( D ) Measurement of chlorophyll in leaves third leaves in rosette were used . Four independent experiments were conducted . Data are presented as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t .

O O O O O B-SMALL_MOLECULE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O 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) Measurement of chlorophyll in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test.


( E ) H2O2 concentrations in leaves third leaves in rosette were used . Four independent experiments were conducted . Data are presented as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O 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) H2O2 concentrations in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test.


( A ) Observation of inflorescences of 7 - week - old wild - type ( WT ) Arabidopsis CDF4 transgenic . asterisk represents flower organ attached along inflorescence . Scale bar indicates 1 . 5 cm .

O O O O O B-TISSUE O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O

(A) Observation of inflorescences of 7-week-old wild-type (WT) Arabidopsis and the CDF4 transgenic plants. The asterisk represents the flower organ attached along the inflorescence. Scale bar indicates 1.5 cm.


Selected abscission - related gene expression in ( B ) WT proIDA : : CDF4 lines by using qPCR . expression of these selected genes in wild - type is given as 1 . relative expression level represents only level of expression of gene relative to wild type . ACTIN2 was used as internal control . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Selected abscission-related gene expression levels in (B) WT and proIDA::CDF4 lines by using qPCR analysis. The expression of these selected genes in the wild-type plant is given as 1. The relative expression level represents only the level of expression of the gene relative to the wild type. ACTIN2 was used as the internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


Selected abscission - related gene expression in ( C ) WT proIDA : : CDF4RNAi lines by using qPCR . expression of these selected genes in wild - type is given as 1 . relative expression level represents only level of expression of gene relative to wild type . ACTIN2 was used as internal control . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t .

Selected abscission-related gene expression levels in (C) WT and proIDA::CDF4RNAi lines by using qPCR analysis. The expression of these selected genes in the wild-type plant is given as 1. The relative expression level represents only the level of expression of the gene relative to the wild type. ACTIN2 was used as the internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


E Observation of GUS activity in proPGAZAT : : GUS proPGAZAT : : GUS & proIDA : : CDF4 . Scale bar indicates 0 . 5 cm .

O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O

E Observation of GUS staining activity in the proPGAZAT::GUS and proPGAZAT::GUS & proIDA::CDF4. Scale bar indicates 0.5 cm.


F ChIP - qPCR of relative binding of CDF4 to promoter regions of PGAZAT . An anti - HA monoclonal antibody was used for immunoprecipitation from 6 - week - old pER8 : : CDF4 - HA transgenic after estradiol induction . Black columns indicate enrichment fold changes normalized to ACT2 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t .

O B-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O 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 ChIP-qPCR analysis of the relative binding of CDF4 to the promoter regions of PGAZAT. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from 6-week-old pER8::CDF4-HA transgenic plants after estradiol induction. Black columns indicate the enrichment fold changes normalized to ACT2. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


( A ) lid - 1 ( xd288 ) atgl - 1 ( xd314 ) mutants show LD accumulation in most neurons in C . elegans , including neurons in head region , tail ventral nerve cord . xdIs109 is a stable transgenic with pan - neuronal expression of LD marker PLIN1 : : GFP . arrows indicate PLIN1 : : GFP - positive LDs in ventral nerve cord of mutants . Scale bar : 20 μm . right panels depict head region . Scale bar : 5 μm . insets in right panels are enlarged views of PLIN1 : : GFP rings , representing LDs .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR B-EXP_ASSAY O O B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-SUBCELLULAR O

(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 ) of numbers of LDs in head region neurons ( each dot represents one worm ) . data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison . Asterisks denote significant differences as compared to control xdIs109 . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . ns : not statistically significant . Data show mean ± . n ≥ 9 .

O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-TISSUE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 show dramatically increased neutral lipids in head region of lid - 1 ( xd288 ) atgl - 1 ( xd314 ) compared with N2 . Scale bar : 10 μm .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

(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 of neurons . red dashed marks outline of neurons . red arrows mark LDs . Scale bar : 0 . 5 μm . scale bar in enlarged is 100 nm . ( I ) Serial sections of atgl - 1 ( xd314 ) show two LDs ( indicated by blue red arrows ) in a neuron . Scale bar : 0 . 5 μm .

O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O

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

O O O O O O O O O O B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM O O B-SUBCELLULAR B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) expression pattern of atgl - 1 viewed by Patgl - 1 : : GFP transcriptional fusion reporter . Patgl - 1 : : GFP expression is high in intestine . Relatively low signals are found in nerve ring , motor neuron commissure , ventral nerve cord tail . Scale bar : 20 μm .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O B-EXP_ASSAY O O O B-TISSUE I-TISSUE O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O

(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 signal when Patgl - 1 : : GFP1 - 10 Prab - 3 : : GFP11 are expressed separately . When Patgl - 1 : : GFP1 - 10 Prab - 3 : : GFP11 are co - expressed , GFP signals are found in head region , tail ventral nerve cord , where most neuronal bodies are located . lines mark outline of worms . Scale bar : 50 μm .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-CELL_TYPE O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O

(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 . left panels depict whole worm . Scale bar : 50 μm . insets in right panels are enlarged views of ventral nerve cord in dotted boxes of left panels . signals of mCherry GFP merge very well . Scale bar : 20 μm .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O

(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 xdIs109 reporter in different genetic backgrounds . 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 ) of number of LDs in head neurons in different genetic backgrounds . Each dot represents one worm . data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison . Asterisks denote significant differences as compared to control xdIs109 . ns : not statistically significant . * * * * P < 0 . 0001 . Data show mean ± . n ≥ 7 .

O O O O B-SUBCELLULAR O B-TISSUE B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-TISSUE B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) of number of LDs in head neurons in different genetic backgrounds . Each dot represents one worm . data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison . Asterisks denote significant differences as compared to control xdIs109 . t - was used between groups under crossbars . ns : not statistically significant . * * * * P < 0 . 0001 . Data show mean ± . n ≥ 9

O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) . zdIs5 reporter marks touch neurons . arrows indicate LDs in touch neurons in atgl - 1 ( xd314 ) . Scale bar : 10 μm .

O O O B-SUBCELLULAR B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

(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 ) lid - 1 ( xd288 ) show a gentle touch sensation defect , which is rescued by neuron - specific expression of atgl - 1 lid - 1 , respectively . fat - 4 mutation enhances touch sensation defect in atgl - 1 ( xd314 ) lid - 1 ( xd288 ) . Wild type mec - 4 ( u253 ) act as negative control positive control , respectively . data were analyzed using Kruskal - Wallis with Dunn ' s . 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 ± . # signify significant differences between groups under crossbars . # # P < 0 . 01 . Data show mean ± . Number of worms analyzed for each n = 25 .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O

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

O O O O O O O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-CELL_TYPE O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) of worms that have three or more surviving touch neurons in different genetic backgrounds . Mutations of fat - 1 , fat - 3 fat - 4 significantly enhance suppression effect of atgl - 1 ( xd314 ) in preventing neurodegeneration of mec - 4 ( d ) . data were analyzed using one - way ANOVA with Bonferroni ' s multiple comparison . # # # # # # # 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 ± . Number of experiments n ≥ 3 , with at least 50 per analyzed in each experiment .

