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 .

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

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 .

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

(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 ) .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD 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 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 B-GENEPROD 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 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 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) 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 .

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

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 .

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

O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O 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 , 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 .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O 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 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 B-TISSUE 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 B-GENEPROD B-ORGANISM O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O B-EXP_ASSAY O 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 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 .

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

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 .

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

O O B-TISSUE O B-EXP_ASSAY 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 B-SMALL_MOLECULE O O 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 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 .

O O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O O 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 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 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 B-SMALL_MOLECULE B-EXP_ASSAY O O 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 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 .

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

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 .

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

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

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 .

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

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

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 .

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

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

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

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 .

O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O O O O O B-ORGANISM O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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


( B ) 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 .

O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-TISSUE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( E ) Oil red O 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 .

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

(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 .

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

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


( C ) Overexpressing 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 .

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

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


( A ) 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 .

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

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


( B ) atgl - 1 is expressed in neurons . There is no 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 .

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

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


( C ) atgl - 1 is expressed widely in neurons . 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 .

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

(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 .

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

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


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

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

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


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

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

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


( B ) atgl - 1 ( xd314 ) 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 .

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

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


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

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

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


( B ) 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 .

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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