EMBO/soda-nel huggingface dataset:
CDF4 promotes leaf senescence by regulating ABA and reactive oxygen species pathways in Arabidopsis (2020)
Figure 2

Figure 2. Overexpression or knockdown of CDF4 in transgenic plants affects leaf senescence.

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.

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.

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.

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.

F The chlorophyll concentration in leaves from (D) 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.
CDF4 promotes leaf senescence by regulating ABA and reactive oxygen species pathways in Arabidopsis (2020)
Figure 3

Figure 3. CDF4 is involved in ABA biosynthesis by directly activating the transcription of NCED2 and NCED3.

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.

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.

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.

J, 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.
CDF4 promotes leaf senescence by regulating ABA and reactive oxygen species pathways in Arabidopsis (2020)
Figure 4

Figure 4. Involvement of NCED2 and NCED3 in the progression of leaf senescence in the vector control, pER8::CDF4, pER8::CDF4 & nced2, pER8::CDF4 & nced3 and pER8::CDF4 & nced2nced3 plants.

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.

F, G (F) SAG12 expression level and (G) Chlorophyll concentration in rosette leaves in E 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.
CDF4 promotes leaf senescence by regulating ABA and reactive oxygen species pathways in Arabidopsis (2020)
Figure 5

Figure 5. CDF4 suppresses H2O2 scavenging.

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, C (B) DAB staining and (C) measurement 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. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test. Scale bars indicate 1 cm.

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.
CDF4 promotes leaf senescence by regulating ABA and reactive oxygen species pathways in Arabidopsis (2020)
Figure 6

Figure 6. CDF4 suppresses catalase activity by repressing CAT2 transcription.

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.

C Schematic diagram indicating the locations of the putative CDF4- binding motif clusters (D1 to D5) in the promoter of CAT2.

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.
CDF4 promotes leaf senescence by regulating ABA and reactive oxygen species pathways in Arabidopsis (2020)
Figure 7

Figure 7. Overexpression of CAT2 represses proSAG12::CDF4 early-senescence phenotype.

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 shown in A. Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.

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

D, E (D) Measurement of chlorophyll and (E) H2O2 concentrations in the leaves shown in A. 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.
CDF4 promotes leaf senescence by regulating ABA and reactive oxygen species pathways in Arabidopsis (2020)
Figure 8

Figure 8. Altered floral organ abscission process in the CDF4 transgenic plants.

A-C (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 or (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.

D Schematic diagram indicating the locations of the putative CDF4 binding motif clusters (D1 to D5) in the approximately 1.8-kb promoter of PGAZAT.

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.
A microRNA-signature that correlates with cognition and is a target against cognitive decline (2021)
Figure 4

Fig 4. Increased expression of 3-microRNA signature perturbs synaptic organization and neuronal activity.

A. Primary hippocampal neurons were treated with a mixture of 3-miR mimic or control oligonucleotides and follow up analyses (imaging, electrical recordings) were performed.

B. Functional mature synapses were quantified via co-localizations of pre- (Synatophysin 1) and the post-synaptic (PSD-95) markers and compared between 3-miR-mix and control groups. Scale bar: 10 μm. Two independent methods (SynQuant and Colocalization) were used for quantification. 3-miR-mix reduced the number of functional synapses compared to controls (n = 24-30 images)

C. Dendrite labeling and quantification. Dendritic spines were stained with Dil. Scale bar: 10 μm. Spine density and total spine length are substantially reduced in 3-miR mix treated primary neurons compared to those treated with scramble RNA (n = 49-97 images)

D. Hippocampal neurons were cultured in a multi electrode array (MEA) plate equipped with sixteen electrodes. Spontaneous activity of the neurons was recorded at every 3 hours (10 minutes/session) for 24 hours. Weighted mean firing rate, number of bursts, and network bursts are significantly decreased in neurons treated with 3-miR-mix compared to control.

E. The aberrant neuronal firing activity (weighted mean firing rate) and reduced number of bursts and network bursts were observed across the 24 hour of time period.

Data information: For panel B, C, D, E following statistical test has been applied: Unpaired t-tests, two-tailed. Bars and error bars in these plots indicate mean ± sem. *P<0.05, ****P<0.0001
A microRNA-signature that correlates with cognition and is a target against cognitive decline (2021)
Figure 7

Fig 7. The 3-microRNA signature is a target for RNA therapeutics to treat neuronal dysfunction.

