catalytic dead mutant  (Addgene inc)

Journal: iScience

Article Title: Targeting histone methylation to reprogram the transcriptional state that drives survival of drug-tolerant myeloid leukemia persisters

doi: 10.1016/j.isci.2022.105013

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Article Snippet: MSCV JMJD3 mutant (KDM6B-H1390A) , Addgene , Cat#21214.

Techniques: Recombinant, Modification, Protease Inhibitor, Gene Expression, Western Blot, Reverse Transcription, DNA Library Preparation, Control, Sequencing, Mutagenesis, Software

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: Identification of KDM6B as a key regulator of PARP-1-dependent cell death. ( A ) The scheme of the CRISPR screening. ( B ) Top 18 hits including KDM6B were identified from the screening. Red, >105 000 reads. Pink, > 200 reads. Purple, >10 reads. Gray, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$ \ge$\end{document} 1 read. ( C ) Genotyping of KDM6B KO2 HeLa cells. ( D ) Immunoblot analysis of scrambled control (SC), KDM6B KO, and C-terminal WT-KDM6B (WT-C) as well as its H1390A mutant (mut-C) rescue HeLa cells. Numbers indicate the signal intensity. ( E and F ) Representative cell death images in SC, KDM6B KO2, and rescued HeLa cells 24 h after the treatment with DMSO or MNNG (50 μM, 15 min) (E). PI-positive cells are quantified in (F) (mean ± SEM, n = 4–7). Scale bar, 20 μm. PI/H, propidium iodide/Hoechst staining. TL, transmission light. **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( G and H ) Representative colony survival in SC, KDM6B KO2, and rescued HeLa cells treated with vehicle, MNNG (2 μM), TMZ (250 μM), Cyclophosphamide (CP, 500 μM), and Carmustine (25 μM) for 10 days (G). Colony numbers are quantified in H (mean ± SEM, n = 2–12). * P < 0.05; *** P < 0.001; **** P < 0.0001 versus DMSO by two-way ANOVA Tukey's multiple comparisons test.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: CRISPR, Western Blot, Control, Mutagenesis, Staining, Transmission Assay

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: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

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

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: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Inhibition, Western Blot, Translocation Assay, Staining

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: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Activation Assay, Staining, Western Blot

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: Cell cycle distribution in SC and KDM6B KO cells following alkylating agent MNNG treatment. Cells were treated with 50 μM MNNG for 15 min, stained with EdU/PI, and analyzed by flow cytometry at 2, 4, 6, 8, 15 and 22 h post the treatment. ( A ) Representative flow cytometry images. ( B ) Cell cycle distribution was quantified (mean ± SEM, n = 3). **** P < 0.0001 vs. identical cell cycle in DMSO group, # P < 0.0001 versus S phase in KDM6B KO cells at the identical timepoint, by two-way ANOVA Tukey's multiple comparisons test.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Staining, Flow Cytometry

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 KO promotes sustained Chk1 activation for DNA repair following alkylating agent treatment. ( A ) Immunoblot analysis of checkpoint response and DNA damage in SC and KDM6B KO2 HeLa cells 0–6 h after MNNG treatment. ( B and C ) Effects of MGMT and MGMT C145A mutant overexpression (B) and MGMT KO (C) on checkpoint response in SC and KDM6B KO HeLa cells 1 h and 4 h after MNNG treatment. Asterisk indicates endogenous MGMT and the top band in the MGMT blot designates ectopic MGMT (B). ( D ) Effects of checkpoint inhibition by GDC0575 (50 nM) on checkpoint response and DNA damage in SC, KDM6B KO2, and KDM6B/MGMT DKO HeLa cells 1 h and 6 h after MNNG treatment. ( E and F ) Effects of checkpoint inhibition by GDC0575 (50 nM) on MNNG-induced cell death in SC, KDM6B KO2, and KDM6B/MGMT DKO HeLa cells 24 h after MNNG treatment. Representative cell death images are in SC, KDM6B KO2, 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 Tukey's multiple comparisons test. ( G ) Immunoblot analysis of checkpoint response in SC and KDM6B KO2 HeLa cells 0–6 h after hydroxyurea (HU, 2 mM) treatment.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Activation Assay, Western Blot, Mutagenesis, Over Expression, Inhibition

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: XRCC1 regulates MNNG-induced DNA damage and cell death. ( A ) Immunoblot analysis of XRCC1 overexpression (OE) and DNA damage in HeLa cells 4 h after MNNG treatment (50 μM, 15 min). SC, scrambled control. ( B ) Immunoblot analysis of XRCC1 KO (mixed clones) and DNA damage in HeLa cells 4 h after MNNG treatment. ( C ) Immunoblot analysis of XRCC1 expression and DNA damage in SC, KDM6B KO and KDM6B/XRCC1 (6B/XR) DKO (mixed clones) HeLa cells 4 h after MNNG treatment. ( D and E ) Effects of XRCC1 on MNNG-induced cell death in SC, XRCC1 OE, XRCC1 KO, KDM6B KO, and KDM6B/XRCC1 (6B/XR) DKO HeLa cells 24 or 72 h after MNNG treatment. Representative cell death images are shown in (D). Cell death is quantified in (E) (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Western Blot, Over Expression, Control, Clone Assay, Expressing

