Journal: Cancers

Article Title: TP-0903 Is Active in Preclinical Models of Acute Myeloid Leukemia with TP53 Mutation/Deletion.

doi: 10.3390/cancers15010029

Figure Lengend Snippet: Figure 3. TP-0903 induces DNA damage response in TP53 mutant AML cell lines. (A) MV4-11 (R248W), (B) HL-60, and (C) Kasumi-1 cells were treated with DMSO or TP-0903 at indicated concentrations for 4 h. Immunoblotting was performed to determine the expression of pChk1 (Ser345), pChk2 (Thr68), and pH2AX (Ser139/Tyr142). Vinculin served as the loading control for pChk1/Chk1 and pChk2/Chk2 blots. GAPDH served as the loading control for pH2AX/H2AX blots. Immunoblots were quantified against respective loading controls (vinculin or GAPDH) using ImageJ. Blots are representative of 2–3 independent experiments.

Article Snippet: Antibodies against p53 (48818; clone DO-7), p21 (2947; clone 12D1), pH2AX (Ser 139) (80312; clone D7T2V), pH2AX (Ser139/Tyr142) (5438; clone N/A), H2AX (7631; clone D17A3), pAURKA(Thr288)/B(Thr232)/C(Thr198) (2914; clone D13A11), AURKA (91590; clone D3V7T), AURKB (3094; clone N/A), pChk1 (Ser345) (2348; clone 133D3), pChk2 (Thr68) (2197; clone C13C1), Chk1 (2360; clone 2G1D5), Chk2 (6334; clone D9C6), vinculin (13901; clone E1E9V), GAPDH (5174S; clone D16H11), and HRP-conjugate secondary antirabbit (7074) were obtained from Cell Signaling Technology (CST) (Danvers, MA, USA).

Techniques: Mutagenesis, Western Blot, Expressing, Control

Journal: Oncoimmunology

Article Title: Uric acid accumulation in DNA-damaged tumor cells induces NKG2D ligand expression and antitumor immunity by activating TGF-β-activated kinase 1

doi: 10.1080/2162402X.2021.2016159

Figure Lengend Snippet: ATM and Chk silencing blocks genotoxic drug-induced ERK phosphorylation, uric acid production, and MICA/B expression . HeLa cells were transfected with scrambled control siRNA, ATM siRNA, Chk1 siRNA, or Chk2 siRNA. After incubation for 24 hr, the cells were left untreated or treated with 5-FU (10 μM) or gemcitabine (2 μM) for another 24 hr. Cell lysates were prepared and analyzed for TAK1 and ERK phosphorylation by Western blot (a) and uric acid levels by using a uric acid assay kit (b). Relative phosphorylation levels were analyzed by quantifying the density of phosphorylated TAK1 and ERK bands normalized by the density of their corresponding total protein bands with NIH Image-J software and presented as bar graphs. Data are the mean ± SD of three experiments. **p < .01, compared to the untreated control. ## p < .01; ns, not significant, compared to the corresponding control siRNA-transfected controls. Single-cell suspensions were prepared and analyzed for the levels of MICA/B expression (c) by flow cytometry. Green line, isotype control; Red line, MICA/B. The data represent one of three independent experiments with similar results.

Article Snippet: TAK1 siRNA (6317S), ATM siRNA (6328S), Chk1 siRNA (6241S), and Chk2 siRNA (6276S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Phospho-proteomics, Expressing, Transfection, Control, Incubation, Western Blot, Uric Acid Assay, Software, Flow Cytometry

Journal:

Article Title: Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-? and Prevents DNA Damage during Replication

doi: 10.1091/mbc.E06-12-1148

Figure Lengend Snippet: Mcm10-depleted cells activate Chk2. HeLa cells were treated with siRNA as described previously or with 3 mM hydroxyurea for 12 h, and then they were harvested at indicated time points. Samples were then immunoblotted for Chk1, phospho-Chk1 (Ser 345), Chk2, and phospho-Chk2 (Thr 68). Ponceau S staining served as a loading control. The Chk1-P immunoblot has a nonspecific band marked by an asterisk (*).

Article Snippet: Anti-phosphorylated Chk2 (Cell Signaling Technology) was used at a 1:1000 dilution for Western blotting.

