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: Molecular Cancer

Article Title: Survivin safeguards chromosome numbers and protects from aneuploidy independently from p53

doi: 10.1186/1476-4598-13-107

Figure Lengend Snippet: Activation of ATM and CHK2 at DNA lesions in SBC-2 cells with knockdown of Survivin. a: Representative immunofluorescence analysis of SBC-2 cells, with knockdown of Survivin and stained for activated ATM (ATM S1981) and ɣH2AX. Note double positive ɣH2AX, ATM S1981 loci indicative for DDR occurring in polyploid nuclei (arrowheads). Inlet showing magnification of indicated multinucleated cell with colocalized ɣH2AX and ATM S1981. b: Representative image depicting ɣH2AX, ATM S1981 staining in shLuc-transduced controls. Magnification bars: 10 μm. c: Representative image of SBC-2 cells after knockdown of Survivin showing the appearance of activated CHK2 (CHK2 T68) in ɣH2AX-positive DNA lesions. d: ɣH2AX and CHK2 T68 do not appear in shLuc-transduced SBC-2 cells. Magnification bars: 10 μm.

Article Snippet: The membranes were probed with primary antibodies in blocking solution over night at 4°C including rabbit polyclonal anti-Survivin (R&D Systems), polyclonal mouse anti-p21 waf/cip (R&D Systems), monoclonal rabbit anti-p21 waf/cip (Cell Signaling), polyclonal goat anti-p53 (R&D Systems), monoclonal rabbit anti-p53 S15 (Abcam), polyclonal anti-p53 S20, polyclonal anti-p53 S37 (Cell Signaling), monoclonal mouse anti-α-Tubulin and monoclonal anti-Actin (Sigma), monoclonal rabbit anti-ATM S1981 (Epitomics), polyclonal rabbit anti-ATM (Merck), monoclonal rabbit anti-Caspase 3 (Cell Signaling), monoclonal rabbit anti-CHK2 T68 (Cell Signaling), polyclonal rabbit anti-Cyclin D1 (Santa Cruz), polyclonal rabbit anti-Cyclin E (Santa Cruz) and monoclonal mouse anti-γH2AX (Millipore).

Techniques: Activation Assay, Knockdown, Immunofluorescence, Staining

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 5 Impaired DNA damage repair in p21-deficient cells after low-dose doxorubicin treatment. A Western blot analysis of phosphorylated ATM (p-ATM), p-Chk1, Chk1, p-Chk2, and Chk2 in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for various time points. β-actin was used as a loading control. B Fluorescence microscopy of Lamin B1 (red) and γ-H2AX foci (green) in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 48 h. Scale Bar = 20 μm. C Fluorescence microscopy showing nuclei (blue) and γ-H2AX foci (green) in HCT116 WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 24 h and then released into fresh media for various time points. Scale bar = 20 μm. Quantification of nuclei with >10 γ-H2AX foci (n = 20 cells per group, repeated 4 times). Data are mean ± SD ****p < 0.0001.

Article Snippet: The following antibodies were used: β-actin (sc-69879), p53 (sc-126), Lamin B1(sc-365214), αtubulin (sc-5286), Noxa (sc-515840, and MKLP1 (sc-136473) (Santa Cruz Biotechnologies, Dallas, TX, USA); p21 (ab109520), phospho-ATM (S1981) (ab81292), and phospho-DNA PKcs (S2056) (ab18192)(Abcam, Cambridge, MA, USA); phospho-Chk1 (Ser345)(# 2348), Chk1 (#2360), phospho-Chk2 (Thr68)(# 2197), Chk2 (#6334), cleaved caspase-3 (#9664), phospho-Histone H2A.X (Ser139)(# 9718), Mcl-1 (#39224), and Aurora B (#3094) (Cell signaling technology, Danvers, MA, USA); Mre11(GTX70212) and α-tubulin (GTX112141) (Genetex, San Antonio, Texas, USA); DNA-PKcs (19983-1-AP) (Proteintech, Rosemont, IL, USA); HRP-conjugated rabbit IgG and mouse IgG (Jackson Immunoresearch, West Grove, PA, USA).

Techniques: Western Blot, Control, Fluorescence, Microscopy

Journal: Nature biomedical engineering

Article Title: Targeted repair of heart injury by stem cells fused with platelet nanovesicles

doi: 10.1038/s41551-017-0182-x

Figure Lengend Snippet: a, A schematic showing the overview of PNV decoration and PNV-CSC therapy. b,c, Red fluorescent DiI-labelled CSCs (b) were fused with green fluorescent DiO-labelled PNVs to form PNV-CSCs (c). d, Co-incubation of CSCs (red) with PNV-CSCs (yellow). Scale bar, 20 μm. e, Western blot analysis revealed the expressions of platelet-specific markers including CD42b (GPIbα), GPVI and CD36 (GPIV) in platelets, PNVs and PNV-CSCs, but not in CSCs. Original western blot images can be found in Supplementary Figs. 2–4. f, Immunocytochemistry staining confirmed CD42b (GPIbα) and GPVI expression in PNV-CSCs (top), but not in CSCs (bottom). Scale bars, 200 μm. g,h, Flow cytometric analysis of platelet and exosome surface marker expressions on PNV-CSCs (n = 3) and CSCs (n = 4). *P< 0.05. All values are mean ± s.d. Two-tailed t-test for comparison.

Article Snippet: Platelet receptor blocking experiment To explore which platelet adhesion molecules contributed to the targeting of PNV-CSCs, PNV-CSCs were pre-treated with anti-CD42b neutralizing antibodies (sc-292722, rabbit polyclonal, Santa Cruz) or isotype control antibodies (ab37415, rabbit polyclonal isotype control, Abcam) for 30 mins.