A. Experimental outline. Anti-miR-mix of the 3 microRNAs was injected into the dorsal hippocampus of the mice as previously described. RNAseq data were generated from dorsal hippocampal tissues and compared to those treated with control scramble oligonucleotides.

B. Weighted gene co-expression analysis of hippocampal RNA-seq data identified the MEblue gene cluster that is decreased when comparing 3-month old mice (young-control) to cognitively impaired 16,5-month old mice (old -control) with a scramble control oligonucleotide injected. Treating old mice with the miR-inhibitor mix (old miR-inhibitor mix) reinstated gene-expression of this cluster, at least in part (n = 6-7, Kruskal-wallis test).

C. Gene ontology reveals that the MEblue cluster is linked to cognition and synapse organization.

D. qPCR assay for several synaptic genes (LRKK2, Cadm3 and Slc6a11) confirms reinstatement of gene expression with anti-miR-mix (n = 5-7, Kruskal-wallis test).

E. Weighted gene co-expression analysis of hippocampal RNA-seq data identified a MElightgreen gene cluster that is decreased when comparing wilt type control (WT control) to APPPS1-21 mice (APP-control) and was reinstated in APPPS1-21 mice treated miR-inhibitor mix (n = 6-7, Kruskal-wallis test).

F. Gene ontology reveals that the MElightgreen cluster is linked to cognition and synapse organization.

G. qPCR data shows rescue of AFF2/FMR2 and Hivep3 expression in APP/PS1 mice treated with inhibitor cocktail (n = 6-7, Kruskal-wallis test).

Data information: *P<0.05, **P<0.01, ***P<0.001. Bars and error bars indicate mean ± SEM. In boxplots (B, E), the center line indicates the median, while the upper and lower lines represent the 75th and 25th percentiles, respectively. The whiskers represent the smallest and largest values in the 1.5× interquartile range.
PTENε suppresses tumor metastasis through regulation of filopodia formation (2021)
Figure 1

Figure 1. Identification of PTEN variants that may be generated by initiation at codons within the PTEN 5′UTR.

A. Western blot of PTEN variants in WT (lane 1), PTENα-/-, β-/- (lane 2), and PTEN-/- (lane 3) Hela cells with a polyclonal antibody against full-length PTEN. Except for PTENα, PTENβ, and canonical PTEN, immunoblotting revealed four additional bands with molecular weights lower than PTENβ in WT and PTENα-/-, β-/- Hela cells. Hela PTEN-/- cells have no expression of PTEN variants or canonical PTEN. The asterisks shown here and in other immunoblot images throughout this manuscript indicate the corresponding protein band of PTENε.

B. Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C-terminal region of PTEN (Cell Signaling Technology, 138G6). The PTEN null prostate cancer cell line PC3 was used as a negative control.

C. Immunoprecipitation of PTEN variants with a homemade PTENα antibody before immunoblotting with a PTEN monoclonal antibody in Hela and HepG2 cells (Cell Signaling Technology, 138G6).

D. Western blot of PTEN variants in WT (lane 1) and N-terminal extended PTEN isoforms-/- (lane 2) Hela cells with a PTEN monoclonal antibody (Cell Signaling Technology, 138G6).
PTENε suppresses tumor metastasis through regulation of filopodia formation (2021)
Figure 3

Figure 3. The PTENε translation initiation codon was identified by MALDI-TOF Mass Spectrometry.

A. Two pFastBac1 plasmids containing PTENα with a C-terminal His-tag with or without CUG816>CUC mutation was used for in vitro purification and mass spectrometry sequencing. The initiation codon of canonical PTEN (AUG1032) was mutated to AUA and the initiation codons of PTENα (CUG513) and PTENβ (AUU594) were mutated to CUC to avoid co-purification of PTEN, PTENα, and PTENβ with PTENε.

B. His-selected affinity purification of PTENε. Sf9-expressed His-PTENε was purified using nickel affinity chromatography. Combined fractions were separated by SDS-PAGE. The protein band ranked third by molecular weight in the M2 lane can be eliminated by the CUG816>CUC mutation (M1 lane vs. M2 lane), indicating it is PTENε. Mass spectrometry analysis of the band of PTENε protein revealed five segments of peptide that match the N-terminal extended sequence of PTENε, including the most proximal N-terminal peptide of PTENε, MAAEEKQAQSLQPSSSR.