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

O O O O O O O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 relative content of total C20 : 3 is increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . increase is greatest in mutants with fat - 4 mutation . ( B ) Lipidomic data show that relative content of total C20 : 4 is increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . increase is greatest in mutants with fat - 4 mutation . ( C ) Lipidomic data show that 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 ) atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) , which matches role of FAT - 4 in synthesizing C20 : 5 ( EPA ) . Data information data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison . 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 ± . Number of experiments n = 5 , with about 10 , 000 per analyzed in each experiment .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) of C20 : 3 - , C20 : 4 - or C20 : 5 - containing TAGs to total lipids containing same PUFA in different genetic backgrounds . of C20 : 4 - containing TAGs to total lipids containing C20 : 4 is significantly increased in atgl - 1 fat - 4 single mutants . is further increased in atgl - 1 fat - 4 double mutant . Data information : FA : free fatty ; TAG : triacylglycerol ; PL : phospholipids . data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison . 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 ± . Number of experiments n = 5 , with about 10 , 000 per analyzed in each experiment .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) of C20 : 3 - , C20 : 4 - or C20 : 5 - containing phospholipids to total lipids containing same PUFA in different genetic backgrounds . of C20 : 4 - containing phospholipids to total lipids containing C20 : 4 is significantly decreased in atgl - 1 fat - 4 single mutants . is further decreased in atgl - 1 fat - 4 double mutant . Data information : FA : free fatty ; TAG : triacylglycerol ; PL : phospholipids . data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison . 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 ± . Number of experiments n = 5 , with about 10 , 000 per 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 ) of worms that have three or more surviving touch neurons in different genetic backgrounds . mboa - 7 mutation significantly increases of three or more surviving touch neurons in mec - 4 ( d ) , atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . data were analyzed using one - way ANOVA with Bonferroni ' s multiple comparison . Asterisks signify significant differences between groups under crossbars . * * P < 0 . 01 , * * * * P < 0 . 0001 . Data show mean ± . Number of experiments n ≥ 4 , with at least 50 per 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 ) 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 ) . data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison . Asterisks signify significant differences as compared to mec - 4 ( d ) . * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± . Number of experiments n = 5 , with about 10 , 000 per analyzed in each experiment .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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) 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.


B . of PNRC1 expression in normal ( blue circles ) cancer ( red circles ) samples by Real Time PCR .

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B. Quantification of PNRC1 expression levels in primary normal (blue circles) and cancer (red circles) samples by Real Time PCR.


Immunofluorescence stainings of PNRC1 ( red ) Ki - 67 ( green ) performed on a healthy colon ( C ) DAPI was used to stain nuclei . Scale bars : 50µm .

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Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a primary healthy colon (C) DAPI was used to stain cell nuclei. Scale bars:50µm.


Immunofluorescence stainings of PNRC1 ( red ) Ki - 67 ( green ) performed on a malignant lymphoma ( D ) . DAPI was used to stain nuclei . Scale bars : 50µm .

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Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a malignant lymphoma (D). DAPI was used to stain cell nuclei. Scale bars:50µm.


E . Proliferation curves of HeLa transfected with HRASG12V ( R ) , PNRC1 ( P ) , their combination ( RP ) or a LacZ control ( L ) . results shown are average ± SD of 2 biological replicates with 3 technical replicates each . A one - way ANOVA with Tukey multiple comparison was performed for 72h dataset ; statistically significant comparisons are as follows : L vs R ( p < 0 . 002 ) , L vs P ( p < 0 . 02 ) , R vs P ( p < 0 . 0002 ) R vs RP ( p < 0 . 0004 )

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E. Proliferation curves of HeLa cells transfected with HRASG12V (R), PNRC1 (P), their combination (RP) or a LacZ control (L). The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each. A one-way ANOVA with Tukey multiple comparison test was performed for 72h dataset; the statistically significant comparisons are as follows: L vs R (p<0.002), L vs P (p<0.02), R vs P (p<0.0002) and R vs RP (p<0.0004)


F . Proliferation curves of MCF7 transfected with HRASG12V ( R ) , PNRC1 ( P ) , their combination ( RP ) or a LacZ control ( L ) . results shown are average ± SD of 2 biological replicates with 3 technical replicates each .

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F. Proliferation curves of MCF7 cells transfected with HRASG12V (R), PNRC1 (P), their combination (RP) or a LacZ control (L). The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each.


G . Proliferation curves of KRASG12S - mutated A549 transfected with PNRC1 or LacZ control . A representative experiment with average ± SD of 3 technical replicates is shown . An unpaired t - was performed for 144h dataset ( p < 0 . 0003 ) .

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G. Proliferation curves of KRASG12S-mutated A549 cells transfected with PNRC1 or LacZ control. A representative experiment with the average ± SD of 3 technical replicates is shown. An unpaired t-test was performed for 144h dataset (p<0.0003).


H . Proliferation curves of BJ - T transduced with a non - targeting or a PNRC1 - specific shRNA construct . Results are shown as average ± SD of 3 biological replicates with 3 technical replicates each . An unpaired t - was performed for each time point ( * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 ) .

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H. Proliferation curves of BJ-T cells transduced with a non-targeting or a PNRC1-specific shRNA construct. Results are shown as the average ± SD of 3 biological replicates with 3 technical replicates each. An unpaired t-test was performed for each time point (** p<0.01, *** p<0.005, **** p<0.001).


A . Click - iT imaging performed on HeLa expressing RFP - PNRC1 or RFP control . Confocal of transfected pulsed with EU for 16 hours were collected at indicated time points after EU removal ( green : Alexa 488 - EU , red : RFP , blue : DAPI , scale bar : 5µm ) . RFP - PNRC1 - transfected are indicated with arrowheads .

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A. Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1 or RFP control. Confocal images of transfected cells pulsed with EU for 16 hours were collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1-transfected cells are indicated with arrowheads.


Confocal microscopy of HeLa expressing GFP - PNRC1 stained with antibodies against NPM1 ( B ) nucleolar ( nuclei are stained in blue with DAPI , scale bar : 5µm ) .

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Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against NPM1 (B) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm).


