Journal: Cancer Cell

Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

doi: 10.1016/j.ccell.2017.08.010

Figure Lengend Snippet:

Article Snippet: p-Chk2 [p Thr68] Rabbit polyclonal Antibody , Novus Biologicals , Cat# NB100-92502; RRID: AB_1216474.

Techniques: Purification, Imaging, Virus, Control, Mutagenesis, Recombinant, Staining, Reverse Transcription, SYBR Green Assay, Microarray, RNA Expression, RNA Sequencing, Methylation, Labeling, Software, Light Microscopy

Journal: Aging (Albany NY)

Article Title: Chronic stress induces Alzheimer’s disease-like pathologies through DNA damage-Chk1-CIP2A signaling

doi: 10.18632/aging.205862

Figure Lengend Snippet: Stress hormone activates DNA damage-Chk1-CIP2A pathway, results in tau hyperphosphorylation, Aβ overproduction and synaptic impairments in primary neurons. ( A – E ) Primary neurons were treated with 100 nM corticotropin-releasing factor (CRF) for 24 hours. ( A ) Representative immunoblots of γH2A.X, Chk1-pS317, Chk1-pS345, Chk1, CIP2A, Tau-pT231, Tau-pS396, Tau-pS404, Tau-5 (total tau), APP-pT668, APP and β-actin. ( B ) Quantification of the relative protein levels; non-phosphorylated proteins such as γH2A.X and CIP2A were normalized to the β-actin levels; phosphorylated Chk1-pS317, Chk1-pS345, Tau-pT231, Tau-pS396, Tau-pS404 and APP-pT668 were normalized to corresponding total Chk1, tau (Tau-5) and APP respectively. n = 3 or n = 6 per group. ( C ) The relative concentration of Aβ 40 and Aβ 42 in supernatant and cell lysate of primary neurons detected by ELISA. n = 3 or n = 4 per group. ( D ) Representative immunoblots of Synapsin I, PSD95, GluA1 and β-actin. ( E ) Quantification of the relative protein levels, which were normalized to the β-actin levels. n = 3 per group. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies used in this research were as follows: anti-γH2A.X (ab26350, Abcam; 80312S, Cell Signaling Technology), anti-NeuN (26975-1-AP, Proteintech), anti-GFAP (12389S, Cell Signaling Technology), anti-Iba1 (ab178846, Abcam), anti-Chk1-pS317 (12302S, Cell Signaling Technology), anti-Chk1-pS345 (39233, Gene Tex; A21009, Abclonal), anti-Chk1 (A7653, Abclonal), anti-CIP2A (A12267, Abclonal), anti-Tau-pT231 (R381181, Zen-Bioscience), anti-Tau-pS396 (381213, Zen-Bioscience), anti-Tau-pS404 (11112, SAB), Tau-5 (ab80579, Abcam), anti-APP-pT668 (6986S, Cell Signaling Technology), anti-APP (60,342–1-Ig, Proteintech), anti-PSD95 (36233S, Cell Signaling Technology; 30255-1-AP, Proteintech), anti-Synapsin- I (5297S, Cell Signaling Technology), anti-GluA1 (13185S, Cell Signaling Technology), anti-GluA2 (13607S, Cell Signaling Technology), anti-β-actin (AC026, Abclonal).

Techniques: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Chronic stress induces Alzheimer’s disease-like pathologies through DNA damage-Chk1-CIP2A signaling

doi: 10.18632/aging.205862

Figure Lengend Snippet: Chronic stress induces cognitive deficits in mice, which could be prevented by vitamin C and Chk1 inhibitor. ( A ) Schematic diagram for establishing chronic restraint stress model and treatment with vitamin C or Chk1 inhibitor. ( B ) The total time (s, left), distance (cm, middle) and time in central area (s, right) in open field test. ( C ) The time spending in open arm (%) in elevated plus maze test. ( D ) The sucrose preference ratio (%) in sucrose preference test. ( E ) The immobility time (s) in tail suspension test. ( F ) The immobility time (s) in forced swimming test. ( G ) The experimental design of novel object recognition test. Above is the training trial, below is the testing trial (after 24 hours). ( H ) Left: the recognition index to object A and object B in the training trial; Right: the recognition index to object A and object C in the testing trial. ( I ) The latency of the mice to find the target holes on different training day in Barnes maze test. ( J ) Errors (explore incorrect holes) (% in total) in the probe trial of Barnes maze test. All data represent mean ± SEM, n=10 per group, * P <0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used in this research were as follows: anti-γH2A.X (ab26350, Abcam; 80312S, Cell Signaling Technology), anti-NeuN (26975-1-AP, Proteintech), anti-GFAP (12389S, Cell Signaling Technology), anti-Iba1 (ab178846, Abcam), anti-Chk1-pS317 (12302S, Cell Signaling Technology), anti-Chk1-pS345 (39233, Gene Tex; A21009, Abclonal), anti-Chk1 (A7653, Abclonal), anti-CIP2A (A12267, Abclonal), anti-Tau-pT231 (R381181, Zen-Bioscience), anti-Tau-pS396 (381213, Zen-Bioscience), anti-Tau-pS404 (11112, SAB), Tau-5 (ab80579, Abcam), anti-APP-pT668 (6986S, Cell Signaling Technology), anti-APP (60,342–1-Ig, Proteintech), anti-PSD95 (36233S, Cell Signaling Technology; 30255-1-AP, Proteintech), anti-Synapsin- I (5297S, Cell Signaling Technology), anti-GluA1 (13185S, Cell Signaling Technology), anti-GluA2 (13607S, Cell Signaling Technology), anti-β-actin (AC026, Abclonal).

