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

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.


C ) 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 .

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

C) 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 .

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

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

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.


B ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


B ) 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 .

B) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


( 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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


TPLSM 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 ) .

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

TPLSM 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 ) .

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

(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 .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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).


Active 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Active 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.


( 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

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


( A ) CD11c - GFP + DC cluster formation near venous vessel with ( B ) protrusions attracting 2d2 . tdRFP Th17 cells .

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(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 .

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(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.


A / 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 .

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A/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 .

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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 .

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR 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-CONTROLLED_VAR O

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.


C 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 .

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C 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 .

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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 .

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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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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.


D 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 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O

D 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 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

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.


A 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 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A 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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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.


B 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 .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


B - 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 ) .

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B-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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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.


A 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 .

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

A 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.


A - 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 .

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A-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 .

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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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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.


A 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 .

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A 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 .

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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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.


A - 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 ) .

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A-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 .

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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 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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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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.


A , 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 ) .

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A, 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 .

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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 .

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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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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.


A , 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 ) .

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A, 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 ) .

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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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.


( 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 .

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(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 .

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(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 .

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(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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(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.


( 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 .

(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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

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(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


( 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 .

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(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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(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.


( A ) Image of touch neurons viewed by zdIs5 in different genetic backgrounds . Scale bar : 20 μm .

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(A) Image of touch neurons viewed by zdIs5 in different genetic backgrounds. Scale bar: 20 μm.


( B ) Quantification of the percentage of animals with 0 , 1 , 2 , 3 , 4 , 5 and 6 surviving touch neurons in different genetic backgrounds . Data show mean ± SEM . At least 50 animals per strain were analyzed in each experiment . Number of experiments n = 5 .

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(B) Quantification of the percentage of animals with 0, 1, 2, 3, 4, 5 and 6 surviving touch neurons in different genetic backgrounds. Data show mean ± SEM. At least 50 animals per strain were analyzed in each experiment. Number of experiments n = 5.


( C ) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds in ( B ) . The lid - 1 ( xd288 ) and atgl - 1 ( xd314 ) mutations significantly increase the number of surviving neurons . Neuron - specific expression of atgl - 1 or lid - 1 reverses the protective effect of atgl - 1 or lid - 1 mutation . The data were analyzed using one - way ANOVA with Turkey ' s multiple comparison test . Asterisks signify significant differences between the groups under the crossbars . * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with at least 50 animals per strain analyzed in each experiment .

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(C) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds in (B). The lid-1(xd288) and atgl-1(xd314) mutations significantly increase the number of surviving neurons. Neuron-specific expression of atgl-1 or lid-1 reverses the protective effect of atgl-1 or lid-1 mutation. The data were analyzed using one-way ANOVA with Turkey's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n = 5, with at least 50 animals per strain analyzed in each experiment.


( D ) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds . Mutations of fat - 1 , fat - 3 and fat - 4 significantly enhance the suppression effect of atgl - 1 ( xd314 ) in preventing the neurodegeneration of mec - 4 ( d ) . The data were analyzed using one - way ANOVA with Bonferroni ' s multiple comparison test . # # # and # # # # denote significant differences as compared to atgl - 1 ( xd314 ) ; mec - 4 ( d ) . Asterisks signify significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * * * * P < 0 . 0001 . # # # P < 0 . 001 , # # # P < 0 . 0001 . Data show mean ± SEM . Number of experiments n ≥ 3 , with at least 50 animals per strain analyzed in each experiment .

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(D) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds. Mutations of fat-1, fat-3 and fat-4 significantly enhance the suppression effect of atgl-1(xd314) in preventing the neurodegeneration of mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. ### and #### denote significant differences as compared to atgl-1(xd314); mec-4(d). Asterisks signify significant differences as compared to mec-4(d). ns: not statistically significant. ****P < 0.0001. ###P < 0.001, ###P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 3, with at least 50 animals per strain analyzed in each experiment.


