anti p53 ps15  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: EXO1 as a therapeutic target for Fanconi Anaemia, ZRSR2 and BRCA1-A complex deficient cancers

doi: 10.1038/s41467-025-63349-7

Figure Lengend Snippet: a Validation of EXO1 and FANCG synthetic lethal interaction in eHAP iCas9 cells by clonogenic assay (representative images, n = 3, well diameter 15.5 mm). b Quantification of clonogenic assay ( n = 3, mean with SD) in ( a ) with ordinary one-way ANOVA statistical analysis ( F = 158.8). c Validation of EXO1 and FANCG synthetic lethal interaction in eHAP iCas9 cells by CellTitre-Glo viability assay ( n = 3, mean with SD) with ordinary one-way ANOVA statistical analysis ( F = 132.5). d Apoptosis of EXO1 and FANCG double KO cells ( n = 3). e Quantification of cell cycle phases ( n = 4, based on gating shown in Supplementary Fig. , mean with SD). f Micronuclei increase in double KO cells. Paired t -test analyses were performed for each of the double KO to compare to ‘ EXO1 KO + sgNT’ ( n = 3, mean with SD, with each percentage calculated on the basis of >2181 nuclei; two-tailed P values). g Increase in phosphorylated RPA at serine 33 (RPA-pS33) foci in double KO cells. Dots in orange, blue and green represent the mean of a biological repeat, with the red line as their mean. Paired t -test analyses were performed on the means of biological repeat for each of the double KO cell lines in comparison to the ‘ EXO1 KO + sgNT’ cell line ( n = 3, mean with SD, with each percentage calculated on the basis of >754 nuclei; RPA-pS33 signal was measured within nuclei; two-tailed P values). h Immunoblotting of total cell extracts and chromatin for ‘WT + sgNT’, ‘WT + sg FANCG ’, ‘ EXO1 KO + sgNT’ and ‘ EXO1 KO + sg FANCG ’ eHAP iCas9 cells at day 5 post Cas9 expression induction. Samples were probed for phosphorylated levels of KAP1, p53, CHK2 and H2AX with controls, and EXO1, alpha-tubulin and histone H3 to control for sample identity and equal loading (representative experiment, n = 3). Source data for b – h are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-EXO1 (ab95068, Abcam, rabbit, 1:1000), anti-alpha-tubulin (clone B-5-1-2, T6074, Sigma, mouse, 1:10000), anti-vinculin (clone hVIN-1, ab11194, Abcam, mouse, 1:20000), anti-FANCG (clone F-8, sc-393382, Santa Cruz, mouse, 1:1000), anti-KAP1-pS824 (ab70369, Abcam, rabbit, 1:1000), anti-KAP1 (clone 20C1, ab22553, Abcam, mouse, 1:1000), anti-p53-pS15 (9284S, CST, rabbit, 1:1000), anti-p53 (clone 1C12, 2524S, CST, mouse, 1:1000), anti-CHK2-pT68 (2661S, CST, rabbit, 1:1000), anti-CHK2 (clone 7, 05-649, Millipore, mouse, 1:500), anti-H2AX-pS139 (gamma-H2AX, clone JBW301, 05-636, Millipore, mouse, 1:1000), anti-histone H3 (ab1791, Abcam, rabbit, 1:5000), anti-RPA-pS33 (A300-246A, Bethyl Laboratories, rabbit, 1:1000), anti-RPA (clone 9H8, ab2175, Abcam, mouse, 1:1000), anti-FANCD2 (clone EPR2302, ab108928, Abcam, rabbit, 1:1000), anti-RMI2 (ab122685, Abcam, rabbit, 1:500), anti-FAM175A/Abraxas1 (clone EPR6310(2), ab139191, Abcam, rabbit, 1:1000), anti-BRCC36 (A302-517A-M, Bethyl Laboratories, rabbit, 1:1000), anti-FANCC (clone 8F3, MABC524, Sigma, mouse, 1:1000).