Confocal microscopy of HeLa expressing GFP - PNRC1 stained with antibodies against UBF1 ( C ) nucleolar ( nuclei are stained in blue with DAPI , scale bar : 5µm ) . A magnification of merged channel is provided for UBF1 .

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Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against UBF1 (C) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm). A magnification of the merged channel images is provided for UBF1 staining.


D . Agarose / formaldehyde gel separation of neotranscribed rRNAs collected from LacZ PNRC1 - expressing pulsed for 15 minutes with 3H - uridine chased for reported time points ( minutes ) . Samples were loaded according to amount of incorporated 3H , quantified by liquid scintillation . FU was included as a control of a blocked rRNA processing .

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D. Agarose/formaldehyde gel separation of neotranscribed rRNAs collected from LacZ and PNRC1-expressing cells pulsed for 15 minutes with 3H-uridine and chased for the reported time points (minutes). Samples were loaded according to the amount of incorporated 3H, quantified by liquid scintillation. FU was included as a control of a blocked rRNA processing.


E . Ratios between of 47S pre - rRNA band bands belonging to 32S or 28S rRNA species relative to panel ( D ) measured at 60 minutes after chase for both LacZ or PNRC1 - expressing .

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E. Ratios between the intensity of 47S pre-rRNA band and bands belonging to 32S or 28S rRNA species relative to panel (D) and measured at 60 minutes after the chase for both LacZ or PNRC1-expressing cells.


F . Real Time PCR of 5 ' - ETS steady - state in HeLa expressing PNRC1 or GFP control . average ± SD of 3 biological replicates is shown .

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F. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1 or GFP control. The average ± SD of 3 biological replicates is shown.


A - B . Coomassie blue western blot of SDS - PAGE gel corresponding to PNRC1 IP . Red arrows represent gel areas excised both from IgG IP lanes for mass spectrometry . Input , unbound IP fractions were loaded as reported . band corresponding to PNRC1 is indicated .

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A-B. Coomassie blue staining and western blot analysis of the SDS-PAGE gel corresponding to PNRC1 IP. Red arrows represent the gel areas excised both from the IgG and IP lanes for mass spectrometry analysis. Input, unbound and IP fractions were loaded as reported. The protein band corresponding to PNRC1 is indicated.


C . Co - immunoprecipitation performed on HeLa expressing HA - PNRC1 with a specific anti - HA antibody in absence or presence of 1mg / ml RNaseA .

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C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1 with a specific anti-HA antibody in absence or presence of 1mg/ml RNaseA.


D . Co - immunoprecipitation of DCP1α - interacting performed on HA - PNRC1 - expressing HeLa with a specific anti - DCP1α antibody .

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D. Co-immunoprecipitation of DCP1α-interacting proteins performed on HA-PNRC1-expressing HeLa cells with a specific anti-DCP1α antibody.


E . Co - immunoprecipitation performed on wild - type HeLa with an anti - PNRC1 antibody probed for DCP1α .

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E. Co-immunoprecipitation performed on wild-type HeLa cells with an anti-PNRC1 antibody and probed for DCP1α.


F - G . Co - immunoprecipitation performed on HeLa expressing HA - PNRC1 with a specific anti - HA antibody probed with antibodies specific for involved in decapping . arrow indicates specific DCP2 band , as reported in datasheet of antibody employed .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

F-G. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1 with a specific anti-HA antibody and probed with antibodies specific for proteins involved in RNA decapping. The white arrow indicates the specific DCP2 band, as reported in the datasheet of the antibody employed.


A . Confocal microscopy of RFP or RFP - PNRC1 - expressing stained for DCP1α , DDX6 LSM1 . RFP - PNRC1 - transfected are indicated with arrowheads . Scale bar : 10µm . B . of classified according to P - localization of three stained . average ± SD of 3 independent experiments is reported .

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A. Confocal microscopy images of RFP or RFP-PNRC1-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1-transfected cells are indicated with arrowheads. Scale bar: 10µm. B. Quantification of cells classified according to the P-body localization of the three stained proteins. The average ± SD of 3 independent experiments is reported.


C . Nucleolar fractionation performed on LacZ or PNRC1 - expressing HeLa . Total lysates , ( Cyto ) , nucleoplasmic ( Nucl ) nucleolar ( No ) fractions were collected . UBF1 , PolII MEK2 were used as markers for nucleolar , non - nucleolar fractions , respectively . electrophoretic mobility of in nucleoplasmic lanes is slightly diminished due to lysis buffer composition . D . Densitometric of DCP1α nucleolar pool obtained from western blot shown in panel ( C ) normalized upon amount of nucleolar UBF1 of DCP1α in input fraction .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-GENEPROD O B-SUBCELLULAR B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O B-GENEPROD O O O O

C. Nucleolar fractionation performed on LacZ or PNRC1-expressing HeLa cells. Total lysates, cytoplasmic (Cyto), nucleoplasmic (Nucl) and nucleolar (No) fractions were collected. UBF1, RNA PolII and MEK2 were used as markers for the nucleolar, non-nucleolar and cytoplasmic fractions, respectively. The electrophoretic mobility of proteins in the nucleoplasmic lanes is slightly diminished due to nuclear lysis buffer composition. D. Densitometric quantification of DCP1α nucleolar pool obtained from the western blot shown in panel (C) and normalized upon the amount of nucleolar UBF1 and the levels of DCP1α in the input fraction.


E . Confocal microscopy of HeLa silenced for endogenous DCP1α expressing a shRNA - resistant GFP - DCP1α in combination with RFP or RFP - PNRC1 transgenes ( green : GFP , red : RFP , blue : DAPI , scale bar : 5µm ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O

E. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing a shRNA-resistant GFP-DCP1α in combination with RFP or RFP-PNRC1 transgenes (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).


A . Co - immunoprecipitation performed on HeLa expressing HA - PNRC1WT , HA - PNRC1W300A or HA - PNRC1ΔNLS with a specific anti - HA antibody probed with anti - NPM1 anti DCP1α antibodies .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O

A. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS with a specific anti-HA antibody and probed with anti-NPM1 and anti DCP1α antibodies.


B - C . Co - immunoprecipitation performed on HeLa expressing HA - PNRC1W300A mutant with a specific anti - HA antibody probed with antibodies specific for PNRC1 - interacting . arrow indicates specific DCP2 band .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O

B-C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1W300A mutant with a specific anti-HA antibody and probed with antibodies specific for PNRC1-interacting proteins. The white arrow indicates the specific DCP2 band.


