D . Spleen , mesenteric and lamina propria lymph node cells were isolated from the mice 10 weeks after adoptive T cell transfer , and the number of Teff cells was determined by flow cytometry .

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D. Spleen, mesenteric and lamina propria lymph node cells were isolated from the mice 10 weeks after adoptive T cell transfer, and the number of Teff cells was determined by flow cytometry.


E . Expression of FOXP3 protein and mRNA by adoptively transferred Tregs . Mesenteric lymph nodes were excised from the recipients of WT and USP44 - / - Tregs in A . FOXP3 protein was detected by intracellular immunostaining and flow cytometry ( left ) . Shown are events within the gate for transferred Tregs ( CD4 + / CD45 . 1 + ) . Transferred Tregs were also recovered from mesenteric lymph node cell suspensions by staining for the congenic marker and CD4 followed by FACS , and Foxp3 mRNA was measured in these recovered Tregs by RT - PCR ( right ) .

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E. Expression of FOXP3 protein and mRNA by adoptively transferred Tregs. Mesenteric lymph nodes were excised from the recipients of WT and USP44-/- Tregs in A. FOXP3 protein was detected by intracellular immunostaining and flow cytometry (left). Shown are events within the gate for transferred Tregs (CD4+/CD45.1+). Transferred Tregs were also recovered from mesenteric lymph node cell suspensions by staining for the congenic marker and CD4 followed by FACS, and Foxp3 mRNA was measured in these recovered Tregs by RT-PCR (right).


F . Cell numbers of injected ( CD45 . 2 + ) Treg cells and FOXP3 + cells were determined by flow cytometry .

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F. Cell numbers of injected (CD45.2+) Treg cells and FOXP3+ cells were determined by flow cytometry.


G . CD4 + T cells were recovered from suspensions of lamina propria - infiltrating leukocytes . These lymphocytes were stimulated ex vivo by PMA / Ionomycin cocktail in the presence of brefeldin A and IFN - γ and IL - 17 production by these lymphocytes was analyzed by ELISA .

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G. CD4+ T cells were recovered from suspensions of lamina propria-infiltrating leukocytes. These lymphocytes were stimulated ex vivo by PMA/Ionomycin cocktail in the presence of brefeldin A and IFN-γ and IL-17 production by these lymphocytes was analyzed by ELISA.


MC38 colon carcinoma , B16F10 melanoma cell lines were s . c . injected into the shaved flanks of Usp44fl / flFoxp3Cre + mice and their wild type ( Usp44fl / flFoxp3Cre - ) littermates ( 1x105 cells / mouse ; n = 5 - 9 mice / group ) . Tumor dimensions were measured every 2 days , and changes in the mean tumor volume over time were observed ( lower panels ) . Upper panels depict representative photographs of excised tumors .

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MC38 colon carcinoma, B16F10 melanoma cell lines were s.c. injected into the shaved flanks of Usp44fl/flFoxp3Cre+ mice and their wild type (Usp44fl/flFoxp3Cre-) littermates (1x105 cells/mouse; n=5-9 mice/group). Tumor dimensions were measured every 2 days, and changes in the mean tumor volume over time were observed (lower panels). Upper panels depict representative photographs of excised tumors.


EL4 thymoma cell lines were s . c . injected into the shaved flanks of Usp44fl / flFoxp3Cre + mice and their wild type ( Usp44fl / flFoxp3Cre - ) littermates ( 1x105 cells / mouse ; n = 5 - 9 mice / group ) . Tumor dimensions were measured every 2 days , and changes in the mean tumor volume over time were observed ( lower panels ) . Upper panels depict representative photographs of excised tumors .

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EL4 thymoma cell lines were s.c. injected into the shaved flanks of Usp44fl/flFoxp3Cre+ mice and their wild type (Usp44fl/flFoxp3Cre-) littermates (1x105 cells/mouse; n=5-9 mice/group). Tumor dimensions were measured every 2 days, and changes in the mean tumor volume over time were observed (lower panels). Upper panels depict representative photographs of excised tumors.


D . 21 days post - implantation , proportions of FOXP3 + Tregs in the CD4 + compartment of all MC38 tumor - bearing mice in each experiment were found by flow cytometry . Suspensions of cells obtained from spleen , tumor - draining lymph nodes , and tumor - infiltrating leukocytes ( TILs ) were stained for surface CD4 and intracellular FOXP3 prior to analysis by flow cytometry .

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D. 21 days post-implantation, proportions of FOXP3+ Tregs in the CD4+ compartment of all MC38 tumor-bearing mice in each experiment were found by flow cytometry. Suspensions of cells obtained from spleen, tumor-draining lymph nodes, and tumor-infiltrating leukocytes (TILs) were stained for surface CD4 and intracellular FOXP3 prior to analysis by flow cytometry.


E , F . Frequencies of CD4 + and CD8 + cells producing the proinflammatory cytokine IFNγ were found among the tissues of tumor - bearing mice in the MC38 modelby intracellular cytokine staining Kralovics et al ( , 2005 ) and flow cytometry after ex vivo re - stimulation of recovered leukocytes by PMA and ionomycin in the presence of brefeldin A .

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E, F. Frequencies of CD4+ and CD8+cells producing the proinflammatory cytokine IFNγ were found among the tissues of tumor-bearing mice in the MC38 modelby intracellular cytokine staining Kralovics et al(, 2005) and flow cytometry after ex vivo re-stimulation of recovered leukocytes by PMA and ionomycin in the presence of brefeldin A.


G , H . The capacity of CD8 + cells for effector function in MC38 tumors was further assessed by measuring ( G ) production of TNFα by ICS and ( H ) intracellular straining of the cytotoxic mediator granzyme B followed by flow cytometry .

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G, H. The capacity of CD8+ cells for effector function in MC38 tumors was further assessed by measuring (G) production of TNFα by ICS and (H) intracellular straining of the cytotoxic mediator granzyme B followed by flow cytometry.


Subcellular localization of dLap1mGFP and dTormGFP - E177Q in the wild - type ( w - ) or dLap1 + / ∆ 11 fat body of 3rd instar fly larvae , when expressed as UAS transgenes .

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Subcellular localization of dLap1mGFP and dTormGFP-E177Q in the wild-type (w-) or dLap1+/∆11 fat body of 3rd instar fly larvae, when expressed as UAS transgenes.


Neutral lipid staining of the 5DO larval fat body . White arrows ( O ) indicate nuclear - localized neutral lipid staining , blue signal shows DAPI .

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Neutral lipid staining of the 5DO larval fat body. White arrows (O) indicate nuclear-localized neutral lipid staining, blue signal shows DAPI.


GFP , Phalloidin and LipidTOX detect the expression of dTormGFP chimeras ( green ) , cell boundaries ( red ) , and LD ( red ) , respectively , in dTorKO fat body cells .

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GFP, Phalloidin and LipidTOX detect the expression of dTormGFP chimeras (green), cell boundaries (red), and LD (red), respectively, in dTorKO fat body cells.


Bars show the fraction ( mean ± SEM ) of membrane GL and GPL lipids with each fatty acyl chain characteristic . n = 3 MS analyses of 4DO fat body samples for each genotype . MUFA , mono - unsaturated ; PUFA , poly - unsaturated . ‡ indicate a significant difference between w - and dTorKO expressing Luciferase RNAi , * indicate a significant difference compared to dTorKO expressing Luciferase RNAi . Two - way ANOVA , Dunnett ' s multiple comparison test . ‡ p < 0 . 05 and * p < 0 . 05 .

