Journal: Molecular Cell

Article Title: Genome homeostasis defects drive enlarged cells into senescence

doi: 10.1016/j.molcel.2023.10.018

Figure Lengend Snippet:

Article Snippet: phospho-histone H2AX (rabbit) , Cell Signaling Technology , #2566.

Techniques: Recombinant, Staining, Lysis, Flow Cytometry, Imaging, Fractionation, Cell Culture, Extraction, Electrophoresis, Software

Journal: Nature Communications

Article Title: Site-selected in situ polymerization for living cell surface engineering

doi: 10.1038/s41467-023-43161-x

Figure Lengend Snippet: a Cell membrane integrity and cellular activity of different Cell-P were measured by FCM using LIVE/DEAD™ fixable far-red dead cell stain kit. b Cell proliferation of native cells and Cell-P measured by CCK-8 kit ( n = 8 independent samples, mean ± SD). c , d Orientation diagrams ( c , left) and corresponding histograms ( d ) of F-actin in different Cell-P and native cells by phalloidin-iFluor 594 staining. The circular color map coding to indicate the orientation of F-actin is shown ( c , right). Scale bar, 10 μm. e Effect of the SSP process on intracellular DNA. Representative FCM immunostaining signals of phosphorylated histone H2AX (γ-H2AX) in Cell-P immediately after polymerization and after 48 h of culture, respectively. Native cells were used as controls. γ-H2AX is a sensitive marker for DNA damage. f Western blotting of proteins associated with ROS and hypoxia damage (caspase-3, caspase-9, and cytochrome c). Cropped blots are shown, and the full scans are supplied in the Source Data file. g Effect of the SSP process on cellular metabolism. Oxygen consumption rates (OCRs) of Cell-P were measured using the Seahorse XF Cell Mito Stress Test, and statistically normalized to basal and maximal respiration levels ( n = 3 independent samples, mean ± SEM). FCCP, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone. h , i Immunostaining images ( h ) and quantitative FCM data ( i ) of Cell-P and native cells undergoing anti-CD3-FITC incubation. Scale bar, 25 μm. Enlarged images show a single stained cell; Scale bar, 3 μm. Data are presented as the mean ± SD of n = 3 individual experiments. j Immunostaining images and FCM histograms of CD69 expressed on Cell Sia -P after stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IO). Native cells with and without stimulation were used as controls. Scale bar, 25 μm. In g and i statistical differences were determined by one-way ANOVA with Tukey’s multiple comparison test. P < 0.05 was considered statistically significant. In a – h and j data are representative of three independent experiments with similar results. Source data are provided as a Source Data file.

Article Snippet: Cells were then incubated in 1% FBS-containing PBS supplemented with monoclonal antibody (mAb) specific for phosphorylated histone H2AX (γ-H2AX) (1:200 dilution, Cell Signaling Technology #9718) for 60 min at r.t. After centrifugation and five washes with 1% FBS-containing PBS, cells were incubated in 1% FBS-containing PBS supplemented with secondary antibody goat anti-rabbit IgG H&L (Alexa Fluor 488, pre-adsorbed, 1:1000 dilution, Abcam #ab150081) for 30 min at r.t.

Techniques: Membrane, Activity Assay, Staining, CCK-8 Assay, Immunostaining, Marker, Western Blot, Incubation, Comparison

Journal: Nucleic Acids Research

Article Title: ATM, KAP1 and the Epstein–Barr virus polymerase processivity factor direct traffic at the intersection of transcription and replication

doi: 10.1093/nar/gkad823

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-phospho-Histone H2AX (Ser139) antibody , Cell Signaling Technology , 9718S.

Techniques: Purification, Gel Extraction, DNA Extraction, RNA Extraction, Plasmid Preparation, SYBR Green Assay, Electroporation, In Vitro, Transfection, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: The Biological Assessment of Shikonin and β,β-dimethylacrylshikonin Using a Cellular Myxofibrosarcoma Tumor Heterogeneity Model

doi: 10.3390/ijms242115910

Figure Lengend Snippet: Apoptotic induction. ( A ) Protein expression of cleaved caspase-8, -9, and -3, as well as cleaved PARP, Noxa, and the DNA damage marker γH2AX. The apoptotic key players were evaluated by immunoblotting under control conditions (0) and after treatment with 0.5, 1.5, and 3 µM DMAS respectively 0.25, 0.5, and 1.0 µM shikonin. β-actin was used as loading control. Δ ratio, fold change was normalized to non-treated controls (mean ± SD of n = 3). Full-length blots are presented in . ( B ) Relative gene expression of the pro-apoptotic markers Bak, Bax and Bim , the anti-apoptotic marker Bcl-2 , and the antagonists Puma and Noxa after treatment with DMAS or shikonin for 24 h in MUG-Myx2a and MUG-Myx2b cells (mean ± SD, n = 6, measured in triplicate). Statistical significances to the untreated controls are defined as follows: * p < 0.05; *** p < 0.001. Statistical significances between the two cell lines MUG-Myx2a and MUG-Myx2b are presented as ## p < 0.01; ### p < 0.001.