D . Confocal microscopy of HeLa silenced for endogenous DCP1α expressing an shRNA - resistant GFP - DCP1α in combination with RFP , RFP - PNRC1WT or RFP - PNRC1W300A mutant ( green : GFP , red : RFP , blue : DAPI , scale bar : 5µm ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O

D. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing an shRNA-resistant GFP-DCP1α in combination with RFP, RFP-PNRC1WT or RFP-PNRC1W300A mutant (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).


E . Confocal microscopy of RFP , RFP - PNRC1 or RFP - PNRC1W300A - expressing stained for DCP1α , DDX6 LSM1 . RFP - PNRC1WT or RFP - PNRC1W300A - transfected are indicated with arrowheads . Scale bar :

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O

E. Confocal microscopy images of RFP, RFP-PNRC1 or RFP-PNRC1W300A-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads. Scale bar: 10µm


F . of HeLa expressing RFP - PNRC1W300A classified according to P - localization of three stained . average of 3 independent experiments ± SD is reported .

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F. Quantification of HeLa cells expressing RFP-PNRC1W300A classified according to the P-body localization of the three stained proteins. The average of 3 independent experiments ± SD is reported.


G . Co - immunoprecipitation performed on HeLa expressing LacZ control , HA - PNRC1WT , HA - PNRC1W300A or HA - PNRC1ΔNLS constructs with a specific anti - DCP1α antibody probed with anti - PNRC1 anti - NPM1 antibodies .

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G. Co-immunoprecipitation performed on HeLa cells expressing LacZ control, HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS constructs with a specific anti-DCP1α antibody and probed with anti-PNRC1 and anti-NPM1 antibodies.


A . Confocal of a Click - iT imaging performed on HeLa expressing RFP - PNRC1W300A pulsed with EU for 16 hours collected at indicated time points after EU removal ( green : Alexa 488 - EU , red : RFP , blue : DAPI , scale bar : 5µm ) . RFP - PNRC1WT or RFP - PNRC1W300A - transfected are indicated with arrowheads .

O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O

A. Confocal images of a Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1W300A pulsed with EU for 16 hours and collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads.


B . Real Time PCR of 5 ' - ETS steady - state in HeLa expressing PNRC1W300A or GFP control . average of 3 independent experiments ± SD is reported .

B. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1W300A or GFP control. The average of 3 independent experiments ± SD is reported.


C . of 5 ' - ETS in HeLa infected with an anti - DCP2 or a non - targeting shRNA expressing GFP , PNRC1WT or PNRC1W300A transgenes . average of 2 independent experiments ± SD is reported .

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C. Quantification of 5'-ETS RNA levels in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average of 2 independent experiments ± SD is reported.


of U3 ( D ) snoRNAs immunoprecipitated by anti - m3G cap antibody measured by Real Time PCR . Values represents average of two biological replicates with three technical replicates

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Levels of U3 (D) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR. Values represents the average of two biological replicates with three technical replicates and are


of U8 ( E ) snoRNAs immunoprecipitated by anti - m3G cap antibody measured by Real Time PCR .

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Levels of U8 (E) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR.


F . of U3 snoRNA immunoprecipitated by anti - m3G cap antibody in HeLa infected with an anti - DCP2 or a non - targeting shRNA expressing GFP , PNRC1WT or PNRC1W300A transgenes . average ± SD of 2 independent experiments with three technical replicates is reported .

O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

F. Levels of U3 snoRNA immunoprecipitated by the anti-m3G cap antibody in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average ± SD of 2 independent experiments with three technical replicates is reported.


G . Western blot of input Flag - IP fractions of RIP experiment . H . of U3 , U8 U1 co - immunoprecipitating with GFP , PNRC1WT or PNRC1W300A . Results are shown as average ± SD of two biological replicates .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

G. Western blot analysis of input and Flag-IP fractions of RIP experiment. H. RNA levels of U3, U8 and U1 co-immunoprecipitating with GFP, PNRC1WT or PNRC1W300A. Results are shown as the average ± SD of two biological replicates.


( A ) Yeast two - hybrid assays to for interaction between Munc18 - 1 ERK1 / 2 . pGAL4 - Munc18 - 1 ( M18 ) pGAL4 - Empty ( control ) were used as baits ERK1 ERK2 as prey . Both ERK1 ERK2 bind Munc18 - 1 allow growth on selection plates ( minus Histidine ( - HIS ) minus Histidine Adenosine ( - ADE ) ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O

(A) Yeast two-hybrid assays to test for interaction between Munc18-1 and ERK1/2. pGAL4-Munc18-1 (M18) and pGAL4-Empty (control) were used as baits and ERK1 and ERK2 as prey. Both ERK1 and ERK2 bind Munc18-1 and allow growth on selection plates (minus Histidine (-HIS) and minus Histidine and Adenosine (-ADE)).


( B ) In vitro kinase assays showing Munc18 - 1 phosphorylation by ERK . Radioactive phosphoblot ( 32P ) immunoblot ( IB ) of in vitro ERK kinase using immunoprecipitatedMunc18WT , Munc184A ( M18T78A , S158A , S241A , T574A ) , Munc183A ( M18S158A , S241A , T574A ) , Munc18AA ( M18S241A , T574A ) from HEK293T incubated with recombinant ERK2 . phosphoblot shows phosphorylated Munc18 - 1 ( pMunc18 - 1 ) auto - phosphorylated ERK2 ( pERK2 ) . ( C ) of phospho - Munc18 - 1 in wild - type Munc18AA mutant expressed as amount of 32P incorporation in Munc18 - 1 normalized to total Munc18 - 1 . Phosphorylation of Munc18WT was set to 100 % . ( Munc18WT : 100 ± 3 % ; Munc18AA : 24 ± 2 % , n = 3 , * p = 0 . 01 ) .

O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-CELL_LINE O O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(B) In vitro kinase assays showing Munc18-1 phosphorylation by ERK. Radioactive phosphoblot (32P) and immunoblot (IB) of in vitro ERK kinase assay using immunoprecipitatedMunc18WT, Munc184A (M18T78A,S158A,S241A,T574A), Munc183A (M18S158A,S241A,T574A), and Munc18AA (M18S241A,T574A) from HEK293T cells incubated with recombinant ERK2. The phosphoblot shows phosphorylated Munc18-1 (pMunc18-1) and auto-phosphorylated ERK2 (pERK2).(C) Quantification of phospho-Munc18-1 intensity in wild-type and Munc18AA mutant expressed as the amount of 32P incorporation in Munc18-1 normalized to total Munc18-1 levels. Phosphorylation of Munc18WT was set to 100%. (Munc18WT: 100±3%; Munc18AA: 24±2%, n=3, * p = 0.01).


( D ) ERK1 in vitro kinase showing ERK - dependent 32P - incorporation in Munc18WT immuno - precipitated from HEK293T . Western Blot stained with Munc18 - 1 antibody shows total precipitated Munc18 - 1 were identical in conditions .

