Journal: Oncotarget

Article Title: Dietary NiCl 2 causes G 2 /M cell cycle arrest in the broiler's kidney

doi:

Figure Lengend Snippet: Antibodies used in immunohitochemistry

Article Snippet: p-Chk2 , Bioss, China , bs-3721R , 1:100.

Techniques:

Journal: Oncotarget

Article Title: Dietary NiCl 2 causes G 2 /M cell cycle arrest in the broiler's kidney

doi:

Figure Lengend Snippet: Sequence of primers used in qRT-PCR

Article Snippet: p-Chk2 , Bioss, China , bs-3721R , 1:100.

Techniques: Sequencing

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Western blotting of HIF-1α, p-CHK2 Thr68, CHK2, VEGF. For all Western blotting, β-actin was used as a loading control. 1% O 2 was performed in HEK293 cells and HEK293-CHK2-KO cells for indicated times. B Western blotting of HIF-1α, p-CHK2 Thr68 and CHK2. HEK293 cells were stimulated by 1% O 2 , CHK2 inhibitor and combination for 6 h. C , D Flow cytometric analysis of ROS levels and median fluorescence intensity of ROS expression. H1299 cells were stimulated by 1% O 2 with or without NAC for 6 h. ** p < 0.01. E Western blotting of p-ATM S1981, ATM, HIF-1α, p-CHK2 Thr68 and CHK2. HEK293 cells were pretreated with NAC for 4 h and then cultured in 1% O 2 for 6 h. F Western blotting of p-ATM S1981, ATM, HIF-1α, p-CHK2 Thr68 and CHK2. H1299 cells were transfected with the indicated shRNA and exposed to 1% O 2 for indicated times. G Western blotting of p-ATM S1981, ATM, HIF-1α, p-CHK2 Thr68 and CHK2. HEK293 and HEK293-CHK2-KO cells were exposed to 1% O 2 or combination with ATM inhibitor for 6 h. RT-qPCR analysis of PKM2 ( H ), PDK1 ( I ) and BNIP3 ( J ) mRNA in CHK2-deficient or wildtype HEK293 cells treated with or without hypoxia for 6 h. ** p < 0.01, n.s. not significant. K Dual-luciferase reporter gene experiment of PKM2, PDK1 and BNIP3. HEK293-CHK2-KO cells were co-transfected with reporter gene and CHK2 plasmid under normoxia (21% O 2 ) and hypoxia (1% O 2 ) . ** p < 0.01, n.s. not significant.

Article Snippet: Antibodies targeting phosphorylated CHK2 (Thr68, #2661), ATM (#2873), p-ATM (Ser1981, #13050), VEGF-A (#50661), ubiquitin (#3933S), β-actin (#3700), human influenza hemagglutinin (HA)-tag (#2367) and His-tag (#2365) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Western Blot, Control, Fluorescence, Expressing, Cell Culture, Transfection, shRNA, Quantitative RT-PCR, Luciferase, Plasmid Preparation

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Western blotting of exogenous HA-HIF-1α and Flag-tagged CHK2. HA-tagged HIF-1α (3 μg) and Flag-tagged CHK2 (1 μg, 3 μg) was transiently transfected into H1299 cells and then exposed to 1% O 2 . B Western blotting of exogenous HA-HIF-1α and CHK2. HA-tagged HIF-1α was transiently transfected into HEK293 and HEK293-CHK2-KO cells under hypoxic microenvironment. MG132 and CQ was then added to above cells respectively or together as indicated. C Endogenous HIF-1α ubiquitination in HEK293 and HEK293-CHK2-KO cells under hypoxia (1% O 2 ) for 6 h. Exogenous HA-HIF-1α ubiquitination under normoxia ( D ) and hypoxia ( E ) in HEK293-CHK2-KO cells transiently transfected with indicated plasmids. F Western blotting of exogenous double mutant HIF-1α protein at both hydroxylation sites P402A/P564A (HA-HIF-1α-DM). HEK293 cells were transfected with an increasing amount of Flag-CHK2 expression plasmid and exposed to hypoxia. G Western blotting of exogenous triple mutant HIF-1α protein at P402A/P564A/N803A (HA-HIF-1α-TM). HEK293 cells were transfected with an increasing amount of Flag-CHK2 expression plasmid and exposed to hypoxia.