Techniques: Incubation, Western Blot, Immunocytochemistry, Staining, Expressing, Marker, Two Tailed Test, Comparison

Journal: Nature biomedical engineering

Article Title: Targeted repair of heart injury by stem cells fused with platelet nanovesicles

doi: 10.1038/s41551-017-0182-x

Figure Lengend Snippet: a, Representative fluorescent micrographs showing the adherence of anti-CD42b or isotype antibody pre-treated PNV-CSCs on denuded rat aortas. b, Quantitation of binding (n = 3 samples per group). HPF, high-power field. c, Representative ex vivo fluorescent imaging of ischaemia/reperfusion rat hearts 24 hrs after intracoronary infusion of anti-CD42b or isotype antibody pre-treated PNV-CSCs. d, Quantitation of cell retention by qPCR (n = 3 animals per group). e, Representative Masson’s trichrome-stained myocardial sections 4 weeks after treatment (blue, scar tissue; red, viable myocardium). Scale bar, 2 mm. Quantitative analyses of viable myocardium and scar size from the Masson’s trichrome images (n = 5 animals per group). Left ventricular ejection fractions (LVEFs) measured by echocardiography at baseline (4 hrs post-MI) and 4 weeks later (n = 6 animals per group). *P< 0.05 when compared to the PNV-CSC + iso. Ab group. All values are mean ± s.d. Two-tailed t-test for comparison.

Article Snippet: Platelet receptor blocking experiment To explore which platelet adhesion molecules contributed to the targeting of PNV-CSCs, PNV-CSCs were pre-treated with anti-CD42b neutralizing antibodies (sc-292722, rabbit polyclonal, Santa Cruz) or isotype control antibodies (ab37415, rabbit polyclonal isotype control, Abcam) for 30 mins.

Techniques: Quantitation Assay, Binding Assay, Ex Vivo, Imaging, Staining, Two Tailed Test, Comparison

Journal: Cell Death & Disease

Article Title: Ablation of beta subunit of protein kinase CK2 in mouse oocytes causes follicle atresia and premature ovarian failure

doi: 10.1038/s41419-018-0505-1

Figure Lengend Snippet: a Immunoblotting analysis of DNA damage response signaling in ovaries of Ck2β fl/fl and Ck2β fl/fl ;GCre + mice at 2 weeks after birth. The ovary lysates were collected at least from three mice of each genotype and immunoblotted for γH2AX, H2AX, p-CHK2 (T68), CHK2, p63, p-p53 (S15), p53 and β-actin. Level of β-actin was detected as internal control. Each experiment was repeated at least 2–3 times. Molecular mass is given in kilo Daltons. b Immunoblotting analysis of the expression of p53 and p63 in ovaries of Ck2β fl/fl and Ck2β fl/fl ;GCre + at 4 weeks after birth. The ovary lysates were obtained from at least three mice of each genotype and immunoblotted for p63, p-p53 (S15), p53 and β-actin. Level of β-actin was used as internal control. Each experiment was repeated at least three times. Molecular mass is given in kilo Daltons. c MVH and γH2AX immunofluorescent staining of 2-week-old ovarian sections from Ck2β fl/fl and Ck2β fl/fl ;GCre + mice. Green: γH2AX; Red: MVH; Blue: DAPI. At least three mice of each genotype were used in this assay. Scale bar: 50 μm

Article Snippet: Rabbit monoclonal anti-CK2β antibody (AJ1128b; ABGENT); mouse monoclonal anti-β-actin antibody (sc-47778; Santa Cruz); mouse monoclonal anti-MVH antibody (ab27591; abcam); rabbit monoclonal anti-p-Akt (S473) antibody (4046; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-Akt (T308) (13038; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-Akt (S129) (ab133458; abcam); rabbit monoclonal anti-AKT1/2/3 antibody (ab32505; abcam); rabbit monoclonal anti-PTEN antibody (9559; Cell Signaling Technology, Inc.); rabbit polyclonal anti-p-TSC2 (S1387) antibody (5584; Cell Signaling Technology, Inc.); rabbit monoclonal anti-TSC2 antibody (4308; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-mTOR (S2448) antibody (5536; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-S6K (T389) antibody (9234; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-rpS6 (S240/244) antibody (5364; Cell Signaling Technology, Inc.); rabbit polyclonal anti-GSK3α/β (S21/9) antibody (9331; Cell Signaling Technology, Inc.); rabbit monoclonal anti-GSK3α/β antibody (5676; Cell Signaling Technology, Inc.); rabbit polyclonal anti-p-FOXO1 (T24)/FOXO3a (T32) antibody (9464; Cell Signaling Technology, Inc.); rabbit monoclonal anti-FOXO1 antibody (2880; Cell Signaling Technology, Inc.); rabbit monoclonal anti-γH2AX antibody (9718; Cell Signaling Technology, Inc.); rabbit monoclonal anti-H2AX antibody (7631; Cell Signaling Technology, Inc.); rabbit polyclonal anti-p-CHK2 (T68) antibody (BS4043; Bioworld Technology, Inc.); rabbit polyclonal anti-CHK2 antibody (BS6791; Bioworld Technology, Inc.); rabbit monoclonal anti-p-p53 (S15) (12571; Cell Signaling Technology, Inc.); rabbit polyclonal anti-p53 antibody (BS3156; Bioworld Technology, Inc.); rabbit monoclonal anti-p63 (ab124762; abcam); mouse monoclonal anti-CK2α antibody (ab70774; abcam); rabbit polyclonal anti-CK2α’ antibody (BS6571; Bioworld Technology, Inc.); rabbit monoclonal anti-phospho-CK2 substrate [(pS/pT)DXE] (8738; Cell Signaling Technology, Inc.); secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co, Ltd (Beijing).

Techniques: Western Blot, Expressing, Staining