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(D) ERK1 in vitro kinase assay showing ERK-dependent 32P-incorporation in Munc18WT immuno-precipitated from HEK293T cells. Western Blot stained with Munc18-1 antibody shows total precipitated Munc18-1 levels were identical in all conditions.


( A ) Experimental setup of fear conditioning paradigm . Mice placed in a fear - conditioning box received no shock ( NS ) , an immediate shock ( IS ) or delayed shock ( DS ) were decapitated 30 min after returning to home cage . Dorsal ventral hippocampi were subjected to fractionation ( P2 ) . DS IS significantly increased of phosphorylated ERK1 / 2 , a hallmark of ERK activation . ( B ) Activity values across three different training paradigms to ascertain that foot shock was received in IS DS group .

O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O

(A) Experimental setup of fear conditioning paradigm. Mice placed in a fear- conditioning box received no shock (NS), an immediate shock (IS) or delayed shock (DS) and were decapitated 30 min after returning to the home cage. Dorsal and ventral hippocampi were subjected to cellular fractionation (P2). DS and IS significantly increased the levels of phosphorylated ERK1/2, a hallmark of ERK activation.(B) Activity values across three different training paradigms to ascertain that foot shock was received in the IS and DS group.


( D ) Western Blot of P2 synaptosomal fractions of ventral hippocampi from fear conditioning paradigm stained for pM18S241 , total Munc18 - 1 , pSynapsin , total synapsin , pERK GAPDH as loading control . ( E ) Munc18 - 1 phosphorylation in dorsal hippocampus expressed as pM18S241 over total Munc18 - 1 . No shock was set to 1 ( NS : 1 . 0 ± 0 . 19 , n = 5 ; DS : 4 . 01 ± 0 . 39 , n = 5 ; IS : 2 . 88 ± 0 . 37 , n = 5 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . ( F ) of Munc18 - 1 phosphorylation in ventral hippocampus expressed as pM18S241 divided by total Munc18 - 1 . ( NS : 1 . 0 ± 0 . 32 , n = 5 ; DS : 4 . 39 ± 0 . 55 , n = 5 ; IS : 3 . 62 ± 0 . 50 , n = 5 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . ( G ) of synapsin phosphorylation in dorsal hippocampus expressed as pSynapsin divided by total synapsin . ( NS : 1 . 0 ± 0 . 31 , n = 5 ; DS : 1 . 88 ± 0 . 43 , n = 5 ; IS : 1 . 18 ± 0 . 34 , n = 5 ; n . s . ) . Inset shows of ERK2 phosphorylation in dorsal hippocampus expressed as pERK2 divided by total ERK2 . ( NS : 1 . 0 ± 0 . 11 , n = 5 ; DS : 3 . 51 ± 0 . 41 , n = 5 ; IS : 3 . 98 ± 0 . 24 , n = 5 ; * * p < 0 . 01 ) . ( H ) of synapsin phosphorylation in ventral hippocampus expressed as pSynapsin divided by total synapsin . ( NS : 1 . 0 ± 0 . 20 , n = 5 ; DS : 3 . 32 ± 0 . 46 , n = 5 ; IS : 3 . 30 ± 0 . 60 , n = 5 ; * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western Blot analysis of P2 synaptosomal fractions of ventral hippocampi from the fear conditioning paradigm stained for pM18S241, total Munc18-1, pSynapsin, total synapsin, pERK and GAPDH as loading control.(E) Quantification Munc18-1 phosphorylation in dorsal hippocampus expressed as ratio pM18S241 over total Munc18-1 levels. No shock was set to 1 (NS: 1.0±0.19, n=5; DS: 4.01±0.39, n=5; IS: 2.88±0.37, n=5; **p<0.01; ***p<0.001).(F) Quantification of Munc18-1 phosphorylation in ventral hippocampus expressed as ratio pM18S241 divided by total Munc18-1 levels. (NS: 1.0±0.32, n=5; DS: 4.39±0.55, n=5; IS: 3.62±0.50, n=5; **p<0.01; ***p<0.001).(G) Quantification of synapsin phosphorylation in dorsal hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.31, n=5; DS: 1.88±0.43, n=5; IS: 1.18±0.34, n=5; n.s.). Inset shows quantification of ERK2 phosphorylation in dorsal hippocampus expressed as ratio pERK2 divided by total ERK2 levels. (NS: 1.0±0.11, n=5; DS: 3.51±0.41, n=5; IS: 3.98±0.24, n=5; **p<0.01).(H) Quantification of synapsin phosphorylation in ventral hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.20, n=5; DS: 3.32±0.46, n=5; IS: 3.30±0.60, n=5; **p<0.01).


( A ) Western Blot of whole lysate of adult mousebrain slices treated with proteasome inhibitor MG132 ( 10 M ) DMSO , GABAA receptor blocker GABAzine ( GBZ , 10 M ) or voltage - gated sodium channel blocker TTX ( 2 M ) for 4 h . Blots were stained for pMunc18S241 , Munc18 - 1 tubulin as loading control . ( B ) of ( A ) . ERK phosphorylation of Munc18 - 1 in adult mousebrain slices expressed as pM18S241 divided by total Munc18 - 1 . Vehicle ( DMSO ) was set to 1 ( DMSO : 100 ± 0 . 4 % ; GBZ : 1 . 73 ± 0 . 12 ; TTX : 0 . 052 ± 0 . 002 , n = 4 , * p < 0 . 05 , * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O 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) Western Blot of whole cell lysate of adult mousebrain slices treated with the proteasome inhibitor MG132 (10 M) and DMSO, the GABAA receptor blocker GABAzine (GBZ, 10 M) or the voltage-gated sodium channel blocker TTX (2 M) for 4 h. Blots were stained for pMunc18S241, Munc18-1 and tubulin as loading control.(B) Quantification of (A). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.4%; GBZ: 1.73 ± 0.12; TTX: 0.052 ± 0.002, n = 4, * p<0.05, ** p<0.01).