( E ) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds supplemented with different fatty acids . AA and EPA but not LA significantly enhance the neurodegeneration triggered by mec - 4 ( d ) . The data were analyzed using one - way ANOVA with Bonferroni ' s multiple comparison test . Asterisks signify significant differences between the groups under the crossbars . ns : not statistically significant . * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n ≥ 3 , with at least 50 animals per strain analyzed in each experiment .

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(E) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds supplemented with different fatty acids. AA and EPA but not LA significantly enhance the neurodegeneration triggered by mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ns: not statistically significant. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 3, with at least 50 animals per strain analyzed in each experiment.


( A ) Lipidomic data show that the relative content of total C20 : 3 is increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) and atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . The increase is greatest in mutants with the fat - 4 mutation . ( B ) Lipidomic data show that the relative content of total C20 : 4 is increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) and atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . The increase is greatest in mutants with the fat - 4 mutation . ( C ) Lipidomic data show that the relative content of total C20 : 5 is slightly increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) but is dramatically decreased in fat - 4 ( wa14 ) ; mec - 4 ( d ) and atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) , which matches the role of FAT - 4 in synthesizing C20 : 5 ( EPA ) . Data information The data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with about 10 , 000 animals per strain analyzed in each experiment .

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(A) Lipidomic data show that the relative content of total C20:3 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation. (B) Lipidomic data show that the relative content of total C20:4 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation. (C) Lipidomic data show that the relative content of total C20:5 is slightly increased in atgl-1(xd314); mec-4(d) but is dramatically decreased in fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d), which matches the role of FAT-4 in synthesizing C20:5 (EPA). Data information The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.


( D ) The percentage of C20 : 3 - , C20 : 4 - or C20 : 5 - containing TAGs to total lipids containing the same PUFA in different genetic backgrounds . The percentage of C20 : 4 - containing TAGs to total lipids containing C20 : 4 is significantly increased in atgl - 1 and fat - 4 single mutants . The percentage is further increased in atgl - 1 and fat - 4 double mutant . Data information : FA : free fatty acids ; TAG : triacylglycerol ; PL : phospholipids . The data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with about 10 , 000 animals per strain analyzed in each experiment .

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(D) The percentage of C20:3-, C20:4- or C20:5-containing TAGs to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing TAGs to total lipids containing C20:4 is significantly increased in atgl-1 and fat-4 single mutants. The percentage is further increased in atgl-1 and fat-4 double mutant. Data information: FA: free fatty acids; TAG: triacylglycerol; PL: phospholipids. The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.


( E ) The percentage of C20 : 3 - , C20 : 4 - or C20 : 5 - containing phospholipids to total lipids containing the same PUFA in different genetic backgrounds . The percentage of C20 : 4 - containing phospholipids to total lipids containing C20 : 4 is significantly decreased in atgl - 1 and fat - 4 single mutants . The percentage is further decreased in atgl - 1 and fat - 4 double mutant . Data information : FA : free fatty acids ; TAG : triacylglycerol ; PL : phospholipids . The data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with about 10 , 000 animals per strain analyzed in each experiment .

(E) The percentage of C20:3-, C20:4- or C20:5-containing phospholipids to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing phospholipids to total lipids containing C20:4 is significantly decreased in atgl-1 and fat-4 single mutants. The percentage is further decreased in atgl-1 and fat-4 double mutant. Data information: FA: free fatty acids; TAG: triacylglycerol; PL: phospholipids. The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.


( F ) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds . mboa - 7 mutation significantly increases the percentage of three or more surviving touch neurons in mec - 4 ( d ) , atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) and atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . The data were analyzed using one - way ANOVA with Bonferroni ' s multiple comparison test . Asterisks signify significant differences between the groups under the crossbars . * * P < 0 . 01 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n ≥ 4 , with at least 50 animals per strain analyzed in each experiment .

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(F) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds. mboa-7 mutation significantly increases the percentage of three or more surviving touch neurons in mec-4(d), atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. **P < 0.01, ****P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 4, with at least 50 animals per strain analyzed in each experiment.