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 is a key cell death regulator of PARthanatos induced by alkylating agents. KDM6B regulates two sequential DNA repair processes including MGMT direct DNA repair and checkpoint associated repair. Loss of KDM6B on one hand enhances MGMT direct DNA repair to promote cell survival, on the other hand triggers sustained Chk1 activation and increases checkpoint associated DNA repair to fix DNA evading from the first step MGMT repair process leading to cell survival. High levels of KDM6B suppress both DNA repair processes leading to cell death. Blocking MGMT and checkpoint associated repair re-sensitizes cancer cells to alkylating agents.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Activation Assay, Blocking Assay

Journal: Genes & Development

Article Title: UTX inhibition as selective epigenetic therapy against TAL1-driven T-cell acute lymphoblastic leukemia

doi: 10.1101/gad.276790.115

Figure Lengend Snippet: The H3K27 demethylase inhibitor GSK-J4 represses TAL1/UTX target genes and selectively kills TAL1-positive T-ALL cells through UTX inhibition. ( A , B ) TAL1-positive T-ALL cells are more sensitive to GSK-J4 treatment. ( A ) Dose-dependent effect of GSK-J4 on the viability of primary blasts from T-ALL patients. Data are presented as mean values of the percentage of CD7-positive Annexin V-negative cells ±SEM. n = 3. The reduction of apoptosis in TAL1-positive T-ALL is statistically significant compared with TAL1-negative T-ALL (e.g., at 5 µM GSK-J4, P -value = 0.016; at 10 µM GSK-J4, P -value = 8.8 × 10 −3 by t -test [unpaired, two-tail, nonequal variance]). ( B ) GSK-J4 (5 µM) decreases growth ( top panel) and increases apoptosis ( bottom panel) of primary blasts from TAL1-positive T-ALL patients. ( Top panel) Viable cell numbers after GSK-J4 treatment relative to viable cell numbers after vehicle control treatment are reported as mean values ± SEM. n = 4. ( Bottom panel) Apoptosis is reported as the percentage of Annexin V-positive cells. Data are shown as mean ± SEM. n = 4. (*) P < 0.05; (***) P < 0.001; (ns) not significant. Representative FACS plots are shown at the right . ( C , D ) In TAL1-positive T-ALL, GSK-J4 acts predominantly through UTX inhibition. ( C ) Correlation of changes in gene expression between UTX knockdown (shUTX) and GSK-J4 treatment. RNA-seq was performed with pairs of duplicates for (1) control and UTX knockdown Jurkat cells and (2) vehicle-treated and GSK-J4-treated Jurkat cells. ( D ) Overexpression (OE) of UTX, but not JMJD3, rescues GSK-J4-mediated apoptosis in TAL1-positive T-ALL. Overexpression of UTX (wild type or enzymatically dead mutant) or JMJD3 (wild type or enzymatically dead mutant) was induced in TAL1-positive Jurkat cells ( top ) or TAL1-negative DND41 cells ( bottom ) followed by treatment with GSK-J4 or a vehicle control. ( Left panels) Western blot analyses of UTX and JMJD3 overexpression. Representative examples of three biological replicates are shown. ( Right panels) The percentage of apoptotic cells (Annexin V-positive) after GSK-J4 treatment is reported relative to the percentage of apoptotic cells after treatment with vehicle control. Data are shown as mean ± SEM. n = 4. ( E ) GSK-J4 treatment increases the repressive histone mark H3K27me3 globally (as measured by Western blot; representative example of three biological replicates) ( left panel) and on specific TAL1/UTX target genes (as measured by ChIP-qPCR) ( right panel). Mean values are presented as a fraction of input ± SD. n = 3. (**) P < 0.01; (***) P < 0.001). ( F ) GSK-J4 treatment leads to down-regulation of the TAL1–UTX transcriptional regulatory network. ( Left panel) Volcano plot showing genome-wide changes in gene expression upon GSK-J4 treatment in Jurkat cells as measured by RNA-seq. Adjusted P -value (−log 10 ) versus fold change (log 2 ). The entire set of TAL1/UTX target genes (defined as TAL1/UTX-cobound genes that are significantly down-regulated upon TAL1 knockdown and UTX knockdown) are highlighted in red, with gene names shown for representative examples. ( Right panel) Validation of RNA-seq by qRT–PCR for a subset of genes. Results are reported as mean values relative to the internal control ß 2M ±SD. n = 3. (**) P < 0.01.

Article Snippet: Retroviral particles expressing JMJD3 (Addgene plasmid no. 21212) or an enzymatically dead JMJD3 mutant (H1390A) (Addgene plasmid no. 21214) ( ) were generated in 293-GP cells using the Retro-X universal packaging system with the Ampho envelope protein vector (Clontech).

Techniques: Inhibition, Control, Gene Expression, Knockdown, RNA Sequencing, Over Expression, Mutagenesis, Western Blot, ChIP-qPCR, Genome Wide, Biomarker Discovery, Quantitative RT-PCR