL - 17hi2d2Th17 cell ( arrow , double positive - > green : IL - 17EGFP and red : 2d2 . tdRFP ) rolling cell towards a CD11c - GFP + cell ( arrowhead ) . White dotted lines mark a venous vessel . ( B ) Time - lapse TPLSM ( maximal intensity projections ) of the boxed area in ( A ) shows that the perivascular elongated CD11c - GFP + cell ( asterisk ) enters into contact with IL - 17 - expressing ( GFP - green and 2d2 . tdRFP - red double - positive ; arrow and arrow head ) Th17 cells ( scale bar : 20 µm ) . ( C ) XYZ - resolved TPLSM depiction reveals that the elongated perivascular CD11c - GFP + cell makes contact with the intravascular 2d2Th17 cells via a filopodium - like dendrite ( xy - plane , 300 × 300 µm , z - depth 70 µm ) . ( D ) Strong interaction of a perivascular IL - 17hi2d2Th17 with a perivascular CD11c - GFP + cell ( insert upper left contact ) . ( E ) Time lapse of the insert in ( D ) : Stopping motility of the round - shaped IL - 17hi2d2Th17 cell is followed by entry into the CNSparenchyma . ( F ) Magnified image of interaction of the upper left and ( G ) lower right perivascular T cell - DC contacts as shown in ( D ) and ( E ) . ( H ) Time - lapse imaging of parenchymal CD11c - GFP + cells ( arrowhead ) , targeted migration of an IL - 17hi 2d2 Th17 cell ( arrow , double positive - > green : IL - 17EGFP and red : 2d2 . tdRFP ) towards the CD11c - GFP + cell ( arrowhead ) . Scale bar is 20 µm . ( I ) Quantification of the motility pattern of IL - 17hi 2d2 . tdRFP vs . all 2d2 . tdRFP in CD11c - DTR / GFP brainstem lesions before and after ( 2 - 5 h ) intraperitoneal DTX injection . Evaluation of mean track velocity of the single cell tracking ( each dot represents a track ) ; pooled data from at least two independent experiments ( > 3 imaging areas ) . For the statistical analysis , the Mann - Whitney - U - test was performed ( * * * p < 0 . 0001 , n . s . = not significant ) . ( K ) Percentage of stopping cells ( red bar ; instantaneous velocity < 2 µm / min ) of the data set as shown in ( I ) .

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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3TPLSM of EAE lesions in the brainstem of adoptive transfer EAE at the onset of the disease. EAE was induced by transfer of in vitro differentiated 2d2Th17 cells (tdRFP-red, IL-17-EGFP-green) into CD11c-DTR/GFPmice (CD11c-GFP - green); imaging area 300 × 300 µm.(A) Intravascular IL-17hi2d2Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) rolling cell towards a CD11c-GFP+ cell (arrowhead). White dotted lines mark a venous vessel.(B) Time-lapse TPLSM (maximal intensity projections) of the boxed area in (A) shows that the perivascular elongated CD11c-GFP+ cell (asterisk) enters into contact with IL-17-expressing (GFP - green and 2d2.tdRFP - red double-positive; arrow and arrow head) Th17 cells (scale bar: 20 µm).(C) XYZ-resolved TPLSM depiction reveals that the elongated perivascular CD11c-GFP+ cell makes contact with the intravascular 2d2Th17 cells via a filopodium-like dendrite (xy-plane, 300 × 300 µm, z-depth 70 µm).(D) Strong interaction of a perivascular IL-17hi2d2Th17 with a perivascular CD11c-GFP+ cell (insert upper left contact).(E) Time lapse of the insert in (D): Stopping motility of the round-shaped IL-17hi2d2Th17 cell is followed by entry into the CNSparenchyma.(F) Magnified image of interaction of the upper left and (G) lower right perivascular T cell-DC contacts as shown in (D) and (E).(H) Time-lapse imaging of parenchymal CD11c-GFP+ cells (arrowhead), targeted migration of an IL-17hi 2d2 Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) towards the CD11c-GFP+ cell (arrowhead). Scale bar is 20 µm.(I) Quantification of the motility pattern of IL-17hi 2d2.tdRFP vs. all 2d2.tdRFP in CD11c-DTR/GFP brainstem lesions before and after (2-5 h) intraperitoneal DTX injection. Evaluation of mean track velocity of the single cell tracking (each dot represents a track); pooled data from at least two independent experiments (>3 imaging areas). For the statistical analysis, the Mann-Whitney-U-test was performed (*** p < 0.0001, n.s. = not significant).(K) Percentage of stopping cells (red bar; instantaneous velocity <2 µm/min) of the data set as shown in (I).


Figure 4Active EAE was induced by subcutaneous immunization with CFA / MOG35 - 55 . Cells were isolated from the CNS of EAE animals at different stages of the disease ( pre - onset [ day 8 - 9 ] , peak [ day 14 - 17 ] , chronic [ day 22 - 27 ] ; data from at least two independent experiments ) . Representative samples are shown , which were analyzed by flow cytometry for the expression of the markers CD11c , CD11b and Ly6C / G ( pre - gated on lympho - monocytic cells by FSC / SSC ; IAb + expression was confirmed for CD11c + cells in separate stainings ) . ( A ) In the CNS , there was a strong increase of CD11c + proportions over the different phases of the disease . The composition of CD11c + subpopulations changed over time , starting with a preponderance of CD11c + Ly6C / G - conventional DCs before onset of EAE . In the peak and chronic phase there was a continuous increase in the proportion of monocyte - derived CD11c + cells . ( B ) For the CNS , the quantification of the absolute numbers shows a strong increase in CD11c + cells during the course of EAE . ( C ) In the CNS , the proportions of the subpopulations show an increasing proportion of monocyte - derived CD11c + cells .

