D . Naïve murine CD4 + T were isolated by FACS from lymph node spleen suspension of USP44fl / fl CD4Cre + mice that of their wild type ( USP44fl / fl CD4Cre - mice ; n = 2 - 3 / group / experiment ) . iTreg were generated from these mice as described for Fig . 1 before incubation on a microscope slide pre - coated with poly - L lysine for 1h . Adhered were then fixed by PFA for 0 . 5 followed by blocking with 1 % BSA for 1h , then incubation with specified antibodies . Representative confocal microscopy ( 40X ) were visualized for endogenous USP44 ( red ) FOXP3 Baxter et al ( ) . DAPI was used to visualize nuclei ( blue ) ; scale bar 50μm .

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

D. Naïve murine CD4+ T cells were isolated by FACS from lymph node and spleen cell suspension of USP44fl/fl CD4Cre+ mice and that of their wild type littermates (USP44fl/fl CD4Cre- mice; n=2-3/group/experiment). iTreg cells were generated from these mice as described for Fig. 1 before incubation on a microscope slide pre-coated with poly-L lysine for 1h. Adhered cells were then fixed by PFA for 0.5 followed by blocking with 1% BSA for 1h, then incubation with the specified antibodies. Representative confocal microscopy images (40X) were visualized for endogenous USP44 (red) and FOXP3 Baxter et al(). DAPI was used to visualize cell nuclei (blue); scale bar 50μm.


A . HEK293T were transfected with expression constructs encoding MYC - FOXP3 , His - Ubiquitin , either a wild type or catalytically inactive ( C282S , " cs " ) version of USP44 tagged with FLAG . Transfected were treated with 20 μM MG132 for 4 hrs subsequently harvested for lysis . Pull - down of His - labeled using Ni - NTA beads allowed recovery of ubiquitinated FOXP3 species that were then visualized by immunobot probing with antibodies specific for MYC .

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A. HEK293T cells were transfected with expression constructs encoding MYC-FOXP3, His-Ubiquitin, and either a wild type or catalytically inactive (C282S, "cs") version of USP44 tagged with FLAG. Transfected cells were treated with 20 μM MG132 for 4 hrs and subsequently harvested for cell lysis. Pull-down of His-labeled proteins using Ni-NTA beads allowed recovery of ubiquitinated FOXP3 species that were then visualized by immunobot probing with antibodies specific for MYC.


B . Effect of USP44 knock down on FOXP3 ubiquitination . Two distinct shRNA lentiviral constructs each containing a G418 resistance cassette were delivered into HEK293T . After selected for 7d , were transfected with MYC - FOXP3 His - Ubiquitin , then treated with 20 μM MG132 for 4 hrs before harvest lysis . Ubiquitinated FOXP3 were visualized as in A .

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B. Effect of USP44 knock down on FOXP3 ubiquitination. Two distinct shRNA lentiviral constructs each containing a G418 resistance cassette were delivered into HEK293T cells. After selected for 7d, cells were transfected with MYC-FOXP3 and His-Ubiquitin, then treated with 20 μM MG132 for 4 hrs before harvest and lysis. Ubiquitinated FOXP3 proteins were visualized as in A.


C . of polyubiquitinated FOXP3 in presence or absence of USP44 . As in Fig 2b , murine iTregs were generated from naïve CD4 + precursors isolated from wild type mice globally deficient in USP44 - / - mice ( n = 3 / group / experiment ) . Ubiquitinated were extracted from iTreg lysates with anti - ubiquitin antibodies ( anti - Ubi ) prior to resolution by SDS PAGE immunoblot , probing for FOXP3 .

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C. Levels of polyubiquitinated FOXP3 in the presence or absence of USP44. As in Fig 2b, murine iTregs were generated from naïve CD4+ precursors isolated from wild type and mice globally deficient in USP44-/- mice (n=3/group/experiment). Ubiquitinated proteins were extracted from iTreg lysates with anti-ubiquitin antibodies (anti-Ubi) prior to resolution by SDS PAGE and immunoblot analysis, probing for FOXP3.


D , E . HEK293T were transfected with plasmids encoding MYC - FOXP3 , FLAG - USP44 ( either wild type USP44 , " wt " or C282S mutant ) . These were subsequently treated with cycloheximide ( CHX ; 5μg / ml ) for indicated time points before harvested lysis . FOXP3 relative turnover rate of this factor was determined by immunoblotting with anti - MYC antibodies .

