Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Tabulated summary of IHC stainings indicating the number of premalignant (green) and malignant (blue) gastric lesions scored for each staining criterion. DDR/replication stress markers-53BP1, H2AX, pH2AX in red; Cell cycle regulators-p53, p16, p21 in purple. B. Summary of the stainings (DDR markers and the cell cycle regulation markers) in paired premalignant and malignant gastric lesions from gastric cancer patient8. PM= Premalignant gastric lesion, M= Malignant gastric lesion. C. Representative IHC images of paired premalignant and malignant gastric lesions from a gastric cancer patient (patient8) stained for DDR markers and cell cycle regulation markers, including H&E staining. Insets represent the zoomed area for each image.

Article Snippet: Cells were then washed and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, blocked and permeabilized PBS-BSA + 0.3% Triton X-100 for 15 minutes at room temperature, and incubated with primary antibodies (rabbit anti-pTIF1β/pKAP1 (Ser824) (1:500, CST # 4127S) and Mouse anti-pH2AX (1:600, CST #80312) in PBS-BSA overnight at +4°C.

Techniques: Staining

Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Summary of dysplasia grade and staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal (grey), premalignant (green), and malignant (blue) gastric lesions of four MNU treated mice. B. Representative IHC images of the staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal, premalignant, and malignant gastric lesions of MNU treated mouse4 (dysplasia score 5).

Article Snippet: Cells were then washed and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, blocked and permeabilized PBS-BSA + 0.3% Triton X-100 for 15 minutes at room temperature, and incubated with primary antibodies (rabbit anti-pTIF1β/pKAP1 (Ser824) (1:500, CST # 4127S) and Mouse anti-pH2AX (1:600, CST #80312) in PBS-BSA overnight at +4°C.

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Correlation plot between H2AX gene expression (log2(TPM+1)) and CIN25 score (DNA damage checkpoint expression) for the CCLE gastric cancer cell lines; R squared value and p-value calculated by simple linear correlation analysis. B. H2AX expression (log2(TPM+1)) between ‘No WGD (0)’ (grey) and ‘with WGD (1/2)’ (green) groups of gastric cancer cell lines available in the BROAD institute PRISM repurposing drug screen dataset; Difference between the H2AX expression is represented as the mean±S.D.; P-value calculated by unpaired t-test. C. Ploidy abnormalities scores for the five gastric cancer cell lines. D. Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in untreated five gastric cancer cell lines and RPE1 cells (non-neoplastic cell line) representing intrinsic replication stress/double-stranded DNA breaks (DSBs); Nucleus stained with DAPI (blue). E. Quantification of data in D., expressed as mean fluorescence intensity per cell (MFI) ± S.D from the following number of cells-RPE1-718, AGS-2080, HGC27, 1031, NUGC3-2119, GSU-2092, KE39-577. F. Quantification of pH2AX staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with ‘intrinsic low-replication stress’-RPE1, AGS (grey) and ‘intrinsic high-replication stress’-GSU, KE39 (red). Data expressed as mean fluorescence intensity (MFI) of pH2AX signal per cell ± S.D from the following number of cells-RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM-350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM-1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. G. Quantification of pKAP1 staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with ‘intrinsic low-replication stress’-RPE1, AGS (grey) and ‘intrinsic high-replication stress’-GSU, KE39 (green). Data expressed as mean fluorescence intensity of pKAP1 signal per cell ± S.D from the following number of cells-RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM-350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM-1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. H. Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in prexasertib (20nM) treated cell lines with intrinsic ‘low-replication stress’-RPE1, AGS, and intrinsic ‘high-replication stress’-GSU, KE39.

Article Snippet: Cells were then washed and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, blocked and permeabilized PBS-BSA + 0.3% Triton X-100 for 15 minutes at room temperature, and incubated with primary antibodies (rabbit anti-pTIF1β/pKAP1 (Ser824) (1:500, CST # 4127S) and Mouse anti-pH2AX (1:600, CST #80312) in PBS-BSA overnight at +4°C.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: iScience

Article Title: Telomeres cooperate in zygotic genome activation by affecting DUX4 / Dux transcription

doi: 10.1016/j.isci.2023.106158

Figure Lengend Snippet:

Article Snippet: Afterward, the embryos were permeabilized in 0.5% Triton X-100 for 25 min, washed three times in PBS, and then blocked with 5% donkey serum for 1 h. Samples were incubated with SIN3A antibody (Cat #3479, Abcam), TRF2 antibody (Cat #ab108997, Abcam), or rabbit polyclonal anti-γH2AX antibody (Cat #80312, Cell Signaling Technology) diluted 1: 100 in blocking solution at 4°C overnight, washed three times, incubated with FITC donkey anti-mouse IgG or FITC anti-rabbit IgG for 1 h at room temperature in the dark, washed again, and finally mounted with VECTASHIELD mounting medium and exposed to 0.5 μg/mL 4, 6-diamidino-2-phenylindole (DAPI, 1 μg/mL, Thermo Fisher Scientific).