Article Snippet: Antibodies targeting phosphorylated CHK2 (Thr68, #2661), ATM (#2873), p-ATM (Ser1981, #13050), VEGF-A (#50661), ubiquitin (#3933S), β-actin (#3700), human influenza hemagglutinin (HA)-tag (#2367) and His-tag (#2365) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Ubiquitin Proteomics, Mutagenesis, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Colocalization of CHK2 and HIF-1α shown by representative confocal microscopic images of NSCLC tissues and H1299 cells. For H1299 cells, experiments were conducted under normoxia (21% O 2 ) and hypoxia (1% O 2 ), respectively. B , C . Co-IP of endogenous CHK2 with HIF-1α in H1299 cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ). IgG, immunoglobulin G. D Co-IP of endogenous CHK2 with HIF-1α in H1299 cells pretreated with NAC for 4 h and then cultured in 1% O 2 for 6 h. E GST-pull-down assay of His-HIF-1α fusion proteins with bacterially expressed GST-CHK2 fragments. Coomassie Brilliant Blue (CBB) staining shows expression of GST and GST-CHK2 fragments. F GST-pull-down assay of His-CHK2 fusion proteins with bacterially expressed GST-HIF-1α fragments. CBB staining shows expression of GST and GST-HIF-1α fragments. G , H Co-IP of exogenous HA-HIF-1α with Flag-CHK2 under normoxia (21% O 2 ) and hypoxia (1% O 2 ) in HEK293-CHK2-KO cells transiently transfected with indicated plasmids.

Article Snippet: Antibodies targeting phosphorylated CHK2 (Thr68, #2661), ATM (#2873), p-ATM (Ser1981, #13050), VEGF-A (#50661), ubiquitin (#3933S), β-actin (#3700), human influenza hemagglutinin (HA)-tag (#2367) and His-tag (#2365) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Co-Immunoprecipitation Assay, Cell Culture, Pull Down Assay, Staining, Expressing, Transfection

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Mass spectrogram of phosphorylation sites of HIF-1α protein T645. B Sequence alignment surrounding the T645 residue of HIF-1α homologs in various species. C Western blotting of HA-HIF-1α, p-CHK2 and CHK2. CHK2 inhibitor was added to HEK2923 cells transiently transfected with plasmids as indicated under hypoxia (1% O 2 ). D Western blotting of HA-HIF-1α and CHK2. shCHK2, HA-HIF-1α and HA-HIF-1α Thr645A plasmids was transiently transfected into HEK293 cells as indicated under hypoxia (1% O 2 ). E Representative images of HUVEC tube formation assay after 24 h incubation in the indicated conditioned media from H1299 cells. F Mean tube length in E was quantified. ** p < 0.01, n.s. not significant. G Exogenous HA-HIF-1α ubiquitination was detected by Co-IP. Flag-CHK2, HA-HIF-1α and HA-HIF-1α Thr645A plasmids were transiently transfected into HEK293 cells as indicated under hypoxia (1% O 2 ). H Co-IP of endogenous HIF-1α with USP7 in HEK293 cells and HEK293-CHK2-KO cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ). I Co-IP of endogenous HIF-1α with USP7. Flag-CHK2 plasmids was transiently transfected into HEK293 cells as indicated under normoxia (21% O 2 ) and hypoxia (1% O 2 ). J Ubiquitination of Flag-HIF-1α WT and Flag-HIF-1α T645A. Flag-HIF-1α and Flag-HIF-1α Thr645A plasmids were transiently transfected into HEK293 cells as indicated under hypoxia (1% O 2 ).