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Bars show the fraction (mean ± SEM) of membrane GL and GPL lipids with each fatty acyl chain characteristic. n = 3 MS analyses of 4DO fat body samples for each genotype. MUFA, mono-unsaturated; PUFA, poly-unsaturated. ‡ indicate a significant difference between w- and dTorKO expressing Luciferase RNAi, * indicate a significant difference compared to dTorKO expressing Luciferase RNAi. Two-way ANOVA, Dunnett's multiple comparison test. ‡ p < 0.05 and * p < 0.05.


Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg - Gal4 driver , and labeled with anti - Calnexin ( red ) and a neutral lipid dye ( LipidTOX , magenta ) . Note that dTorKO cells are smaller . White arrowhead indicates NE enrichment of LipinmGFP .

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Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg-Gal4 driver, and labeled with anti-Calnexin (red) and a neutral lipid dye (LipidTOX, magenta). Note that dTorKO cells are smaller. White arrowhead indicates NE enrichment of LipinmGFP.


Lipin1mGFP localization in HEK293T cells transfected with a plasmid that co - expresses CTDNEP1myc - WT or - CD ( red ) and NEP1R1V5 ( magenta ) . ( G ' ) Upper : enlargement of panel G , white arrow highlights NE enrichment . Lower : plot showing the intensity of Lipin1mGFP signal ( mean ± SD , n = 5 cells ) along a 3 µm profile that transects the NE at 0 µm .

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Lipin1mGFP localization in HEK293T cells transfected with a plasmid that co-expresses CTDNEP1myc-WT or -CD (red) and NEP1R1V5 (magenta). (G') Upper: enlargement of panel G, white arrow highlights NE enrichment. Lower: plot showing the intensity of Lipin1mGFP signal (mean ± SD, n = 5 cells) along a 3 µm profile that transects the NE at 0 µm.


Percentage of MEFs with primarily cytosolic or nuclear Lipin1mGFP . Chi - square test . * * * * p < 0 . 0001 .

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Percentage of MEFs with primarily cytosolic or nuclear Lipin1mGFP. Chi-square test. **** p < 0.0001.


Control ( w - ) and dTorKO fat body nuclei labeled with anti - LaminDm0 , Megator / Tpr , mAb414 , or expressing UAS Nup107GFP or UAS Nup358GFP with the Cg - Gal4 driver .

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Control (w-) and dTorKO fat body nuclei labeled with anti-LaminDm0, Megator/Tpr, mAb414, or expressing UAS Nup107GFP or UAS Nup358GFP with the Cg-Gal4 driver.


mAb414 labeling of 5DO dTorKO fat body cells expressing the dTor cDNA , Luciferase RNAi , or RNAi against Nep1r1 , Ctdnep1 , or Lipin . Nuc , nucleus . The percentage of fat body cells with NE - localized mAb414 . * * and * * * * indicate that Lipin , Ctdnep1 , and Nep1r1 RNAi significantly increased the number of dTorKO cells with NE - specific mAb414 labeling compared to the Luciferase RNAi ( Luc ) . Chi - square test followed by individual post hoc Chi - square tests . * * p < 0 . 01 and * * * * p < 0 . 0001 . NE ultrastructure in 5DO dTorKO cells . Red arrows , nuclear pores ; yellow arrows , abnormal INM herniations . Nuc , nucleus ; Cyto , cytosol .

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mAb414 labeling of 5DO dTorKO fat body cells expressing the dTor cDNA, Luciferase RNAi, or RNAi against Nep1r1, Ctdnep1, or Lipin. Nuc, nucleus. The percentage of fat body cells with NE-localized mAb414. ** and **** indicate that Lipin, Ctdnep1, and Nep1r1 RNAi significantly increased the number of dTorKO cells with NE-specific mAb414 labeling compared to the Luciferase RNAi (Luc). Chi-square test followed by individual post hoc Chi-square tests. ** p < 0.01 and **** p < 0.0001. NE ultrastructure in 5DO dTorKO cells. Red arrows, nuclear pores; yellow arrows, abnormal INM herniations. Nuc, nucleus; Cyto, cytosol.


The percentage that Lipin , Ctdnep1 , and Nep1r1 RNAi normalize the abundance of membrane GL and GPL classes in the dTorKO fat body . 0 % " normal " reflects lipid abundance in dTorKO expressing Luciferase RNAi , while 100 % " normal " reflects lipid abundance in w - expressing Luciferase RNAi . Percentage normalization was separately calculated for PI , PA , DAG , PC , PE and PS ( for dTorKO expressing Lipin , Ctdnep1 , and Nep1r1 RNAi ) . Points show the mean of all lipid class normalization per sample , bars show mean ± SEM for the group .

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The percentage that Lipin, Ctdnep1, and Nep1r1 RNAi normalize the abundance of membrane GL and GPL classes in the dTorKO fat body. 0% "normal" reflects lipid abundance in dTorKO expressing Luciferase RNAi, while 100% "normal" reflects lipid abundance in w- expressing Luciferase RNAi. Percentage normalization was separately calculated for PI, PA, DAG, PC, PE and PS (for dTorKO expressing Lipin, Ctdnep1, and Nep1r1 RNAi). Points show the mean of all lipid class normalization per sample, bars show mean ± SEM for the group.


NE ultrastructure of 5DO fat body cells overexpressing UAS transgenes , or from dLap1 - / - animals . Yellow dotted lines highlight NE regions with subtly abnormal membrane architecture . Red arrows indicate nuclear pores . R1 , region 1 ; R2 , region2 ; Nuc , nucleus ; Cyto ,

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NE ultrastructure of 5DO fat body cells overexpressing UAS transgenes, or from dLap1-/- animals. Yellow dotted lines highlight NE regions with subtly abnormal membrane architecture. Red arrows indicate nuclear pores. R1, region 1; R2, region2; Nuc, nucleus; Cyto, cytosol


Data from FIB - SEM imaging of fat body nuclei expressing Sec61b ( control ) , or CTDNEP1mGFP . ( J - L ) 3D reconstructions , ( M ) distance between the INM and ONM at NPC in wild - type cells versus CTDNEP1mGFP - induced structures . Kolmogorov - Smirnov non - parametric T - Test . * * * * p < 0 .

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Data from FIB-SEM imaging of fat body nuclei expressing Sec61b (control), or CTDNEP1mGFP. (J - L) 3D reconstructions, (M) distance between the INM and ONM at NPC in wild-type cells versus CTDNEP1mGFP-induced structures. Kolmogorov-Smirnov non-parametric T-Test. **** p < 0.0001


mAb414 localization in 5DO fat body cells expressing the indicated UAS transgenes or from a dLap1 - / - animal . * * * and * * * * indicate significant difference compared to control mGFPSec61b expressing cells . Chi - square test followed by individual post hoc Chi - square tests . * * * p < 0 . 001 and * * * * p < 0 . 0001 .

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mAb414 localization in 5DO fat body cells expressing the indicated UAS transgenes or from a dLap1-/- animal. *** and **** indicate significant difference compared to control mGFPSec61b expressing cells. Chi-square test followed by individual post hoc Chi-square tests. *** p < 0.001 and **** p < 0.0001.


NE ultrastructure of 5DO fat body cells from a ( I ) control animal , ( J ) homozygous Nup35BG01311 and ( K ) homozygous Nup35MB02683 animals . Nuc , nucleus ; Cyto , cytosol .