Techniques: Biomarker Discovery, Clonogenic Assay, Viability Assay, Two Tailed Test, Comparison, Western Blot, Expressing, Control

Journal: Experimental Neurobiology

Article Title: KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells

doi: 10.5607/en25017

Figure Lengend Snippet: KCC-07 suppresses growth of U-87MG and SH-SY5Y cell lines. (A) Dose dependent proliferation responses of U-87MG and SH-SY5Y cell lines to KCC-07 as measured by MTT assay. Both U-87MG (glioma cell line) and SH-SY5Y (neuroblastoma cell line) are sensitive to KCC-07 treatment in a dose dependent manner. Representative data are shown in the graphs (n=12). The readouts of the NT group were set as 100%, then the survival rates were calculated as a relative values to the NT group. Each experiment was repeated twice independently. (B) Dose dependent proliferation responses of U-87MG and SH-SY5Y cell lines to DNA damage induced by either phleomycin (Phleo) or etoposide (ETP) in combination with KCC-07 exposure as measured by MTT assay. Dose dependency of DNA damage inducing drugs is not affected by KCC-07 treatment. Representative data are shown in the graphs (n=4~6). The readouts of the NT group were set as 100%, then the survival rates were calculated as a relative values to the NT group. Each experiment was repeated twice independently. (C) Detection of p53 stabilization in the nucleus following KCC-07 treatment and DNA damage induction, indicating the activation of p53 signaling in both U-87MG and SH-SY5Y cell lines. The distribution of MDM2, an E3 ubiquitin ligase for p53, was not changed; its localization is restricted to the cytosol. Representative data are shown in the figure. Each experiment was repeated three times independently. The experimental conditions of drug treatments are indicated in the figure. NT, Not treated; NS, not significant; C, cytosolic fraction; N, nuclear fraction.

Article Snippet: The antibodies used for Western blot analysis were ATM (Abcam, Cambridge, UK), ATM-pS1981 (Cell Signaling Technology, MA, USA), KAP1 (Novus Biologicals, CO, USA), KAP1-pS824 (Novus Biologicals), CHK2 (Millipore, MA, USA), CHK2-pT68 (Novus Biologicals), γ-H2AX (Cell Signaling Technology), p53 (Santa Cruz Biotechnology, TX, USA), p53-pS15 (Cell Signaling Technology), MDM2 (Santa Cruz Biotechnology), GAPDH (Genetex, CA, USA), and histone H3 (Abcam).

Techniques: MTT Assay, Activation Assay, Ubiquitin Proteomics

Journal: Experimental Neurobiology

Article Title: KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells

doi: 10.5607/en25017

Figure Lengend Snippet: Both KCC-07 and DNA damage induction result in known p53 dependent gene expression. (A) Induction of CDKN1A expression, which is p53 dependent and involved in cell cycle regulation, by either KCC-07 or DNA damaging reagent treatments measured by real-time PCR. Combined treatment with KCC-07 and DNA damaging reagents did not further increase CDKN1A expression in both cell lines. The SH-SY5Y cell line is more sensitive to DNA damaging reagents. The gene expression levels were normalized by β-actin real-time PCR readout. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (B) Induction of BBC3 expression, which is p53 dependent and involved in apoptosis, by DNA damaging reagents. KCC-07 treatment did not induce BBC3 expression in both cell lines. The gene expression levels were normalized by β-actin real-time PCR readout. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (C) Consistent DNA damage response (DDR) following combined treatment with KCC-07 and DNA damaging reagents analyzed by Western blots. Both cell lines displayed well-established DDR including ATM phosphorylation, KAP1 and CHK2 phosphorylation (ATM-dependent), and p53 activation (ATM-dependent) followed by induction of apoptotic and cell cycle arrest factors (p53-dependent, see B and C in this figure) upon DNA damage induction by Phleo or ETP treatments. KCC-07 addition does not change the proper DDR upon DNA damage, but sustained H2AX phosphorylation indicates defective DNA damage repair. Ponceau S staining and GAPDH western blot served as loading controls. Representative data are shown. Each experiment was repeated twice independently. The experimental conditions of drug treatments are indicated in the figure. NS, not significant.

Article Snippet: The antibodies used for Western blot analysis were ATM (Abcam, Cambridge, UK), ATM-pS1981 (Cell Signaling Technology, MA, USA), KAP1 (Novus Biologicals, CO, USA), KAP1-pS824 (Novus Biologicals), CHK2 (Millipore, MA, USA), CHK2-pT68 (Novus Biologicals), γ-H2AX (Cell Signaling Technology), p53 (Santa Cruz Biotechnology, TX, USA), p53-pS15 (Cell Signaling Technology), MDM2 (Santa Cruz Biotechnology), GAPDH (Genetex, CA, USA), and histone H3 (Abcam).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Activation Assay, Staining

