mgmt methylation test Search Results


mgmt promoter methylation detection kit  (EntroGen Inc)
90
EntroGen Inc mgmt promoter methylation detection kit
Commercially available IVD tests by the type of <t> methylation-based </t> biomarker use
Mgmt Promoter Methylation Detection Kit, supplied by EntroGen Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mgmt+methylation+test/mgmt+methylation+detection+kit/pmc07336678-255-5-11
Average 90 stars, based on 1 article reviews
mgmt promoter methylation detection kit - by Bioz Stars, 2026-10
90/100 stars
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anti mgmt antibody  (Proteintech)
93
Proteintech anti mgmt antibody
KDM6B regulates PARP-1 activation and <t>MGMT</t> expression. ( A and B ) Volcano plots of KDM6B target genes in HeLa cells. Log 2 FC represents the fold change of mRNA expression in SC/KDM6B KO. n = 2. ( C ) Venn diagram of KDM6B rescued target genes in HeLa cells. ( D ) Immunoblot analysis <t>of</t> <t>PAR</t> and DNA damage-related proteins in SC and KDM6B KO2 HeLa cells treated with vehicle (−) or MNNG for indicated time. ( E ) RT-qPCR analysis of indicated genes in SC and KDM6B KO2 HeLa cells (mean ± SEM, n = 3–6). ** P < 0.01 by unpaired two-tailed Student's t test. ( F ) RNA-seq analysis of MGMT expression in SC, KDM6B KO2, and rescued HeLa cells (mean ± SEM, n = 4). **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. ( G ) Immunoblot analysis of MGMT in SC, KDM6B KO2, and rescued HeLa cells. ( H ) RT-qPCR analysis of MGMT expression in HeLa cells expressing EV, WT KDM6B, or catalytically mutant (mut) KDM6B (mean ± SEM, n = 6–12). * P < 0.05; **** P < 0.0001 vs . EV by one-way ANOVA Dunnett's multiple comparisons test. ( I ) Immunoblot analysis of indicated proteins in HeLa cells expressing full-length WT or H1390A KDM6B. ( J ) Representative KDM6B and MGMT immunostaining images in HeLa cells expressing WT or catalytically mutant KDM6B. Scale bar, 20 μm. ( K ) Immunoblot analysis of MGMT in HeLa cells treated with or without GSK-J4 for 72 h. ( L and M ) Representative DNA PAGE gels of methylated and unmethylated MGMT promoters in WT and KDM6B KO2 HeLa cells (L). DNA intensity is quantified in M (mean ± SEM, n = 4). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( N ) In vitro MGMT activity assay. The assay strategy is shown on the top. Immunoblot analysis of biotin is shown at the bottom. Numbers indicate the signal intensity.
Anti Mgmt Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mgmt+methylation+test/MGMT+Antibody/pmc09226499-67-17-19
Average 93 stars, based on 1 article reviews
anti mgmt antibody - by Bioz Stars, 2026-10
93/100 stars
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mgmt methylation test  (MDxHealth)
90
MDxHealth mgmt methylation test
KDM6B regulates PARP-1 activation and <t>MGMT</t> expression. ( A and B ) Volcano plots of KDM6B target genes in HeLa cells. Log 2 FC represents the fold change of mRNA expression in SC/KDM6B KO. n = 2. ( C ) Venn diagram of KDM6B rescued target genes in HeLa cells. ( D ) Immunoblot analysis <t>of</t> <t>PAR</t> and DNA damage-related proteins in SC and KDM6B KO2 HeLa cells treated with vehicle (−) or MNNG for indicated time. ( E ) RT-qPCR analysis of indicated genes in SC and KDM6B KO2 HeLa cells (mean ± SEM, n = 3–6). ** P < 0.01 by unpaired two-tailed Student's t test. ( F ) RNA-seq analysis of MGMT expression in SC, KDM6B KO2, and rescued HeLa cells (mean ± SEM, n = 4). **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. ( G ) Immunoblot analysis of MGMT in SC, KDM6B KO2, and rescued HeLa cells. ( H ) RT-qPCR analysis of MGMT expression in HeLa cells expressing EV, WT KDM6B, or catalytically mutant (mut) KDM6B (mean ± SEM, n = 6–12). * P < 0.05; **** P < 0.0001 vs . EV by one-way ANOVA Dunnett's multiple comparisons test. ( I ) Immunoblot analysis of indicated proteins in HeLa cells expressing full-length WT or H1390A KDM6B. ( J ) Representative KDM6B and MGMT immunostaining images in HeLa cells expressing WT or catalytically mutant KDM6B. Scale bar, 20 μm. ( K ) Immunoblot analysis of MGMT in HeLa cells treated with or without GSK-J4 for 72 h. ( L and M ) Representative DNA PAGE gels of methylated and unmethylated MGMT promoters in WT and KDM6B KO2 HeLa cells (L). DNA intensity is quantified in M (mean ± SEM, n = 4). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( N ) In vitro MGMT activity assay. The assay strategy is shown on the top. Immunoblot analysis of biotin is shown at the bottom. Numbers indicate the signal intensity.