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

Figure 4Active EAE was induced by subcutaneous immunization with CFA/MOG35-55. Cells were isolated from the CNS of EAE animals at different stages of the disease (pre-onset [day 8-9], peak [day 14-17], chronic [day 22-27]; data from at least two independent experiments). Representative samples are shown, which were analyzed by flow cytometry for the expression of the markers CD11c, CD11b and Ly6C/G (pre-gated on lympho-monocytic cells by FSC/SSC; IAb+ expression was confirmed for CD11c+ cells in separate stainings).(A) In the CNS, there was a strong increase of CD11c+ proportions over the different phases of the disease. The composition of CD11c+ subpopulations changed over time, starting with a preponderance of CD11c+Ly6C/G- conventional DCs before onset of EAE. In the peak and chronic phase there was a continuous increase in the proportion of monocyte-derived CD11c+ cells.(B) For the CNS, the quantification of the absolute numbers shows a strong increase in CD11c+ cells during the course of EAE.(C) In the CNS, the proportions of the subpopulations show an increasing proportion of monocyte-derived CD11c+ cells.


Figure 5 ( A ) CD11c + cells , CD4 + cells and CX3CR1 + CD45intIAb + ( microglia ) cells were isolated from peak EAE animals from the CNS . Samples from 6 - 11 animals were pooled in each experiment ; at least two experiments were performed for each subset . Depicted are normalized mRNA expression ( to expression in spleen CD11c + cells ) levels as determined by qRT - PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 ( Eef1a1 ) and Peptidylprolyl isomerase A ( Ppia ) . CNS CD11c + cells show gene expression of the inflammatory chemokines Ccl2 , Ccl5 , Cxcl9 , Cxcl10 but not Il23 ( mean ± SEM ) . ( C ) Quantitative expression of EAE - relevant factors Il23 and Csf2 were investigated in the CNS samples as described above ( B ) CD11c + cells , CD4 + cells and CD11c - CD11b + IAb + ( monocytes / macrophages ) were isolated from EAE affected mice as described in ( A ) . Samples from 6 - 11 animals were pooled in each experiment ; at least two experiments were performed for each subset . Depicted are normalized mRNA expression ( to expression in spleenCD11c + cells ) levels as determined by qRT - PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 ( Eef1a1 ) and Peptidylprolyl isomerase A ( Ppia ) . Relevantly regulated chemokines in other cell populations than CNS CD11c + cells are shown ( mean ± SEM ) . ( D ) Quantitative expression of EAE - relevant factors Il23 and Csf2 were investigated in the spleen samples . Figure 5Chemokine expression profiling of CD11c + cells in the spleen and CNS reveal a key role of CNSCD11c + cells for inflammatory chemokines . ( A ) CD11c + cells , CD4 + cells and CX3CR1 + CD45intIAb + ( microglia ) cells were isolated from peak EAE animals from the CNS . Samples from 6 - 11 animals were pooled in each experiment ; at least two experiments were performed for each subset . Depicted are normalized mRNA expression ( to expression in spleenCD11c + cells ) levels as determined by qRT - PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 ( Eef1a1 ) and Peptidylprolyl isomerase A ( Ppia ) . CNSCD11c + cells show gene expression of the inflammatory chemokines Ccl2 , Ccl5 , Cxcl9 , Cxcl10 but not Ccl20 ( mean ± SEM ) . ( B ) CD11c + cells , CD4 + cells and CD11c - CD11b + IAb + ( monocytes / macrophages ) were isolated from EAE affected mice as described in ( A ) . Relevantly regulated chemokines in other cell populations than CNSCD11c + cells are shown ( mean ± SEM ) . ( C ) Quantitative expression of EAE - relevant factors Il23 and Csf2 were investigated in the CNS samples as described above and ( D ) the spleen samples . Statistical significance was determined using Kruskal - Wallis test . P - values < 0 . 05 were regarded as statistically significant . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD11c+ cells, CD4+ cells and CX3CR1+CD45intIAb+ (microglia) cells were isolated from peak EAE animals from the CNS. Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleen CD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). CNS CD11c+ cells show gene expression of the inflammatory chemokines Ccl2, Ccl5, Cxcl9, Cxcl10 but not Il23 (mean ± SEM).(C) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the CNS samples as described above(B) CD11c+ cells, CD4+ cells and CD11c-CD11b+IAb+ (monocytes/macrophages) were isolated from EAE affected mice as described in (A). Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleenCD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). Relevantly regulated chemokines in other cell populations than CNS CD11c+ cells are shown (mean ± SEM).(D) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the spleen samples.Figure 5Chemokine expression profiling of CD11c+ cells in the spleen and CNS reveal a key role of CNSCD11c+ cells for inflammatory chemokines.(A) CD11c+ cells, CD4+ cells and CX3CR1+CD45intIAb+ (microglia) cells were isolated from peak EAE animals from the CNS. Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleenCD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). CNSCD11c+ cells show gene expression of the inflammatory chemokines Ccl2, Ccl5, Cxcl9, Cxcl10 but not Ccl20 (mean ± SEM).(B) CD11c+ cells, CD4+ cells and CD11c-CD11b+IAb+ (monocytes/macrophages) were isolated from EAE affected mice as described in (A). Relevantly regulated chemokines in other cell populations than CNSCD11c+ cells are shown (mean ± SEM).(C) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the CNS samples as described above and (D) the spleen samples.Statistical significance was determined using Kruskal-Wallis test. P-values < 0.05 were regarded as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.