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NE ultrastructure of 5DO fat body cells from a (I) control animal, (J) homozygous Nup35BG01311 and (K) homozygous Nup35MB02683 animals. Nuc, nucleus; Cyto, cytosol.


( a ) p35 interacts with EndoB1 in a GST pulldown assay .

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(a) p35 interacts with EndoB1 in a GST pulldown assay.


( b , c ) EndoB1 co - immunoprecipitates ( IP ) with p35 in COS - 7 cell lysates expressing p35 and full - length EndoB1 ( b ) and adult rat brain lysate ( c ) .

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(b,c) EndoB1 co-immunoprecipitates (IP) with p35 in COS-7 cell lysates expressing p35 and full-length EndoB1 (b) and adult rat brain lysate (c).


( e ) Dose - dependent phosphorylation of EndoB1 by Cdk5 / p35 in an in vitro kinase assay .

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(e) Dose-dependent phosphorylation of EndoB1 by Cdk5/p35 in an in vitro kinase assay.


( f ) Cdk5 / p35 phosphorylates EndoB1 at Thr 145 in an in vitro kinase assay .

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(f) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in an in vitro kinase assay.


( g ) Cdk5 / p35 phosphorylates EndoB1 at Thr 145 in COS - 7 cells .

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(g) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in COS-7 cells.


( h ) Cdk5 phosphorylates EndoB1 at Thr 145 in Cdk5 + / + , but not Cdk5 - / - mouse brains . Quantification of p - Thr145 - EndoB1 level is shown in the right panel . Data are means ± s . e . m . ; n = 3 . Uncropped images of blots are shown in Supplementary Fig .

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(h) Cdk5 phosphorylates EndoB1 at Thr 145 in Cdk5+/+, but not Cdk5-/- mouse brains. Quantification of p-Thr145-EndoB1 level is shown in the right panel. Data are means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8


( a ) Left : starvation increases the level of RFP - LC3 puncta ( arrows ) in rat cortical neurons . Middle : quantification of the percentage of cells with RFP - LC3 puncta . Right : pretreatment with 10 mM 3 - MA attenuates the starvation - induced increase in LC3 - II level in neurons . Data are means ± s . e . m . ; n = 3 . Samples were run on the same gel and spliced together to create the image shown .

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(a) Left: starvation increases the level of RFP-LC3 puncta (arrows) in rat cortical neurons. Middle: quantification of the percentage of cells with RFP-LC3 puncta. Right: pretreatment with 10 mM 3-MA attenuates the starvation-induced increase in LC3-II level in neurons. Data are means ± s.e.m.; n=3. Samples were run on the same gel and spliced together to create the image shown.


( b ) Knockdown of EndoB1 abolishes the starvation - induced increase in LC3 - II levels ( left ) and the percentage of cells with RFP - LC3 puncta ( right ) in neurons . Data are means ± s . e . m . ; n = 3 .

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(b) Knockdown of EndoB1 abolishes the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP-LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=3.


( c ) Starvation enhances Cdk5 activity ( top ) and increases the level of Thr 145 - phosphorylated EndoB1 in neurons ( top and bottom ) . Histone H1 was used as a Cdk5 substrate . Data are means ± s . e . m . ; n = 3 .

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(c) Starvation enhances Cdk5 activity (top) and increases the level of Thr 145-phosphorylated EndoB1 in neurons (top and bottom). Histone H1 was used as a Cdk5 substrate. Data are means ± s.e.m.; n=3.


( d , e ) Pretreatment with Ros ( d ) or knockdown of Cdk5 ( e ) attenuates starvation - induced EndoB1 phosphorylation at Thr 145 and autophagy induction in neurons . Data are means ± s . e . m . ; n = 3 for d and n = 8 for e . DMSO , dimethylsulphoxide .

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(d,e) Pretreatment with Ros (d) or knockdown of Cdk5 (e) attenuates starvation-induced EndoB1 phosphorylation at Thr 145 and autophagy induction in neurons. Data are means ± s.e.m.; n=3 for d and n=8 for e. DMSO, dimethylsulphoxide.


( f ) Overexpression of EndoB1T145A significantly reduces the starvation - induced increase in LC3 - II levels ( left ) and the percentage of cells with RFP - LC3 puncta ( right ) in neurons . Data are means ± s . e . m . ; n = 6 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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(f) Overexpression of EndoB1T145A significantly reduces the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP-LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=6. Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) Left : lipid binding of EndoB1 is inhibited by mutating the five lysine / arginine residues between amino acids 176 and 183 of EndoB1 to glutamic acid ( 5E mutant ) . Right : purified GST protein was included as a negative control . S , supernatant fraction ; P , pellet fraction that contains the lipid - bound proteins .

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(a) Left: lipid binding of EndoB1 is inhibited by mutating the five lysine/arginine residues between amino acids 176 and 183 of EndoB1 to glutamic acid (5E mutant). Right: purified GST protein was included as a negative control. S, supernatant fraction; P, pellet fraction that contains the lipid-bound proteins.


( b , c ) The 5E mutant of EndoB1 fails to dimerize ( b ) in 293T cells .

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(b,c) The 5E mutant of EndoB1 fails to dimerize (b) in 293T cells.


b , c ) The 5E mutant of EndoB1 fails to dimerize ( b ) and interact with UVRAG ( c ) in 293T cells .

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b,c) The 5E mutant of EndoB1 fails to dimerize (b) and interact with UVRAG (c) in 293T cells.


( d ) Overexpression of the 5E mutant of EndoB1 blocks starvation - induced autophagy in cortical neurons . Data are means ± s . e . m . ; n = 3 .

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(d) Overexpression of the 5E mutant of EndoB1 blocks starvation-induced autophagy in cortical neurons. Data are means ± s.e.m.; n=3.


( e ) Mutation of Thr 145 to alanine ( T145A ) or glutamic acid ( T145E ) has negligible effect on the lipid - binding property of EndoB1 . Conversely , the 5E mutant is less able to bind lipid . The right panel shows quantified data as means ± s . e . m . ; n = 3 .

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(e) Mutation of Thr 145 to alanine (T145A) or glutamic acid (T145E) has negligible effect on the lipid-binding property of EndoB1. Conversely, the 5E mutant is less able to bind lipid. The right panel shows quantified data as means ± s.e.m.; n=3.


( f ) Starvation increases co - localization of GFP - EndoB1 ( green ) with Atg5 ( red ) in neurons . The T145A mutation of GFP - EndoB1 does not affect its co - localization with Atg5 - positive entities . Arrows denote GFP - EndoB1 - and Atg5 - positive vesicles . The right panel shows quantified data as means ± s . e . m . ; n = 9 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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(f) Starvation increases co-localization of GFP-EndoB1 (green) with Atg5 (red) in neurons. The T145A mutation of GFP-EndoB1 does not affect its co-localization with Atg5-positive entities. Arrows denote GFP-EndoB1- and Atg5- positive vesicles. The right panel shows quantified data as means ± s.e.m.; n=9. Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) Overexpression of Cdk5 / p35 enhances EndoB1 dimerization in 293T cells . Samples were run on the same gel and spliced together to create the image

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(a) Overexpression of Cdk5/p35 enhances EndoB1 dimerization in 293T cells. Samples were run on the same gel and spliced together to create the image shown


( b ) Thr 145 phosphorylation of EndoB1 facilitates its dimerization in 293T cells .

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(b) Thr 145 phosphorylation of EndoB1 facilitates its dimerization in 293T cells.


( c ) Diminished interaction between EndoB1 and UVRAG in Cdk5 − / − mouse brain lysates .