Mgmt Methylation Test, supplied by MDxHealth, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mgmt+methylation+test/mgmt+promoter+methylation+testing/pm29776209-176-22-18
Average 90 stars, based on 1 article reviews
mgmt methylation test - by Bioz Stars, 2026-10
90/100 stars
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temozolomide  (MDxHealth)
90
MDxHealth temozolomide
KDM6B regulates PARP-1 activation and <t>MGMT</t> expression. ( A and B ) Volcano plots of KDM6B target genes in HeLa cells. Log 2 FC represents the fold change of mRNA expression in SC/KDM6B KO. n = 2. ( C ) Venn diagram of KDM6B rescued target genes in HeLa cells. ( D ) Immunoblot analysis <t>of</t> <t>PAR</t> and DNA damage-related proteins in SC and KDM6B KO2 HeLa cells treated with vehicle (−) or MNNG for indicated time. ( E ) RT-qPCR analysis of indicated genes in SC and KDM6B KO2 HeLa cells (mean ± SEM, n = 3–6). ** P < 0.01 by unpaired two-tailed Student's t test. ( F ) RNA-seq analysis of MGMT expression in SC, KDM6B KO2, and rescued HeLa cells (mean ± SEM, n = 4). **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. ( G ) Immunoblot analysis of MGMT in SC, KDM6B KO2, and rescued HeLa cells. ( H ) RT-qPCR analysis of MGMT expression in HeLa cells expressing EV, WT KDM6B, or catalytically mutant (mut) KDM6B (mean ± SEM, n = 6–12). * P < 0.05; **** P < 0.0001 vs . EV by one-way ANOVA Dunnett's multiple comparisons test. ( I ) Immunoblot analysis of indicated proteins in HeLa cells expressing full-length WT or H1390A KDM6B. ( J ) Representative KDM6B and MGMT immunostaining images in HeLa cells expressing WT or catalytically mutant KDM6B. Scale bar, 20 μm. ( K ) Immunoblot analysis of MGMT in HeLa cells treated with or without GSK-J4 for 72 h. ( L and M ) Representative DNA PAGE gels of methylated and unmethylated MGMT promoters in WT and KDM6B KO2 HeLa cells (L). DNA intensity is quantified in M (mean ± SEM, n = 4). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( N ) In vitro MGMT activity assay. The assay strategy is shown on the top. Immunoblot analysis of biotin is shown at the bottom. Numbers indicate the signal intensity.
Temozolomide, supplied by MDxHealth, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mgmt+methylation+test/temozolomide/pm23033952-45-49-65
Average 90 stars, based on 1 article reviews
temozolomide - by Bioz Stars, 2026-10
90/100 stars
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pearson’s chi-squared test  (CH Instruments)
90
CH Instruments pearson’s chi-squared test
KDM6B regulates PARP-1 activation and <t>MGMT</t> expression. ( A and B ) Volcano plots of KDM6B target genes in HeLa cells. Log 2 FC represents the fold change of mRNA expression in SC/KDM6B KO. n = 2. ( C ) Venn diagram of KDM6B rescued target genes in HeLa cells. ( D ) Immunoblot analysis <t>of</t> <t>PAR</t> and DNA damage-related proteins in SC and KDM6B KO2 HeLa cells treated with vehicle (−) or MNNG for indicated time. ( E ) RT-qPCR analysis of indicated genes in SC and KDM6B KO2 HeLa cells (mean ± SEM, n = 3–6). ** P < 0.01 by unpaired two-tailed Student's t test. ( F ) RNA-seq analysis of MGMT expression in SC, KDM6B KO2, and rescued HeLa cells (mean ± SEM, n = 4). **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. ( G ) Immunoblot analysis of MGMT in SC, KDM6B KO2, and rescued HeLa cells. ( H ) RT-qPCR analysis of MGMT expression in HeLa cells expressing EV, WT KDM6B, or catalytically mutant (mut) KDM6B (mean ± SEM, n = 6–12). * P < 0.05; **** P < 0.0001 vs . EV by one-way ANOVA Dunnett's multiple comparisons test. ( I ) Immunoblot analysis of indicated proteins in HeLa cells expressing full-length WT or H1390A KDM6B. ( J ) Representative KDM6B and MGMT immunostaining images in HeLa cells expressing WT or catalytically mutant KDM6B. Scale bar, 20 μm. ( K ) Immunoblot analysis of MGMT in HeLa cells treated with or without GSK-J4 for 72 h. ( L and M ) Representative DNA PAGE gels of methylated and unmethylated MGMT promoters in WT and KDM6B KO2 HeLa cells (L). DNA intensity is quantified in M (mean ± SEM, n = 4). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( N ) In vitro MGMT activity assay. The assay strategy is shown on the top. Immunoblot analysis of biotin is shown at the bottom. Numbers indicate the signal intensity.
Pearson’s Chi Squared Test, supplied by CH Instruments, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mgmt+methylation+test/pearson+chi+square+test/pm20627446-139-14-18
Average 90 stars, based on 1 article reviews
pearson’s chi-squared test - by Bioz Stars, 2026-10
90/100 stars
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Image Search Results


