Journal: Nucleic Acids Research

Article Title: Acetylation of MORC2 by NAT10 regulates cell-cycle checkpoint control and resistance to DNA-damaging chemotherapy and radiotherapy in breast cancer

doi: 10.1093/nar/gkaa130

Figure Lengend Snippet: NAT10 acetylates MORC2 at K767. ( A , B ) Cells were treated with 5 μM TSA and 5 mM NAM for 6 h. Lysates were subjected to IP assays with control IgG, an anti-MORC2 (A) or anti-Ac-K (B) antibody, followed by immunoblotting analysis with the indicated antibodies. ( C ) Cells were treated with or without 5 mM NAM or 5 μM TSA alone or in combination for 6 h and subjected to IP and immunoblotting analysis with the indicated antibodies. MORC2 acetylation levels were normalized to those of total MORC2 protein. ( D ) Analysis of MORC2 acetylation sites in publicly available databases. ( E , F ) HEK293T cells stably expressing pCDH, Flag-MORC2, and Flag-MORC2 K767R were treated or without NAM at the indicated concentrations for 6 h (E) or 5 mM NAM for the indicated times (F). IP and immunoblotting analyses were performed with the indicated antibodies. ( G ) Alignment of MORC2 protein sequence across different species. ( H ) HEK293T cells stably expressing pCDH and Flag-MORC2 (WT, K767R and K767Q) were treated with or without 5 mM NAM for 6 h and subjected to IP and immunoblotting analyses with the indicated antibodies. ( I ) MCF-7 and BT549 cells were treated with or without 5 mM NAM for 6 h and subjected to IP and immunoblotting analyses with the indicated antibodies. MORC2 K767Ac levels were normalized to those of total MORC2 protein. ( J ) MCF-7 and BT549 cells were transfected with pCDH, HA-NAT10, or HA-NAT10 G641E. After 48 h of transfection, lysates were subjected to IP and immunoblotting analysis. ( K–M ) HEK293T cells stably expressing pCDH and Flag-MORC2 (K), MCF-7 (L), or BT549 (M) cells were transfected with negative control siRNA (siNC) or two siRNAs targeting NAT10 (siNAT10). After 48 h of transfection, cells with treated with or without 5 mM NAM for 6 h and subjected to IP and immunoblotting analysis. In L, cells were pretreated with or without 5 μM Remodelin for 3 h prior to NAM treatment. ( N ) Purified His-MORC2 was incubated with or without purified NAT10, 2 mM acetyl-CoA in reaction buffer at 37°C for 1 h. MORC2 K767Ac was detected by immunoblotting. His-MORC2 was visualized by Coomassie blue staining. ( O ) Purified His-MORC2 (WT and K767R) were incubated with or without purified NAT10, 2 mM acetyl-CoA in reaction buffer at 37°C for 1 h. MORC2 K767Ac was detected by immunoblotting. His-MORC2 was visualized by Coomassie blue staining.

Article Snippet: Myc-DDK-tagged MORC2 (#RC200518) and GFP-tagged NAT10 (#RG207082) cDNAs were obtained from Origene.

Techniques: Western Blot, Stable Transfection, Expressing, Sequencing, Transfection, Negative Control, Purification, Incubation, Staining

Journal: Nucleic Acids Research

Article Title: Acetylation of MORC2 by NAT10 regulates cell-cycle checkpoint control and resistance to DNA-damaging chemotherapy and radiotherapy in breast cancer

doi: 10.1093/nar/gkaa130

Figure Lengend Snippet: DNA-damaging agents stimulate MORC2 K767Ac in a NAT10-dependent manner. ( A ) MCF-7 and BT549 cells were treated with or without 1 mM MMS, 1 mM H 2 O 2 , 1 μM ADR, 100 μM CDDP or 6 Gy IR. After 2 h of treatment, lysates were subjected to IP and immunoblotting analyses with the indicated antibodies. MORC2 K767Ac levels were normalized to those of total MORC2 protein. ( B , C ) HEK293T cells were treated with or without 1mM MMS (B) or 6 Gy IR (C). After 2 h of treatment, cells were stained with MORC2 K767Ac antibody (green) and γH2AX (red). DNA was counterstained with DAPI (blue). For peptide blocking assays, K767 acetylated peptide (final concentration: 0.1 μg/μL) was added into the diluted K767Ac antibody. Quantitative results for K767Ac- and γH2AX-positively stained cells are shown in the right panel. ***, P < 0.01, **, P < 0.01. Scale bar, 25 μm. ( D , F ) Cells were transfected with siNC or two siNAT10s. After 48 h of transfection, cells were treated with or without 1mM MMS or 6 Gy IR for 2 h and harvested for IP and immunoblotting analysis with the indicated antibodies. ( G ) WT and SIRT2 KO MCF-7 cells were treated with or without 1 mM MMS or 6 Gy IR for 2 h and harvested for IP and immunoblotting analysis with the indicated antibodies. MORC2 K767Ac levels were normalized to those of total MORC2 protein. ( H–M ) MCF-7 and BT549 cells were treated with or without 1mM MMS or 6 Gy IR for 2 h. IP and immunoblotting analysis was conducted with the indicated antibodies. ( N ) MCF-7 cells were transfected with HA-NAT10 and Flag-MORC2. After 48 h of transfection, cells were treated with or without 1 mM MMS or 6 Gy IR for 2 h and stained with anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Typical co-location between NAT10 and MORC2 was indicated by arrows. Quantitative results for cells with NAT10 nucleoplasm translocation are shown in the right panel. *** P < 0.01, ** P < 0.01. Scale bar, 2.5 μm.