Article Snippet: Antibodies targeting phosphorylated CHK2 (Thr68, #2661), ATM (#2873), p-ATM (Ser1981, #13050), VEGF-A (#50661), ubiquitin (#3933S), β-actin (#3700), human influenza hemagglutinin (HA)-tag (#2367) and His-tag (#2365) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Phospho-proteomics, Sequencing, Residue, Western Blot, Transfection, HUVEC Tube Formation Assay, Incubation, Ubiquitin Proteomics, Co-Immunoprecipitation Assay

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: A Ten mice (n = 5/group) were injected with H1299 cells. DMSO or the CHK2 inhibitor (1 mg/kg) was administered intraperitoneally on alternate days for 20 days. B Representative CD31 and Ki-67 staining of harvested xenografts of mice treated by DMSO or CHK2 inhibitor. Scale bar = 50 μm. C Quantification of IHC. ** p < 0.01. D Western blotting of HA-tagged protein in HIF-1α-WT and HIF-1α-T645A stable transduced H1299 cells. E , F Flow cytometry and statistical analysis showing cell apoptosis exposed to hypoxia of the H1299 cells with indicated transfection. ** p < 0.01. G H1299 cells stably expressing HIF-1α WT or HIF-1α T645A were injected subcutaneously into the rear flanks of athymic nude mice ( n = 6). H IHC of harvested tumors in G with antibodies specific for CD31 and Ki-67 to assess angiogenesis and proliferation respectively. I Measurement of tumor volume of mice in ( G ). ** p < 0.01. J Representative CHK2 and CD31 staining in NSCLC tissues, n = 50. K Scattering plot depicting the correlation between CHK2 and CD31 score.

Article Snippet: Antibodies targeting phosphorylated CHK2 (Thr68, #2661), ATM (#2873), p-ATM (Ser1981, #13050), VEGF-A (#50661), ubiquitin (#3933S), β-actin (#3700), human influenza hemagglutinin (HA)-tag (#2367) and His-tag (#2365) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Injection, Staining, Paraffin-embedded Immunohistochemistry, Western Blot, Flow Cytometry, Transfection, Stable Transfection, Expressing

Journal: Oncogene

Article Title: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment

doi: 10.1038/s41388-025-03336-w

Figure Lengend Snippet: Model showing CHK2 interaction with the HIF-1α inhibitory domain and phosphorylate HIF-1α at Thr645 site, enhancing HIF-1α binding to its deubiquitinating enzyme USP7, prohibiting HIF-1α ubiquitination and thus triggering angiogenesis by upregulating VEGF.

Article Snippet: Antibodies targeting phosphorylated CHK2 (Thr68, #2661), ATM (#2873), p-ATM (Ser1981, #13050), VEGF-A (#50661), ubiquitin (#3933S), β-actin (#3700), human influenza hemagglutinin (HA)-tag (#2367) and His-tag (#2365) were sourced from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: Science Advances

Article Title: Molecular portraits of cell cycle checkpoint kinases in cancer evolution, progression, and treatment responsiveness

doi: 10.1126/sciadv.adf2860

Figure Lengend Snippet: Breast cancer subtypes and known associations with cell cycle checkpoint kinase dysregulation based on epidemiological studies in the literature.

Article Snippet: Antibodies used include phosphor-Chk2 (Cell Signaling Technology, catalog no. 2197S), ER (EMD Millipore, catalog no. 04820MI), 53BP1 (Novus Biologicals, catalog no. NB100-304), gH2AX (Cell Signaling Technology, catalog no. 9718), and Ki67 (Abcam, catalog no. ab16667).

Techniques: Variant Assay, Mutagenesis

Journal: Science Advances

Article Title: Molecular portraits of cell cycle checkpoint kinases in cancer evolution, progression, and treatment responsiveness