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D, E. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, FLAG-USP44 (either wild type USP44, "wt" or the C282S mutant). These cells were subsequently treated with cycloheximide (CHX; 5μg/ml) for the indicated time points before harvested and cell lysis. FOXP3 levels and the relative turnover rate of this factor was determined by immunoblotting analysis with anti-MYC antibodies.


F , USP44 knock down in human Tregs . Naïve CD4 + T ( CD4 + CD25 - CD45RA + ) were isolated from peripheral blood of healthy donors by FACS , iTreg were generated after 7 days of in vitro skewing conditions . As in Fig . 3b , shRNA constructs targeting USP44 were delivered by lentivirus . Here human iTregs received either shCK ( control ) , or one of two different shUSP44 constructs . Endogenous FOXP3 USP44 were visualized by immunoblot ( F )

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F, USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. Endogenous FOXP3 and USP44 protein levels were visualized by immunoblot (F)


G . USP44 knock down in human Tregs . Naïve CD4 + T ( CD4 + CD25 - CD45RA + ) were isolated from peripheral blood of healthy donors by FACS , iTreg were generated after 7 days of in vitro skewing conditions . As in Fig . 3b , shRNA constructs targeting USP44 were delivered by lentivirus . Here human iTregs received either shCK ( control ) , or one of two different shUSP44 constructs . FOXP3 expression was assessed by intracellular followed by flow cytometry ( G ) .

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

G. USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. FOXP3 expression was assessed by intracellular staining followed by flow cytometry (G).


H . Murine naïve CD4 T ( CD62L + / CD25 - / CD4 + ) from WT USP44 - / - mice ( n = 2 - 3 / group / experiment ) were FACS purified activated in vitro for four days by CD3 / CD28 cross - linking antibodies ( 1ug 4ug / ml , respectively ) in presence of 100 U / ml IL - 2 indicated dose of TGF - β . Down - regulation of FOXP3 was observed in USP44 - / - mice by intracellular immunostaining flow cytometry .

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H. Murine naïve CD4 T cells (CD62L+/CD25-/CD4+) from WT and USP44-/- mice (n=2-3/group/experiment) were FACS purified and activated in vitro for four days by CD3/CD28 cross-linking antibodies (1ug and 4ug/ml, respectively) in the presence of 100 U/ml IL-2 and the indicated dose of TGF-β. Down-regulation of FOXP3 was observed in USP44-/- mice by intracellular immunostaining and flow cytometry.


A . Reciprocal Co - IP of USP44 USP7 . HEK293T were transfected with constructs encoding MYC - USP44 FLAG - USP7 . These were lysed pertinent factors were immunoprecipitated using either anti - FLAG or anti - MYC . Pulled - down were observed by immunoblot using either anti - MYC or anti - FLAG antibodies .

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

A. Reciprocal Co-IP of USP44 and USP7. HEK293T cells were transfected with constructs encoding MYC-USP44 and FLAG-USP7. These cells were lysed and pertinent factors were immunoprecipitated using either anti-FLAG or anti-MYC. Pulled-down proteins were observed by immunoblot analysis using either anti-MYC or anti-FLAG antibodies.


B . Effect of USP44 on USP7 . HEK293T were transfected with MYC - FOXP3 . also received combinations of FLAG - USP7 varying doses of pLVX - USP44 encoding plasmids . FOXP3 was immunoprecipitated using anti - MYC , immunoblots were analyzed using anti - FLAG , anti - USP44 or anti - MYC antibodies .

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

B. Effect of USP44 on USP7 levels. HEK293T cells were transfected with MYC-FOXP3. Cells also received combinations of FLAG-USP7 and varying doses of pLVX-USP44 encoding plasmids. FOXP3 was immunoprecipitated using anti-MYC, and immunoblots were analyzed using anti-FLAG, anti-USP44 or anti-MYC antibodies.