( C ) Western Blot of whole lysate of adult mousebrain slices treated with proteasome inhibitor MG132 ( 10 M ) MEK inhibitor PD98059 ( 10 M ) for 4 h . Proteasome block increases of phosphorylated Munc18 - 1 ERK1 / 2 , which can be prevented by MEK inhibition . Blots were stained for pMunc18S241 , Munc18 , pERK1 / 2 ERK1 / 2 . ( D ) of ( C ) . ERK phosphorylation of Munc18 - 1 in adult mousebrain slices expressed as pM18S241 divided by total Munc18 - 1 . Vehicle ( DMSO ) was set to 1 ( DMSO : 100 ± 0 . 8 % ; MG132 : 375 ± 14 . 2 % ; MG132 + PD98059 : 24 ± 3 % , n = 3 , * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(C) Western Blot of whole cell lysate of adult mousebrain slices treated with proteasome inhibitor MG132 (10 M) and MEK inhibitor PD98059 (10 M) for 4 h. Proteasome block increases the levels of phosphorylated Munc18-1 and ERK1/2, which can be prevented by MEK inhibition. Blots were stained for pMunc18S241, Munc18, pERK1/2 and ERK1/2.(D) Quantification of (C). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.8%; MG132: 375 ± 14.2%; MG132 + PD98059: 24 ± 3%, n = 3, ** p<0.01).


( E ) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 ( 1 M ) for 5 min or BDNF ( 100ng / ml ) for 48 h in presence of 2 M TTX stained for pMunc18S241 , Munc18 , pERK tubulin as loading control .

O O O O B-ORGANISM B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O O O O

(E) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 (1 M) for 5 min or BDNF (100ng/ml) for 48 h in the presence of 2 M TTX and stained for pMunc18S241, Munc18, pERK and tubulin as loading control.


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( A ) Amplitude of excitatory synaptic response ( EPSC ) ( M18WT : 1 . 37 ± 0 . 21 nA , n = 35 ; M18S241A : 2 . 53 ± 0 . 31 nA , n = 45 ; M18S241D : 0 . 24 ± 0 . 05 nA , n = 17 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ) .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(A) Amplitude of excitatory synaptic response (EPSC) (M18WT: 1.37±0.21 nA, n=35; M18S241A: 2.53±0.31 nA, n=45; M18S241D: 0.24±0.05 nA, n=17, ***p<0.001, **p<0.01, *p<0.05).


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( B ) of that do not respond to electrical stimulation ( M18WT : 15 . 5 % ; M18S241A : 0 % ; M18S241D : 56 . 9 % ) . These are not included in ( A ) . Insert : representative recording of single EPSCs . Scale bar represents 10 ms 0 . 5 nA .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(B) Percentage of cells that do not respond to electrical stimulation (M18WT: 15.5%; M18S241A: 0%; M18S241D: 56.9%). These cells are not included in (A). Insert: representative recording of single EPSCs. Scale bar represents 10 ms and 0.5 nA.


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( C ) Typical examples of spontaneous vesicle release . Scale bar represents 0 . 1 s 25 pA . ( D ) Spontaneous release frequency ( M18WT : 7 . 05 ± 1 . 28 Hz , n = 32 ; M18S241A : 14 . 26 ± 2 . 04 Hz , n = 37 ; M18S241D : 0 . 64 ± 0 . 29 Hz , n = 11 ; * * * p < 0 . 001 , * * p < 0 . 01 ) . ( E ) Amplitude of spontaneous release events ( M18WT : 15 . 40 ± 1 . 06 pA , n = 17 ; M18S241A : 15 . 03 ± 0 . 61 pA , n = 20 ; M18S241D : 10 . 98 ± 0 . 72 pA , n = 10 ; * * p < 0 . 01 ) .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(C) Typical examples of spontaneous vesicle release. Scale bar represents 0.1 s and 25 pA.(D) Spontaneous release frequency (M18WT: 7.05 ± 1.28 Hz, n=32; M18S241A: 14.26 ± 2.04 Hz, n=37; M18S241D: 0.64 ± 0.29 Hz, n=11; ***p<0.001, **p<0.01).(E) Amplitude of spontaneous release events (M18WT: 15.40 ± 1.06 pA, n=17; M18S241A: 15.03 ± 0.61 pA, n=20; M18S241D: 10.98 ± 0.72 pA, n=10; **p<0.01).


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( F ) of readily releasable pool ( RRP ) assessed with single hyperosmotic sucrose application ( 500 mM , 3 . 5 s , M18WT : 0 . 74 ± 0 . 15 nC , n = 7 ; M18S241A : 1 . 6 ± 0 . 33 nC , n = 10 ; M18S241D : could not be determined ; * p < 0 . 05 ) .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(F) Size of the readily releasable pool (RRP) assessed with single hyperosmotic sucrose application (500 mM, 3.5 s, M18WT: 0.74 ± 0.15 nC, n=7; M18S241A: 1.6 ± 0.33 nC, n=10; M18S241D: could not be determined; * p < 0.05).


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( G ) Neurons were stimulated with two consecutive pulses with varying inter - stimulus interval to assess release probability . Paired calculated as of second to first synaptic response is plotted as function of inter - stimulus interval ( M18WT , n = 25 ; M18S241A , n = 19 ; M18S241D , n = 17 ) ; * p < 0 . 05 , * * p < 0 . 01 ) . data are expressed as mean ± . See also Figure EV2 EV3 .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(G) Neurons were stimulated with two consecutive pulses with varying inter-stimulus interval to assess release probability. Paired pulse ratio calculated as ratio of the second to the first synaptic response is plotted as function of inter-stimulus interval (M18WT, n=25; M18S241A, n=19; M18S241D, n=17); * p < 0.05, ** p < 0.01). All data are expressed as mean ± SEM. See also Figure EV2 and EV3.


( A - F ) Munc18 - 1 were measured in glia free cultures of munc18 - 1null mutant neurons expressing M18WT , M18S241A or M18S241D . ( A ) Typical examples showing Munc18 - 1 localization in neurites of M18WT , M18S241A or M18S241Dneurons . MAP2 in blue , VAMP2 in red Munc18 - 1 in green . Scale bar represents 2 m . ( B ) Mean somatic Munc18 - 1 ( M18WT : 1682 ± 206 a . u . , n = 46 ; M18S241A : 1719 ± 238 a . u . , n = 36 ; M18S241D : 582 ± 98 a . u . , n = 33 ; * * * p < 0 . 001 ) . ( C ) Mean synapticMunc18 - 1 ( M18WT : 1281 ± 161 a . u . ; M18S241A : 1390 ± 180 a . u . ; M18S241D : 210 ± 30 a . u . ; * * * p < 0 . 001 ) . ( D ) Mean synapticMunc18 - 1 as a function of radial from soma .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR B-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O

(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1null mutant neurons expressing M18WT, M18S241A or M18S241D.(A) Typical examples showing Munc18-1 protein localization and intensity in neurites of M18WT, M18S241A or M18S241Dneurons. MAP2 in blue, VAMP2 in red and Munc18-1 in green. Scale bar represents 2 m.(B) Mean somatic Munc18-1 intensity (M18WT: 1682 ± 206 a.u., n = 46; M18S241A: 1719 ± 238 a.u., n = 36; M18S241D: 582 ± 98 a.u., n = 33; *** p < 0.001).(C) Mean synapticMunc18-1 intensity (M18WT: 1281 ± 161 a.u.; M18S241A: 1390 ± 180 a.u.; M18S241D: 210 ± 30 a.u.; *** p < 0.001).(D) Mean synapticMunc18-1 intensity as a function of radial distance from the soma.