Article Snippet: Myc-DDK-tagged MORC2 (#RC200518) and GFP-tagged NAT10 (#RG207082) cDNAs were obtained from Origene.

Techniques: Western Blot, Staining, Blocking Assay, Concentration Assay, Transfection, Translocation Assay

Journal: Nucleic Acids Research

Article Title: Acetylation of MORC2 by NAT10 regulates cell-cycle checkpoint control and resistance to DNA-damaging chemotherapy and radiotherapy in breast cancer

doi: 10.1093/nar/gkaa130

Figure Lengend Snippet: MORC2 K767Ac binds to H3T11P. ( A ) HEK293T cells stably expressing Flag-MORC2 were pretreated with DMSO or 5 mM NAM for 6 h and then incubated with 100 μg/ml CHX for the indicated times. Immunoblotting analysis was performed with the indicated antibodies. Relative MORC2 levels (MORC2/Vinculin) are shown in lower panels. ( B ) HEK293T cells stably expressing Flag-MORC2 or Flag-MORC2 K767R were pretreated with 5 mM NAM for 6 h and then incubated with 100 μg/ml CHX for the indicated times. Immunoblotting analysis was performed with the indicated antibodies. Relative MORC2 levels (MORC2/Vinculin) are shown in lower panels. ( C ) HEK293T cells stably expressing Flag-MORC2 or Flag-MORC2 K767R were treated with or without 1 mM MMS for 2 h and then stained with an anti-Flag (green) or an anti-γH2AX antibody. DNA was counterstained with DAPI (blue). Quantitative results for MORC2- and γH2AX-positively stained cells are shown in right panel ( n = 100). *** P < 0.001, ** P < 0.01. Scale bar, 2.5 μm. ( D ) Histone binding assays were performed using the MODified Histone Peptide Arrays (Active motif) and purified Flag-MORC2 K767R and Flag-MORC2 K767Q proteins from HEK293T cells according to the manufacturer's instructions. Signals were deleted by ECL visualization and analyzed by Array Analysis Software. The results were quantitated according to specificity factor (right panel). ( E ) HET293T cells were transfected with the indicated expression vectors. After 48 h of transfection, lysates were subjected to IP analysis with anti-Flag antibody, followed by immunoblotting analysis. ( F ) HEK293T cells were transfected with pCDH, Flag-MORC2, Flag-MORC K767R, and Flag-MORC K767Q. After 48 h of transfection, lysates were incubated with Biotin-H3 or Blotin-H3T11P peptides and then subjected to pull-down assays with Fag-beads or Streptavidin-beads, followed by immunoblotting analysis. ( G ) His, His-MORC2 WT, and His-MORC2 K767Q were purified from E. coli strain BL21 (DE3) and incubated with Biotin-H3 or Biotin-H3T11P peptides. The mixture was subjected to pull-down with Flag-beads or Streptavidin beads, followed by immunoblotting analysis. ( H ) His-MORC2 WT and His-MORC2 K767R were pre-incubated with NAT10 in HAT buffer to be acetylated and then incubated with Biotin-H3 or Biotin-H3T11P peptides, followed by pull-down and immunoblotting analysis.

Article Snippet: Myc-DDK-tagged MORC2 (#RC200518) and GFP-tagged NAT10 (#RG207082) cDNAs were obtained from Origene.

Techniques: Stable Transfection, Expressing, Incubation, Western Blot, Staining, Binding Assay, Modification, Purification, Software, Transfection

Journal: Nucleic Acids Research

Article Title: Acetylation of MORC2 by NAT10 regulates cell-cycle checkpoint control and resistance to DNA-damaging chemotherapy and radiotherapy in breast cancer