Figure 6 ( A ) CD11c - GFP + DC cluster formation near venous vessel with ( B ) protrusions attracting 2d2 . tdRFP Th17 cells . ( C ) Low - velocity motility of 2d2 . tdRFPT cells in DC cluster compared to outside of the cluster . SHG in upper panel on the left ( arrow ) . In addition , intercellular matrix fibers ( arrow ) are visualized that are only found in inflamed CNSparenchyma and are due to second harmonic generation ( SHG ) at 1110 nm wavelength excitation and detection at 535 ± 50nm . Tracking of 2d2 . tdRFPTh17 in right panel ( color - coded tracks , low velocity : red , high velocity : yellow ) . ( D ) Quantitative analysis of T cell tracking : higher mean velocity of 2d2 . tdRFPTh17 cells in the onset of the disease with differential distribution at the peak of the disease . Cluster associated tracks have significantly lower mean velocities than Th17 cells outside the CD11c - GFP clusters . ( E ) Analogously for track straightness ; Kruskal - Wallis Test and Dunn ' s Multiple Comparison Test ( * * * p < 0 . 0001 ) were performed ; pooled data from at least three independent experiments per group .

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 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

Figure 6(A) CD11c-GFP+ DC cluster formation near venous vessel with (B) protrusions attracting 2d2.tdRFP Th17 cells.(C) Low-velocity motility of 2d2.tdRFPT cells in DC cluster compared to outside of the cluster. SHG in upper panel on the left (arrow). In addition, intercellular matrix fibers (arrow) are visualized that are only found in inflamed CNSparenchyma and are due to second harmonic generation (SHG) at 1110 nm wavelength excitation and detection at 535 ± 50nm. Tracking of 2d2.tdRFPTh17 in right panel (color-coded tracks, low velocity: red, high velocity: yellow). (D) Quantitative analysis of T cell tracking: higher mean velocity of 2d2.tdRFPTh17 cells in the onset of the disease with differential distribution at the peak of the disease. Cluster associated tracks have significantly lower mean velocities than Th17 cells outside the CD11c-GFP clusters. (E) Analogously for track straightness; Kruskal-Wallis Test and Dunn's Multiple Comparison Test (*** p < 0.0001) were performed; pooled data from at least three independent experiments per group.


Figure 1A / B Lysates from parental , SMCR8 KO ( A ) or C9orf72 KO ( B ) 293T cells were analyzed by immunoblotting . * indicates unspecific protein band detected by C9orf72 antibody . Calnexin served as loading control . C Rescue of C9orf72 protein levels in SMCR8 KO cells after transient overexpression of untagged - or C - terminal tagged SMCR8 analyzed by immunoblotting . * indicates unspecific protein band detected by C9orf72 antibody . Calnexin served as loading control . G Parental and SMCR8 KO cells were subjected to a cycloheximide ( CHX ) chase in the absence or presence of DMSO or Btz . * indicates unspecific protein band detected by C9orf72 antibody . α ‑ tubulin served as loading control . NRF2 confirmed proteasomal inhibition by Btz .

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Figure 1A/B Lysates from parental, SMCR8 KO (A) or C9orf72 KO (B) 293T cells were analyzed by immunoblotting. * indicates unspecific protein band detected by C9orf72 antibody. Calnexin served as loading control.C Rescue of C9orf72 protein levels in SMCR8 KO cells after transient overexpression of untagged- or C-terminal tagged SMCR8 analyzed by immunoblotting. * indicates unspecific protein band detected by C9orf72 antibody. Calnexin served as loading control.G Parental and SMCR8 KO cells were subjected to a cycloheximide (CHX) chase in the absence or presence of DMSO or Btz. * indicates unspecific protein band detected by C9orf72 antibody. α‑tubulin served as loading control. NRF2 confirmed proteasomal inhibition by Btz.


Figure 2C SMCR8 KO cells were treated with DMSO or MLN7243 prior to fixation and immunostaining with an anti - C9orf72 antibody . Untreated parental and C9orf72 KO cells served as controls . Scale bar , 10 µm . D Empty or stably C9orf72 - HA expressing SMCR8 KO cells were treated with Btz or DMSO followed by lysis and immunoblotting . Untreated parental cells served as control . Calnexin served as loading control . NRF2 confirmed proteasomal inhibition by Btz . E Parental and SMCR8 KO cells stably expressing C9orf72 - HA were subjected to CHX chase in absence and presence of DMSO or Btz . NRF2 confirmed proteasomal inhibition by Btz . F Lysates from empty or stably C9orf72 - HA expressing SMCR8 KO cells treated with DMSO or Btz were subjected to HA - IP under denaturing conditions in the absence and presence of the catalytic domain of the deubiquitinase USP2 .

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Figure 2C SMCR8 KO cells were treated with DMSO or MLN7243 prior to fixation and immunostaining with an anti-C9orf72 antibody. Untreated parental and C9orf72 KO cells served as controls. Scale bar, 10 µm.D Empty or stably C9orf72-HA expressing SMCR8 KO cells were treated with Btz or DMSO followed by lysis and immunoblotting. Untreated parental cells served as control. Calnexin served as loading control. NRF2 confirmed proteasomal inhibition by Btz.E Parental and SMCR8 KO cells stably expressing C9orf72-HA were subjected to CHX chase in absence and presence of DMSO or Btz. NRF2 confirmed proteasomal inhibition by Btz.F Lysates from empty or stably C9orf72-HA expressing SMCR8 KO cells treated with DMSO or Btz were subjected to HA-IP under denaturing conditions in the absence and presence of the catalytic domain of the deubiquitinase USP2.