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(c) Diminished interaction between EndoB1 and UVRAG in Cdk5−/− mouse brain lysates.


( d , e ) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG ( d ) in 293T cells .

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(d,e) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG (d) in 293T cells.


( d , e ) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG ( d ) and Beclin 1 ( e ) in 293T cells .

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(d,e) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG (d) and Beclin 1 (e) in 293T cells.


( f , g ) Gel - filtration analysis on the membrane fraction of mouse brain lysates . Fractions obtained were subjected to immunoblot analysis ( f ) , or immunoprecipitation followed by immunoblotting ( g ) . Uncropped images of blots are shown in Supplementary Fig . S8 .

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(f,g) Gel-filtration analysis on the membrane fraction of mouse brain lysates. Fractions obtained were subjected to immunoblot analysis (f), or immunoprecipitation followed by immunoblotting (g). Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) MPTP administration decreases the numbers of tyrosine hydroxylase ( TH ) - and cresyl violet ( CV ) - positive neurons in the substantia nigra . Representative images of tyrosine hydroxylase - stained brain sections are shown . Right panels show quantified data as means ± s . e . m . ; n = 9 for tyrosine hydroxylase and n = 3 for cresyl violet .

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(a) MPTP administration decreases the numbers of tyrosine hydroxylase (TH)- and cresyl violet (CV)-positive neurons in the substantia nigra. Representative images of tyrosine hydroxylase-stained brain sections are shown. Right panels show quantified data as means ± s.e.m.; n=9 for tyrosine hydroxylase and n=3 for cresyl violet.


( b ) MPTP administration upregulates the levels of LC3 - II and Thr 145 - phosphorylated EndoB1 in the midbrain . Lower panels show quantified data as means ± s . e . m . ; n = 3 .

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(b) MPTP administration upregulates the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the midbrain. Lower panels show quantified data as means ± s.e.m.; n=3.


( c ) Knockdown of EndoB1 or Cdk5 inhibits MPP + - induced autophagy in cultured neurons . Data are means ± s . e . m . ; n = 5 .

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(c) Knockdown of EndoB1 or Cdk5 inhibits MPP+-induced autophagy in cultured neurons. Data are means ± s.e.m.; n=5.


( d ) Cdk5 mediates the MPP + - induced EndoB1 phosphorylation at Thr 145 and the MPP + - induced increase in the LC3 - II level in neurons .

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(d) Cdk5 mediates the MPP+-induced EndoB1 phosphorylation at Thr 145 and the MPP+-induced increase in the LC3-II level in neurons.


( e ) Overexpression of EndoB1T145A attenuates MPP + - induced autophagy in neurons . Data are means ± s . e . m . ; n = 7 .

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(e) Overexpression of EndoB1T145A attenuates MPP+-induced autophagy in neurons. Data are means ± s.e.m.; n=7.


( f ) MPTP fails to increase LC3 - II and Thr 145 - phosphorylated EndoB1 levels in the midbrain of p35 − / − mice . The concomitant reduction in tyrosine hydroxylase level triggered by MPTP injection is also markedly attenuated . Right panels show quantified data as means ± s . e . m . ; n = 3 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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(f) MPTP fails to increase LC3-II and Thr 145-phosphorylated EndoB1 levels in the midbrain of p35−/− mice. The concomitant reduction in tyrosine hydroxylase level triggered by MPTP injection is also markedly attenuated. Right panels show quantified data as means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) Upregulation of the levels of LC3 - II and Thr 145 - phosphorylated EndoB1 in the cerebellum and striatum of α - synucleinA53T transgenic mice . Right panels show quantified data as means ± s . e . m . ; n = 3 .

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(a) Upregulation of the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the cerebellum and striatum of α-synucleinA53T transgenic mice. Right panels show quantified data as means ± s.e.m.; n=3.


b ) Overexpression of α - synucleinA53T mutant upregulates LC3 - II level and Thr 145 - phosphorylated EndoB1 in cultured neurons . Lower panel shows quantified data as means ± s . e . m . ; n = 3 .

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b) Overexpression of α-synucleinA53T mutant upregulates LC3-II level and Thr 145-phosphorylated EndoB1 in cultured neurons. Lower panel shows quantified data as means ± s.e.m.; n=3.


( c ) Knockdown of EndoB1 or Cdk5 inhibits α - synucleinA53T mutant - induced autophagy induction in neurons . Data are means ± s . e . m . ; n = 7 .

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(c) Knockdown of EndoB1 or Cdk5 inhibits α-synucleinA53T mutant-induced autophagy induction in neurons. Data are means ± s.e.m.; n=7.


( d ) Cdk5 mediates α - synucleinA53T mutant - induced Thr 145 phosphorylation of EndoB1 and the increase of LC3 - II in neurons .

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(d) Cdk5 mediates α-synucleinA53T mutant-induced Thr 145 phosphorylation of EndoB1 and the increase of LC3-II in neurons.


( e ) Overexpression of EndoB1T145A attenuates α - synucleinA53T mutant - induced autophagy in neurons . Data are means ± s . e . m . ; n = 7 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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(e) Overexpression of EndoB1T145A attenuates α-synucleinA53T mutant-induced autophagy in neurons. Data are means ± s.e.m.; n=7. Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) Pretreatment with 3 - MA , but not z - VAD - fmk , significantly reduces MPP + - induced neuronal death . Data are means ± s . e . m . ; n = 6 .

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(a) Pretreatment with 3-MA, but not z-VAD-fmk, significantly reduces MPP+-induced neuronal death. Data are means ± s.e.m.; n=6.


( b ) Knockdown of Atg5 expression abrogates MPP + - induced neuronal loss , whereas it reduces neuronal survival in untreated cells . Data are means ± s . e . m . ; n = 3 .

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(b) Knockdown of Atg5 expression abrogates MPP+-induced neuronal loss, whereas it reduces neuronal survival in untreated cells. Data are means ± s.e.m.; n=3.


( c ) Knockdown of EndoB1 or Cdk5 expression abolishes MPP + - induced neuronal death . Data are means ± s . e . m . ; n = 8 .

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(c) Knockdown of EndoB1 or Cdk5 expression abolishes MPP+-induced neuronal death. Data are means ± s.e.m.; n=8.


( d ) Overexpression of EndoB1T145A inhibits MPP + - induced neuronal death . Data are means ± s . e . m . ; n = 5 .

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(d) Overexpression of EndoB1T145A inhibits MPP+-induced neuronal death. Data are means ± s.e.m.; n=5.


( e ) Pretreatment with 3 - MA , but not z - VAD - fmk , reduces α - synucleinA53T mutant - induced neuronal death . Data are means ± s . e . m . ; n = 6 .

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(e) Pretreatment with 3-MA, but not z-VAD-fmk, reduces α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6.


( f ) Knockdown of Atg5 expression abolishes α - synucleinA53T mutant - induced neuronal death , whereas it reduces neuronal survival in vector - transfected cells . Data are means ± s . e . m . ; n = 3 .

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(f) Knockdown of Atg5 expression abolishes α-synucleinA53T mutant-induced neuronal death, whereas it reduces neuronal survival in vector-transfected cells. Data are means ± s.e.m.; n=3.


( g ) Knockdown of EndoB1 or Cdk5 expression abolishes α - synucleinA53T mutant - induced neuronal death . Data are means ± s . e . m . ; n = 4 .

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(g) Knockdown of EndoB1 or Cdk5 expression abolishes α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=4.