Techniques: Suspension

Journal: Aging (Albany NY)

Article Title: Chronic stress induces Alzheimer’s disease-like pathologies through DNA damage-Chk1-CIP2A signaling

doi: 10.18632/aging.205862

Figure Lengend Snippet: Vitamin C and Chk1 inhibitor reduce DNA damage, Chk1 activation and CIP2A expression, decrease tau phosphorylation and Aβ levels in hippocampus of mice exposed to chronic stress. ( A ) Representative immunoblots of γH2A.X, Chk1-pS317, Chk1-pS345, Chk1, CIP2A, β-actin in hippocampal tissues of mice in different groups. ( B ) Quantification of the relative protein levels; non-phosphorylated proteins such as γH2A.X and CIP2A were normalized to the β-actin levels; phosphorylated Chk1-pS317, Chk1-pS345 were normalized to total Chk1. n = 3 per group. ( C ) Representative immunoblots of Tau-pT231, Tau-pS396, Tau-pS404, Tau-5, APP-pT668, APP, β-actin in hippocampus of mice in different groups. ( D ) Quantification of the relative protein expression levels; phosphorylated Tau-pT231, Tau-pS396, Tau-pS404 and APP-pT668 were normalized to Tau-5 and total APP respectively. n = 3 per group. ( E ) The Aβ 40 and Aβ 42 in soluble fraction of hippocampal tissues in different groups were detected by ELISA kit. n = 3 per group. ( F ) The Aβ 40 and Aβ 42 in insoluble fraction of hippocampal tissues in different groups were detected by ELISA kit. n = 3 per group. All data represent mean ± SEM * P <0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used in this research were as follows: anti-γH2A.X (ab26350, Abcam; 80312S, Cell Signaling Technology), anti-NeuN (26975-1-AP, Proteintech), anti-GFAP (12389S, Cell Signaling Technology), anti-Iba1 (ab178846, Abcam), anti-Chk1-pS317 (12302S, Cell Signaling Technology), anti-Chk1-pS345 (39233, Gene Tex; A21009, Abclonal), anti-Chk1 (A7653, Abclonal), anti-CIP2A (A12267, Abclonal), anti-Tau-pT231 (R381181, Zen-Bioscience), anti-Tau-pS396 (381213, Zen-Bioscience), anti-Tau-pS404 (11112, SAB), Tau-5 (ab80579, Abcam), anti-APP-pT668 (6986S, Cell Signaling Technology), anti-APP (60,342–1-Ig, Proteintech), anti-PSD95 (36233S, Cell Signaling Technology; 30255-1-AP, Proteintech), anti-Synapsin- I (5297S, Cell Signaling Technology), anti-GluA1 (13185S, Cell Signaling Technology), anti-GluA2 (13607S, Cell Signaling Technology), anti-β-actin (AC026, Abclonal).

Techniques: Activation Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Chronic stress induces Alzheimer’s disease-like pathologies through DNA damage-Chk1-CIP2A signaling

doi: 10.18632/aging.205862

Figure Lengend Snippet: Vitamin C and Chk1 inhibitor rescue neuron loss and synaptic impairments in mice exposed to chronic stress. ( A ) Representative immunoblots of Synapsin I, PSD95, GluA1, GluA2, β-actin in hippocampus (left) and cortex (right) of mice in different groups. ( B ) Quantification of the relative protein expression levels, which were normalized to the β-actin levels. n = 3 per group. ( C ) Representative images of Nissl staining in the CA1, CA3 and DG region of mice hippocampus. Scale bar: 50 μm (CA1 and CA3) or 100 μm (DG). ( D ) The quantitative analysis of the area occupied by neurons in the CA1, CA3 and DG region. n = 8-11 per group. All data represent mean ± SEM, * P <0.05, ** P <0.01, **** P < 0.0001.