Figure 3D Lysates from C9orf72 - HA expressing parental and SMCR8 KO cells treated with DMSO or Btz were subjected to HA - IP followed by immunoblot analysis . Empty parental and SMCR8 KO served as controls . : NRF2 confirmed proteasomal inhibition by Btz . E SMCR8 KO cells expressing C9orf72 - HA were transiently transfected with GFP - UBR5 or GFP and treated with DMSO or Btz . Lysates were incubated with Streptavidin agarose coupled to biotinylated GFP VHH nanobodies and bound proteins were analyzed by immunoblotting .

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Figure 3D Lysates from C9orf72-HA expressing parental and SMCR8 KO cells treated with DMSO or Btz were subjected to HA-IP followed by immunoblot analysis. Empty parental and SMCR8 KO served as controls. : NRF2 confirmed proteasomal inhibition by Btz.E SMCR8 KO cells expressing C9orf72-HA were transiently transfected with GFP-UBR5 or GFP and treated with DMSO or Btz. Lysates were incubated with Streptavidin agarose coupled to biotinylated GFP VHH nanobodies and bound proteins were analyzed by immunoblotting.


Figure 4A SMCR8 KO cells stably overexpressing C9orf72 - HA were treated with Btz and lysates were subjected to HA - IP under mild conditions in the absence and presence of the catalytic domain of the deubiquitinase USP2 . FK2 confirmed deubiquitination by USP2 . D SMCR8 KO cells transiently overexpressing GFP , GFP - C9orf72 WT or GFP - C9orf72 mutant K156R , K388R , K414R , K422R , K424R , K425R , K428R ( KR7 ) were treated with Btz followed by streptavidin pulldown with biotinylated GFP VHH nanobodies under denaturing conditions .

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Figure 4A SMCR8 KO cells stably overexpressing C9orf72-HA were treated with Btz and lysates were subjected to HA-IP under mild conditions in the absence and presence of the catalytic domain of the deubiquitinase USP2. FK2 confirmed deubiquitination by USP2.D SMCR8 KO cells transiently overexpressing GFP, GFP-C9orf72 WT or GFP-C9orf72 mutant K156R, K388R, K414R, K422R, K424R, K425R, K428R (KR7) were treated with Btz followed by streptavidin pulldown with biotinylated GFP VHH nanobodies under denaturing conditions.


Figure 5B SMCR8 KO cells stably expressing GFP - C9orf72 were treated with DMSO or Btz followed by denaturing lysis and differential USP2 treatment . Lysates were incubated with biotinylated GFP VHH nanobodies coupled to Streptavidin agarose and enriched proteins were analyzed by immunoblotting . D SMCR8 KO cells were transiently transfected with siUBR4 / 5 , siCTRL or left untreated but grown in the presence of Btz . Lysates were subjected to immunoblotting . Quantification of C9orf72 levels in SMCR8 KO cells treated with siUBR4 / 5 or siCTRL . n = 3 biological replicates . Data represent mean ± SD . Statistical analysis of C9orf72 / α ‑ tubulin ratio was performed using one - tailed , unpaired Student ' s t - test . * p < 0 . 05 . Representative Western blot is shown beside . α ‑ tubulin served as loading control .

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Figure 5B SMCR8 KO cells stably expressing GFP-C9orf72 were treated with DMSO or Btz followed by denaturing lysis and differential USP2 treatment. Lysates were incubated with biotinylated GFP VHH nanobodies coupled to Streptavidin agarose and enriched proteins were analyzed by immunoblotting.D SMCR8 KO cells were transiently transfected with siUBR4/5, siCTRL or left untreated but grown in the presence of Btz. Lysates were subjected to immunoblotting. Quantification of C9orf72 levels in SMCR8 KO cells treated with siUBR4/5 or siCTRL. n = 3 biological replicates. Data represent mean ± SD. Statistical analysis of C9orf72/α‑tubulin ratio was performed using one-tailed, unpaired Student's t-test. * p < 0.05. Representative Western blot is shown beside. α‑tubulin served as loading control.


Figure 1B - B2 Largely non ‐ overlapping distribution of AVP + , oxytocin + and secretagogin + ( sgcn + ) neurons in the magnocellular PVN . The mousesupraoptic nucleus ( SON ) harbored vasopressin + and oxytocin + but not secretagogin + neurons . Open arrowheads pinpoint single ‐ labeled neurons . Solid arrowhead denotes AVP / secretagogin dual ‐ labeling . lv , lateral ventricle ; PVNm , magnocellular part of the paraventricular nucleus ; scgn , secretagogin . B3 , B4 Secretagogin + neurons had smaller somatic diameters than AVP + or oxytocin + neurons yet without a difference in their diameter quotient , a measure of ovoid profiles ( * P 0 . 05 , Student ' s t ‐ test ) . C - D3 Terminal ‐ like profiles in the posterior pituitary ( arrowheads in C ) suggesting that secretagogin can co ‐ exist , even if infrequently with oxytocin . Solanum tuberosum lectin ( Sol . tub ) was used to identify blood vessels .

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Figure 1B-B2 Largely non‐overlapping distribution of AVP+, oxytocin+ and secretagogin+ (sgcn+) neurons in the magnocellular PVN. The mousesupraoptic nucleus (SON) harbored vasopressin+ and oxytocin+ but not secretagogin+neurons. Open arrowheads pinpoint single‐labeled neurons. Solid arrowhead denotes AVP/secretagogin dual‐labeling. lv, lateral ventricle; PVNm, magnocellular part of the paraventricular nucleus; scgn, secretagogin.B3, B4 Secretagogin+neurons had smaller somatic diameters than AVP+ or oxytocin+neurons yet without a difference in their diameter quotient, a measure of ovoid profiles (*P 0.05, Student's t‐test).C-D3 Terminal‐like profiles in the posterior pituitary (arrowheads in C) suggesting that secretagogin can co‐exist, even if infrequently with oxytocin. Solanum tuberosum lectin (Sol. tub) was used to identify blood vessels.