( G ) The percentage of C20 : 4 - containing phospholipids to total lipids that have C20 : 4 is dramatically decreased in mboa - 7 ( gk399 ) ; atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . The data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison test . Asterisks signify significant differences as compared to mec - 4 ( d ) . * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with about 10 , 000 animals per strain analyzed in each experiment .

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(G) The percentage of C20:4-containing phospholipids to total lipids that have C20:4 is dramatically decreased in mboa-7(gk399); atgl-1(xd314); fat-4(wa14); mec-4(d). The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks signify significant differences as compared to mec-4(d). ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.


Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies .

Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies.


Fluorescence analyses of GFP - ATG5 or ATG16L1 - GFP stably expressed in ATG5 - / - or ATG16L1 - / - . Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence . Scale bar : 10 μm .

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Fluorescence analyses of GFP-ATG5 or ATG16L1-GFP stably expressed in ATG5-/- or ATG16L1-/-. Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence. Scale bar: 10 μm.


Assessment of ATG16L1 levels in the cytosolic ( C ) and membrane ( M ) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1 . ATG16L1 - / - and ATG5 - / - stably expressed ATG16L1 - GFP while ATG3 - / - stably expressed Flag - S - ATG16L1 . Antibodies against α - tubulin and integrin β1 were used as controls for fractionation . Exogenous ( exo ) ATG16L1 was detected using antibodies against ATG16L1 .

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Assessment of ATG16L1 levels in the cytosolic (C) and membrane (M) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1. ATG16L1-/- and ATG5-/- stably expressed ATG16L1-GFP while ATG3-/- stably expressed Flag-S-ATG16L1. Antibodies against α-tubulin and integrin β1 were used as controls for fractionation. Exogenous (exo) ATG16L1 was detected using antibodies against ATG16L1.


Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs ( WIPI2 + / + ) and WIPI2 - / - cells .

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Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs (WIPI2+/+) and WIPI2-/- cells.


Immunofluorescence analyses of ATG16L1 - / - and WIPI2 - / - stably expressing Flag - S - ATG16L1 . Cells were amino acid starved ( AA starve ) for 2 hr in the presence or absence of 3 ' MA ( and additional pretreatment for 30 min ) followed by fixation and immuno - staining using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

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Immunofluorescence analyses of ATG16L1-/- and WIPI2-/- stably expressing Flag-S-ATG16L1. Cells were amino acid starved (AA starve) for 2 hr in the presence or absence of 3'MA (and additional pretreatment for 30 min) followed by fixation and immuno-staining using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Fragments depicted in ( A ) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S - ATG16L1 . Scale bar : 10 μm .

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Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S-ATG16L1. Scale bar: 10 μm.


U2OS cells expressing Flag - ATG16L1 ∆ 2 or Flag - ATG16L1 ∆ 2 ∆ 182 - 205 were treated as in ( B ) and stained using antibodies against Flag tag ( to detect ATG16L1 , green ) and FIP200 ( red ) . Scale bar : 10 μm .

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U2OS cells expressing Flag-ATG16L1∆2 or Flag-ATG16L1∆2∆182-205 were treated as in (B) and stained using antibodies against Flag tag (to detect ATG16L1, green) and FIP200 (red). Scale bar: 10 μm.


Protein - protein interaction assay in 293T cells transiently transfected with the indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies .

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Protein-protein interaction assay in 293T cells transiently transfected with the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


Homodimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies .

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Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


Flag - ATG16L1WT or Flag - ATG16L1 ∆ 182 - 205 were stably expressed in ATG5 - / - cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

Flag-ATG16L1WT or Flag-ATG16L1∆182-205 were stably expressed in ATG5-/- cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Microscopy - based protein - liposome binding assay . ATG16L1 - GFP immobilised on beads incubated in the presence of rhodamine - labelled liposome preparations containing the indicated phosphoinositides . Scale bar : 50 μm .