C . Effect of USP44 USP7 coexpression on FOXP3 ubiquitination . HEK293T were transfected with plasmids encoding MYC - FOXP3 , either FLAG - USP44 or FLAG - USP7 or both , His - ubiquitin . were treated with 20 μM MG132 for 4 hrs before being harvested , lysed , incubated on Ni - NTA beads to pull down ubiquitinated FOXP3 , which was visualized by immunoblotting using antibodies specific for MYC .

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

C. Effect of USP44 and USP7 coexpression on FOXP3 ubiquitination. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, either FLAG-USP44 or FLAG-USP7 or both, His-ubiquitin. Cells were treated with 20 μM MG132 for 4 hrs before being harvested, lysed, and incubated on Ni-NTA beads to pull down ubiquitinated FOXP3, which was visualized by immunoblotting using antibodies specific for MYC.


D . ( left panel ) iTreg were polarized from human naïve CD4 + T over 7 days ( n = 3 donors / experiment ) receiving control ( shCK ) , or shRNA knockdown constructs targeting USP44 ( shUSP44 - 1 , - 2 ) , or USP7 ( shUSP7 - 1 , - 2 ) , or both shRNA constructs . Freshly isolated nTregs were treated with either control or shRNA constructs . After transduction with lentivirus for 48 hours , were harvested run SDS - PAGE gels . were visualized by immunoblotting using antibodies specific for FOXP3 , USP44 , USP7 GAPDH . Shown are representative findings from 3 independent experiments ( biological replicates ) .

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

D. (left panel) iTreg were polarized from human naïve CD4+ T cells over 7 days (n=3 donors /experiment) receiving control (shCK), or shRNA knockdown constructs targeting USP44 (shUSP44-1, -2), or USP7 (shUSP7-1, -2), or both shRNA constructs. Freshly isolated nTregs were treated with either control or shRNA constructs. After transduction with lentivirus for 48 hours, cells were harvested and run SDS-PAGE gels. Protein levels were visualized by immunoblotting using antibodies specific for FOXP3, USP44, USP7 and GAPDH. Shown are representative findings from 3 independent experiments (biological replicates).


A . Jurkat T were transfected with indicated combinations relative doses of expression constructs encoding MYC - FOXP3 , FLAG - USP44 along with an IL - 2 promoter - driven firefly luciferase reporter . After 8 hours stimulation with PMA / Inomycin , were lysed luciferase activity , which was observed normalized to renilla luciferase activity .

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A. Jurkat T cells were transfected with the indicated combinations and relative doses of expression constructs encoding MYC-FOXP3, FLAG-USP44 along with an IL-2 promoter-driven firefly luciferase reporter. After 8 hours stimulation with PMA/Inomycin, cells were lysed and luciferase activity, which was observed and normalized to renilla luciferase activity.


B . Tregs ( CD4 + / CD25High ) were recovered from lymph nodes spleens of wild type C57BL / 6 ( WT ) USP44 - / - mice by FACS purification . were activated with anti - CD3 / CD28 agonist antibodies overnight , was isolated , cDNA was generated from these samples for measurement of several signature Treg - associated transcripts by qRT - PCR . relative expression level of these by WT KO - derived Tregs is shown .

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B. Tregs (CD4+/CD25High) were recovered from the lymph nodes and spleens of wild type C57BL/6 (WT) and USP44-/- mice by FACS purification. Cells were activated with anti-CD3/CD28 agonist antibodies overnight, and RNA was isolated, and cDNA was generated from these samples for the measurement of several signature Treg-associated transcripts by qRT-PCR. The relative expression level of these by WT and KO-derived Tregs is shown.


C , D . of in vitro suppressive function of Tregs isolated from WT mice or - sex - matched USP44 - / - mice ( n = 3 / group / experiment ) . Tregs were mixed with CFSE - labeled responder naïve T from CD45 . 1 + C57BL / 6 mice ( n = 3 / experiment ) for three days . CFSE dilution in CD45 . 1 + T was observed by flow cytometry suppression of responder proliferation by co - cultured Tregs was determined .

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C, D. Analysis of the in vitro suppressive function of Tregs isolated from WT mice or age- and sex-matched USP44-/- mice (n=3/group/experiment). Tregs were mixed with CFSE-labeled responder naïve T cells from CD45.1+ C57BL/6 mice (n=3/experiment) for three days. CFSE dilution in CD45.1+ T cells was observed by flow cytometry and the percent suppression of responder cell proliferation by the co-cultured Tregs was determined.