Figure 2A Secretagogin ( scgn ) distribution at the ultrastructural level as revealed by pre ‐ embedding silver ‐ enhanced immunogold labeling . Secretagogin ( arrowheads ) was localized to membranous organelles in the perikarya ( A ) , particularly the plasmalemma ( A1 ) and endoplasmic reticulum ( ER ) in neuronal soma ( s ; A2 ) . Open rectangles in ( A ) denote the location of insets . Semi ‐ transparent shading is used to visually dissociate subcellular compartments in ( A - C ) . B Pre ‐ embedding secretagogin labeling ( arrowheads ) was also seen in dendrite ( d ) segments . C In axo ‐ dendritic terminals ( ax ) , secretagogin was closely associated with synaptic vesicles along the plasmalemma ( arrowheads ) . D Quantitative analysis of subcellular secretagogin distribution upon electron microscopy detection of silver ‐ enhanced gold particles . Particles were considered as membrane bound when they were at 50 nm of a membrane ( plasma membrane or endomembrane ; i . e . , secretory vesicle , Golgi or endoplasmic reticulum ) . In the soma of PVNneurons , significantly more particles were found in the cytosol as along the plasma membrane ( * * P 0 . 01 ) . In contrast , membrane association predominated in axonal nerve endings in the median eminence ( * P 0 . 05 ) . Note that a significant proportion of particles was found adjacent to endomembranes in all subcellular compartments studied .

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Figure 2A Secretagogin (scgn) distribution at the ultrastructural level as revealed by pre‐embedding silver‐enhanced immunogold labeling. Secretagogin (arrowheads) was localized to membranous organelles in the perikarya (A), particularly the plasmalemma (A1) and endoplasmic reticulum (ER) in neuronal soma (s; A2). Open rectangles in (A) denote the location of insets. Semi‐transparent shading is used to visually dissociate subcellular compartments in (A-C).B Pre‐embedding secretagogin labeling (arrowheads) was also seen in dendrite (d) segments.C In axo‐dendritic terminals (ax), secretagogin was closely associated with synaptic vesicles along the plasmalemma (arrowheads).D Quantitative analysis of subcellular secretagogin distribution upon electron microscopy detection of silver‐enhanced gold particles. Particles were considered as membrane bound when they were at 50 nm of a membrane (plasma membrane or endomembrane; i.e., secretory vesicle, Golgi or endoplasmic reticulum). In the soma of PVNneurons, significantly more particles were found in the cytosol as along the plasma membrane (**P 0.01). In contrast, membrane association predominated in axonal nerve endings in the median eminence (*P 0.05). Note that a significant proportion of particles was found adjacent to endomembranes in all subcellular compartments studied.


Figure 3A - A3 Biocytin ‐ filled ( arrow ) neuron immunonegative for secretagogin yet containing AVP / oxytocin ( a mixture of magnocellular markers was used in triple ‐ labeling experiments ) . Scale bars : 50 μm ( A ) , 8 μm ( A3 ) . lv , lateral ventricle ; PVN , paraventricular nucleus . B - B2 Secretagogin + biocytin ‐ filled neuron ( arrow ) lacking AVP / oxytocin immunosignal . Scale bars : 8 μm . D Electrophysiology characteristics of magnocellular and parvocellular neurons . The waveform of repetitive action potential firing is shown on the left , while differential channel characteristics are depicted on the right . Examples of secretagogin + neurons are shown in red .

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Figure 3A-A3 Biocytin‐filled (arrow) neuron immunonegative for secretagogin yet containing AVP/oxytocin (a mixture of magnocellular markers was used in triple‐labeling experiments). Scale bars: 50 μm (A), 8 μm (A3). lv, lateral ventricle; PVN, paraventricular nucleus.B-B2 Secretagogin+ biocytin‐filled neuron (arrow) lacking AVP/oxytocin immunosignal. Scale bars: 8 μm.D Electrophysiology characteristics of magnocellular and parvocellular neurons. The waveform of repetitive action potential firing is shown on the left, while differential channel characteristics are depicted on the right. Examples of secretagogin+neurons are shown in red.


Figure 4A Differential clustering based on secretagogin ( Scgn ) , neuropeptide , hormone and hormone receptor mRNA expression . Secretagogin ‐ expressing ( + ) neurons typically contained corticotropin ‐ releasing hormone ( Crh ) and Nr3c1 mRNA transcripts . A1 Clusters of gene transcripts from 130 cells reveal the phenotypic segregation of PVN neurons . Increasing mRNA copy numbers were depicted by a color gradient from deep blue ( not detected ) to red ( high numbers ) . Secretagogin + neurons are indicated by red arrows . C , C1 Secretagogin co ‐ localization with GFP in the PVN ( solid arrowheads ) of adult CRH ‐ GFP ( BAC ) reporter mice ( Alon et al , ) . Open arrowhead pinpoints a single ‐ labeled neuron . D - I3 We found secretagogin ( scgn ) co ‐ expressed with CRH in both neuronal soma ( solid arrowheads ; D1 - D3 ) and axon terminal ‐ like specializations ( arrowheads ; E - E2 ) in the PVN . Likewise , secretagogin + neurons harbored , yet infrequently , tyrosine hydroxylase ( TH ; solid arrowheads ; F - F3 ) , GABA ( G - G2 ) , somatostatin ( Sst ; H - H3 ) and galanin ( I - I2 ) . Open rectangles denote the general location of insets . Open arrowheads indicate the lack of co ‐ localization .