Article Snippet: These included cleaved-caspase-8, -9, and -3, cleaved-PARP, Noxa, phosphorylated histone H2AX (γH2AX), phospho-AKT Ser473 , AKT, phospho-STAT3 Tyr705 , STAT3, phospho-ERK Thr202/Tyr204 , ERK, phospho-JNK Thr183/Tyr185 , JNK, phospho-p38 Thr180/Tyr182 , and p38 as well as the DNA damage key proteins pATR, pATM, MSH3, XPC, and pChK1/2 (all Cell Signaling Technology).

Techniques: Expressing, Marker, Western Blot

Journal: Antioxidants

Article Title: Light Quality Potentiates the Antioxidant Properties of Brassica rapa Microgreen Extracts against Oxidative Stress and DNA Damage in Human Cells

doi: 10.3390/antiox12101895

Figure Lengend Snippet: Confocal immunofluorescence to detect YB-1 subcellular localization and γ-H2AX foci in basal and oxidative stress conditions. ( A – D ) Confocal analysis of HaCaT cells treated with indicated concentrations of W (white light) and RB (red–blue light) extracts. Hydrogen peroxide was used to induce oxidative and/or genotoxic. Cells were stained with α-YB1 and α-γ-H2AX antibodies. Nuclei were stained with DAPI. ( E ) The quantitation of the foci number was performed by Fiji—ImageJ software version 1.52 and values were compared to the baseline/physiologic number of foci of the DMSO (cells in 0.1% DMSO, negative control) and Trolox 100 μg/mL (positive control). Data are the means ± standard error (n = 6) of three independent biological replicates. Statistical analysis was carried out by two-way ANOVA followed by Sidak’s multiple comparison tests ( p -value * < 0.01, ** < 0.05, *** p < 0.001; **** p < 0.0001).

Article Snippet: After 1 h incubation at RT with primary antibodies, anti-YB-1 (Abcam 12148), and anti-γ-H2AX histone (Cell signaling 9718S), cells were incubated with, Alexa Fluor 488 anti-rabbit donkey and Alexa Fluor 594 donkey secondary antibodies (Thermo Fisher Scientific) for 45 min in darkness.

Techniques: Immunofluorescence, Staining, Quantitation Assay, Software, Negative Control, Positive Control, Comparison

Journal: Antioxidants

Article Title: Light Quality Potentiates the Antioxidant Properties of Brassica rapa Microgreen Extracts against Oxidative Stress and DNA Damage in Human Cells

doi: 10.3390/antiox12101895

Figure Lengend Snippet: Confocal immunofluorescence to detect YB-1 subcellular localization and γ-H2AX foci in basal and oxidative stress conditions. ( A – D ) Confocal analysis of HaCaT cells treated with indicated concentrations of W (white light) and RB (red–blue light) extracts. Hydrogen peroxide was used to induce oxidative and/or genotoxic. Cells were stained with α-YB1 and α-γ-H2AX antibodies. Nuclei were stained with DAPI. ( E ) The quantitation of the foci number was performed by Fiji—ImageJ software version 1.52 and values were compared to the baseline/physiologic number of foci of the DMSO (cells in 0.1% DMSO, negative control) and Trolox 100 μg/mL (positive control). Data are the means ± standard error (n = 6) of three independent biological replicates. Statistical analysis was carried out by two-way ANOVA followed by Sidak’s multiple comparison tests ( p -value * < 0.01, ** < 0.05, *** p < 0.001; **** p < 0.0001).

Article Snippet: Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Chemical Co., St. Louis, MO, USA), FBS, Hyclone Laboratories, Inc. Logan, UT, USA, 3-(4,5-methylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT, neoFroxx Einhausen Deutschland, Germany), Dimethyl Sulfoxide for cell culture, Applichem GmbH ITW Reagents, Glenview, IL, USA, 2′-7′ dichlorofluorescein diacetate (DCFDA, Sigma Chemical Co., St. Louis, MO, USA), Hydrogen peroxide solution 30%, Carlo Erba Reagents GmbH, Milan, Italy, Trolox, 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid (Sigma Chemical Co., St. Louis, MO, USA), PBS Dulbecco’s Phosphate-Buffered Saline (Euroclone S.p.A, Milan, Italy), DAPIsolution (1:50,000) (Sigma Chemical Co., St. Louis, MO, USA), Dako mounting medium (Agilent, Santa Clara, CA, USA), anti-YB-1 (Abcam 12148, Cambridge, UK), anti-g-H2AX histone (Cell signaling 9718S, Danvers, MA, USA), Alexa Fluor 488 anti-rabbit donkey and Alexa Fluor 594 donkey (Thermo Fisher Scientific, Waltham, MA, USA), anti-β-actin (C4) and anti-NRF-2 (A-10) were from Santa Cruz Biotechnology (Dallas, TX, USA), while p21WAF were from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Immunofluorescence, Staining, Quantitation Assay, Software, Negative Control, Positive Control, Comparison