Techniques: Staining, Control, Western Blot

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 5 Impaired DNA damage repair in p21-deficient cells after low-dose doxorubicin treatment. A Western blot analysis of phosphorylated ATM (p-ATM), p-Chk1, Chk1, p-Chk2, and Chk2 in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for various time points. β-actin was used as a loading control. B Fluorescence microscopy of Lamin B1 (red) and γ-H2AX foci (green) in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 48 h. Scale Bar = 20 μm. C Fluorescence microscopy showing nuclei (blue) and γ-H2AX foci (green) in HCT116 WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 24 h and then released into fresh media for various time points. Scale bar = 20 μm. Quantification of nuclei with >10 γ-H2AX foci (n = 20 cells per group, repeated 4 times). Data are mean ± SD ****p < 0.0001.

Article Snippet: The following antibodies were used: β-actin (sc-69879), p53 (sc-126), Lamin B1(sc-365214), αtubulin (sc-5286), Noxa (sc-515840, and MKLP1 (sc-136473) (Santa Cruz Biotechnologies, Dallas, TX, USA); p21 (ab109520), phospho-ATM (S1981) (ab81292), and phospho-DNA PKcs (S2056) (ab18192)(Abcam, Cambridge, MA, USA); phospho-Chk1 (Ser345)(# 2348), Chk1 (#2360), phospho-Chk2 (Thr68)(# 2197), Chk2 (#6334), cleaved caspase-3 (#9664), phospho-Histone H2A.X (Ser139)(# 9718), Mcl-1 (#39224), and Aurora B (#3094) (Cell signaling technology, Danvers, MA, USA); Mre11(GTX70212) and α-tubulin (GTX112141) (Genetex, San Antonio, Texas, USA); DNA-PKcs (19983-1-AP) (Proteintech, Rosemont, IL, USA); HRP-conjugated rabbit IgG and mouse IgG (Jackson Immunoresearch, West Grove, PA, USA).

Techniques: Western Blot, Control, Fluorescence, Microscopy

Journal: Carcinogenesis

Article Title: Chemopreventive properties of pinoresinol-rich olive oil involve a selective activation of the ATM-p53 cascade in colon cancer cell lines.

doi: 10.1093/carcin/bgm255

Figure Lengend Snippet: Fig. 5. Evaluation of the involvement of ATM axis in the EVOO-A effects on p53-proficient cells. (A) Significant changes were observed after treatment of RKO and HCT116 for ATM, Chk1, Chk2 and cdc2 (increased expression) and cyclin E (decreased expression). A slight decrease in cyclin B and no change in cyclin D expression was evident in the p53-proficient cell lines. No changes in protein expression were detected after treatment of the p53-deficient cell lines. U (untreated), control, T (treated). (B) Flow cytometry cell cycle profiles in RKO and HCT116 cells with and without pretreatment with caffeine. Pretreatment with caffeine was able to abrogate the G2/M arrest determined by EVOO-A treatment, confirming the involvement of the ATM axis in EVOO-A effects (no differences compared with untreated controls; P . 0.05).

Article Snippet: The primary antibodies, including anti-cyclin D1 (clone A-12), anticyclin E (clone HE 12), anti-cyclin B1 (clone D11), anti-p53 (clone DO-1), anti-p21 (clone F5), anti-GADD45 (C4), anti-14-3-3r (N14), Chk1 (G4) and Chk2 (B4) were obtained from Santa Cruz Biotechnology and incubated for 3 h at room temperature.

Techniques: Expressing, Control, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Oncogene PRR14 promotes breast cancer through activation of PI3K signal pathway and inhibition of CHEK2 pathway