doi: 10.1126/sciadv.adf2860

Figure Lengend Snippet: ( A and B ) Stacked columns representing frequency of mutations in ESR1 and TP53 (A, control genes) and ATM and CHEK2 (B) in ER + /HER2 − primary (Pri) versus metastatic (Met) breast cancer and TNBC. Darker shading indicates incidence of mutations in multiple genes of interest, while lighter shading indicates mutation of only the specified gene of interest. P values were derived by comparing light shaded columns between the three breast cancer subtypes in a Fisher’s exact test. Holm’s adjustment for multiple comparisons was conducted. Sample sizes in parentheses. ( C ) Western blots demonstrating impact of indicated inhibitors and activators on phosphorylation of target proteins in MCF7 cells. ( D and E ) Representative images of wound healing assay of ER + /HER2 − breast cancer cell line, MCF7, treated with vehicle (Veh), 100 nM Fulvestrant (Fulv), or the combination of 10 μM DIM, a CHK2 activator and fulvestrant (D), or 100 nM CHK2 inhibitor dihydrate (CHK2i) (E) at 48 hours. Dot plots representing quantification of area of scratch at 0, 24, and 48 hours with error bars depicting SD. ( F and G ) Representative images of transwell migration (top) and invasion (G, bottom) assays at 48 hours after treatment with vehicle, DIM, or CHK2i, along with bar graphs depicting quantification. DIM treatments were done in media with charcoal-stripped serum + 10 pM β-estradiol, while CHK2i assays were done in media with full serum. Error bars represent SD. Two-tailed Student’s t test determined P values for (D) to (G). # P ≤ 0.1, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Supporting data validating activity of compounds used and independent confirmation in a second cell line are presented in fig. S4.

Article Snippet: Antibodies used include phosphor-Chk2 (Cell Signaling Technology, catalog no. 2197S), ER (EMD Millipore, catalog no. 04820MI), 53BP1 (Novus Biologicals, catalog no. NB100-304), gH2AX (Cell Signaling Technology, catalog no. 9718), and Ki67 (Abcam, catalog no. ab16667).

Techniques: Control, Mutagenesis, Derivative Assay, Western Blot, Phospho-proteomics, Wound Healing Assay, Migration, Two Tailed Test, Activity Assay

Journal: Science Advances

Article Title: Molecular portraits of cell cycle checkpoint kinases in cancer evolution, progression, and treatment responsiveness

doi: 10.1126/sciadv.adf2860

Figure Lengend Snippet: ( A , B , and H ) Kaplan-Meier survival curves measuring specified outcomes in patients with mutated (A) and (H) or down-regulated (B) CHK2 and ATM (H) in breast (A) and (B) and other cancers (H). Log-rank test determined P values. ( C ) Stacked column graph representing incidence of CHK2 down-regulation measured by reverse phase proteomics array in ER + /HER2 − breast cancer samples from TCGA. Fisher’s exact test determined the P values. ( D and F ) Bar graphs quantifying percentage of pCHK2 + and proliferating cells (Ki67 + ) in xenograft tumors derived from ER + /HER2 − breast cancer cell line, T47D, grown in mice with β-estradiol supplementation in drinking water (Veh), estrogen deprivation (−E 2 ), and CHK2 activator, DIM incorporated in chow. ( E ) Dot plot depicting mean tumor volumes in mice xenografted with T47D cells and treated as specified. Three mice per group. Representative images of tumors from specified treatment conditions at harvest. ( G ) Heatmaps indicating protein levels of pCHK2, phospho-ATM (pATM), and phospho-ATR (pATR) across a panel of ER + /HER2 − breast cancer PDXs. Ability of tumors to grow in the absence of estrogen supplementation and age of diagnosis are represented. Low (mean − SD) and high (mean + SD) phosphorylated/total protein levels were compared for statistically significant differences in age at diagnosis and estrogen-independent growth using Fisher’s exact test. ( I ) Working model depicting the impact of mutations in different cell cycle checkpoint kinase genes on the type of breast cancer a patient may develop and clinical consequences. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Unless otherwise specified, two-tailed Student’s t test determined P values and error bars represent SD. Associated data are presented in fig. S6.

Article Snippet: Antibodies used include phosphor-Chk2 (Cell Signaling Technology, catalog no. 2197S), ER (EMD Millipore, catalog no. 04820MI), 53BP1 (Novus Biologicals, catalog no. NB100-304), gH2AX (Cell Signaling Technology, catalog no. 9718), and Ki67 (Abcam, catalog no. ab16667).

Techniques: Derivative Assay, Biomarker Discovery, Two Tailed Test

Journal: Leukemia & lymphoma

Article Title: Synergistic cytotoxicity of busulfan, melphalan, gemcitabine, panobinostat, and bortezomib in lymphoma cells

doi: 10.3109/10428194.2016.1157871

Figure Lengend Snippet: List of primary antibodies, their sources, dilutions and molecular weights.