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Microscopy-based protein-liposome binding assay. ATG16L1-GFP immobilised on beads incubated in the presence of rhodamine-labelled liposome preparations containing the indicated phosphoinositides. Scale bar: 50 μm.


Quantification of relative liposome binding


Microscopy - based protein - liposome binding assay as in ( B ) . ATG16L1WT - and ATG16L1LD - GFP immobilised on beads were incubated with rhodamine - labelled , PI3P - positive liposome preparations . Scale bar : 50 μm . Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values .

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Microscopy-based protein-liposome binding assay as in (B). ATG16L1WT- and ATG16L1LD-GFP immobilised on beads were incubated with rhodamine-labelled, PI3P-positive liposome preparations. Scale bar: 50 μm. Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values.


Protein - protein interaction assay in 293T cells transiently transfected with the indicated S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies .

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Protein-protein interaction assay in 293T cells transiently transfected with the indicated S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


by immunoblotting using the indicated antibodies . Dimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated S - tagged ATG16L1 constructs and

by immunoblotting using the indicated antibodies. Dimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated S-tagged ATG16L1 constructs and analysed


ATG16L1 - / - stably expressing the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) and ATG5 ( red ) . Scale bar : 9 μm . Right panels show quantifications of average number of Flag - and ATG5 - positive dots per cell .

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ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and ATG5 (red). Scale bar: 9 μm. Right panels show quantifications of average number of Flag- and ATG5-positive dots per cell.


ATG16L1 - / - stably expressing the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag ( to detect ATG16L1 , red ) and WIPI2 ( green ) . Scale bar : 9 μm . Lower panel shows quantification of average number of WIPI2 positive dots per cell .

ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag (to detect ATG16L1, red) and WIPI2 (green). Scale bar: 9 μm. Lower panel shows quantification of average number of WIPI2 positive dots per cell.


Average intensities of individual Flag - ATG16L1 dots in ( A ) . Underlying grey circles represent individual data points . Dots were quantified ( n = 1225 for ATG16L1WT ; n = 334 for ATG16L1LD ) .

Average intensities of individual Flag-ATG16L1 dots in (A). Underlying grey circles represent individual data points. Dots were quantified (n=1225 for ATG16L1WT; n=334 for ATG16L1LD).


WIPI2 - / - cells reconstituted with the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) and FIP200 ( red ) . Scale bar : 9 μm . Lower panel shows quantification of average number of Flag positive dots per puncta - positive cell .

WIPI2-/- cells reconstituted with the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and FIP200 (red). Scale bar: 9 μm. Lower panel shows quantification of average number of Flag positive dots per puncta-positive cell.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Cells were amino acid starved ( AA starved ) to induce mTORC1 - dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Cells were amino acid starved (AA starved) to induce mTORC1-dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Mitophagy was induced by CCCP ( 10 μM ) treatment for 6 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Mitophagy was induced by CCCP (10 μM) treatment for 6 hr prior to lysis.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Glucose starvation ( Glu starved ) for 20 hr was used to induce mTORC1 - independent autophagy . BafA1 was added to all conditions 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Glucose starvation (Glu starved) for 20 hr was used to induce mTORC1-independent autophagy. BafA1 was added to all conditions 2 hr prior to lysis.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . LC3 - associated phagocytosis ( LAP ) - like LC3 lipidation was induced by treating cells with CCCP ( 100 μM ) for 2 hr prior to lysis . Right panel shows quantification of LC3 - II / LC3 - I ratio .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LC3-associated phagocytosis (LAP)-like LC3 lipidation was induced by treating cells with CCCP (100 μM) for 2 hr prior to lysis. Right panel shows quantification of LC3-II/LC3-I ratio.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . LAP - like LC3 lipidation induced by monensin or ammonium chloride ( NH4Cl ) for 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LAP-like LC3 lipidation induced by monensin or ammonium chloride (NH4Cl) for 2 hr prior to lysis.