Journal: The Journal of Neuroscience

Article Title: DAPK1–p53 Interaction Converges Necrotic and Apoptotic Pathways of Ischemic Neuronal Death

doi: 10.1523/JNEUROSCI.5119-13.2014

Figure Lengend Snippet: Direct binding of DAPK1DD to p53DM. A, Yeast two-hybrid screen of p53 as an interactive partner of DAPK1DD was found in a mouse whole-brain cDNA library. Strains were spotted at decreasing cell density on plates that allow for growth without (+His) or only with (−His+ 3-AT). B, Coimmunoprecipitation of 500 μg proteins from the adult mouse cerebral cortex with nonspecific IgG (IgG), anti-DAPK1 (DAPK1), or anti-p53 (p53), as indicated. The precipitates were probed with antibodies against DAPK1, p53, ERK, or Tau protein, as indicated. Input: 30 μg of protein of the extracts without IP was loaded. C, The constructs showing GST-p53 and GST-p53 mutants from a mouse full-length p531–390. D, The affinity binding assay showing that Flag-tagged DAPK1DD binds to GST-p53DM241–281. E, The affinity binding assay showing a minimal binding region of DAPK1DD in a sequence consisting of amino acids 270–281 of p53DM. F, An illustration showing that the p53DM peptide inhibits the DAPK1–p53 association. G, The p53DM peptide at 5 μm incubated in affinity binding assay buffer and completely eliminates a direct binding of Flag-tagged DAPK1DD with GST-p53. Data are mean ± SEM (ANOVA, F(3,11) = 5.26, *p < 0.01, n = 5/group). H, Application of p53DM disrupts DAPK1 association with p53, but not with ERK and Tau proteins. Coimmunoprecipitation of 500 μg proteins from the adult mouse cerebral cortex was performed with nonspecific IgG (IgG) or anti-DAPK1 (DAPK1). The precipitates were then incubated with 5 μm p53DM (lane 1) or a scrambled control (lane 2). The precipitates were probed with antibodies against DAPK1, p53, ERK, or Tau proteins, as indicated. B, E, H, Similar results were observed in each of the five experiments.

Article Snippet: To determine a binding sequence of DAPK1 DD with p53 protein, the GST-p53 deletion mutants (GST-p53 1–61 , GST-p53 61–126 , GST-p53 126–241 , GST-p53 241–281 , and GST-p53 281–390 ) were generated from full-length cDNA mouse p53 1–390 (Addgene, p LCRcala (p53), plasmid 12136), as we described previously ( Tu et al., 2010 ; Yang et al., 2012 ).

Techniques: Binding Assay, Two Hybrid Screening, cDNA Library Assay, Construct, Affinity Binding Assay, Sequencing, Incubation

Journal: The Journal of Neuroscience

Article Title: DAPK1–p53 Interaction Converges Necrotic and Apoptotic Pathways of Ischemic Neuronal Death

doi: 10.1523/JNEUROSCI.5119-13.2014

Figure Lengend Snippet: DAPK1 induces pS23 accumulation and nuclear translocation. A, cDAPK1 activates p53 reporter genes via a direct binding of cDAPK1DD to p53DM in HEK293 cells. Data are mean ± SEM; n = 5. *p < 0.01 (compared with the vector). B, The p53 protein unchanged in the presence of cDAPK1. HEK293 cells were transfected with 2 μg plasmids, as indicated. At 72 h after transfection, cell lysates (30 μg proteins) were prepared and probed with antibodies against p53 or GAPDH, as indicated. Similar results were observed in each of the four experiments. C, The cellular extracts from p53−/− cells coexpressing cDAPK1 with p53 or p53A23 are blotted with anti-pS23 or anti-p53 after treatment with control buffer (CTR) or 100 units of alkaline phosphatase (PPA). Similar results were observed in each of the five experiments. D, cDAPK1DD-p53DM binding induces pS23 accumulation. cDAPK1 in the presence of 5 μm of a membrane-permeable p53DM (Tat-p53DM) peptide or scrambled control (Tat-s-p53DM), or vehicle, or cDAPK1DDΔ alone was expressed in DAPK1−/− cortical neurons. At 72 h after the expression, the cellular proteins were prepared and blotted with antibodies against pS23, p53, DAPK1, or GAPDH, as indicated. The pS23 band intensities are expressed as the ratio of the respective p53 protein. Data are mean ± SEM; n = 5. *p < 0.01. E, Inhibition of ERK or Tau does not affect DAPK1–p53 interaction. Cultured cortical neurons (DIV10) were transfected with cDNA encoding wDAPK1, cDAPK1DDΔ, or cDAPK1 with a scrambled control small interference RNA (s-siRNA), siRNA that specifically targets to ERK (E-siRNA) or Tau (T-siRNA) using rAAV1/2 viral vector. At 72 h after transfection, cell lysates were prepared and blotted with antibodies, as indicated. Similar results were observed in each of the four experiments. F, The expression of exogenous p53 does not cause pS23 accumulation. Cultured cortical p53+/+ and p53−/− neurons (DIV10) were transfected with cDNA encoding p53 using a rAAV1/2 viral vector. At 72 h after transfection, cell lysates were prepared and blotted with antibodies against pS23, p53, or tubulin, as indicated. Similar results were observed in each of the four experiments. G, The pS23 undergoes nuclear translocation. The nuclear proteins were purified from p53+/+ cortical neurons expressing an empty vector, wDAPK1, cDAPK1, or cDAPK1DDΔ using individual rAAV1/2 viral vector. At 72 h after expression, cell lysates were prepared and probed with antibodies, as indicated. pS23 band intensity is expressed as the ratio of the respective p53 protein. Data are mean ± SEM; n = 4. *p < 0.01. H, The pS23 is expressed in the nucleus. The cultured cortical neurons (DIV10) were transfected with wDAPK1 or cDAPK1 using the individual rAAV1/2 virus vector. At 72 h after the transfection, the cultures were stained with antibodies against p53 (green), or pS23 (red) and DAPI (blue), as indicated. Similar results were seen in each of the five experiments. Scale bar, 50 μm. I, pS23 is colocalized with mitochondrial protein, COX-IV. The cultured cortical neurons (DIV10) were transfected with cDAPK1 using the rAAV1/2 viral vector. At 72 h after the transfection, the cultures were stained with antibodies against pS23 and COX-IV, respectively.