Article Snippet: The primary antibodies, their sources, dilutions, and antigen molecular weights are listed in (Supplementary Materials). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibodies Source (Catalogue #) Clone type Dilutions Molecular weight (kDa) AcH3K9 Active Motif/39137 pAb 3500 17 AKT Cell Signaling/4691 pAb 2000 60 ATM Santa Cruz/23921 mAb 800 250 BCL-2 DAKO/124 mAb 1500 26 c-MYC Cell Signaling/9402 pAb 2500 57–70 Cleaved Caspase 3 Cell Signaling/9661 pAb 2000 17,19 Caspase 8 Cell Signaling/9746 mAb 2000 43 Caspase 9 Cell Signaling/9502 pAb 2000 35–47 CHK2 Cell Signaling/2662 pAb 1000 62 HDAC4 Cell Signaling/5392 mAb 3000 140 KAP1 Bethyl Lab A300-275A pAb 2500 100–117 MCL-1 Santa Cruz/819 pAb 700 40 mTOR Cell Signaling/2983 pAb 2500 289 NOXA Calbiochem/0180 mAb 1500 15 p-ATM (Ser1981) Rockland/200–301-400 mAb 2000 250 p-AKT (Ser 473) Cell Signaling/4060 pAb 2500 60 p-CHK2 (Thr68) Cell Signaling/2661 pAb 2000 62 p-KAP1 (Ser824) Cell Signaling/4127 pAb 2500 100 p-mTOR (Ser2448) Cell Signaling/5536 pAb 1500 289 p-P53 (Ser 15) Cell Signaling/9284 pAb 2000 53 p-PI3K (Tyr199/Tyr458) Cell Signaling /4228 pAb 2500 60, 85 p-SMC1 (Ser957) Novus Biologicals/100–205 pAb 1500 140 P53 Santa Cruz/126 pAb 2000 53 PARP1 Santa Cruz /8007 mAb 1000 116 PI3K Santa Cruz/423 pAb 500 85 SMC1 Cell Signaling/4802 pAb 2500 140 XIAP Cell Signaling/2045 pAb 2000 53 α-Tubulin Cell Signaling/2144 pAb 1000 57 β-ACTIN Sigma/A5316 mAb 10000 42 γ-H2AX Millipore/2554898 mAb 3000 15 3MeH3K27 Active Motif/39155 pAb 3500 17 Open in a separate window List of primary antibodies, their sources, dilutions and molecular weights.

Techniques: Molecular Weight

Journal: Biochemical pharmacology

Article Title: Mechanisms of tolvaptan-induced toxicity in HepG2 cells ☆

doi: 10.1016/j.bcp.2015.03.015

Figure Lengend Snippet: HepG2 cells were treated with tolvaptan (0–100 μM) for 24 or 48 h. (A) Tail moment in alkaline comet assays with or without post-incubation with Fpg. The results shown are the mean and standard deviation of three independent experiments. * Significantly (p < 0.05) different from 0 μM tolvaptan. (B) Relative levels of 8-oxoguanine and (C) ROS in the HepG2 cells treated with tolvaptan. The results shown are the mean and standard deviation of three independent experiments. * Significantly (p < 0.05) different from 0 μM tolvaptan. (D) Representative Western blot images of γ-H2AX, p-Chk1, and p-Chk2 in the HepG2 cells treated with tolvaptan. β-Actin was used as a loading control.

Article Snippet: Mouse monoclonal antibodies against cyclin D1, cyclin D3, cyclin-dependent kinase (CDK) 4 (CDK4), and CDK6, rabbit monoclonal antibodies against CDK2, phosphorylated checkpoint kinase 1 at Ser345 (p-Chk1), Bcl-2, Bad, Bim, Bax, phosphorylated-ERK1/2 at Thr202/Tyr204 (p-ERK1/2), ERK1/2, phosphorylated-JNK at Thr183/Tyr185 (p-JNK), JNK, phosphorylated-p38 at Thr180/Tyr182 (p-p38), and p38, and rabbit polyclonal antibodies against phosphorylated checkpoint kinase 2 at Thr68 (p-Chk2), cytochrome C, and Bid were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Standard Deviation, Western Blot