Journal: Experimental Neurobiology

Article Title: KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells

doi: 10.5607/en25017

Figure Lengend Snippet: KCC-07 suppresses growth of U-87MG and SH-SY5Y cell lines. (A) Dose dependent proliferation responses of U-87MG and SH-SY5Y cell lines to KCC-07 as measured by MTT assay. Both U-87MG (glioma cell line) and SH-SY5Y (neuroblastoma cell line) are sensitive to KCC-07 treatment in a dose dependent manner. Representative data are shown in the graphs (n=12). The readouts of the NT group were set as 100%, then the survival rates were calculated as a relative values to the NT group. Each experiment was repeated twice independently. (B) Dose dependent proliferation responses of U-87MG and SH-SY5Y cell lines to DNA damage induced by either phleomycin (Phleo) or etoposide (ETP) in combination with KCC-07 exposure as measured by MTT assay. Dose dependency of DNA damage inducing drugs is not affected by KCC-07 treatment. Representative data are shown in the graphs (n=4~6). The readouts of the NT group were set as 100%, then the survival rates were calculated as a relative values to the NT group. Each experiment was repeated twice independently. (C) Detection of p53 stabilization in the nucleus following KCC-07 treatment and DNA damage induction, indicating the activation of p53 signaling in both U-87MG and SH-SY5Y cell lines. The distribution of MDM2, an E3 ubiquitin ligase for p53, was not changed; its localization is restricted to the cytosol. Representative data are shown in the figure. Each experiment was repeated three times independently. The experimental conditions of drug treatments are indicated in the figure. NT, Not treated; NS, not significant; C, cytosolic fraction; N, nuclear fraction.

Article Snippet: The antibodies used for Western blot analysis were ATM (Abcam, Cambridge, UK), ATM-pS1981 (Cell Signaling Technology, MA, USA), KAP1 (Novus Biologicals, CO, USA), KAP1-pS824 (Novus Biologicals), CHK2 (Millipore, MA, USA), CHK2-pT68 (Novus Biologicals), γ-H2AX (Cell Signaling Technology), p53 (Santa Cruz Biotechnology, TX, USA), p53-pS15 (Cell Signaling Technology), MDM2 (Santa Cruz Biotechnology), GAPDH (Genetex, CA, USA), and histone H3 (Abcam).

Techniques: MTT Assay, Activation Assay, Ubiquitin Proteomics

Journal: Experimental Neurobiology

Article Title: KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells

doi: 10.5607/en25017

Figure Lengend Snippet: Both KCC-07 and DNA damage induction result in known p53 dependent gene expression. (A) Induction of CDKN1A expression, which is p53 dependent and involved in cell cycle regulation, by either KCC-07 or DNA damaging reagent treatments measured by real-time PCR. Combined treatment with KCC-07 and DNA damaging reagents did not further increase CDKN1A expression in both cell lines. The SH-SY5Y cell line is more sensitive to DNA damaging reagents. The gene expression levels were normalized by β-actin real-time PCR readout. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (B) Induction of BBC3 expression, which is p53 dependent and involved in apoptosis, by DNA damaging reagents. KCC-07 treatment did not induce BBC3 expression in both cell lines. The gene expression levels were normalized by β-actin real-time PCR readout. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (C) Consistent DNA damage response (DDR) following combined treatment with KCC-07 and DNA damaging reagents analyzed by Western blots. Both cell lines displayed well-established DDR including ATM phosphorylation, KAP1 and CHK2 phosphorylation (ATM-dependent), and p53 activation (ATM-dependent) followed by induction of apoptotic and cell cycle arrest factors (p53-dependent, see B and C in this figure) upon DNA damage induction by Phleo or ETP treatments. KCC-07 addition does not change the proper DDR upon DNA damage, but sustained H2AX phosphorylation indicates defective DNA damage repair. Ponceau S staining and GAPDH western blot served as loading controls. Representative data are shown. Each experiment was repeated twice independently. The experimental conditions of drug treatments are indicated in the figure. NS, not significant.

Article Snippet: The antibodies used for Western blot analysis were ATM (Abcam, Cambridge, UK), ATM-pS1981 (Cell Signaling Technology, MA, USA), KAP1 (Novus Biologicals, CO, USA), KAP1-pS824 (Novus Biologicals), CHK2 (Millipore, MA, USA), CHK2-pT68 (Novus Biologicals), γ-H2AX (Cell Signaling Technology), p53 (Santa Cruz Biotechnology, TX, USA), p53-pS15 (Cell Signaling Technology), MDM2 (Santa Cruz Biotechnology), GAPDH (Genetex, CA, USA), and histone H3 (Abcam).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Activation Assay, Staining