A . Colitis was induced by intravenous coinjection of CD4 + CD25 - CD62Lhigh T congenically distinct ( CD45 . 2 ) CD4 + CD25high Treg into Rag2 - / - mice ( n = 8 / group ; 1x106 2x105 per Rag2 - / - recipient , respectively ) . Changes in over time were monitored are expressed as a of original .

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A. Colitis was induced by intravenous coinjection of CD4+CD25-CD62Lhigh T cells and congenically distinct (CD45.2) CD4+CD25high Treg cells into Rag2-/- mice (n=8/group; 1x106 and 2x105 cells per Rag2-/- recipient, respectively). Changes in body weight over time were monitored and are expressed as a percentage of the original weight.


B . Representative photomicrographs of distal colon of Rag2 - / - mice after T transfer . 10 after transfer , mice were euthanized , colons were harvested , fixed in 10 % buffered formalin , processed for standard H & E prior to histological . ( i ) - ( iv ) present bright - field ( 100x ) .

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B. Representative photomicrographs of the distal colon of Rag2-/- mice after T cell transfer. 10 weeks after transfer, mice were euthanized, and colons were harvested, fixed in 10% buffered formalin, and processed for standard H&E staining prior to histological analysis. (i)- (iv) present bright-field micrographs (100x).


C . H & E slides were scored in a blinded fashion , colon pathology was scored as described in Methods Section . Shown are mean scores for each treatment group .

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C. H&E slides were scored in a blinded fashion, and colon pathology was scored as described in the Methods Section. Shown are mean scores for each treatment group.


D . Spleen , mesenteric lamina propria lymph node were isolated from mice 10 after adoptive T transfer , number of Teff 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 mRNA by adoptively transferred Tregs . Mesenteric lymph nodes were excised from recipients of WT USP44 - / - Tregs in A . FOXP3 was detected by intracellular immunostaining flow cytometry ( left ) . Shown are events within gate for transferred Tregs ( CD4 + / CD45 . 1 + ) . Transferred Tregs were also recovered from mesenteric lymph node suspensions by for congenic marker CD4 followed by FACS , 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 . numbers of injected ( CD45 . 2 + ) Treg FOXP3 + 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 were recovered from suspensions of lamina propria - infiltrating leukocytes . These lymphocytes were stimulated ex vivo by PMA / Ionomycin cocktail in presence of brefeldin A IFN - γ 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 lines were s . c . injected into shaved flanks of Usp44fl / flFoxp3Cre + mice their wild type ( Usp44fl / flFoxp3Cre - ) ( 1x105 / mouse ; n = 5 - 9 mice / group ) . dimensions were measured every 2 days , changes in mean volume over time were observed ( lower panels ) . Upper panels depict representative 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 lines were s . c . injected into shaved flanks of Usp44fl / flFoxp3Cre + mice their wild type ( Usp44fl / flFoxp3Cre - ) ( 1x105 / mouse ; n = 5 - 9 mice / group ) . dimensions were measured every 2 days , changes in mean volume over time were observed ( lower panels ) . Upper panels depict representative of excised tumors .

B-CELL_LINE B-DISEASE O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O 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

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 CD4 + compartment of MC38 - bearing mice in each experiment were found by flow cytometry . Suspensions of obtained from spleen , - draining lymph nodes , - infiltrating leukocytes ( TILs ) were stained for surface CD4 intracellular FOXP3 prior to 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 + CD8 + producing proinflammatory cytokine IFNγ were found among tissues of - bearing mice in MC38 modelby intracellular cytokine Kralovics et al ( , 2005 ) flow cytometry after ex vivo re - stimulation of recovered leukocytes by PMA ionomycin in presence of brefeldin A .

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

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 . capacity of CD8 + for effector function in MC38 tumors was further assessed by measuring ( G ) production of TNFα by ICS ( H ) intracellular straining of 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 dTormGFP - E177Q in wild - type ( w - ) or dLap1 + / ∆ 11 fat 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 of 5DO larval fat . arrows ( O ) indicate - localized neutral lipid , 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 LipidTOX detect expression of dTormGFP chimeras ( green ) , boundaries ( red ) , LD ( red ) , respectively , in dTorKO fat .