( h ) Expression of EndoB1T145A inhibits α - synucleinA53T mutant - induced neuronal death . Data are means ± s . e . m . ; n = 6 .

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(h) Expression of EndoB1T145A inhibits α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6.


( A ) FAK + / + and FAK − / − murine SCC cells were lysed and FAK , Ret or Src immunoprecipitated and immunoblotting carried out using anti ‐ FAK , anti ‐ Ret , anti ‐ Src and anti ‐ actin .

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(A) FAK+/+ and FAK−/− murine SCC cells were lysed and FAK, Ret or Src immunoprecipitated and immunoblotting carried out using anti‐FAK, anti‐Ret, anti‐Src and anti‐actin.


( B ) Cells were fixed and stained with either anti ‐ Ret ( green in the upper left panels ) or anti ‐ Ret PY1062 ( green in the lower left panels ) and DAPI ( blue ) . Merged and zoomed images are shown . Solid arrows indicate localization at adhesions while broken arrows show localization in puncta . Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . Lysates were immunoblotted with anti ‐ Ret PY1062 and anti ‐ actin ( lower right panels ) .

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(B) Cells were fixed and stained with either anti‐Ret (green in the upper left panels) or anti‐Ret PY1062 (green in the lower left panels) and DAPI (blue). Merged and zoomed images are shown. Solid arrows indicate localization at adhesions while broken arrows show localization in puncta. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). Lysates were immunoblotted with anti‐Ret PY1062 and anti‐actin (lower right panels).


( C ) Cells were fixed and stained with anti ‐ Ret ( green ) and anti ‐ FAK ( red ) . Merged images are also shown . Solid arrows indicate co ‐ localization between Ret and FAK at adhesions , while broken arrows indicate its absence in puncta . Scale bars , 20 μM . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection ; SCC , squamous cell carcinoma .

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(C) Cells were fixed and stained with anti‐Ret (green) and anti‐FAK (red). Merged images are also shown. Solid arrows indicate co‐localization between Ret and FAK at adhesions, while broken arrows indicate its absence in puncta. Scale bars, 20 μM. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection; SCC, squamous cell carcinoma.


Ret localizes to Src ‐ positive autophagosomes in FAK ‐ deficient SCC cells . Cells were fixed and stained with either ( A ) anti ‐ Ret ( green ) and anti ‐ PY416 Src ( red ) or ( B ) anti ‐ PY416 Src ( red ) and anti ‐ LC3B ( green ) . Merged and zoomed images are shown . Solid arrows indicate co ‐ localization at adhesions , while broken arrows show co ‐ localization in intracellular puncta .

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Ret localizes to Src‐positive autophagosomes in FAK‐deficient SCC cells. Cells were fixed and stained with either (A) anti‐Ret (green) and anti‐PY416 Src (red) or (B) anti‐PY416 Src (red) and anti‐LC3B (green). Merged and zoomed images are shown. Solid arrows indicate co‐localization at adhesions, while broken arrows show co‐localization in intracellular puncta.


( C ) Cells were transfected with scrambled , Atg5 or Atg7 siRNA , trypsinized and allowed to readhere before immunoblotting with anti ‐ Atg5 , anti ‐ Atg7 and anti ‐ actin ( right panels ) . Cells were fixed and stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) and DAPI ( blue ) . Merged images are shown . Solid arrows indicate co ‐ localization between Ret and paxillin at adhesions , while broken arrows indicate its absence in puncta .

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(C) Cells were transfected with scrambled, Atg5 or Atg7 siRNA, trypsinized and allowed to readhere before immunoblotting with anti‐Atg5, anti‐Atg7 and anti‐actin (right panels). Cells were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Merged images are shown. Solid arrows indicate co‐localization between Ret and paxillin at adhesions, while broken arrows indicate its absence in puncta.


( D ) Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) .

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(D) Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3).


( E ) Cells , either untreated or treated with chloroquine for 24 h , were fixed and stained with anti ‐ Ret ( green ) and DAPI ( blue ) . Scale bars , 20 μM . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection ; SCC , squamous cell carcinoma ; siRNA , short interfering RNA .

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(E) Cells, either untreated or treated with chloroquine for 24 h, were fixed and stained with anti‐Ret (green) and DAPI (blue). Scale bars, 20 μM. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection; SCC, squamous cell carcinoma; siRNA, short interfering RNA.


( A ) FAK − / − cells stably re ‐ expressing FAK ‐ wt , FAK ‐ Y397F , FAK ‐ KD or FAK ‐ Y4F ‐ Y9F were immunoblotted using anti ‐ FAK , anti ‐ FAK Y397 , anti ‐ FAK Y861 , anti ‐ FAK Y925 , anti ‐ Src , anti ‐ Ret and anti ‐ actin . FAK and Ret were immunoprecipitated from these cells and then samples were immunoblotted with anti ‐ Src , anti ‐ FAK or anti ‐ Ret .

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(A) FAK−/− cells stably re‐expressing FAK‐wt, FAK‐Y397F, FAK‐KD or FAK‐Y4F‐Y9F were immunoblotted using anti‐FAK, anti‐FAK Y397, anti‐FAK Y861, anti‐FAK Y925, anti‐Src, anti‐Ret and anti‐actin. FAK and Ret were immunoprecipitated from these cells and then samples were immunoblotted with anti‐Src, anti‐FAK or anti‐Ret.


( B ) Cells were fixed and stained with anti ‐ Ret ( green ) and either anti ‐ FAK ( red in the upper panels ) or anti ‐ PY416 Src ( red in the lower panels ) . Merged images are shown . Solid arrows indicate Ret in adhesions , while broken arrows show Ret in puncta . Scale bars , 20 μM .

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(B) Cells were fixed and stained with anti‐Ret (green) and either anti‐FAK (red in the upper panels) or anti‐PY416 Src (red in the lower panels). Merged images are shown. Solid arrows indicate Ret in adhesions, while broken arrows show Ret in puncta. Scale bars, 20 μM.


( C ) Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . FAK , focal adhesion kinase ; Ret , rearranged during transfection .

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(C) Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). FAK, focal adhesion kinase; Ret, rearranged during transfection.


( A ) FAK + / + cells were treated with dasatinib , then lysates were immunoblotted with anti ‐ FAK , anti ‐ FAK Y397 , anti ‐ FAK Y925 , anti ‐ PY416 Src , anti ‐ Src and anti ‐ actin . FAK was immunoprecipitated and then immunoblotting performed using anti ‐ Src , anti ‐ FAK or anti ‐ Ret .

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(A) FAK+/+ cells were treated with dasatinib, then lysates were immunoblotted with anti‐FAK, anti‐FAK Y397, anti‐FAK Y925, anti‐PY416 Src, anti‐Src and anti‐actin. FAK was immunoprecipitated and then immunoblotting performed using anti‐Src, anti‐FAK or anti‐Ret.


( B ) Cells were treated with dasatinib , then fixed and stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) and DAPI ( blue ) . Solid arrows indicate co ‐ localization in adhesions . Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . ( C ) FAK − / − cells treated with dasatinib were fixed and stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) and DAPI ( blue ) . Solid arrows indicate co ‐ localization in adhesions , while broken arrows show lack of co ‐ localization in puncta . Scale bars , 20 μM . Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) .

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

(B) Cells were treated with dasatinib, then fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). (C) FAK−/− cells treated with dasatinib were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions, while broken arrows show lack of co‐localization in puncta. Scale bars, 20 μM. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3).


