Journal: Journal of Advanced Research

Article Title: Multi-omics approach reveals TGF-β signaling-driven senescence in periodontium stem cells

doi: 10.1016/j.jare.2024.12.037

Figure Lengend Snippet: TGF-β1-induced ROS accumulation leads to G2 arrest through ATM signaling. (A). RT-qPCR analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (B). Western blot analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (C). Western blot analysis of ATM signaling phosphorylated activation in the condition of ATM or CHK2 knockdown; (D). Western blot analysis of ATM signaling phosphorylated activation under N-acetyl-l-cysteine (NAC) treatment; (E). Western blot analysis of ATM signaling phosphorylated activation under decitabine treatment. Reagent doses and duration: TGF-β1 (10 ng/ml, 48 h), NAC (4 mM, 48 h), decitabine (10uM, 48 h). Predicted molecular weight: ATM 350 kDa, pATM 370 kDa, CDC25C&pCDC25C 60 kDa, CHK1&pCHK1 54 kDa, CHK2&pCHK2 62 kDa, β-Tubulin 55 kDa; GAPDH 37 kDa. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ns denotes not significant by one-way analysis of variance followed by the Tukey’s test. Error bars represent means ± SEM of at least three independent experiments.

Article Snippet: Antibodies used in this study were listed as follows: antibodies from Abmart: anti-β-Tubulin ( M20005 ); antibodies from Huabio: anti-GAPDH recombinant rabbit monoclonal antibody (ET1601-4), anti-DNMT1 recombinant rabbit monoclonal antibody (ET1702-77), anti-DNMT3A recombinant rabbit monoclonal antibody (ET1609-31), anti-DNMT3B recombinant rabbit monoclonal antibody (ET1605-9), anti-p16 INK4A recombinant rabbit monoclonal antibody (ET1608-62), anti-AMPKγ1 recombinant mouse monoclonal antibody (EM2001-06), anti-AMPK alpha 1 recombinant rabbit monoclonal antibody (ET1608-40), anti-phospho-AMPK alpha 1 (S496) recombinant rabbit monoclonal antibody (ET1612-72), HRP-conjugated goat anti-rabbit IgG goat polyclonal antibody (HA1001), HRP-conjugated goat anti-mouse IgG polyclonal antibody (HA1006); antibodies from Abcam: anti-ATM antibody (ab32420), anti-phospho-ATM (S1981) antibody (ab81292), anti-CHK1 antibody (ab40866), anti-phospho-CHK1 (S296) antibody (ab79758), anti-CDC25C antibody (ab32444), anti-p21 antibody (ab109199), anti-CHK2 antibody (ab109413), anti-histone H3 (tri-methyl K9) (ab176916), anti-phospho-γ-H2AX (S139) antibody (ab81299), anti-HMGB1 antibody (ab79823); antibodies from CST: anti-Ki67 (D3B5) rabbit mAb (#9129), anti-phospho-CHK2 (Thr68) rabbit mAb (#2197), anti-phospho-CDC25C (Ser216) rabbit mAb (#4901); antibodies from Bioss: goat anti-rabbit IgG antibody (H + L), FITC-conjugated (bs-0295G-FITC); goat anti-rat IgG antibody (H + L), cyanine 3-conjugated (bs-0293G-Cy3); antibodies from Biolegend: APC anti-human CD34 antibody (#343509), FITC anti-human CD146 antibody (#361011).

Techniques: Quantitative RT-PCR, Western Blot, Activation Assay, Knockdown, Molecular Weight

Journal: Cancers

Article Title: TP-0903 Is Active in Preclinical Models of Acute Myeloid Leukemia with TP53 Mutation/Deletion.

doi: 10.3390/cancers15010029

Figure Lengend Snippet: Figure 3. TP-0903 induces DNA damage response in TP53 mutant AML cell lines. (A) MV4-11 (R248W), (B) HL-60, and (C) Kasumi-1 cells were treated with DMSO or TP-0903 at indicated concentrations for 4 h. Immunoblotting was performed to determine the expression of pChk1 (Ser345), pChk2 (Thr68), and pH2AX (Ser139/Tyr142). Vinculin served as the loading control for pChk1/Chk1 and pChk2/Chk2 blots. GAPDH served as the loading control for pH2AX/H2AX blots. Immunoblots were quantified against respective loading controls (vinculin or GAPDH) using ImageJ. Blots are representative of 2–3 independent experiments.

Article Snippet: Antibodies against p53 (48818; clone DO-7), p21 (2947; clone 12D1), pH2AX (Ser 139) (80312; clone D7T2V), pH2AX (Ser139/Tyr142) (5438; clone N/A), H2AX (7631; clone D17A3), pAURKA(Thr288)/B(Thr232)/C(Thr198) (2914; clone D13A11), AURKA (91590; clone D3V7T), AURKB (3094; clone N/A), pChk1 (Ser345) (2348; clone 133D3), pChk2 (Thr68) (2197; clone C13C1), Chk1 (2360; clone 2G1D5), Chk2 (6334; clone D9C6), vinculin (13901; clone E1E9V), GAPDH (5174S; clone D16H11), and HRP-conjugate secondary antirabbit (7074) were obtained from Cell Signaling Technology (CST) (Danvers, MA, USA).