( A - F ) Munc18 - 1 were measured in glia free cultures of munc18 - 1 null mutant neurons expressing M18WT , M18S241A or M18S241D . ( E ) Mean synaptic Munc18 - 1 in cultures with 3 times standard number of neurons to increase network activity ( M18WT : 1072 ± 123 a . u . , n = 36 ; M18S241A : 1480 ± 170 a . u . , n = 32 ; M18S241D : 251 ± 70 a . u . , n = 34 ; * p < 0 . 05 , * * * p < 0 . 001 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(E) Mean synaptic Munc18-1 intensity in cultures with 3 times the standard number of neurons to increase network activity (M18WT: 1072 ± 123 a.u., n = 36; M18S241A: 1480 ± 170 a.u., n = 32; M18S241D: 251 ± 70 a.u., n = 34; * p < 0.05, *** p < 0.001).


( A - F ) Munc18 - 1 were measured in glia free cultures of munc18 - 1 null mutant neurons expressing M18WT , M18S241A or M18S241D . ( F ) Mean synaptic Munc18 - 1 in cultures treated with GABAA receptor antagonist Bicuculline ( 40 μM , 48 h ) . ( M18WT : 761 ± 121 a . u . , n = 26 ; M18S241A : 1482 ± 150 a . u . , n = 31 , * * p < 0 . 01 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(F) Mean synaptic Munc18-1 intensity in cultures treated with GABAA receptor antagonist Bicuculline (40 μM, 48 h). (M18WT: 761 ± 121 a.u., n = 26; M18S241A: 1482 ± 150 a.u., n = 31, ** p < 0.01).


O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mean synaptic syntaxin intensity in autaptic cultures (M18WT: 875 ± 35, n = 51; M18S241A: 1058 ± 42, n = 46; M18S241D: 458 ± 19, n=40, ** p < 0.01, ***p<0.001).


( H ) Western Blot of lysates from high - density neuronal cultures from munc18 - 1 null mutant mice rescued with M18WT , M18S241A or M18S241D . Prior to lysis , were treated for 6 h with MG132 ( 10 M ) or DMSO as control . Western Blots were stained for Munc18 - 1 tubulin as loading control . ( I ) of ( H ) . Munc18 - 1 were normalized to tubulin . Effect of proteasome inhibition was quantified as between MG132 DMSO treatment of each mutant ( M18WT : 1 . 62 ± 0 . 41 ; M18S241A : 1 . 26 ± 0 . 16 ; M18S241D : 7 . 53 ± 1 . 17 , n = 3 ; * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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) Western Blot of lysates from high-density neuronal cultures from munc18-1 null mutant mice rescued with M18WT, M18S241A or M18S241D. Prior to lysis, cells were treated for 6 h with MG132 (10 M) or DMSO as control. Western Blots were stained for Munc18-1 and tubulin as loading control.(I) Quantification of (H). Munc18-1 levels were normalized to tubulin levels. Effect of proteasome inhibition was quantified as ratio between MG132 and DMSO treatment of each mutant (M18WT: 1.62 ± 0.41; M18S241A: 1.26 ± 0.16; M18S241D: 7.53 ± 1.17, n = 3; **p<0.01).


( J ) Ubiquitination of different Munc18 - 1 mutants in HEK293T . Western Blot of denatured Munc18 - 1 immuno - precipitations from expressing Munc18 - 1 mutants HA - tagged ubiquitin . Prior to lysis , were treated for 8 h with MG132 ( 10 M ) . Ubiquitin covalently attached to Munc18 - 1 can be observed as a smear above characteristic 68 kD Munc18 - 1 band on Western blots stained for HA - tag . ( K ) of immuno - precipitation in ( J ) . Level of Munc18 - 1 ubiquitination was quantified as of immunoprecipitated ubiquitin Munc18 - 1 . M18WT was set to 1 ( M18WT : 1 ± 0 . 06 ; M18S241A : 0 . 84 ± 0 . 57 ; M18S241D : 2 . 70 ± 0 . 07 , n = 3 ; * * p < 0 . 01 ) . data are expressed as mean ± .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Ubiquitination of different Munc18-1 mutants in HEK293T cells. Western Blot of denatured Munc18-1 immuno-precipitations from cells expressing Munc18-1 mutants and HA-tagged ubiquitin. Prior to lysis, cells were treated for 8 h with MG132 (10 M). Ubiquitin covalently attached to Munc18-1 can be observed as a smear above the characteristic 68 kD Munc18-1 band on Western blots stained for HA-tag.(K) Quantification of immuno-precipitation in (J). Level of Munc18-1 ubiquitination was quantified as ratio of immunoprecipitated ubiquitin and Munc18-1 levels. M18WT was set to 1 (M18WT: 1 ± 0.06; M18S241A: 0.84 ± 0.57; M18S241D: 2.70 ± 0.07, n = 3; **p<0.01). All data are expressed as mean ± SEM.


( A ) Western blot from whole lysate of munc18 - 1 null mutant neurons rescued with M18WT incubated with WIN55 , 212 - 2 ( 5 M ) for 30 s . were harvested after indicated time . CB1R activation leads to a rapid transient increase in pERK activity .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(A) Western blot from whole cell lysate of munc18-1 null mutant neurons rescued with M18WT and incubated with WIN55,212-2 (5 M) for 30 s. Cells were harvested after the indicated time. CB1R activation leads to a rapid and transient increase in pERK activity.


( B - D ) Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A . CB1R agonist WIN55 , 212 - 2 ( 5 M ) was applied at t = 60 s for 30 s . ( B ) Amplitude of excitatory synapse response normalized to t = 0 s . Neurons were stimulated by a single action potential every 30 s for 4 min ( M18WTn = 21 , M18S241An = 24 , * p < 0 . 05 ) . Note broken y - axis . ( C ) of frequency of spontaneous release events after ( t = 180 s ) before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 58 . 0 ± 2 . 3 % , n = 21 ; M18S241A : 84 . 2 ± 5 . 6 % , n = 24 , * p < 0 . 05 ) . ( D ) of amplitude of spontaneous release events after ( t = 240 s ) before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 85 . 0 ± 2 . 7 % , n = 21 ; M18S241A : 83 . 1 ± 2 . 6 % , n = 24 ) .