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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 fraction ( mean ± ) of GL GPL lipids with each fatty acyl chain characteristic . n = 3 MS analyses of 4DO fat samples for each genotype . MUFA , mono - unsaturated ; PUFA , poly - unsaturated . ‡ indicate a significant difference between w - dTorKO expressing Luciferase RNAi , * indicate a significant difference compared to dTorKO expressing Luciferase RNAi . Two - way ANOVA , Dunnett ' s multiple comparison . ‡ p < 0 . 05 * 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 dTorKO 5DO fat expressing UAS GFP fusion with Cg - Gal4 driver , labeled with anti - Calnexin ( red ) a neutral lipid dye ( LipidTOX , magenta ) . Note that dTorKO are smaller . 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 transfected with a plasmid that co - expresses CTDNEP1myc - WT or - CD ( red ) NEP1R1V5 ( magenta ) . ( G ' ) Upper : enlargement of panel G , arrow highlights NE enrichment . Lower : plot showing of Lipin1mGFP signal ( mean ± SD , n = 5 ) along a 3 µm profile that transects 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.


of MEFs with primarily or Lipin1mGFP . Chi - square . * * * * p < 0 . 0001 .

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


Control ( w - ) dTorKO fat nuclei labeled with anti - LaminDm0 , Megator / Tpr , mAb414 , or expressing UAS Nup107GFP or UAS Nup358GFP with 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 expressing dTor cDNA , Luciferase RNAi , or RNAi against Nep1r1 , Ctdnep1 , or Lipin . Nuc , nucleus . of fat with NE - localized mAb414 . * * * * * * indicate that Lipin , Ctdnep1 , Nep1r1 RNAi significantly increased number of dTorKO with NE - specific mAb414 labeling compared to Luciferase RNAi ( Luc ) . Chi - square followed by post hoc Chi - square . * * p < 0 . 01 * * * * p < 0 . 0001 . NE ultrastructure in 5DO dTorKO . Red arrows , pores ; yellow arrows , abnormal INM herniations . Nuc , nucleus ; Cyto , cytosol .

O O O O B-GENEPROD B-TISSUE O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O B-TISSUE O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD 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 B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O

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.


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

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

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 overexpressing UAS transgenes , or from dLap1 - / - . Yellow dotted lines highlight NE regions with subtly abnormal architecture . Red arrows indicate 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 - imaging of fat nuclei expressing Sec61b ( control ) , or CTDNEP1mGFP . ( J - L ) 3D reconstructions , ( M ) between INM ONM at NPC in wild - type versus CTDNEP1mGFP - induced structures . Kolmogorov - Smirnov non - parametric T - . * * * * p < 0 .

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

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 expressing indicated UAS transgenes or from a dLap1 - / - . * * * * * * * indicate significant difference compared to control mGFPSec61b expressing . Chi - square followed by post hoc Chi - square . * * * p < 0 . 001 * * * * p < 0 . 0001 .

O B-EXP_ASSAY O O B-TISSUE 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 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

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 from a ( I ) control , ( J ) homozygous Nup35BG01311 ( K ) homozygous Nup35MB02683 . Nuc , nucleus ; Cyto , cytosol .

B-SUBCELLULAR O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O

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 .

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

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


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

O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O O O O

(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 .

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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 .

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

(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 .

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

(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 . of p - Thr145 - EndoB1 level is shown in right panel . Data are means ± s . e . m . ; n = 3 . Uncropped 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 level of RFP - LC3 ( arrows ) in rat cortical neurons . Middle : of of with RFP - LC3 . Right : pretreatment with 10 mM 3 - MA attenuates starvation - induced increase in LC3 - II level in neurons . Data are means ± s . e . m . ; n = 3 . Samples were run on same gel spliced together to create 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 starvation - induced increase in LC3 - II ( left ) of with RFP - LC3 ( 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 ) increases level of Thr 145 - phosphorylated EndoB1 in neurons ( top 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 autophagy induction in neurons . Data are means ± s . e . m . ; n = 3 for d 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 starvation - induced increase in LC3 - II ( left ) of with RFP - LC3 ( right ) in neurons . Data are means ± s . e . m . ; n = 6 . Uncropped 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 five lysine / arginine residues between 176 183 of EndoB1 to glutamic acid ( 5E mutant ) . Right : purified GST was included as a negative control . S , supernatant fraction ; P , pellet fraction that contains lipid - bound .