Article Snippet: The primary antibodies used in this research were as follows: anti-γH2A.X (ab26350, Abcam; 80312S, Cell Signaling Technology), anti-NeuN (26975-1-AP, Proteintech), anti-GFAP (12389S, Cell Signaling Technology), anti-Iba1 (ab178846, Abcam), anti-Chk1-pS317 (12302S, Cell Signaling Technology), anti-Chk1-pS345 (39233, Gene Tex; A21009, Abclonal), anti-Chk1 (A7653, Abclonal), anti-CIP2A (A12267, Abclonal), anti-Tau-pT231 (R381181, Zen-Bioscience), anti-Tau-pS396 (381213, Zen-Bioscience), anti-Tau-pS404 (11112, SAB), Tau-5 (ab80579, Abcam), anti-APP-pT668 (6986S, Cell Signaling Technology), anti-APP (60,342–1-Ig, Proteintech), anti-PSD95 (36233S, Cell Signaling Technology; 30255-1-AP, Proteintech), anti-Synapsin- I (5297S, Cell Signaling Technology), anti-GluA1 (13185S, Cell Signaling Technology), anti-GluA2 (13607S, Cell Signaling Technology), anti-β-actin (AC026, Abclonal).

Techniques: Western Blot, Expressing, Staining

Journal: Translational Cancer Research

Article Title: Fumarate hydratase deficiency induces chronic myeloid leukemia progression

doi: 10.21037/tcr.2019.03.23

Figure Lengend Snippet: Five Gy γ-IR was used to induce DNA damage. (A) The cell morphology under white light; (B) a nucleus stained with DAPI; (C) immunofluorescence staining for phosphorylated H2AX; (D) merged image of staining with DAPI and an anti-phosphorylated H2AX antibody; (E) the protein expression of phosphorylated H2AX and Chk2 after IR induction in K562 cells and the FH overexpression and knockdown groups. FH, fumarate hydratase.

Article Snippet: Primary antibodies were added (anti-Fumarase/FH mouse monoclonal antibody, anti-HIF-1α mouse monoclonal antibody, anti-HK2, anti-PKM2, anti-PFK2, anti-LDHA, anti-β-actin, anti-p-H2AX and anti-p-Chk2 antibodies) (EnoGene, USA) and incubated overnight on a rocker at 4 °C.

Techniques: Staining, Immunofluorescence, Expressing, Over Expression, Knockdown

Journal: Aging (Albany NY)

Article Title: Histone methyltransferase Smyd2 drives vascular aging by its enhancer-dependent activity

doi: 10.18632/aging.204449

Figure Lengend Snippet: Inhibition of Smyd2 alleviates Ang II-induced senescence associated phenotypes in vitro . ( A , B ) SA-β-gal staining ( A ) and EdU incorporation ( B ) assays in Ang II-induced RAECs with LLY-507 (3 μM) pretreatment. Representative staining images are shown in the figure left, and the statistical analysis of positive cells is shown in the figure right. Scale bars, 100 μm. ( C ) Expressions of senescence markers (p53, p21 and p16), pro-inflammatory molecules (VCAM-1 and COX-2) and DNA damage markers (p-Chk2 and γH2AX) were detected by western blot analysis upon LLY-507 pretreatment in Ang II-induced RAECs. Representative western blot images are shown on the left, and the statistical analysis of relative protein expressions is shown on the right. GAPDH was used as the loading control. ( D ) The mRNA levels of Nos2 (encoding the iNOS protein) in Ang II-induced RAECs with LLY-507 pretreatment was examined by RT-qPCR. ( E , F ) Immunofluorescence double staining of Smyd2 and the senescence markers (p53 and p16) upon LLY-507 pretreatment in Ang II-induced RAECs. Scale bars, 100 μm or 50 μm. Data are presented as the mean ± SEMs, * p < 0.05, ** p < 0.01, *** p < 0.001, each acquired from three individual experiments ( n = 3).

Article Snippet: Reagents and antibodies used in this study were obtained as follows: angiotension II (Ang II) was purchased from Meilunbio (Meilun Biotechnology, Dalian, China); the Smyd2 specific inhibitor LLY-507 was purchased from MedChem Express (MCE, USA); Smyd2, p53, p21, p16 and γ-H2AX antibodies were obtained from Cell Signaling Biotechnology (Danvers, MA, USA); H3K4me3, H3K4me1 and H3K27ac antibodies were purchased from Abcam (Cambridge, MA, USA); COX-2, VCAM-1 and GAPDH were purchased from Proteintech (Rosemont, IL, USA); p-ATM and p-Chk2 antibodies were purchased from Absci (MD, USA); IL-6 and iNOS antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Inhibition, In Vitro, Staining, Western Blot, Quantitative RT-PCR, Immunofluorescence, Double Staining