( D ) Lysates from untreated and dasatinib ‐ treated FAK − / − cells were immunoblotted with anti ‐ Ret , anti ‐ PY416 Src , anti ‐ Src and anti ‐ actin . Ret was also immunoprecipitated from cells and then immunoblotted using anti ‐ Src and anti ‐ Ret . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection .

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(D) Lysates from untreated and dasatinib‐treated FAK−/− cells were immunoblotted with anti‐Ret, anti‐PY416 Src, anti‐Src and anti‐actin. Ret was also immunoprecipitated from cells and then immunoblotted using anti‐Src and anti‐Ret. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection.


B Trans - activation activity assays of CDF4 in Arabidopsis protoplasts . The full - length ORF of CDF4 and its four deletion constructs Dof ( gray boxes ) represent the DNA - binding domains of CDF4 . Negative control was the effector vector without gene inserts . The effector vector used is shown in the left panel . The GAL4 transient expression assays were performed using Arabidopsis protoplasts , shown in the right panel . Deletion of the CDF4 N - terminal domain compromised the transcriptional repression by CDF4 in our assay . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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

B Trans-activation activity assays of CDF4 in Arabidopsis protoplasts. The full-length ORF of CDF4 and its four deletion constructs Dof (gray boxes) represent the DNA-binding domains of CDF4. Negative control was the effector vector without gene inserts. The effector vector used is shown in the left panel. The GAL4 transient expression assays were performed using Arabidopsis protoplasts, shown in the right panel. Deletion of the CDF4 N-terminal domain compromised the transcriptional repression by CDF4 in our assay. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


C Localized expression of CDF4 in a senescing rosette leaf . The ~ 50 % senesced rosette leaf was cut into three parts as indicated by the red lines . The level of CDF4 mRNA was determined in the three sections of the senescing leaf . Base ; Middle ; Tip . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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C Localized expression of CDF4 in a senescing rosette leaf. The ~50% senesced rosette leaf was cut into three parts as indicated by the red lines. The level of CDF4 mRNA was determined in the three sections of the senescing leaf. Base; Middle; Tip. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


D CDF4 expression level at various developmental stages of rosette leaves . YL , young leaf ; NS , fully expanded , non - senescent leaf ; ES , early - senescent leaf , with 1 / 4 leaf area turned yellow ; LS , late - senescent leaf , with > 50 % leaf area turned yellow . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D CDF4 expression level at various developmental stages of rosette leaves. YL, young leaf; NS, fully expanded, non-senescent leaf; ES, early-senescent leaf, with 1/4 leaf area turned yellow; LS, late-senescent leaf, with >50% leaf area turned yellow. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


E Effect of plant growth hormones ( 1 mM SA , 1 μM ABA , 10 μM JA and 10 μM 1 - aminocyclopropane - 1 - carboxylic acid ( ACC ) on CDF4 expression . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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E Effect of plant growth hormones (1 mM SA, 1 μM ABA, 10 μM JA and 10 μM 1-aminocyclopropane -1-carboxylic acid (ACC) on CDF4 expression. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


F Effects of abiotic stress treatments on CDF4 expression . Rosette leaves were treated with salt ( 100 mM NaCl ) , drought , H2O2 ( 2mM ) or darkness . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Effects of abiotic stress treatments on CDF4 expression. Rosette leaves were treated with salt (100 mM NaCl), drought, H2O2 (2mM) or darkness. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


G Effects of SA , H2O2 , and dark treatment on CDF4 expression in the aba2 - 1 mutant background . AtACT2 was used as an internal control . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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G Effects of SA, H2O2, and dark treatment on CDF4 expression in the aba2-1 mutant background. AtACT2 was used as an internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


A The age - dependent leaf senescence symptoms in the vector control and 35S : : CDF4 lines grown under long - day conditions were shown at 21 - days - old . The 35S : : CDF4 plants had a small leaf size and extremely early leaf senescent phenotype , while the control vector leaves were fully green . Scale bars indicate 1 cm .

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

A The age-dependent leaf senescence symptoms in the vector control and 35S::CDF4 lines grown under long-day conditions were shown at 21-days-old. The 35S::CDF4 plants had a small leaf size and extremely early leaf senescent phenotype, while the control vector leaves were fully green. Scale bars indicate 1 cm.


B The increase in SAG12 , SAG13 , PR1 and PR5 gene expression levels were investigated in 35S : : CDF4 plants . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 in comparison with the vector control by Student ' s t test .

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B The increase in SAG12, SAG13, PR1 and PR5 gene expression levels were investigated in 35S::CDF4 plants. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 in comparison with the vector control by Student's t test.


C Phenotypes of vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi and pER8 : : amiCDF4 plants , after treatment with 20 µM estradiol for seven days at 18 - days - old . Scale bars indicate 1 . 5 cm .

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C Phenotypes of vector control, pER8::CDF4, pER8::CDF4-RNAi and pER8::amiCDF4 plants, after treatment with 20 µM estradiol for seven days at 18-days-old. Scale bars indicate 1.5 cm.


E Analysis of the relative chlorophyll concentration in the sixth and seventh rosette leaves from the vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi and pER8 : : amiCDF4 lines shown at various development stages . Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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

E Analysis of the relative chlorophyll concentration in the sixth and seventh rosette leaves from the vector control, pER8::CDF4, pER8::CDF4-RNAi and pER8::amiCDF4 lines shown at various development stages. Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.


D The leaf senescence phenotypes of detached rosette leaves in vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi and pER8 : : amiCDF4 lines before and after treatment with 2 mΜ H2O2 in the dark for four days . Scale bars indicate 0 . 7 cm .

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D The leaf senescence phenotypes of detached rosette leaves in vector control, pER8::CDF4, pER8::CDF4 -RNAi and pER8::amiCDF4 lines before and after treatment with 2 mΜ H2O2 in the dark for four days. Scale bars indicate 0.7 cm.


F The chlorophyll concentration in leaves was analyzed . The percentage indicates the chlorophyll content relative to vector control before treatment . Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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F The chlorophyll concentration in leaves was analyzed. The percentage indicates the chlorophyll content relative to vector control before treatment. Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.


A Measurement of free ABA levels in the third and fourth rosette leaves from 21 - day - old transgenic lines with altered CDF4 expression after estradiol induction for three days ( once every day ) . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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 Measurement of free ABA levels in the third and fourth rosette leaves from 21-day-old transgenic lines with altered CDF4 expression after estradiol induction for three days (once every day). Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


B , C qPCR analysis of ABA synthesis and signaling - related genes in Col - 0 and 35S : : CDF4 plants . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t - test .

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B, C qPCR analysis of ABA synthesis and signaling-related genes in Col-0 and 35S::CDF4 plants. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.


D , E Relative expression of NCED2 and NCED3 in 14 - day - old CDF4GR transgenic plants treated with 20 μM β - estradiol or mock treatment for 0 , 2 , or 4 h . The expression of the corresponding genes in mock - treated plants was set to 1 . 0 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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D, E Relative expression of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM β-estradiol or mock treatment for 0, 2, or 4 h. The expression of the corresponding genes in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


F , G Relative expression level of NCED2 and NCED3 in 14 - day - old CDF4GR transgenic plants treated with 20 μM DEX , 100μM CHX , DEX plus 100μM CHX , or mock . The gene expression in mock - treated plants was set to 1 . 0 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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

F, G Relative expression level of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM DEX, 100μM CHX, DEX plus 100μM CHX, or mock. The gene expression in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


H Schematic diagram indicating the locations of six DOF motif clusters ( D1 to D6 ) in the NCED2 and NCED3 gene promoters . I ChIP - qPCR analysis of the ability of CDF4 to bind to the promoters of NCED2 and NCED3 . An anti - HA monoclonal antibody was used for DNA immunoprecipitation from three - week - old pER8 : : CDF4 - HA transgenic plants . Black bars indicate the enrichment fold - changes normalized to that of ACT2 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t - test . Transient dual - luciferase reporter assay . The pGreenII - 0800 LUC construct containing the CDF4 promoter and the p62 - SK construct with or without the CDF4 coding region and CDF4 - VP16 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) and Renilla luciferase ( REN ) activities were measured after culturing the protoplasts under low light conditions for 16 h . The ProNCED2 / 3 : LUC / Pro35S : REN ratio represents the relative activities of NCED2 and NCED3 transcription . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t - test .