Techniques: Mutagenesis, Western Blot, Expressing, Control

Journal:

Article Title: Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

doi: 10.1038/sj.embor.embor901

Figure Lengend Snippet: Checkpoint kinase 2 docking to the DNA-binding domain of p53. (A) Low homology between p53 and checkpoint kinase 2 (CHK2) substrates. Consensus phosphorylation sites have been defined for both CHK1 and CHK2 (CHK1-tide and CHK2-tide; O'Neill et al., 2002). The homology of these consensus-site peptides to cell division cycle 25 (CDC25) and p53 are as indicated. The key residues required for p300 and MDM2 (mouse double minute 2) binding to the p53 BOX-I domain (Dornan et al., 2003) are shown in blue. Green shading indicates homolgous sites, red shading indicates phosphoacceptor sites. (B) CHK2 modifies two sites on p53. An immunochemical assay using phospho-specific monoclonal antibodies to the BOX-I phosphorylation sites (Craig et al., 1999) was used to assay CHK2. The addition of CHK2 to reactions with p53 tetramers (compare lane 1 with lane 2) results in the specific phosphorylation of p53 at Ser 20 and Thr 18, but not at Ser 15. (C) Two peptides derived from the core domain of p53 bind to CHK2. (Ca) Overlapping peptides derived from p53 were tested for binding to CHK2. The five conserved BOX domains of p53 are shown in black. The CHK2 phosphorylation sites are in the BOX-I domain, whereas the CHK2-docking peptides are in the BOX-II and BOX-V domains. (Cb) Direct binding of CHK2 was tested for all p53 peptides, and relative binding of CHK2 to the BOX-II and BOX-V peptides in an ELISA (enzyme-linked immunosorbent assay) format (ELISA method as in Dornan et al., 2003) is shown as relative light units (RLUs). (D,E) The BOX-II-domain and BOX-V-domain peptides stimulate CHK2 activity. Kinase reactions containing p53 were assembled without CHK2 (lane 1) or with CHK2 (lanes 2–9). The indicated overlapping peptides from the BOX-I, BOX-II and BOX-V domains were added, and reaction products were analysed for p53 phosphorylation at Ser 20 (D) and Thr 18 (E) using an immunochemical blotting assay. (F) The BOX-II-domain and BOX-V-domain peptides stimulate CHK2 activity. Kinase reactions containing [32Pγ]ATP, full-length p53 and CHK2 were assembled without peptide (lane 1) or with the indicated peptides from BOX-II and BOX-V (lanes 2 and 3). Reaction products were analysed for p53 phosphorylation after electrophoresis by autoradiography. The upper band shows CHK2 autophosphorylation and the lower band shows p53 phosphorylation. (G) The minimal adjacent locations of the BOX-II-domain and BOX-V-domain peptides (in the S2′ and H2 motifs) within the core domain of p53 are shown in green. (H) The entire BOX-II and BOX-V peptide sequences that bind CHK2 (C) extend further into the core domain and reside within the same plane (shown in red, including part of the S10 β-sheet, the S2 and S2' β-sheets and the helix H2).

Article Snippet: Antibodies to CHK2 and the Thr-68-modified CHK2 were obtained from Santa Cruz Biotechnology. p21 protein was purified as indicated and the Ser-146-phospho-specific antibody was used as a probe for p21 phosphorylation ( Scott et al ., 2002 ).

Techniques: Binding Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Electrophoresis, Autoradiography

Journal:

Article Title: Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

doi: 10.1038/sj.embor.embor901

Figure Lengend Snippet: Checkpoint kinase 2 is activated in trans by p53 DNA-binding-domain peptides. (A) Two models account for the stimulation of checkpoint kinase 2 (CHK2) by the BOX-II-domain and BOX-V-domain peptides. According to the anchoring model (model 1), the BOX-II-domain and BOX-V-domain peptides only anchor CHK2 and should have no effect in trans on CHK2 activity towards the p53 fragments. Alternatively, according to the allosteric model (model 2), the BOX-II-domain and BOX-V-domain peptides can activate CHK2 towards the p53 activation-domain fragment that lacks the CHK2-core domain sites. (B) The BOX-II-domain and BOX-V-domain peptides activate CHK2. Kinase reactions containing [32Pγ]ATP, p53N1–66 and CHK2 were assembled without peptide (lane 1) or with the indicated peptides from BOX-II and BOX-V (lanes 2 and 3). Reaction products were analysed after electrophoresis by autoradiography for p53N1–66 phosphorylation and CHK2 autophosphorylation. The upper band shows CHK2 autophosphorylation and the lower band shows p53N1–66 phosphorylation. (C) The BOX-II-domain and BOX-V-domain peptides synergistically activate CHK2. Kinase reactions contained [32Pγ]ATP and CHK2 only (lane 1), or contained [32Pγ]ATP, CHK2 and p53N1–66 either without peptide (lane 2), with the indicated peptides from BOX-II and BOX-V (lanes 3 and 4), or with both peptides from BOX-II and BOX-V (lane 5). Reaction products were analysed after electrophoresis by autoradiography for p53N1–66 phosphorylation (lower band) and for CHK2 autophosphorylation (upper band). (D) The BOX-II-doman and BOX-V-domain peptides do not affect CHK2 phosphorylation of CDC25. Kinase reactions contained [32Pγ]ATP and CHK2 only (lane 1), or contained [32Pγ]ATP, CHK2 and CDC25C either without peptide (lane 2), with the indicated peptides from BOX-II and BOX-V (lanes 3 and 4), or with both peptides from BOX-II and BOX-V (lane 5). Reaction products were analysed for CDC25 phosphorylation (lower band) and CHK2 autophosphorylation (upper band) after electrophoresis by autoradiography. (E) The BOX-II-domain and BOX-V-domain peptides activate CHK2 phosphorylation of p53 at Thr 18. Kinase reactions containing p53N1–66 and CHK2 were assembled without peptide (lanes 1 and 4) or with peptides (lanes 2, 3, 5 and 6). Reaction products were analysed for p53N1–66 phosphorylation at Ser 20 (lanes 1–3) or Thr 18 (lanes 4–6) using an immunochemical blotting assay. Protein band 'a' is unphosphorylated p53N1–66 or p53N1–66 phosphorylated at Ser 20, whereas protein band 'b' is a kinase supershift that indicates Thr 18 phosphorylation. (F) The BOX-II-domain and BOX-V-domain peptides activate CHK2 phosphorylation of p53(S15A)N1–66 at Ser 20 and Thr 18. Kinase reactions containing p53(S15A)N1–66 and CHK2 were assembled without peptide (lanes 1 and 4) or with peptides (lanes 2, 3, 5 and 6). Reaction products were analysed for p53 phosphorylation at Ser 20 (lanes 1–3) or Thr 18 (lanes 4–6) phosphorylation using an immunochemical blotting assay. (G) Mutation of the BOX-V peptide attenuates its activity as a CHK2 activator. Reactions were assembled with p53N1–66, [32Pγ]ATP, CHK2 and the BOX-V or BOX-II peptides, as indicated in the figure. BOX-V peptides containing alanine mutations at the indicated codons (270–277) were added, and the relative phosphorylation of p53 and CHK2 is indicated.

Article Snippet: Antibodies to CHK2 and the Thr-68-modified CHK2 were obtained from Santa Cruz Biotechnology. p21 protein was purified as indicated and the Ser-146-phospho-specific antibody was used as a probe for p21 phosphorylation ( Scott et al ., 2002 ).

Techniques: Binding Assay, Activity Assay, Activation Assay, Electrophoresis, Autoradiography, Mutagenesis

Journal:

Article Title: Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

doi: 10.1038/sj.embor.embor901

Figure Lengend Snippet: p53-core-domain peptides induce checkpoint-kinase-2-dependent phosphorylation of p21WAF1. (A) Homology of the p53 BOX-I-homology domain to p21WAF1. A database search of proteins with homology to the BOX-I domain of p53 identified p21WAF1. The Thr 18 and Ser 20 phosphoacceptor sites of p53 (bold) correspond to the Ser 146 and Thr 148 phosphoacceptor sites of p21WAF1. (B) The BOX-II-domain and BOX-V-domain peptides activate checkpoint kinase 2 (CHK2) phosphorylation of a BOX-I domain peptide. Kinase reactions contained the p53 BOX-I peptide, [32Pγ]ATP and CHK2 (all bars), either without CHK2-docking peptides (left bar) or with the BOX-II-domain and BOX-V-domain peptides, individually or together (II + V; right bars). Reaction products were spotted onto filter paper; kinase activity is represented as picomoles of 32P incorporation (Scott et al., 2002). (C) CHK2 activation towards p21WAF1. The BOX-II-domain and BOX-V-domain peptides synergistically activate CHK2 phosphorylation of p21WAF1. Kinase reactions contained [32Pγ]ATP and CHK2 only (lane 1), or [32Pγ]ATP, CHK2 and p21WAF1 with or without the indicated peptides from BOX-II and BOX-V (lanes 2–5). Reaction products were analysed after electrophoresis by autoradiography for p21WAF1 phosphorylation (Scott et al., 2002).

Article Snippet: Antibodies to CHK2 and the Thr-68-modified CHK2 were obtained from Santa Cruz Biotechnology. p21 protein was purified as indicated and the Ser-146-phospho-specific antibody was used as a probe for p21 phosphorylation ( Scott et al ., 2002 ).

Techniques: Activity Assay, Activation Assay, Electrophoresis, Autoradiography