Journal: Clinical and Translational Radiation Oncology

Article Title: Overexpression of JAML in colorectal cancer cells predicts higher radiosensitivity by inactivating ATR pathway

doi: 10.1016/j.ctro.2025.101016

Figure Lengend Snippet: CRC cells with JAML overexpression demonstrate increased apoptosis and DNA damage after irradiation, accompanied by inhibiting the ATR-CHK1 signaling pathway. (A) Representative images of apoptosis of LOVO and DLD-1 cells using flow cytometry after 24 h after X-ray irradiation. (B and C) Quantitative statistics of apoptosis of LOVO and DLD-1 cells. (D) Representative images of fluorescence of γH2AX in LOVO and DLD-1 cells after 12 h of X-ray irradiation or lack of X-ray irradiation. (E and F) Quantitative statistical analysis of the γH2AX-positive rate in LOVO and DLD-1 cells. (G and L) Western blot images of the ATR-CHK1 signaling pathway’s protein expression in LOVO and DLD-1 cells after 24 h of X-ray irradiation/lack of irradiation. (H–K and M−P) Quantitative statistical analysis of the relative ATR-CHK1 signaling pathway’s protein expression in LOVO and DLD-1 cells (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤ 0.0001).

Article Snippet: Antibodies against GAPDH (Proteintech, dilution 1:50000, China), pCHK1 (Cell Signaling Technology, dilution 1:1000, USA), checkpoint kinase 1 (CHK1) (MCE, dilution 1:1000, MCE), pATR (Cell Signaling Technology, dilution 1:1000, USA), and ataxia telangiectasia and Rad3-related protein (ATR) (Proteintech, dilution 1:1000, China) were used.

Techniques: Over Expression, Irradiation, Flow Cytometry, Fluorescence, Western Blot, Protein-Protein interactions, Expressing

Journal: Clinical and Translational Radiation Oncology

Article Title: Overexpression of JAML in colorectal cancer cells predicts higher radiosensitivity by inactivating ATR pathway

doi: 10.1016/j.ctro.2025.101016

Figure Lengend Snippet: CRC tumors with JAML overexpression show lower Ki67 expression after X-ray radiation therapy , accompanied by inhibiting the ATR-CHK1 signaling pathway in vivo. (A)Representative immunofluorescence images of Ki67 in LOVO and DLD-1 tumor tissues. (B and C) Quantitative statistical analysis of the expression of Ki67 in LOVO and DLD-1 tumor tissues. (D) Representative western blot images of the ATR-CHK1 signaling pathway in LOVO and DLD-1 tumor tissues. (E–L) Quantitative statistical analysis of the relative ATR-CHK1 signaling pathway’s protein expression in LOVO and DLD-1 tumor tissues (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤ 0.0001).

Article Snippet: Antibodies against GAPDH (Proteintech, dilution 1:50000, China), pCHK1 (Cell Signaling Technology, dilution 1:1000, USA), checkpoint kinase 1 (CHK1) (MCE, dilution 1:1000, MCE), pATR (Cell Signaling Technology, dilution 1:1000, USA), and ataxia telangiectasia and Rad3-related protein (ATR) (Proteintech, dilution 1:1000, China) were used.

Techniques: Over Expression, Expressing, In Vivo, Immunofluorescence, Western Blot, Protein-Protein interactions

Journal: Nature Communications

Article Title: Histone H3 serine-57 is a CHK1 substrate whose phosphorylation affects DNA repair

doi: 10.1038/s41467-023-40843-4

Figure Lengend Snippet: a Radar plot with activity values of 190 kinases in kinase assays with H3 51-72 peptide as substrate ( n = 1 experiment). Only kinases above the threshold are labelled, see Supplementary Data for full results. In vitro kinase assays using γ 33 P-ATP, recombinant CHK1 and H3 peptides, with the indicated modifications ( b ; n = 4) or full-length H3 ( c ; n = 4–5) as substrates; error bars, mean ± SD; results are normalised to, and represented as fold increase over, the control assays without kinase. d In vitro kinase assays as in c but with non-radioactive ATP and analysed by western blotting; n = 1 experiment. The histone extract was used as positive control for the WB. U2OS cells were treated with CHK1 inhibitors (SCH900776, 5 µM; CHIR-124, 2 µM) for indicated times, and analysed by WB of acid-extracted histones ( e ), FACS cell cycle profiles ( f ), or by immunofluorescence of fixed cells ( g ); n = 2 independent experiments. All lanes come from the same membrane/exposure, unrelated lanes were removed. DNA was stained with DAPI; scale bar, 20 µm. Knockdown of CHK1 by RNA interference in U2OS cells, analysed subsequently by WB ( h ) of total cell lysates (top) and acid-extracted histones (bottom), and FACS cell cycle profiles ( i ); amidoblack staining was used for loading control; siCtrl, siRNA targeting the firefly luciferase gene; n = 2 independent experiments. Source data are provided as Source Data file.

Article Snippet: For other kinase assays, commercial kinase CHK1 (0282-0000-1, Proqinase) or DYRK1A (a gift from Sandrine Ruchaud) were used in 20 μl reactions containing 50 μM ATP, γ- 33 P-ATP, 1–2 μg of substrate, and 1× kinase buffer (50 mM Hepes pH 7.6, 10 mM MgCl, 1 mM DTT, 0.02% Triton X-100) at 30 °C for 20–30 min. For substrates, recombinant full length H3, H3 51-72 or H3 5-20 peptides were used; RBER-CHKtide (0581-0000-5; Proqinase) was used for positive control; reactions without substrate or reactions without kinase (to control for kinase autophosphorylation) were used for negative controls.