Commercially available IVD tests by the type of methylation-based biomarker use

Journal: Clinical Epigenetics

Article Title: Current status of development of methylation biomarkers for in vitro diagnostic IVD applications

doi: 10.1186/s13148-020-00886-6

Figure Lengend Snippet: Commercially available IVD tests by the type of methylation-based biomarker use

Article Snippet: The third CE-marked test is MGMT Promoter Methylation Detection Kit from EntroGen Inc. (Los Angeles, CA, USA).

Techniques: Methylation, Biomarker Assay, Diagnostic Assay, Real-time Polymerase Chain Reaction, Next-Generation Sequencing, Multiplex Assay, Methylation Sequencing

KDM6B regulates PARP-1 activation and MGMT expression. ( A and B ) Volcano plots of KDM6B target genes in HeLa cells. Log 2 FC represents the fold change of mRNA expression in SC/KDM6B KO. n = 2. ( C ) Venn diagram of KDM6B rescued target genes in HeLa cells. ( D ) Immunoblot analysis of PAR and DNA damage-related proteins in SC and KDM6B KO2 HeLa cells treated with vehicle (−) or MNNG for indicated time. ( E ) RT-qPCR analysis of indicated genes in SC and KDM6B KO2 HeLa cells (mean ± SEM, n = 3–6). ** P < 0.01 by unpaired two-tailed Student's t test. ( F ) RNA-seq analysis of MGMT expression in SC, KDM6B KO2, and rescued HeLa cells (mean ± SEM, n = 4). **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. ( G ) Immunoblot analysis of MGMT in SC, KDM6B KO2, and rescued HeLa cells. ( H ) RT-qPCR analysis of MGMT expression in HeLa cells expressing EV, WT KDM6B, or catalytically mutant (mut) KDM6B (mean ± SEM, n = 6–12). * P < 0.05; **** P < 0.0001 vs . EV by one-way ANOVA Dunnett's multiple comparisons test. ( I ) Immunoblot analysis of indicated proteins in HeLa cells expressing full-length WT or H1390A KDM6B. ( J ) Representative KDM6B and MGMT immunostaining images in HeLa cells expressing WT or catalytically mutant KDM6B. Scale bar, 20 μm. ( K ) Immunoblot analysis of MGMT in HeLa cells treated with or without GSK-J4 for 72 h. ( L and M ) Representative DNA PAGE gels of methylated and unmethylated MGMT promoters in WT and KDM6B KO2 HeLa cells (L). DNA intensity is quantified in M (mean ± SEM, n = 4). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( N ) In vitro MGMT activity assay. The assay strategy is shown on the top. Immunoblot analysis of biotin is shown at the bottom. Numbers indicate the signal intensity.