Article Snippet: To determine a binding sequence of DAPK1 DD with p53 protein, the GST-p53 deletion mutants (GST-p53 1–61 , GST-p53 61–126 , GST-p53 126–241 , GST-p53 241–281 , and GST-p53 281–390 ) were generated from full-length cDNA mouse p53 1–390 (Addgene, p LCRcala (p53), plasmid 12136), as we described previously ( Tu et al., 2010 ; Yang et al., 2012 ).

Techniques: Translocation Assay, Binding Assay, Plasmid Preparation, Transfection, Expressing, Inhibition, Cell Culture, Purification, Staining

Journal: The Journal of Neuroscience

Article Title: DAPK1–p53 Interaction Converges Necrotic and Apoptotic Pathways of Ischemic Neuronal Death

doi: 10.1523/JNEUROSCI.5119-13.2014

Figure Lengend Snippet: The DAPK1–p53 interaction mediates ischemic neuronal death. A, OGD induces pS23 accumulation via DAPK1 activation. Cultured DAPK1+/+ and DAPK1−/− mouse cortical neurons (DIV14) were treated with 60 min OGD. At 24, 30, 36, or 48 h after OGD treatment, cellular extracts were prepared and blotted with anti-pS23 and anti-p53 antibodies. B, The ratio of pS23 versus p53 was normalized at different time points to the 24 h time point (defined as 100%). Data are mean ± SEM (n = 4 assays). *p < 0.01. C, OGD does not alter p53 expression. Cultured DAPK1+/+ and DAPK1−/− cortical neurons (DIV14) were challenged with 60 min OGD. At 24, 30, 36, or 48 h after OGD, cellular RNAs were prepared and analyzed by qPCR. Data are mean ± SEM (n = 4 assays). D, E, Representative images showing the double labeling of PI and TUNEL in cultured cortical neurons treated with 60 min (D) or 90 min (E) OGD. Cortical cultures (DIV14) were exposed to OGD for 60 min or 90 min. Three days after recovery from the OGD treatment, the cultures were stained with PI (red) and TUNEL (green), as indicated. F, The DAPK1–p53 interaction mediates OGD-induced necrosis and apoptosis. Cultured p53+/+ neurons were challenged with OGD in the presence of 5 μm vehicle, Tat-p53DM, or Tat-s-p53DM. At 3 d after OGD, the cultures were stained with PI and TUNEL. In cultured p53−/− neurons, exogenous wild-type p53 or its mutant p53A23 or empty vector was expressed. Data are expressed as a percentage of PI- and TUNEL-labeled cells relative to DAPI labeled cells. Data are mean ± SEM (n = 5 assays). *p < 0.01. G, A working model: the DAPK1–p53 interaction activates necrotic and apoptotic pathways of ischemic injury through transcription- and mitochondria-dependent pathways. DAPK1 binds to p53DM and catalyzes the pS23, which enters into the nucleus and activates the proapoptotic gene expression and apoptosis. The pS23 also enters into the mitochondrial matrix and interacts with CypD and causes necrosis. ANT, Adenine nucleotide translocase; VDAC, voltage-dependent anion channel; CypD, cyclophilin D; mPTP, mitochondrial permeability transition pore.

Article Snippet: To determine a binding sequence of DAPK1 DD with p53 protein, the GST-p53 deletion mutants (GST-p53 1–61 , GST-p53 61–126 , GST-p53 126–241 , GST-p53 241–281 , and GST-p53 281–390 ) were generated from full-length cDNA mouse p53 1–390 (Addgene, p LCRcala (p53), plasmid 12136), as we described previously ( Tu et al., 2010 ; Yang et al., 2012 ).

Techniques: Activation Assay, Cell Culture, Expressing, Labeling, TUNEL Assay, Staining, Mutagenesis, Plasmid Preparation, Permeability

Journal: Cell stem cell

Article Title: Stress-induced changes in bone marrow stromal cell populations revealed through single-cell protein expression mapping

doi: 10.1016/j.stem.2019.06.003

Figure Lengend Snippet: Multi-Dimensional Single-Cell Mass Cytometry analysis reveals distinguishable clusters of bone marrow stromal cells

Article Snippet: Anti-Mouse CD31/PECAM-1 (390)-165Ho , Fluidigm , Cat# 3165013B RRID:AB_2801434.

Techniques: Mass Cytometry, Purification, Affinity Purification, Functional Assay, Recombinant, Electron Microscopy, Modification, Conjugation Assay, Labeling, Isolation, RNA Library Preparation, Imaging, Gene Expression, Software