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Figure 4A Differential clustering based on secretagogin (Scgn), neuropeptide, hormone and hormone receptor mRNA expression. Secretagogin‐expressing(+) neurons typically contained corticotropin‐releasing hormone (Crh) and Nr3c1 mRNA transcripts.A1 Clusters of gene transcripts from 130 cells reveal the phenotypic segregation of PVN neurons. Increasing mRNA copy numbers were depicted by a color gradient from deep blue (not detected) to red (high numbers). Secretagogin+ neurons are indicated by red arrows.C, C1 Secretagogin co‐localization with GFP in the PVN (solid arrowheads) of adult CRH‐GFP (BAC) reporter mice (Alon et al, ). Open arrowhead pinpoints a single‐labeled neuron.D-I3 We found secretagogin (scgn) co‐expressed with CRH in both neuronal soma (solid arrowheads; D1-D3) and axon terminal‐like specializations (arrowheads; E-E2) in the PVN. Likewise, secretagogin+ neurons harbored, yet infrequently, tyrosine hydroxylase (TH; solid arrowheads; F-F3), GABA (G-G2), somatostatin (Sst; H-H3) and galanin (I-I2). Open rectangles denote the general location of insets. Open arrowheads indicate the lack of co‐localization.


Figure 5A - A2 Secretagogin co ‐ existed in the majority of CRH + nerve endings in the median eminence ( ME ) . Open rectangle denotes the general location of ( B , B1 ) . B , B1 Large axon terminals in the median eminence were immunopositive for secretagogin with silver ‐ intensified immunogold particles ( open arrowheads ) associated with axonal membrane and dense ‐ core vesicles ( see Fig D for quantitative data ) . Solid arrowheads denote silver deposit particles proximal to the plasmalemma . C Immunoprecipitation using an anti ‐ secretagogin antibody in Ca2 + ‐ free and Ca2 + ‐ loaded conditions was subtractively used to decipher the Ca2 + ‐ dependent protein interactions . Silver ‐ staining gel is shown . C1 Ontology classification of the 99 protein hits based on primary function assignments . Unbiased MALDI ‐ TOF proteomics was used to identify interacting proteins . The most abundant hits ( Supplementary Table S3 is referred to for details on individual proteins ) are proteins implicated in vesicle fusion , trafficking , transport and formation and the regulation of vesicle exocytosis . " Other " refers to a group of proteins without known function . E - E3 Rab3 , a family of vesicular fusion and transport proteins ( Schluter et al , ) , was found co ‐ localized with secretagogin ( arrowheads ) in the median eminence . Neuronal soma in the PVN lacked appreciable co ‐ localization . Note that neither our MALDI ‐ TOF analysis nor our histochemical probing allowed the precise identification of individual Rab3C ‐ E family members .

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Figure 5A-A2 Secretagogin co‐existed in the majority of CRH+ nerve endings in the median eminence (ME). Open rectangle denotes the general location of (B, B1).B, B1 Large axon terminals in the median eminence were immunopositive for secretagogin with silver‐intensified immunogold particles (open arrowheads) associated with axonal membrane and dense‐core vesicles (see Fig D for quantitative data). Solid arrowheads denote silver deposit particles proximal to the plasmalemma.C Immunoprecipitation using an anti‐secretagogin antibody in Ca2+‐free and Ca2+‐loaded conditions was subtractively used to decipher the Ca2+‐dependent protein interactions. Silver‐staining gel is shown.C1 Ontology classification of the 99 protein hits based on primary function assignments. Unbiased MALDI‐TOF proteomics was used to identify interacting proteins. The most abundant hits (Supplementary Table S3 is referred to for details on individual proteins) are proteins implicated in vesicle fusion, trafficking, transport and formation and the regulation of vesicle exocytosis. "Other" refers to a group of proteins without known function.E-E3 Rab3, a family of vesicular fusion and transport proteins (Schluter et al, ), was found co‐localized with secretagogin (arrowheads) in the median eminence. Neuronal soma in the PVN lacked appreciable co‐localization. Note that neither our MALDI‐TOF analysis nor our histochemical probing allowed the precise identification of individual Rab3C‐E family members.


Figure 6A , A1 siRNA ‐ mediated secretagogin ( scgn ) knockdown in cultured hypothalamic neurons , as indicated by reduced secretagogin immunoreactivity ( A ) and decreased CRH content in the culture medium ( A1 ) . B - B2 Transient overexpression of secretagogin in immortalized CRH ‐ expressing mHypoE ‐ N44 hypothalamic cells significantly reduced CRHimmunofluorescence microscopy intensity in secretagogin + cell bodies , indirectly supporting enhanced CRH release . All experiments were performed in triplicate . Scale bar : 50 nm . C , C1 siRNA ‐ mediated in vivo silencing of secretagogin mRNA expression in the PVN provoked somatic CRH accumulation ( arrowheads ) . Scale bar : 150 μm . C2 Quantitative analysis demonstrating significantly increased somatic CRH contents . Note that somatic secretagogin levels remained unchanged , which we interpret as data on a neuronal contingent not affected by siRNA silencing . The lack of secretagogin / CRH co ‐ localization suggests that secretagogin expression fell below detection threshold in many CRH + neurons . D , D1 Individual data points show maximal CRH fluorescence intensity ( gray scale arbitrary unit ( a . u . ) expression ) in PVN neurons that have low or no secretagogin expression after siRNA infusion .

