Journal: Nature Communications

Article Title: Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis

doi: 10.1038/s41467-018-02950-5

Figure Lengend Snippet: Cisplatin induces K472 acetylation and S461 phosphorylation of PFKFB3. a DNA damage signals induced PFKFB3 K472 acetylation. Flag-tagged PFKFB3 was expressed in HEK293T cells, which were then treated with etoposide (10 μM), adriamycin (1 μM), UV irradiation (10 J/m 2 ) and cisplatin (50 or 100 μM) for 24 h. Flag-PFKFB3 was immunoprecipitated with Flag beads and immunoblotting was performed with the antibodies indicated. Relative PFKFB3 K472 acetylation and phosphorylation were normalized by Flag protein. b The amino acid sequence near K472 of PFKFB3 displays high similarity with the sequence near K320 of TP53. c Cisplatin treatment induces PFKFB3 cytoplasmic accumulation. HEK293T cells were treated with or without cisplatin (50 μM) for 24 h before harvest. Cells were then suspended in PBS and treated with a gradient concentration of digitonin. Supernatant and precipitate were collected for immunoblotting with indicated antibodies. S, supernatant; P, precipitate. d Cisplatin induces K472 acetylation and S461 phosphorylation of endogenous PFKFB3. Endogenous PFKFB3 protein was purified from HEK293T cells after cisplatin treatment as indicated for 24 h. e Cisplatin or etoposide treatment enhances K472 acetylation in SIRT1 knockout cells. Endogenous PFKFB3 protein were purified from WT or SIRT1 knockout HEK293T cells treated with EX527 (10 μM), cisplatin (50 μM) or etoposide (10 μM) for 24 h. f Combined knockdown of PCAF and GCN5 abolishes cisplatin- or etoposide-induced PFKFB3 K472 acetylation. HEK293T cells were transfected with siRNAs targeting PCAF and GCN5. After 60 h, cells were treated with cisplatin (50 μM) or etoposide (10 μM) for 24 h. g , h Cisplatin treatment induces pan-acetylation of PCAF and GCN5. Flag-tagged PCAF or GCN5 was expressed in HEK293T cells, which were treated with cisplatin for the duration indicated at a concentration of 50 μM. Relative pan-acetylation level of PCAF or GCN5 was normalized by Flag protein. i Cisplatin increases acetyltransferase activity of PCAF and GCN5. Flag-tag PCAF or GCN5 was purified from HEK293T cells treated with or without cisplatin (50 μM) for 24 h, then incubated with recombinant His-PFKFB3 in acetylation assay buffer. Purified proteins visualized by Coomassie blue staining are shown (lower panel). Data are representative of at least two independent experiments

Article Snippet: Antibody against acetyl-p53 (Lys320, 06–1283, 1:1000) was purchased from Merck Millipore.

Techniques: Phospho-proteomics, Irradiation, Immunoprecipitation, Western Blot, Sequencing, Concentration Assay, Purification, Knock-Out, Knockdown, Transfection, Activity Assay, FLAG-tag, Incubation, Recombinant, Acetylation Assay, Staining

Journal: Cell Death & Disease

Article Title: PARP1 regulates the protein stability and proapoptotic function of HIPK2

doi: 10.1038/cddis.2016.345

Figure Lengend Snippet: PARP1 interacted with HIPK2 and decreased its expression. ( a ) Representative images of HEK 293 cells stained for endogenous HIPK2 (green) and PARP1 (red) by indirect immunofluorescence (endo, endogenous). Overlapping localization is shown in yellow (merge). Nucleus was visualized by DAPI (upper panels). HEK 293 cells were transfected with expression vectors encoding GFP-HIPK2 and analyzed for HIPK2 (green) and endogenous PARP1 (red) by indirect immunofluorescence (lower panels). ( b ) Expression plasmid coding for Myc-HIPK2 was transfected into HEK 293 cells. The transfected cells were immunoprecipitated with anti-Myc antibodies, followed by immunoblotting using anti-PARP1 antibodies (upper panel). The blots were also examined for the detection of HIPK2 using anti-Myc antibodies (lower panel). The input lane represents 10% of total cell lysates. ( c ) Endogenous HIPK2 was immunoprecipitated from HEK 293 cell lysates using rabbit polyclonal anti-HIPK2 antibodies (endo) or control normal rabbit serum. The precipitates were analyzed by immunoblotting for the detection of PARP1 (upper panel) or HIPK2 (lower panel). ( d ) In vitro translated HIPK2 was incubated with equal amounts of either GST protein or purified GST-PARP1. Bound proteins were examined by immunoblotting using anti-Myc antibodies (upper panel). Affinity-purified GST or GST-PARP1 used in this assay is shown in the lower panel following blotting using anti-PARP1 and anti-GST antibodies. Input indicates 10% of in vitro translated Myc-HIPK2 used in the binding reaction. ( e ) Increasing amounts (0.125, 0.25, 0.5 μ g) of PARP1 expression vectors were transfected into HEK 293 cells with Myc-HIPK2 plasmids (0.5 μ g) and then examined by immunoblotting. ( f ) HEK 293 cells co-transfected with expression vectors for HIPK2 and PARP1 were incubated for the indicated times and then examined by immunoblotting. ( g ) PARP1 expression vector was transfected into the cell line stably expressing low level of kinase-dead Myc-HIPK2 (KD, K221R) and then analyzed by immunoblotting. ( h ) Myc-HIPK2 expression vector was transfected into HEK 293 cells with expression vector or shRNA vector (pLKO.1) for PARP1 to examine the effect of PARP1 knockdown on HIPK2 expression. ( i ) Wild-type (WT) and PARP1 knockout (KO) MEFs were prepared from three different mice per each genotype and pooled for culture. The levels of endogenous HIPK2 were analyzed by immunoblotting using anti-HIPK2 antibodies