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H Schematic diagram indicating the locations of six DOF motif clusters (D1 to D6) in the NCED2 and NCED3 gene promoters. I ChIP-qPCR analysis of the ability of CDF4 to bind to the promoters of NCED2 and NCED3. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from three-week-old pER8::CDF4-HA transgenic plants. Black bars indicate the enrichment fold-changes normalized to that of ACT2. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test. Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CDF4 promoter and the p62-SK construct with or without the CDF4 coding region and CDF4-VP16 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured after culturing the protoplasts under low light conditions for 16 h. The ProNCED2/3:LUC /Pro35S:REN ratio represents the relative activities of NCED2 and NCED3 transcription. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.


K Transient dual - luciferase reporter assay . The pGreenII - 0800 LUC construct containing the CDF4 promoter and the p62 - SK construct with or without the CDF4 coding region and CDF4 - VP16 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) and Renilla luciferase ( REN ) activities were measured after culturing the protoplasts under low light conditions for 16 h . The ProNCED2 / 3 : LUC / Pro35S : REN ratio represents the relative activities of NCED2 and NCED3 transcription . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t - test .

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

K Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CDF4 promoter and the p62-SK construct with or without the CDF4 coding region and CDF4-VP16 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured after culturing the protoplasts under low light conditions for 16 h. The ProNCED2/3:LUC /Pro35S:REN ratio represents the relative activities of NCED2 and NCED3 transcription. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.


A - D Transcript levels were determined by qPCR and normalized to AtACT2 . ( A , C ) Localized NCED2 and NCED3 expression in the wild - type senescing leaf . Half - senesced leaves were split into three parts , as shown in Fig . 1A , C . ( B , D ) NCED2 and NCED3 expression during leaf senescence in the vector control and CDF4 - RNAi - 1 plants . Transgenic plant leaves were analyzed from 7 to 56 d ( d , days after planting ) . Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t - test .

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A-D Transcript levels were determined by qPCR and normalized to AtACT2. (A, C) Localized NCED2 and NCED3 expression in the wild-type senescing leaf. Half-senesced leaves were split into three parts, as shown in Fig. 1A, C. (B, D) NCED2 and NCED3 expression during leaf senescence in the vector control and CDF4- RNAi-1 plants. Transgenic plant leaves were analyzed from 7 to 56 d (d, days after planting). Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.


E Observation of rosette leaf longevity from the 26 - day - old vector control , pER8 : : CDF4 , pER8 : : CDF4 & nced2 , pER8 : : CDF4 & nced3 and pER8 : : CDF4 & nced2nced3 transgenic plants after 20 μM estradiol induction for two weeks . Phenotype of detached rosette leaves arranged from oldest to youngest . Scale bars indicate 1 . 5 cm .

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E Observation of rosette leaf longevity from the 26-day-old vector control, pER8::CDF4, pER8::CDF4 & nced2, pER8::CDF4 & nced3 and pER8::CDF4 & nced2nced3 transgenic plants after 20 μM estradiol induction for two weeks. Phenotype of detached rosette leaves arranged from oldest to youngest. Scale bars indicate 1.5 cm.


( F ) SAG12 expression level in rosette leaves at various development stages . Three independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t - test .

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(F) SAG12 expression level in rosette leaves at various development stages. Three independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.


( G ) Chlorophyll concentration in rosette leaves at various development stages . Three independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t - test .

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(G) Chlorophyll concentration in rosette leaves at various development stages. Three independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.


A The senescence phenotype of the vector control , pER8 : : CDF4 and pER8 : : CDF4 : : RNAi detached leaves treated with H2O2 and incubated in the dark for three days . The third leaf in rosettes of 3 - week - old plants was detached and incubated in MES buffer ( 2 mM MES , pH 5 . 8 ) treated with 2 mM H2O2 and 20 μM β - estradiol under dark conditions for three days . Picture shows the leaves before and after treatment . Scale bars indicate 1 cm .

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

A The senescence phenotype of the vector control, pER8::CDF4 and pER8::CDF4::RNAi detached leaves treated with H2O2 and incubated in the dark for three days. The third leaf in rosettes of 3-week-old plants was detached and incubated in MES buffer (2 mM MES, pH 5.8) treated with 2 mM H2O2 and 20 μM β-estradiol under dark conditions for three days. Picture shows the leaves before and after treatment. Scale bars indicate 1 cm.


( B ) DAB staining of H2O2 before and after 2 mM H2O2 plus 20 µM β - estradiol treatment under dark conditions for two days . The brown color represents H2O2 accumulation . Scale bars indicate 1 cm .

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(B) DAB staining of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. The brown color represents H2O2 accumulation. Scale bars indicate 1 cm.


( C ) measurement of H2O2 before and after 2 mM H2O2 plus 20 µM β - estradiol treatment under dark conditions for two days . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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(C) measurement of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


D Chlorophyll concentrations in the leaves shown in A . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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D Chlorophyll concentrations in the leaves shown in A. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


A Measurement of catalase activity in vector control and CDF4RNAi plants . Ten - day - old green seedlings ( " Young " ) and the third and fourth rosette leaves from 36 - day - old plants ( " Old " ) were used , respectively . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t test , * p < 0 . 05 .

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A Measurement of catalase activity in vector control and CDF4RNAi plants. Ten-day-old green seedlings ("Young") and the third and fourth rosette leaves from 36-day-old plants ("Old") were used, respectively. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.


B qPCR analysis of CAT genes expression in the third and fourth rosette leaves from 36 - day - old plants . The expression of CAT genes in the wild - type plant is given as 1 . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t test , * p < 0 . 05 .

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B qPCR analysis of CAT genes expression in the third and fourth rosette leaves from 36-day-old plants. The expression of CAT genes in the wild-type plant is given as 1. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.


D ChIP - qPCR analysis of the relative binding of CDF4 to the promoter of CAT2 . An anti - HA monoclonal antibody was used for DNA immunoprecipitation from 32 - d - old pER8 : : CDF4 - HA transgenic plants . Black columns indicate the enrichment fold - changes normalized to that of ACT2 . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t test , * p < 0 . 05 .

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D ChIP-qPCR analysis of the relative binding of CDF4 to the promoter of CAT2. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from 32-d-old pER8::CDF4-HA transgenic plants. Black columns indicate the enrichment fold-changes normalized to that of ACT2. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.


E Transient dual - luciferase reporter assay . The pGreenII - 0800 LUC construct containing the CAT2 promoter and the p62 - SK construct with or without CDF4 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) and Renilla luciferase ( REN ) activity were measured after culturing the protoplasts under low light conditions for 16 h . six independent experiments were conducted . Values are given as mean ± SD , n = 6 . * p < 0 . 05 by Student ' s t test .