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Figure 6A, A1 siRNA‐mediated secretagogin (scgn) knockdown in cultured hypothalamic neurons, as indicated by reduced secretagogin immunoreactivity (A) and decreased CRH content in the culture medium (A1).B-B2 Transient overexpression of secretagogin in immortalized CRH‐expressing mHypoE‐N44 hypothalamic cells significantly reduced CRHimmunofluorescence microscopy intensity in secretagogin+cell bodies, indirectly supporting enhanced CRH release. All experiments were performed in triplicate. Scale bar: 50 nm.C, C1 siRNA‐mediated in vivo silencing of secretagogin mRNA expression in the PVN provoked somatic CRH accumulation (arrowheads). Scale bar: 150 μm.C2 Quantitative analysis demonstrating significantly increased somatic CRH contents. Note that somatic secretagogin levels remained unchanged, which we interpret as data on a neuronal contingent not affected by siRNA silencing. The lack of secretagogin/CRH co‐localization suggests that secretagogin expression fell below detection threshold in many CRH+ neurons.D, D1 Individual data points show maximal CRH fluorescence intensity (gray scale arbitrary unit (a.u.) expression) in PVN neurons that have low or no secretagogin expression after siRNA infusion.


Figure 7A , A1 Paraventricular secretagogin ‐ immunoreactive ( scgn + ) neurons weakly expressed CRH ( open arrowheads ) but not c ‐ fos under control conditions . Scale bars : 300 μm ( A ) , 25 μm ( A1 ) . B , B1 Stress induced by subcutaneous injection of formalin ‐ triggered co ‐ expression of c ‐ fos and CRH in secretagogin + paraventricular neurons ( arrowheads ) . Scale bars : 300 μm ( B ) , 25 μm ( B1 ) . C , D In vivo siRNA ‐ mediated silencing of secretagogin expression in the PVN occluded the stress ‐ induced surge of serum ACTH levels ( C ) and significantly reduced the increase in plasma corticosterone ( D ) . * P 0 . 05 versus control .

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Figure 7A, A1 Paraventricular secretagogin‐immunoreactive (scgn+) neurons weakly expressed CRH (open arrowheads) but not c‐fos under control conditions. Scale bars: 300 μm (A), 25 μm (A1).B, B1 Stress induced by subcutaneous injection of formalin‐triggered co‐expression of c‐fos and CRH in secretagogin+ paraventricular neurons (arrowheads). Scale bars: 300 μm (B), 25 μm (B1).C, D In vivo siRNA‐mediated silencing of secretagogin expression in the PVN occluded the stress‐induced surge of serum ACTH levels (C) and significantly reduced the increase in plasma corticosterone (D). *P 0.05 versus control.


Figure 1 ( A ) lid - 1 ( xd288 ) and atgl - 1 ( xd314 ) mutants show LD accumulation in most neurons in C . elegans , including neurons in the head region , tail and ventral nerve cord . xdIs109 is a stable transgenic line with pan - neuronal expression of the LD marker PLIN1 : : GFP . The white arrows indicate PLIN1 : : GFP - positive LDs in the ventral nerve cord of mutants . Scale bar : 20 μm . The right panels depict the head region . Scale bar : 5 μm . The insets in the right panels are enlarged views of PLIN1 : : GFP rings , representing LDs . ( B ) Quantification of the numbers of LDs in head region neurons ( each dot represents one worm ) . The data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to the control xdIs109 . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . ns : not statistically significant . Data show mean ± SEM . n ≥ 9 . ( E ) Oil red O staining show dramatically increased neutral lipids in the head region of lid - 1 ( xd288 ) and atgl - 1 ( xd314 ) compared with N2 . Scale bar : 10 μm . ( F - H ) EM images of neurons . The red dashed line marks the outline of neurons . The red arrows mark LDs . Scale bar : 0 . 5 μm . The scale bar in enlarged images is 100 nm . ( I ) Serial sections of atgl - 1 ( xd314 ) show two LDs ( indicated by blue and red arrows ) in a neuron cell body . Scale bar : 0 . 5 μm .

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Figure 1(A) lid-1(xd288) and atgl-1(xd314) mutants show LD accumulation in most neurons in C. elegans, including neurons in the head region, tail and ventral nerve cord. xdIs109 is a stable transgenic line with pan-neuronal expression of the LD marker PLIN1::GFP. The white arrows indicate PLIN1::GFP-positive LDs in the ventral nerve cord of mutants. Scale bar: 20 μm. The right panels depict the head region. Scale bar: 5 μm. The insets in the right panels are enlarged views of PLIN1::GFP rings, representing LDs.(B) Quantification of the numbers of LDs in head region neurons (each dot represents one worm). The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: not statistically significant. Data show mean ± SEM. n ≥ 9.(E) Oil red O staining show dramatically increased neutral lipids in the head region of lid-1(xd288) and atgl-1(xd314) compared with N2. Scale bar: 10 μm.(F-H) EM images of neurons. The red dashed line marks the outline of neurons. The red arrows mark LDs. Scale bar: 0.5 μm. The scale bar in enlarged images is 100 nm. (I) Serial sections of atgl-1(xd314) show two LDs (indicated by blue and red arrows) in a neuron cell body. Scale bar: 0.5 μm.


Figure 2 ( C ) Overexpressing the homologs of DGAT1 / 2 in neurons of C . elegans leads to LD accumulation in neurons . Each dot represents one worm . The data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to the control xdIs109 . * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . n ≥ 8 .

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Figure 2(C) Overexpressing the homologs of DGAT1/2 in neurons of C. elegans leads to LD accumulation in neurons. Each dot represents one worm. The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. n ≥ 8.