Techniques: Recombinant, Isolation, SYBR Green Assay, Lysis, Stripping Membranes, Chromatin Immunoprecipitation, CRISPR, Software, Fluorescence, Microscopy

Journal: iScience

Article Title: Telomeres cooperate in zygotic genome activation by affecting DUX4 / Dux transcription

doi: 10.1016/j.isci.2023.106158

Figure Lengend Snippet:

Article Snippet: Mouse anti-γH2AX , Cell Signaling Technology , Cat # 80312.

Techniques: Recombinant, Isolation, SYBR Green Assay, Lysis, Stripping Membranes, Chromatin Immunoprecipitation, CRISPR, Software, Fluorescence, Microscopy

Journal: Cancers

Article Title: Inhibition of Checkpoint Kinase 1 (CHK1) Upregulates Interferon Regulatory Factor 1 (IRF1) to Promote Apoptosis and Activate Anti-Tumor Immunity via MICA in Hepatocellular Carcinoma (HCC)

doi: 10.3390/cancers15030850

Figure Lengend Snippet: CHK1 inhibition induces DNA damage to cause apoptosis in HCC cells. ( a ) Western blot and quantitative analysis for γ-H2AX level is shown in Huh-7 cells induced by cisplatin (5 µM) for 24 h. The γ-H2AX expression is measured by western blot in HepG2 ( b ) and Huh-7 cells ( c ) induced by prexasertib with a dose of 5 µM for 24 h. ( d ) Representative image of FACS analysis of apoptotic Huh-7 cell rate treated with DMSO or prexasertib with a dose of 5 µM for 24 h are shown. ( e ) The statistical summary of apoptotic Huh-7 cell rate is shown with t test ( n = 3). Data represent mean ± SD, * p < 0.05. Western blot image shown are representative of 3 experiments. FACS assay shown are representative of 3 independent experiments. The uncropped blots are shown in .

Article Snippet: Antibodies used for western blot were antibodies against CHK1 (25887-1-AP), MICA (12619-1-AP), and β-actin (20536-1-AP) (Proteintech, Wuhan, China); Anti-IRF1 (ab243895, abcam, Burlingame, CA, USA); anti-γ-H2AX (#80312) and GAPDH (CST, Danfoss, MA, USA).

Techniques: Inhibition, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Perinatal Obesity Sensitizes for Premature Kidney Aging Signaling

doi: 10.3390/ijms24032508

Figure Lengend Snippet: ( A ): Study design: Perinatal obesity induces aging-associated processes, e.g., DNA damage response, and causes premature aging of the kidneys with increased susceptibility for chronic kidney diseases later in life. Identification of converging signaling pathways using transcriptomics of kidneys from male offspring at postnatal day 21 (P21) after perinatal obesity, naturally-aged kidneys and prematurely-aged kidneys of genetic modified mice with an ablation or Ercc1 . ( B ): Scheme illustrating the experimental mouse model of perinatal obesity, in which male offspring of high-fat diet (HFD, perinatal obesity) or standard diet (SD, control) fed dams are investigated at P21. ( C ): Body weight (g), kidney weight (g) to body weight (g) ratio, and white adipose tissue (g) to body weight (g) ratio in male offspring at P21. ( D , E ): Assessment of DNA damage response (DDR) using γH2AX as an indicator in kidneys at P21. Representative immunohistochemical γH2AX staining of cortical and medullary compartments ( D ); black arrows indicate γH2AX positive nuclei. Percentage of γH2AX positive cells related to all cells is shown for medulla, cortex, and glomeruli ( E ). ( F , G ): Immunofluorescence staining for 8-Oxo-dG (RNA/DNA damage) in kidney medulla and cortex as an indicator of oxidative stress in kidneys at P21. Relative 8-Oxo-dG intensity per cell is indicated in the graph under the respective images. n = 5–6 per group; Mean ± SEM; Unpaired or Mann-Whitney test; * p < 0.05, ** p < 0.01.

Article Snippet: Afterwards, tissue was blocked (Sea Block, Thermo Scientific™, #37527, Waltham, MA, USA) at room temperature for 1 h. Primary antibody rabbit anti-mouse γH2AX (S139, γH2AX; Cell signaling, #2577, 1:1000, Danvers, MA, USA) was applied overnight at 4 °C.

Techniques: Modification, Immunohistochemical staining, Staining, Immunofluorescence, MANN-WHITNEY