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E Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CAT2 promoter and the p62-SK construct with or without CDF4 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activity were measured after culturing the protoplasts under low light conditions for 16 h. six independent experiments were conducted. Values are given as mean ± SD, n=6. *p<0.05 by Student's t test.


A The senescence phenotypes of five - week - old vector control , proSAG12 : : CDF4 and proSAG12 : : CDF4 & 35S : : CAT2 plants , from old to young . Scale bars indicate 1 . 5 cm .

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A The senescence phenotypes of five-week-old vector control, proSAG12::CDF4 and proSAG12::CDF4 & 35S::CAT2 plants, from old to young. Scale bars indicate 1.5 cm.


B qPCR analysis of the expression of CDF4 and CAT2 in the leaves Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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B qPCR analysis of the expression of CDF4 and CAT2 in the leaves Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.


qPCR analysis of the expression of CAT2 in the leaves Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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qPCR analysis of the expression of CAT2 in the leaves Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.


( 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 .

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(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 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 .

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(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 and the CDF4 transgenic plants . The asterisk represents the flower organ attached along the inflorescence . Scale bar indicates 1 . 5 cm .

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(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 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 .

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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 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 .

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 staining activity in the proPGAZAT : : GUS and proPGAZAT : : GUS & proIDA : : CDF4 . Scale bar indicates 0 . 5 cm .

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


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 .

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

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


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

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(B) Quantification of the numbers of LDs in head region neurons (each dot represents one worm). The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: not statistically significant. Data show mean ± SEM. n ≥ 9.


( E ) Oil red O staining show dramatically increased neutral lipids in the head region of lid - 1 ( xd288 ) and atgl - 1 ( xd314 ) compared with N2 . Scale bar : 10 μm .

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(E) Oil red O staining show dramatically increased neutral lipids in the head region of lid-1(xd288) and atgl-1(xd314) compared with N2. Scale bar: 10 μm.


( F - H ) EM images of neurons . The red dashed line marks the outline of neurons . The red arrows mark LDs . Scale bar : 0 . 5 μm . The scale bar in enlarged images is 100 nm . ( I ) Serial sections of atgl - 1 ( xd314 ) show two LDs ( indicated by blue and red arrows ) in a neuron cell body . Scale bar : 0 . 5 μm .

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(F-H) EM images of neurons. The red dashed line marks the outline of neurons. The red arrows mark LDs. Scale bar: 0.5 μm. The scale bar in enlarged images is 100 nm. (I) Serial sections of atgl-1(xd314) show two LDs (indicated by blue and red arrows) in a neuron cell body. Scale bar: 0.5 μm.


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

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


( A ) The expression pattern of atgl - 1 viewed by Patgl - 1 : : GFP transcriptional fusion reporter . Patgl - 1 : : GFP expression is high in intestine . Relatively low fluorescent signals are found in nerve ring , motor neuron commissure , ventral nerve cord and tail . Scale bar : 20 μm .

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(A) The expression pattern of atgl-1 viewed by Patgl-1::GFP transcriptional fusion reporter. Patgl-1::GFP expression is high in intestine. Relatively low fluorescent signals are found in nerve ring, motor neuron commissure, ventral nerve cord and tail. Scale bar: 20 μm.


( B ) atgl - 1 is expressed in neurons . There is no fluorescent signal when Patgl - 1 : : GFP1 - 10 and Prab - 3 : : GFP11 are expressed separately . When Patgl - 1 : : GFP1 - 10 and Prab - 3 : : GFP11 are co - expressed , the GFP signals are found in the head region , tail and ventral nerve cord , where most neuronal cell bodies are located . The white lines mark the outline of worms . Scale bar : 50 μm .

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(B) atgl-1 is expressed in neurons. There is no fluorescent signal when Patgl-1::GFP1-10 and Prab-3::GFP11 are expressed separately. When Patgl-1::GFP1-10 and Prab-3::GFP11 are co-expressed, the GFP signals are found in the head region, tail and ventral nerve cord, where most neuronal cell bodies are located. The white lines mark the outline of worms. Scale bar: 50 μm.


( C ) atgl - 1 is expressed widely in neurons . The left panels depict the whole worm . Scale bar : 50 μm . The insets in the right panels are enlarged views of the ventral nerve cord in the white dotted boxes of left panels . The signals of mCherry and GFP merge very well . Scale bar : 20 μm .

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(C) atgl-1 is expressed widely in neurons. The left panels depict the whole worm. Scale bar: 50 μm. The insets in the right panels are enlarged views of the ventral nerve cord in the white dotted boxes of left panels. The signals of mCherry and GFP merge very well. Scale bar: 20 μm.


( D ) Visualizing LDs in head neurons with the xdIs109 reporter in different genetic backgrounds . The accumulation of LDs in head neurons of atgl - 1 ( xd314 ) mutants can be rescued by expressing wild - type atgl - 1 in neurons ( Punc - 119 : : atgl - 1 ) , but not in intestine ( Pvha - 6 : : atgl - 1 ) , glia ( Phlh - 17 : : atgl - 1 ) or hypodermis ( Pajm - 1 : : atgl - 1 ) . Scale bar : 5 μm . ( E ) Quantification of the number of LDs in head neurons in different genetic backgrounds . Each dot represents one worm . The data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to the control xdIs109 . ns : not statistically significant . * * * * P < 0 . 0001 . Data show mean ± SEM . n ≥ 7 .

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(D) Visualizing LDs in head neurons with the xdIs109 reporter in different genetic backgrounds. The accumulation of LDs in head neurons of atgl-1(xd314) mutants can be rescued by expressing wild-type atgl-1 in neurons (Punc-119::atgl-1), but not in intestine (Pvha-6::atgl-1), glia (Phlh-17::atgl-1) or hypodermis (Pajm-1::atgl-1). Scale bar: 5 μm. (E) Quantification of the number of LDs in head neurons in different genetic backgrounds. Each dot represents one worm. The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. ns: not statistically significant. ****P < 0.0001. Data show mean ± SEM. n ≥ 7.


( F ) Neuron - specific knockout of atgl - 1 causes LD accumulation in neurons , similar to atgl - 1 ( xd314 ) mutants . Scale bar : 5 μm . ( G ) Quantification of the number of LDs in head neurons in different genetic backgrounds . Each dot represents one worm . The data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to the control xdIs109 . t - test was used between the groups under the crossbars . ns : not statistically significant . * * * * P < 0 . 0001 . Data show mean ± SEM . n ≥ 9

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(F) Neuron-specific knockout of atgl-1 causes LD accumulation in neurons, similar to atgl-1(xd314) mutants. Scale bar: 5 μm. (G) Quantification of the number of LDs in head neurons in different genetic backgrounds. Each dot represents one worm. The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. t-test was used between the groups under the crossbars. ns: not statistically significant. ****P < 0.0001. Data show mean ± SEM. n ≥ 9


( A ) LDs accumulate in touch neurons in atgl - 1 ( xd314 ) visualized by SRS ( Stimulated Raman Scattering ) . The zdIs5 reporter marks touch neurons . The white arrows indicate LDs in touch neurons in atgl - 1 ( xd314 ) . Scale bar : 10 μm .

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(A) LDs accumulate in touch neurons in atgl-1(xd314) visualized by SRS (Stimulated Raman Scattering). The zdIs5 reporter marks touch neurons. The white arrows indicate LDs in touch neurons in atgl-1(xd314). Scale bar: 10 μm.


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

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


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

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


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

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