C. The MS/MS spectrum of the peptide (MAAEEKQAQSLQPSSSR) that matches the N-terminal sequence of PTENε.

D. Verification of expression of FLAG-tagged PTENα, PTENβ, and PTENε in PtenFLAG knock-in mice. Various tissues from PtenFLAG knock-in mice or control wild-type mice were lysed for immunoprecipitation with anti-FLAG M2 magnetic beads before being immunoblotted with the anti-PTENα antibody raised in our laboratory.

E. The MS/MS spectrum of the peptide (MAAEEKQAQSLQPSSSR) of endogenous PTENε in mice that matches the N-terminal sequence of PTENε. Protein lysates of PtenFLAG knock-in liver tissues were subjected to immunoprecipitation with anti-FLAG M2 agarose. The bound proteins were separated with SDS-PAGE, and gel slices of the band ranked fifth by molecular weight were analyzed by mass spectrometry.
PTENε suppresses tumor metastasis through regulation of filopodia formation (2021)
Figure 4

Figure 4. EIF2A is critical for non-AUG initiation of PTEN isoforms.

A. Correlation analysis of PTENε protein level with eIF2A or eIF5 mRNA level. The mRNA level of eIF2A and eIF5 in cell lines as listed in Fig 1B was measured by RT-PCR. PTENε protein level in cell lines listed in Fig 1B was measured by evaluating the gray value of corresponding protein bands. PTENε protein level is positively correlated with the eIF2A mRNA level (p < 0.05), while it has no significant correlation with the eIF5 mRNA level (p >0.05). The Pearson correlation test was employed to analysis the correlation between PTENε and eIF2A or eIF5 and data are presented as the mean ± SD based on three independent experiments.

B. Reduction of PTENε in response to eIF2A knockdown. Hela cells were infected with lentivirus expressing eIF2A shRNA, eIF5 shRNA, or scramble shRNA separately. The knockdown of eIF2A or eIF5 was validated by western blot (left panel). Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN, and GAPDH (right panel).

C. Overexpression of eIF2A induces the relative expression of PTENε. FLAG-tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN. GAPDH was used as a loading control.

D. A palindromic motif 14 bp downstream CUG816 in the 5′UTR of PTEN is evolutionarily conserved. (upper panel) Phylogenetic analysis of the 5′UTR of PTEN mRNA in bonobo (Pan paniscus), killer whale (Orcinus orca), and mouse (Mus musculus). The CUG816 initiation codon of PTENε is highlighted in a blue box and the 14 bp downstream palindromic sequence is highlighted in a red box. The putative hairpin structure shown in the lower panel was predicted by the Mfold web server.

(E, F) Abolition of PTENε expression by disruption of the palindromic motif. (E) Disruption of the CUG816 downstream palindromic motif by mutagenesis. C-terminal FLAG-tagged PTENε expression plasmids with or without disruption of the palindromic motif were introduced into HEK293 cells, followed by immunoblotting with FLAG and GAPDH antibodies (F).
PTENε suppresses tumor metastasis through regulation of filopodia formation (2021)
Figure 5

Figure 5. PTENε is localized predominantly in the cell membrane.

A. A set of different constructs of PTEN, PTENα, PTENβ, and PTENε with a C-terminal GFP tag. The AUG start codon of canonical PTEN was mutated to AUA in PTENα, PTENβ, and PTENε constructs to abolish the expression of canonical PTEN and the corresponding start site of these PTEN isoforms was mutated into AUG to enhance their expression.

B. Subcellular localization of C-terminal GFP-tagged PTENα, PTENβ, PTENε, and PTEN. The constructs indicated in the data (A) were introduced into Hela PTEN-/- cells. Thirty-six hours after transfection, cells were stained with DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm.

C. The transfected Hela PTEN-/- cells in (B) were stained with an anti-β-catenin antibody and DAPI before being imaged with confocal microscopy. The scale bars represent 5 μm.

D. HEK293 cells were transfected with C-terminal GFP tagged PTEN and PTENε and subjected to cell fractionation, followed by western blotting with GFP, E-cadherin, and β-tubulin antibodies. C: cytoplasm. M: cell plasma membrane.

E. Prediction of N-terminal structure of PTENε by Swiss Model (left panel); a different set of C-terminal GFP-tagged PTENε plasmids with or without fragment deletion in PTENε N-terminal sequence were constructed.

(F, G) To determine the sequences that are critical for the membrane localization of PTENε. Hela and HEK293 cells were transfected with constructs indicated in (E). Thirty-six hours after transfection, transfected Hela cells were stained with anti-β-catenin antibody and DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm (F). The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP, E-cadherin, and β-tubulin antibodies (G). C: cytoplasm. M: cell plasma membrane.