doi: 10.1038/s41419-020-2640-8

Figure Lengend Snippet: CHEK2 mRNA expression ( a ), CHEK2 protein expression ( b ) and p-CHEK2 (T68) protein expression ( c ) in PRR14 genetically unaltered and altered (amplified and mutated) breast cancer cases in TCGA database are statistically analyzed by two-tailed Student’s t -test. CHEK2 protein expression is detected by immunostaining in xenografts in nude mice from established MCF7 cell lines and MDA-MB-231 cell lines ( d ), as well as human breast cancer ( e ). CHEK2 transcription in human breast cancer is also detected by qRT-PCR ( f ). The data are quantified and two-tailed Student’s t -test is employed to determine the significance of the difference. Established MCF7 and MDA-MB-231 ( g ) PRR14-overexpressing and control cell lines are treated with various of genotoxic chemicals including Bleo, Eto, 5-FU, H2O2 and HU at indicated concentrations for indicated time. Key components of the ATM/CHEK2/P53 signaling pathway are detected by immunostaining. And CHEK2 protein expression ( h ) and mRNA expression ( i ) are detected by immunostaining and qRT-PCR, respectively. The data are analyzed by two-tailed Student’s t -test. Established MCF7 and MDA-MB-231 ( j ) PRR14-overexpressing and control cell lines are treated with Eto at indicated concentration for indicated time to induce p-CHEK2 (T68), which is detected by immunostaining and quantified and normalized by CHEK2 total protein ( k ). The data are analyzed by two-tailed Student’s t -test.

Article Snippet: Chemicals including selective CHEK2 inhibitor BML-277 (BML, HY-13946, MCE) , and genotoxic chemicals, including bleomycin (Bleo, HY-17565, MCE), etoposide (Eto, HY-13629, MCE), 5-fluorouracil (5-FU, HY-90006, MCE), H 2 O 2 (88597, Millipore) and hydroxyurea (HU, HY-B0313, MCE), were used to treat cells.

Techniques: Expressing, Amplification, Two Tailed Test, Immunostaining, Quantitative RT-PCR, Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: Oncogene PRR14 promotes breast cancer through activation of PI3K signal pathway and inhibition of CHEK2 pathway

doi: 10.1038/s41419-020-2640-8

Figure Lengend Snippet: MCF7 cells transfected with indicated siRNA sequences for 48 h are treated w/wo 5 μg/ml Eto for 6 h, then cells are harvested for immunostaining ( a ). PRR14 in cells is depleted by RNAi transfection for 48 h, and CHEK2 mRNA level are quantified by qRT-PCR in both MCF7 and MDA-MB-231 cell lines ( b ). MCF7, 10AKRAS, 7E6 and MDA-MB-231 cell lines are treated with increasing concentration of CHEK2 inhibitor BML for 24 h and followed with a 24 h treatment of Eto at 5 μg/ml. Cells are harvested to stain with PI followed with FACS analysis ( c ). The percentage of the 4N fraction is analyzed by one-way ANOVA analysis ( n = 3) ( d ).

Article Snippet: Chemicals including selective CHEK2 inhibitor BML-277 (BML, HY-13946, MCE) , and genotoxic chemicals, including bleomycin (Bleo, HY-17565, MCE), etoposide (Eto, HY-13629, MCE), 5-fluorouracil (5-FU, HY-90006, MCE), H 2 O 2 (88597, Millipore) and hydroxyurea (HU, HY-B0313, MCE), were used to treat cells.

Techniques: Transfection, Immunostaining, Quantitative RT-PCR, Concentration Assay, Staining

Journal: Cell Death & Disease

Article Title: Oncogene PRR14 promotes breast cancer through activation of PI3K signal pathway and inhibition of CHEK2 pathway

doi: 10.1038/s41419-020-2640-8

Figure Lengend Snippet: KM survival curves of breast cancer patients receiving chemotherapy are stratified by their expression levels of either CHEK2 ( a ) or PRR14 ( b ). For comparison, KM survival curves of breast cancer patients receiving endocrine therapy and stratified by PRR14 expression is also analyzed ( c ). KM survival curves of breast cancer patients receiving chemotherapy with mutant P53 ( d ) or wild-type P53 ( e ) are stratified by their expression level of PRR14.

Article Snippet: Chemicals including selective CHEK2 inhibitor BML-277 (BML, HY-13946, MCE) , and genotoxic chemicals, including bleomycin (Bleo, HY-17565, MCE), etoposide (Eto, HY-13629, MCE), 5-fluorouracil (5-FU, HY-90006, MCE), H 2 O 2 (88597, Millipore) and hydroxyurea (HU, HY-B0313, MCE), were used to treat cells.

Techniques: Expressing, Comparison, Mutagenesis