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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 ) 5E mutant of EndoB1 fails to dimerize ( b ) in 293T .

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


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

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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 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 lipid - binding property of EndoB1 . Conversely , 5E mutant is less able to bind lipid . 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 . T145A mutation of GFP - EndoB1 does not affect its co - localization with Atg5 - positive entities . Arrows denote GFP - EndoB1 - Atg5 - positive vesicles . right panel shows quantified data as means ± s . e . m . ; n = 9 . Uncropped 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 . Samples were run on same gel spliced together to create 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 .

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


( c ) Diminished interaction between EndoB1 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 .

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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 ) Beclin 1 ( e ) in 293T .

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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 on fraction of mouse brain lysates . Fractions obtained were subjected to immunoblot ( f ) , or immunoprecipitation followed by immunoblotting ( g ) . Uncropped 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 numbers of tyrosine hydroxylase ( TH ) - cresyl violet ( CV ) - positive neurons in substantia nigra . Representative of tyrosine hydroxylase - stained brain sections are shown . Right panels show quantified data as means ± s . e . m . ; n = 9 for tyrosine hydroxylase 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 of LC3 - II Thr 145 - phosphorylated EndoB1 in 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 MPP + - induced EndoB1 phosphorylation at Thr 145 MPP + - induced increase in 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 Thr 145 - phosphorylated EndoB1 in midbrain of p35 − / − mice . 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 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 of LC3 - II Thr 145 - phosphorylated EndoB1 in cerebellum 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 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 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 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 . 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 . 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 . 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 . 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 . 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 , whereas it reduces neuronal survival in vector - transfected . 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 . 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 . 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 + / + FAK − / − murine SCC were lysed FAK , Ret or Src immunoprecipitated immunoblotting carried out using anti ‐ FAK , anti ‐ Ret , anti ‐ Src 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 ) were fixed stained with either anti ‐ Ret ( green in upper left panels ) or anti ‐ Ret PY1062 ( green in lower left panels ) DAPI ( blue ) . Merged zoomed are shown . Solid arrows indicate localization at adhesions while broken arrows show localization in . of of that contained Ret in intracellular is shown . Data are presented as mean ± s . d . significance calculated using a Student ' s t ‐ ( n = 3 ) . Lysates were immunoblotted with anti ‐ Ret PY1062 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 ) were fixed stained with anti ‐ Ret ( green ) anti ‐ FAK ( red ) . Merged are also shown . Solid arrows indicate co ‐ localization between Ret FAK at adhesions , while broken arrows indicate its absence in . Scale bars , 20 μM . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection ; SCC , squamous 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 . were fixed stained with either ( A ) anti ‐ Ret ( green ) anti ‐ PY416 Src ( red ) or ( B ) anti ‐ PY416 Src ( red ) anti ‐ LC3B ( green ) . Merged zoomed are shown . Solid arrows indicate co ‐ localization at adhesions , while broken arrows show co ‐ localization in intracellular .

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

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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 ) of of that contained Ret in intracellular is shown . Data are presented as mean ± s . d . significance calculated using a Student ' s t ‐ ( 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 ) , either untreated or treated with chloroquine for 24 h , were fixed stained with anti ‐ Ret ( green ) DAPI ( blue ) . Scale bars , 20 μM . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection ; SCC , squamous carcinoma ; siRNA , short interfering .

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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 − / − 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 anti ‐ actin . FAK Ret were immunoprecipitated from these 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 ) were fixed stained with anti ‐ Ret ( green ) either anti ‐ FAK ( red in upper panels ) or anti ‐ PY416 Src ( red in lower panels ) . Merged are shown . Solid arrows indicate Ret in adhesions , while broken arrows show Ret in . 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 ) of of that contained Ret in intracellular is shown . Data are presented as mean ± s . d . significance calculated using a Student ' s t ‐ ( 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 + / + were treated with dasatinib , then lysates were immunoblotted with anti ‐ FAK , anti ‐ FAK Y397 , anti ‐ FAK Y925 , anti ‐ PY416 Src , anti ‐ Src anti ‐ actin . FAK was immunoprecipitated 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 ) were treated with dasatinib , then fixed stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) DAPI ( blue ) . Solid arrows indicate co ‐ localization in adhesions . of of that contained Ret in intracellular is shown . Data are presented as mean ± s . d . significance calculated using a Student ' s t ‐ ( n = 3 ) . ( C ) FAK − / − treated with dasatinib were fixed stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) DAPI ( blue ) . Solid arrows indicate co ‐ localization in adhesions , while broken arrows show lack of co ‐ localization in . Scale bars , 20 μM . of of that contained Ret in intracellular is shown . Data are presented as mean ± s . d . significance calculated using a Student ' s t ‐ ( n = 3 ) .