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: KDM6B regulates PARP-1 activation and MGMT expression. ( A and B ) Volcano plots of KDM6B target genes in HeLa cells. Log 2 FC represents the fold change of mRNA expression in SC/KDM6B KO. n = 2. ( C ) Venn diagram of KDM6B rescued target genes in HeLa cells. ( D ) Immunoblot analysis of PAR and DNA damage-related proteins in SC and KDM6B KO2 HeLa cells treated with vehicle (−) or MNNG for indicated time. ( E ) RT-qPCR analysis of indicated genes in SC and KDM6B KO2 HeLa cells (mean ± SEM, n = 3–6). ** P < 0.01 by unpaired two-tailed Student's t test. ( F ) RNA-seq analysis of MGMT expression in SC, KDM6B KO2, and rescued HeLa cells (mean ± SEM, n = 4). **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. ( G ) Immunoblot analysis of MGMT in SC, KDM6B KO2, and rescued HeLa cells. ( H ) RT-qPCR analysis of MGMT expression in HeLa cells expressing EV, WT KDM6B, or catalytically mutant (mut) KDM6B (mean ± SEM, n = 6–12). * P < 0.05; **** P < 0.0001 vs . EV by one-way ANOVA Dunnett's multiple comparisons test. ( I ) Immunoblot analysis of indicated proteins in HeLa cells expressing full-length WT or H1390A KDM6B. ( J ) Representative KDM6B and MGMT immunostaining images in HeLa cells expressing WT or catalytically mutant KDM6B. Scale bar, 20 μm. ( K ) Immunoblot analysis of MGMT in HeLa cells treated with or without GSK-J4 for 72 h. ( L and M ) Representative DNA PAGE gels of methylated and unmethylated MGMT promoters in WT and KDM6B KO2 HeLa cells (L). DNA intensity is quantified in M (mean ± SEM, n = 4). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( N ) In vitro MGMT activity assay. The assay strategy is shown on the top. Immunoblot analysis of biotin is shown at the bottom. Numbers indicate the signal intensity.

Article Snippet: The membrane was blocked with 5% milk and incubated overnight with primary antibodies: anti-Flag antibody (Sigma, F3165), anti-MGMT antibody (Proteintech, 17195-1-AP), anti-PAR antibody (Trevigen, 4336-BPC-100), anti-PARP-1 antibody (Proteintech, 22999-1-AP), anti-γH2AX antibody (Millipore, 05-636), anti-H2AX antibody (Proteintech, 10856-1-AP), anti-phospho-Chk1 (Ser345) antibody (Cell Signaling Technology, 2348), anti-phospho-RPA32 (Ser33) (Fisher, 50-155-7198), anti-RPA70 (Santa Cruz, SC166023), anti-phospho-ATR (Thr1989) antibody (Cell Signaling Technology, 30632S), anti-ATR antibody (Cell Signaling Technology, 13934S), anti-phospho-ATM (Ser1981) (D6H9) (Cell Signaling Technology, 5883S), anti-ATM (Cell Signaling Technology, 92356S), anti-phospho-Chk1 (Ser296) antibody (Cell Signaling Technology, 2349S), anti-Chk1 antibody (Santa Cruz, sc-8408), anti-phospho-Chk2 (Thr68) (C13C1) antibody (Cell Signaling Technology, 2197S), anti-phospho-Chk2 (D9C6) antibody (Cell Signaling Technology, 6334S), anti-PP2A antibody (Proteintech, 10321-1-AP), anti-H3K27me3 (Cell Signaling Technology, 9733S), anti-XRCC1 antibody (Proteintech, 21468-1-AP), anti-MSH2 antibody (25D12) (Santa Cruz, sc-56163), anti-MSH6 antibody (F-1) (Santa Cruz, sc-271979), or anti-actin antibody (Proteintech, 66009-1-AP) at 4°C, followed by donkey anti-mouse or goat anti-rabbit IgG conjugated to HRP for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing, Mutagenesis, Immunostaining, Methylation, In Vitro, Activity Assay