O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O 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-D) Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A. CB1R agonist WIN55,212-2 (5 M) was applied at t = 60 s for 30 s.(B) Amplitude of excitatory synapse response normalized to t=0 s. Neurons were stimulated by a single action potential every 30 s for 4 min (M18WTn = 21, M18S241An = 24, *p<0.05). Note the broken y-axis.(C) Ratio of the frequency of spontaneous release events after (t = 180 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 58.0 ± 2.3 %, n = 21; M18S241A: 84.2 ± 5.6 %, n = 24, * p < 0.05).(D) Ratio of amplitude of spontaneous release events after (t=240 s) and before (t=0 s) WIN55,212-2 application (M18WT: 85.0 ± 2.7 %, n = 21; M18S241A: 83.1 ± 2.6 %, n = 24).


( E - G ) Autaptic neurons of munc18 - 1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55 , 212 - 2 ( 1 M ) , WIN55 , 212 - 2 ( 1 M ) plus AM251 ( 1 M ) or vehicle only ( DMSO ) . ( E ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) or vehicle only ( DMSO ) were applied at t = 60 s for 30 s . ( WT + WIN n = 13 ; WT + DMSO n = 16 , * p < 0 . 05 ) . Note broke Y - axis . ( F ) Paired ( 20 ms interval ) at t = 0 t = 240 s in M18WT neurons superfused with CB1R agonist WIN55 , 212 - 2 ( 1 M ) at t = 60 s for 30 s . WIN55 , 212 - 2 application increases PP ratios indicative of a reduction of synaptic release probability ( t = 0 s : 0 . 94 ± 0 . 1 , n = 13 ; t = 240 s : 1 . 36 ± 0 . 2 , n = 16 , * p < 0 . 05 ) . ( G ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) was applied together with CB1R antagonist AM251 ( 1 M ) at t = 60 s for 30 s . ( WT + WIN n = 13 , same trace as 6F ; WT + WIN + AM251 n = 15 , * p < 0 . 05 ) . Note broke Y - axis .

O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-CELL_TYPE O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(E-G) Autaptic neurons of munc18-1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55,212-2 (1 M), WIN55,212-2 (1 M) plus AM251 (1 M) or vehicle only (DMSO).(E) Amplitude of excitatory synapse response normalized to t=0 s. CB1R agonist WIN55,212-2 (1 M) or vehicle only (DMSO) were applied at t=60 s for 30 s. (WT + WIN n = 13; WT + DMSO n = 16, * p < 0.05). Note the broke Y-axis.(F) Paired pulse ratio (20 ms interval) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55,212-2 (1 M) at t = 60 s for 30 s. WIN55,212-2 application increases PP ratios indicative of a reduction of synaptic release probability (t = 0 s: 0.94 ± 0.1, n = 13; t = 240 s: 1.36 ± 0.2, n = 16, * p < 0.05).(G) Amplitude of excitatory synapse response normalized to t = 0 s. CB1R agonist WIN55,212-2 (1 M) was applied together with the CB1R antagonist AM251 (1 M) at t = 60 s for 30 s. (WT + WIN n = 13, same trace as 6F; WT + WIN + AM251 n = 15, * p < 0.05). Note the broke Y-axis.


( H - J ) Hippocampal autaptic neurons of munc18 - 1 null mutant mice were rescued with M18WT or M18S241A . mGluR2 / 3 agonist LY379268 ( 1 M ) was applied by bath - perfusion for 4 min . ( H ) of amplitude of evoked release events after ( t = 240 s ) before ( t = 0 s ) LY379268 application ( M18WT : 55 . 0 ± 4 . 7 % , n = 14 ; M18S241A : 81 . 1 ± 3 . 6 % , n = 16 , * p < 0 . 05 ) . ( I ) of frequency of spontaneous release events after ( t = 240 s ) before ( t = 0 s ) LY379268 application ( M18WT : 63 . 0 ± 5 . 7 % , n = 14 ; M18S241A : 85 . 1 ± 3 . 4 % , n = 16 , * p < 0 . 05 ) . ( J ) of amplitude of spontaneous release events after ( t = 240 s ) before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 81 . 0 ± 1 . 3 % , n = 14 ; M18S241A : 82 . 1 ± 3 . 3 % , n = 16 ) .

O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O 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-J) Hippocampal autaptic neurons of munc18-1 null mutant mice were rescued with M18WT or M18S241A. mGluR2/3 agonist LY379268 (1 M) was applied by bath-perfusion for 4 min.(H) Ratio of amplitude of evoked release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 55.0 ± 4.7 %, n = 14; M18S241A: 81.1 ± 3.6 %, n = 16, * p < 0.05).(I) Ratio of frequency of spontaneous release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 63.0 ± 5.7 %, n = 14; M18S241A: 85.1 ± 3.4 %, n = 16, * p < 0.05).(J) Ratio of the amplitude of spontaneous release events after (t = 240 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 81.0 ± 1.3 %, n = 14; M18S241A: 82.1 ± 3.3 %, n = 16).


( A - D ) DSE duration amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre - treatment with PD98059 ( PD ; 10 µM ) compared to 0 . 1 % DMSO ( control ) . ( A ) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between DMSO PD pretreated groups ( DMSO : 196 . 36 + / - 16 . 51 pA , n = 10 ; PD : 196 . 42 + / - 18 . 61 pA , n = 11 , p = 0 . 9 ) . ( B ) DSE in neurons of DMSO pre - treated slices , induced by a 10s depolarization to 0 mV ( arrow ) . Inset : example traces of 2 averaged EPSCs before ( black ) after depolarization ( grey ) . ( C ) DSE in slices pre - treated with PD . Insets : example traces of 2 averaged EPSCs before ( red ) after depolarization ( grey ) . ( D ) Comparison of DSE between control PD . PD pre - treatment limits amplitude duration of DSE . * * = p < 0 . 01 ; unpaired T - with Welch ' s correction . Numbers in bars represent number of neurons in each group .

O O O O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O 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-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O

(A-D) DSE duration and amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre-treatment with PD98059 (PD; 10 µM) compared to 0.1% DMSO (control).(A) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 196.36 +/- 16.51 pA, n = 10; PD: 196.42 +/- 18.61 pA, n = 11, p = 0.9).(B) DSE in neurons of DMSO pre-treated slices, induced by a 10s depolarization to 0 mV (arrow). Inset: example traces of 2 averaged EPSCs before (black) and after depolarization (grey).(C) DSE in slices pre-treated with PD. Insets: example traces of 2 averaged EPSCs before (red) and after depolarization (grey).(D) Comparison of DSE between control and PD. PD pre-treatment limits amplitude and duration of DSE. ** = p < 0.01; unpaired T-test with Welch's correction. Numbers in bars represent the number of neurons in each group.