Article Snippet: The following antibodies were used: mouse monoclonal anti-PARP1 antibody (BD Bioscience, San Jose, CA, USA), mouse monoclonal anti-HIPK2 antibody (kindly provided by Dr H Koseki, RIKEN Research Center, Yokohama, Japan), rabbit polyclonal anti-PAR (BD Bioscience), mouse monoclonal anti- α -tubulin (Sigma-Aldrich), mouse monoclonal antibodies for Myc, HA, Flag and GST (abm, Richmond, Canada), rabbit polyclonal anti-phospho-p53 (serine 46) (BD Bioscience), mouse monoclonal anti-ubiquitin (Millipore, Billerica, MA, USA), rabbit monoclonal anti-p53 (Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-HSP70 (Stressgen, San Diego, CA, USA), rabbit polyclonal anti-CHIP (kindly provided by Dr S Yoo, Kyung Hee University, Korea).

Techniques: Expressing, Staining, Immunofluorescence, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, In Vitro, Incubation, Purification, Affinity Purification, Binding Assay, Stable Transfection, shRNA, Knock-Out

Journal: eLife

Article Title: Opposing p53 and mTOR/AKT promote an in vivo switch from apoptosis to senescence upon telomere shortening in zebrafish

doi: 10.7554/eLife.54935

Figure Lengend Snippet: ( A-B ) Western blot analysis of DNA damage and senescence-associated proteins in gut and testis of 3 month ( A ) or 9-month-old ( B ) of WT and tert-/- siblings (N >= 5 fish). Representative western blot (left panel) and corresponding quantification (right panel) showing induction of DNA Damage Response (H2A.X-P and p53) in 3-month-old and senescence (p15/16) in 9-month-old tert-/- zebrafish. ( C ) RT-qPCR analysis of senescence associated genes p15/16 and p21. RT-qPCR graphs are representing mean ± SEM mRNA fold increase after normalisation by rpl13a gene expression levels (* p-value<0.05; ** p-value<0.01, using the Mann-Whitney test). Figure 2—source data 1. Western Blot quantifications, as plotted in . Figure 2—source data 2. Real-time qPCR data of p15/16 and p21, as plotted in .

Article Snippet: Antibody , anti-zebrafish p53 (rabbit polyclonal) , Anaspec , #55342; RRID: AB_2287635 , WB (1:1000).

Techniques: Western Blot, Quantitative RT-PCR, Gene Expression, MANN-WHITNEY

Journal: eLife

Article Title: Opposing p53 and mTOR/AKT promote an in vivo switch from apoptosis to senescence upon telomere shortening in zebrafish

doi: 10.7554/eLife.54935

Figure Lengend Snippet: ( A and E ) Representative haematoxylin and eosin-stained sections of gut ( A ) (scale bar: 40 µm) and testis ( E ) (scale bar: 25 µm) from 6-month-old WT, tert-/-, tp53-/ - and tert-/- tp53-/ - siblings (N = 3 fish each);. Mutation of tp53 in tert-/- fish rescues short-telomere induced tissue defects. ( B and F ) Representative western blot analysis of AKT-p and SOD2 in gut ( B ) and testis ( F ) (N = 2 fish each). Mutation of tp53 in tert-/- fish prevents phosphorylation of AKT and downstream downregulation of SOD2 leading to a rescue of increased ROS levels (C and G; N = 3 fish per genotype). ( D and H ) Representative images of SA-β-GAL staining of gut (scale bar: 40 µm) ( D ) and testis (scale bar: 25 µm) ( H ) from 6 month-old WT, tert-/-, p53-/ - and tert-/- p53-/ - siblings (N = 3 fish). Data are represented as mean ± SEM (** p-value<0.01, using t-test). Figure 5—source data 1. ROS levels measurements, as plotted in .

Article Snippet: Antibody , anti-zebrafish p53 (rabbit polyclonal) , Anaspec , #55342; RRID: AB_2287635 , WB (1:1000).

Techniques: Staining, Mutagenesis, Western Blot, Phospho-proteomics

Journal: eLife

Article Title: Opposing p53 and mTOR/AKT promote an in vivo switch from apoptosis to senescence upon telomere shortening in zebrafish

doi: 10.7554/eLife.54935

Figure Lengend Snippet: Early telomere shortening triggers p53-dependent apoptosis and inhibition of cell proliferation. At early age, apoptosis is the predominant cell fate and it mostly counterbalanced by compensatory proliferation of neighboring cells. However, inhibition of cell proliferation results in a progressive loss of tissue cellularity, eventually leading to tissue damage. As age progresses, loss of tissue homeostasis triggers the pro-proliferative mTOR/AKT pathway. Akt phosphorylates FoxO, inducing its translocation from the nucleus to the cytoplasm. Loss of FoxO transcriptional activity reduces mitochondrial SOD2 expression generating mitochondria oxidative stress through increased ROS levels. Mitochondrial dysfunction eventually triggers p15/16 expression and senescence becomes the predominant cell fate.

Article Snippet: Antibody , anti-zebrafish p53 (rabbit polyclonal) , Anaspec , #55342; RRID: AB_2287635 , WB (1:1000).

Techniques: Inhibition, Translocation Assay, Activity Assay, Expressing

Journal: eLife

Article Title: Opposing p53 and mTOR/AKT promote an in vivo switch from apoptosis to senescence upon telomere shortening in zebrafish

doi: 10.7554/eLife.54935

Figure Lengend Snippet:

Article Snippet: Antibody , anti-zebrafish p53 (rabbit polyclonal) , Anaspec , #55342; RRID: AB_2287635 , WB (1:1000).

Techniques: Sequencing, Control, In Situ, Cell Viability Assay, Staining