Pharmacological inhibition or deletion of MGMT sensitizes KDM6BKO cells to alkylating agents. ( A ) Immunoblot analysis of MGMT in SC and KDM6B KO2 HeLa cells 6 h after the treatment of MNNG (50 μM, 15 min) and/or BG (200 μM). ( B and C ) Representative cell death images in SC and KDM6B KO2 HeLa cells 72 h after treatment with MNNG and/or BG (B). PI-positive cells are quantified in C (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Tukey's multiple comparisons test. ns, not significant. ( D ) Immunoblot analysis of MGMT in SC, KDM6B KO2, MGMT KO, and KDM6B/MGMT DKO HeLa cells 4 h after MNNG treatment. Numbers indicate the signal intensity. ( E and F ) Representative cell images in SC, KDM6B KO2, MGMT KO, KDM6B/MGMT DKO HeLa cells 24 h after MNNG treatment (E). Cell death is quantified in F (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( G, H ) Representative images (G) and quantification (H) of AIF nuclear translocation in SC and KDM6B KO cells in the presence or absence of BG (200 μM, pretreatment 24 h) and PARP inhibitor Olaparib (10 μM, 30 min pretreatment) at 4 h post MNNG (50 μM, 15 min) treatment. Red, AIF staining; Blue, DAPI staining; Purple, overlay of AIF and DAPI in the nuclei (mean ± SEM, n = 5). **** P < 0.0001 by two-way ANOVA Tukey's multiple comparisons test. Scale bar, 10 μm.

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: Pharmacological inhibition or deletion of MGMT sensitizes KDM6BKO cells to alkylating agents. ( A ) Immunoblot analysis of MGMT in SC and KDM6B KO2 HeLa cells 6 h after the treatment of MNNG (50 μM, 15 min) and/or BG (200 μM). ( B and C ) Representative cell death images in SC and KDM6B KO2 HeLa cells 72 h after treatment with MNNG and/or BG (B). PI-positive cells are quantified in C (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Tukey's multiple comparisons test. ns, not significant. ( D ) Immunoblot analysis of MGMT in SC, KDM6B KO2, MGMT KO, and KDM6B/MGMT DKO HeLa cells 4 h after MNNG treatment. Numbers indicate the signal intensity. ( E and F ) Representative cell images in SC, KDM6B KO2, MGMT KO, KDM6B/MGMT DKO HeLa cells 24 h after MNNG treatment (E). Cell death is quantified in F (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( G, H ) Representative images (G) and quantification (H) of AIF nuclear translocation in SC and KDM6B KO cells in the presence or absence of BG (200 μM, pretreatment 24 h) and PARP inhibitor Olaparib (10 μM, 30 min pretreatment) at 4 h post MNNG (50 μM, 15 min) treatment. Red, AIF staining; Blue, DAPI staining; Purple, overlay of AIF and DAPI in the nuclei (mean ± SEM, n = 5). **** P < 0.0001 by two-way ANOVA Tukey's multiple comparisons test. Scale bar, 10 μm.

Article Snippet: The membrane was blocked with 5% milk and incubated overnight with primary antibodies: anti-Flag antibody (Sigma, F3165), anti-MGMT antibody (Proteintech, 17195-1-AP), anti-PAR antibody (Trevigen, 4336-BPC-100), anti-PARP-1 antibody (Proteintech, 22999-1-AP), anti-γH2AX antibody (Millipore, 05-636), anti-H2AX antibody (Proteintech, 10856-1-AP), anti-phospho-Chk1 (Ser345) antibody (Cell Signaling Technology, 2348), anti-phospho-RPA32 (Ser33) (Fisher, 50-155-7198), anti-RPA70 (Santa Cruz, SC166023), anti-phospho-ATR (Thr1989) antibody (Cell Signaling Technology, 30632S), anti-ATR antibody (Cell Signaling Technology, 13934S), anti-phospho-ATM (Ser1981) (D6H9) (Cell Signaling Technology, 5883S), anti-ATM (Cell Signaling Technology, 92356S), anti-phospho-Chk1 (Ser296) antibody (Cell Signaling Technology, 2349S), anti-Chk1 antibody (Santa Cruz, sc-8408), anti-phospho-Chk2 (Thr68) (C13C1) antibody (Cell Signaling Technology, 2197S), anti-phospho-Chk2 (D9C6) antibody (Cell Signaling Technology, 6334S), anti-PP2A antibody (Proteintech, 10321-1-AP), anti-H3K27me3 (Cell Signaling Technology, 9733S), anti-XRCC1 antibody (Proteintech, 21468-1-AP), anti-MSH2 antibody (25D12) (Santa Cruz, sc-56163), anti-MSH6 antibody (F-1) (Santa Cruz, sc-271979), or anti-actin antibody (Proteintech, 66009-1-AP) at 4°C, followed by donkey anti-mouse or goat anti-rabbit IgG conjugated to HRP for 1 h at room temperature.