Figure 3 ( A ) The expression pattern of atgl - 1 viewed by Patgl - 1 : : GFP transcriptional fusion reporter . Patgl - 1 : : GFP expression is high in intestine . Relatively low fluorescent signals are found in nerve ring , motor neuron commissure , ventral nerve cord and tail . Scale bar : 20 μm . ( B ) atgl - 1 is expressed in neurons . There is no fluorescent signal when Patgl - 1 : : GFP1 - 10 and Prab - 3 : : GFP11 are expressed separately . When Patgl - 1 : : GFP1 - 10 and Prab - 3 : : GFP11 are co - expressed , the GFP signals are found in the head region , tail and ventral nerve cord , where most neuronal cell bodies are located . The white lines mark the outline of worms . Scale bar : 50 μm . ( C ) atgl - 1 is expressed widely in neurons . The left panels depict the whole worm . Scale bar : 50 μm . The insets in the right panels are enlarged views of the ventral nerve cord in the white dotted boxes of left panels . The signals of mCherry and GFP merge very well . Scale bar : 20 μm . ( D ) Visualizing LDs in head neurons with the xdIs109 reporter in different genetic backgrounds . The accumulation of LDs in head neurons of atgl - 1 ( xd314 ) mutants can be rescued by expressing wild - type atgl - 1 in neurons ( Punc - 119 : : atgl - 1 ) , but not in intestine ( Pvha - 6 : : atgl - 1 ) , glia ( Phlh - 17 : : atgl - 1 ) or hypodermis ( Pajm - 1 : : atgl - 1 ) . Scale bar : 5 μm . ( E ) Quantification of the number of LDs in head neurons in different genetic backgrounds . Each dot represents one worm . The data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to the control xdIs109 . ns : not statistically significant . * * * * P < 0 . 0001 . Data show mean ± SEM . n ≥ 7 . ( F ) Neuron - specific knockout of atgl - 1 causes LD accumulation in neurons , similar to atgl - 1 ( xd314 ) mutants . Scale bar : 5 μm . ( G ) Quantification of the number of LDs in head neurons in different genetic backgrounds . Each dot represents one worm . The data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to the control xdIs109 . t - test was used between the groups under the crossbars . ns : not statistically significant . * * * * P < 0 . 0001 . Data show mean ± SEM . n ≥ 9

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 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 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 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

Figure 3(A) The expression pattern of atgl-1 viewed by Patgl-1::GFP transcriptional fusion reporter. Patgl-1::GFP expression is high in intestine. Relatively low fluorescent signals are found in nerve ring, motor neuron commissure, ventral nerve cord and tail. Scale bar: 20 μm.(B) atgl-1 is expressed in neurons. There is no fluorescent signal when Patgl-1::GFP1-10 and Prab-3::GFP11 are expressed separately. When Patgl-1::GFP1-10 and Prab-3::GFP11 are co-expressed, the GFP signals are found in the head region, tail and ventral nerve cord, where most neuronal cell bodies are located. The white lines mark the outline of worms. Scale bar: 50 μm.(C) atgl-1 is expressed widely in neurons. The left panels depict the whole worm. Scale bar: 50 μm. The insets in the right panels are enlarged views of the ventral nerve cord in the white dotted boxes of left panels. The signals of mCherry and GFP merge very well. Scale bar: 20 μm.(D) Visualizing LDs in head neurons with the xdIs109 reporter in different genetic backgrounds. The accumulation of LDs in head neurons of atgl-1(xd314) mutants can be rescued by expressing wild-type atgl-1 in neurons (Punc-119::atgl-1), but not in intestine (Pvha-6::atgl-1), glia (Phlh-17::atgl-1) or hypodermis (Pajm-1::atgl-1). Scale bar: 5 μm. (E) Quantification of the number of LDs in head neurons in different genetic backgrounds. Each dot represents one worm. The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. ns: not statistically significant. ****P < 0.0001. Data show mean ± SEM. n ≥ 7. (F) Neuron-specific knockout of atgl-1 causes LD accumulation in neurons, similar to atgl-1(xd314) mutants. Scale bar: 5 μm. (G) Quantification of the number of LDs in head neurons in different genetic backgrounds. Each dot represents one worm. The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. t-test was used between the groups under the crossbars. ns: not statistically significant. ****P < 0.0001. Data show mean ± SEM. n ≥ 9


Figure 4 ( A ) LDs accumulate in touch neurons in atgl - 1 ( xd314 ) visualized by SRS ( Stimulated Raman Scattering ) . The zdIs5 reporter marks touch neurons . The white arrows indicate LDs in touch neurons in atgl - 1 ( xd314 ) . Scale bar : 10 μm . ( B ) atgl - 1 ( xd314 ) and lid - 1 ( xd288 ) show a gentle touch sensation defect , which is rescued by the neuron - specific expression of atgl - 1 and lid - 1 , respectively . The fat - 4 mutation enhances the touch sensation defect in atgl - 1 ( xd314 ) and lid - 1 ( xd288 ) . Wild type and mec - 4 ( u253 ) act as the negative control and positive control , respectively . The data were analyzed using Kruskal - Wallis test with Dunn ' s test . Asterisks denote significant differences as compared to wild type . ns : not statistically significant . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . # signify significant differences between the groups under the crossbars . # # P < 0 . 01 . Data show mean ± SEM . Number of worms analyzed for each strain n = 25 .

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Figure 4(A) LDs accumulate in touch neurons in atgl-1(xd314) visualized by SRS (Stimulated Raman Scattering). The zdIs5 reporter marks touch neurons. The white arrows indicate LDs in touch neurons in atgl-1(xd314). Scale bar: 10 μm.(B) atgl-1(xd314) and lid-1(xd288) show a gentle touch sensation defect, which is rescued by the neuron-specific expression of atgl-1 and lid-1, respectively. The fat-4 mutation enhances the touch sensation defect in atgl-1(xd314) and lid-1(xd288). Wild type and mec-4(u253) act as the negative control and positive control, respectively. The data were analyzed using Kruskal-Wallis test with Dunn's test. Asterisks denote significant differences as compared to wild type. ns: not statistically significant. **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. # signify significant differences between the groups under the crossbars. ##P < 0.01. Data show mean ± SEM. Number of worms analyzed for each strain n =25.