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

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

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

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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-TISSUE 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 O O O O O 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 . ~ 50 % senesced rosette leaf was cut into three parts as indicated by red lines . level of CDF4 mRNA was determined in three sections of senescing leaf . Base ; Middle ; Tip . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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

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 turned yellow ; LS , late - senescent leaf , with > 50 % leaf turned yellow . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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

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

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

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

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

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

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

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

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

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

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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

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 of relative chlorophyll in sixth seventh rosette leaves from vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi 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 .

O O O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE 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 O O O O O O O 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 leaf senescence of detached rosette leaves in vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi pER8 : : amiCDF4 lines before after treatment with 2 mΜ H2O2 in for four days . Scale bars indicate 0 . 7 cm .

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

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 chlorophyll in leaves was analyzed . indicates 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 .

O B-SMALL_MOLECULE O B-TISSUE O O O O B-SMALL_MOLECULE 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

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 in third 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 .

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

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

O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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

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 NCED3 in 14 - day - old CDF4GR transgenic treated with 20 μM β - estradiol or mock treatment for 0 , 2 , or 4 h . expression of corresponding genes in mock - treated 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 .

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

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 NCED3 in 14 - day - old CDF4GR transgenic treated with 20 μM DEX , 100μM CHX , DEX plus 100μM CHX , or mock . gene expression in mock - treated 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 .

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

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

O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD I-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 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-TISSUE O B-ORGANISM B-GENEPROD O B-GENEPROD O B-ORGANISM B-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O 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

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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-TISSUE O B-ORGANISM B-GENEPROD O B-GENEPROD O B-ORGANISM B-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O 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

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 were determined by qPCR normalized to AtACT2 . ( A , C ) Localized NCED2 NCED3 expression in wild - type senescing leaf . Half - senesced leaves were split into three parts , as shown in Fig . 1A , C . ( B , D ) NCED2 NCED3 expression during leaf senescence in vector control CDF4 - RNAi - 1 . Transgenic 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 - .

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

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

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

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 - .

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

(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 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 - .

O O O B-SMALL_MOLECULE O B-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 O O O O O

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

O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O B-TISSUE I-TISSUE I-TISSUE 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 B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-TISSUE 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 of H2O2 before after 2 mM H2O2 plus 20 µM β - estradiol treatment under conditions for two days . 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 after 2 mM H2O2 plus 20 µM β - estradiol treatment under conditions for two days . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t .

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

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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 CDF4RNAi . Ten - day - old green seedlings ( " Young " ) third fourth rosette leaves from 36 - day - old ( " Old " ) were used , respectively . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t , * 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 of CAT genes expression in third fourth rosette leaves from 36 - day - old . expression of CAT genes in wild - type is given as 1 . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t , * 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 of relative binding of CDF4 to promoter of CAT2 . An anti - HA monoclonal antibody was used for immunoprecipitation from 32 - d - old pER8 : : CDF4 - HA transgenic . Black columns indicate enrichment fold - changes normalized to that of ACT2 . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t , * 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 . pGreenII - 0800 LUC construct containing CAT2 promoter p62 - SK construct with or without CDF4 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) Renilla luciferase ( REN ) activity were measured after culturing protoplasts under low conditions for 16 h . six independent experiments were conducted . Values are given as mean ± SD , n = 6 . * p < 0 . 05 by Student ' s t .

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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 senescence of five - week - old vector control , proSAG12 : : CDF4 proSAG12 : : CDF4 & 35S : : CAT2 , 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 of expression of CDF4 CAT2 in leaves Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t .

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

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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.