Techniques: Inhibition, Western Blot, Translocation Assay, Staining

MGMT regulates O 6 MeG levels and O 6 MeG-triggered PARP-1 activation. (A and B) Representative images of O 6 MeG staining ( A ) and quantification ( B ) in SC, KDM6B-KO2, MGMT (MT) KO, KDM6B/MGMT DKO cells with or without MGMT inhibitor BG (200 μM) or PARP inhibitor Olaparib (10 μM) at 0 min, 2 h and 6 h after MNNG treatment (25 μM, 15 min). * P < 0.05, *** P < 0.001, **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. Scale bar, 20 μm. ( C ) Immunoblot analysis of PARP-1 activation and γH2AX in SC and MGMT KO HeLa cells treated with vehicle (−) or MNNG (25 μM, 15 min) for indicated time. ( D ) Immunoblot analysis of PARP-1 activation and γH2AX in SC, KDM6B KO2, MGMT KO, KDM6B/MGMT DKO HeLa cells treated with vehicle (−) or MNNG (50 μM, 15 min) for indicated time. Numbers on the blot indicate the relative signal intensity.

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: MGMT regulates O 6 MeG levels and O 6 MeG-triggered PARP-1 activation. (A and B) Representative images of O 6 MeG staining ( A ) and quantification ( B ) in SC, KDM6B-KO2, MGMT (MT) KO, KDM6B/MGMT DKO cells with or without MGMT inhibitor BG (200 μM) or PARP inhibitor Olaparib (10 μM) at 0 min, 2 h and 6 h after MNNG treatment (25 μM, 15 min). * P < 0.05, *** P < 0.001, **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. Scale bar, 20 μm. ( C ) Immunoblot analysis of PARP-1 activation and γH2AX in SC and MGMT KO HeLa cells treated with vehicle (−) or MNNG (25 μM, 15 min) for indicated time. ( D ) Immunoblot analysis of PARP-1 activation and γH2AX in SC, KDM6B KO2, MGMT KO, KDM6B/MGMT DKO HeLa cells treated with vehicle (−) or MNNG (50 μM, 15 min) for indicated time. Numbers on the blot indicate the relative signal intensity.

Article Snippet: The membrane was blocked with 5% milk and incubated overnight with primary antibodies: anti-Flag antibody (Sigma, F3165), anti-MGMT antibody (Proteintech, 17195-1-AP), anti-PAR antibody (Trevigen, 4336-BPC-100), anti-PARP-1 antibody (Proteintech, 22999-1-AP), anti-γH2AX antibody (Millipore, 05-636), anti-H2AX antibody (Proteintech, 10856-1-AP), anti-phospho-Chk1 (Ser345) antibody (Cell Signaling Technology, 2348), anti-phospho-RPA32 (Ser33) (Fisher, 50-155-7198), anti-RPA70 (Santa Cruz, SC166023), anti-phospho-ATR (Thr1989) antibody (Cell Signaling Technology, 30632S), anti-ATR antibody (Cell Signaling Technology, 13934S), anti-phospho-ATM (Ser1981) (D6H9) (Cell Signaling Technology, 5883S), anti-ATM (Cell Signaling Technology, 92356S), anti-phospho-Chk1 (Ser296) antibody (Cell Signaling Technology, 2349S), anti-Chk1 antibody (Santa Cruz, sc-8408), anti-phospho-Chk2 (Thr68) (C13C1) antibody (Cell Signaling Technology, 2197S), anti-phospho-Chk2 (D9C6) antibody (Cell Signaling Technology, 6334S), anti-PP2A antibody (Proteintech, 10321-1-AP), anti-H3K27me3 (Cell Signaling Technology, 9733S), anti-XRCC1 antibody (Proteintech, 21468-1-AP), anti-MSH2 antibody (25D12) (Santa Cruz, sc-56163), anti-MSH6 antibody (F-1) (Santa Cruz, sc-271979), or anti-actin antibody (Proteintech, 66009-1-AP) at 4°C, followed by donkey anti-mouse or goat anti-rabbit IgG conjugated to HRP for 1 h at room temperature.

Techniques: Activation Assay, Staining, Western Blot