Techniques: Activity Assay, In Vitro, Recombinant, Control, Western Blot, Positive Control, Immunofluorescence, Membrane, Staining, Knockdown, Luciferase

Journal: PLoS ONE

Article Title: Genomic structure, expression, and functional characterization of checkpoint kinase 1 from Penaeus monodon

doi: 10.1371/journal.pone.0198036

Figure Lengend Snippet: Primer sequences for amplification.

Article Snippet: The proteins of ovaries at 0–96 h after injection with dsRNA-Chk1 or dsRNA-GFP were detected by a tissue Chk1 kinase activity photometric assay, using a quantitative detection kit (Genmed, USA).

Techniques: Amplification, Sequencing, Clone Assay, Quantitative RT-PCR, Recombinant, Expressing, In Situ Hybridization

Journal: PLoS ONE

Article Title: Genomic structure, expression, and functional characterization of checkpoint kinase 1 from Penaeus monodon

doi: 10.1371/journal.pone.0198036

Figure Lengend Snippet: (A) Full-length cDNA of PmChk1 (schematic). The deduced amino-acid sequence is depicted underneath the nucleotide sequence. Initiation code (ATG) and termination code (TAA) are denoted by boxes. Polyadenylation signal sequence (AATAAA) is emboldened. (B) Multiple alignment of the deduced amino-acid sequences of Chk1 from P . monodon and other species. A sequence logo denoting similarity is shown at the top of alignments, and numbers of amino acids are shown on the right-hand side of alignments. GenBank numbers of Chk1 were: P . monodon : KU958380; Homo sapiens : AAC51736.1; Poeciliopsis prolifica : JAO88875.1; Mus musculus : AAC53334.1; Daphnia pulex : AGN95867.1; Melipona quadrifasciata : KOX69887.1; Cyphomyr mexcostatus : KYN05919.1. The S-TKc domain is indicated with a red box. (C) Neighbor-joining phylogenetic tree of E2F-2s based on amino-acid sequences. Confidence in each node was evaluated by 2000 bootstrap replicates using Mega v5.03. (D) Comparison of the genomic DNA sequence encoding Chk1 in P . monodon . Green-shaded rectangles denote exons, gray horizontal lines denote introns, and the numbers reflect the length (in bp) of exons and introns.

Article Snippet: The proteins of ovaries at 0–96 h after injection with dsRNA-Chk1 or dsRNA-GFP were detected by a tissue Chk1 kinase activity photometric assay, using a quantitative detection kit (Genmed, USA).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Genomic structure, expression, and functional characterization of checkpoint kinase 1 from Penaeus monodon

doi: 10.1371/journal.pone.0198036

Figure Lengend Snippet: (A) Expression of PmChk1 protein in ovaries after dsRNA-Chk1 injection. Lane M = pre-stained molecular-weight marker. (B) Relative expression of PmChk1 in the ovaries of P . monodon after dsRNA-Chk1 treatment. (C) Relative expression of PmChk1 in the hepatopancreas of P . monodon after dsRNA-Chk1 treatment. Vertical bars represent the mean ± SD (n = 3 for each group). Significant differences from controls are denoted: ** P < 0.01, * P < 0.05.

Article Snippet: The proteins of ovaries at 0–96 h after injection with dsRNA-Chk1 or dsRNA-GFP were detected by a tissue Chk1 kinase activity photometric assay, using a quantitative detection kit (Genmed, USA).

Techniques: Expressing, Injection, Staining, Molecular Weight, Marker

Journal: PLoS ONE

Article Title: Genomic structure, expression, and functional characterization of checkpoint kinase 1 from Penaeus monodon

doi: 10.1371/journal.pone.0198036

Figure Lengend Snippet: The hepatopancreas was harvested after dsRNA-Chk1 administration. Brown dots denote positive reactions (arrows). A represents a negative control. B represents the collected hepatopancreas after dsRNA-GFP injection. C represents the collected hepatopancreas after dsRNA-Chk1 injection. Scale bar = 30.

Article Snippet: The proteins of ovaries at 0–96 h after injection with dsRNA-Chk1 or dsRNA-GFP were detected by a tissue Chk1 kinase activity photometric assay, using a quantitative detection kit (Genmed, USA).

Techniques: Negative Control, Injection

Journal: PLoS ONE

Article Title: Genomic structure, expression, and functional characterization of checkpoint kinase 1 from Penaeus monodon

doi: 10.1371/journal.pone.0198036

Figure Lengend Snippet: (A) Chk1 activity after injection of dsRNA-Chk1 and dsRNA-GFP. Vertical bars represent the mean ± SD (n = 3 for each group). Significant differences from controls are denoted: * P < 0.05. (B) The GSI (ovary weight/body weight × 100) of P . monodon after injection of dsRNA-GFP, dsRNA-Chk1, or PBS.

Article Snippet: The proteins of ovaries at 0–96 h after injection with dsRNA-Chk1 or dsRNA-GFP were detected by a tissue Chk1 kinase activity photometric assay, using a quantitative detection kit (Genmed, USA).

Techniques: Activity Assay, Injection