doi: 10.1093/nar/gkaa130

Figure Lengend Snippet: NAT10 regulates H3T11P, CDK1, and Cyclin B1 expression through MORC2 K767Ac. ( A–D ) MCF-7 cells were transfected with siNC or two siNAT10s. After 48 h of transfection, cells were treated with or without 1 mM MMS or 6 Gy IR for 2 h and then subjected to immunoblotting (A-B) and qPCR analysis (C-D). ( E–H ) MCF-7 cells were pretreated with or without 5 μM Remodelin for 3 h, followed by treatment with or without 1 mM MMS or 6 Gy IR for another 2 h. Immunoblotting (E, F) and qPCR analyses (G-H) were performed as indicated. ( I–L ) MORC2 KO MCF-7 cells stably expressing Flag-MORC2 and Flag-MORC2 K767Q were transfected with siNC or two siNAT10s. After 48 h of transfection, cells were treated with or without 1mM MMS or 6 Gy IR for 2 h and then subjected to immunoblotting (I and J) and qPCR analysis (K and L). ( M–P ) MORC2 KO MCF-7 cells expressing Flag-MORC2 and Flag-MORC2 K767Q were pretreated with or without 5 μM Remodelin for 3 h, followed by treatment with or without 1 mM MMS or 6 Gy IR for another 2 h. Immunoblotting (M and N) and qPCR analyses (O and P) were performed as indicated.

Article Snippet: Myc-DDK-tagged MORC2 (#RC200518) and GFP-tagged NAT10 (#RG207082) cDNAs were obtained from Origene.

Techniques: Expressing, Transfection, Western Blot, Stable Transfection

Journal: Nucleic Acids Research

Article Title: Acetylation of MORC2 by NAT10 regulates cell-cycle checkpoint control and resistance to DNA-damaging chemotherapy and radiotherapy in breast cancer

doi: 10.1093/nar/gkaa130

Figure Lengend Snippet: NAT10 is essential for cell survival in response to MMS and IR treatment. ( A–C ) WT and NAT10 KO MCF-7 and BT549 stable cells were treated with or without increasing doses of MMS or IR and subjected to colony formation survival assays. Representative images of survival colonies are shown A, and corresponding quantitative results are shown B and C. ( D and E ) MCF-7 and BT549 cells were transfected with siNC or two siNAT10s. After 24 h of transfection, cells were treated with increasing doses of MMS (D) or IR (E). After 48 h of treatment, cells were subjected to CCK-8 assays. ( F–H ) MCF-7 and BT549 cells were treated with increasing doses of MMS or IR and subjected to colony formation survival assays. DMSO or 5 μM Remodelin was added to culture medium. Representative images of survival colonies are shown in F, and corresponding quantitative results of survival colonies are shown in G and H. ( I and J ) MCF-7 and BT549 cells were treated with increasing doses of MMS or IR. After 48 h of treatment, cells were subjected to CCK-8 assays. DMSO or 5 μM Remodelin was added to culture medium.

Article Snippet: Myc-DDK-tagged MORC2 (#RC200518) and GFP-tagged NAT10 (#RG207082) cDNAs were obtained from Origene.

Techniques: Transfection, CCK-8 Assay

Journal: Nucleic Acids Research

Article Title: Acetylation of MORC2 by NAT10 regulates cell-cycle checkpoint control and resistance to DNA-damaging chemotherapy and radiotherapy in breast cancer

doi: 10.1093/nar/gkaa130

Figure Lengend Snippet: NAT10 inhibitor Remodelin enhances the sensitivity of cells expressing WT MORC2, but not K767R mutant MORC2, to MMS and IR. ( A–D ) MORC2 KO MCF-7 and BT549 cells stably expressing Flag-MORC2 or Flag-MORC2 K767R were treated with increasing doses of MMS (A) or IR (B) and subjected to colony formation survival assays. DMSO or 5 μM Remodelin was added to culture medium. Representative images of survival colonies are shown in A and B, and corresponding quantitative results are shown in C and D.

Article Snippet: Myc-DDK-tagged MORC2 (#RC200518) and GFP-tagged NAT10 (#RG207082) cDNAs were obtained from Origene.

Techniques: Expressing, Mutagenesis, Stable Transfection

Journal: Nucleic Acids Research

Article Title: Acetylation of MORC2 by NAT10 regulates cell-cycle checkpoint control and resistance to DNA-damaging chemotherapy and radiotherapy in breast cancer

doi: 10.1093/nar/gkaa130

Figure Lengend Snippet: MORC2 K767 is positively associated with NAT10 expression levels in clinical breast tumor samples. ( A ) Lysates from 16 pairs of human breast tumor tissues (T) and adjacent noncancerous normal tissues (N) were subjected to immunoblotting analysis with the indicated antibodies. The expression levels of NAT10, MORC2, and MORC2 K767Ac were normalized to those of Vinculin. ( B , C ) Relative expression levels of NAT10 (B) and MORC2 K767Ac (C) in normal breast and breast tumor tissues. The expression levels of MORC2 K767Ac were normalized to those of total MORC2. ( D ) Correlation analysis of expression levels between NAT10 and MORC2 K767Ac. ( E ) IHC staining of NAT10 and MORC2 K767Ac in 128 human breast tumor specimens. Representative images are shown. Scale bars, 50 μm. ( F ) The staining score of MORC2 K767Ac in breast tumor samples correlates with that of NAT10. *** P < 0.001. ( G ) Correction analysis of NAT10 and MORC2 K767Ac expression levels in 128 breast tumor tissues. Pearson correlation test was used. ( H ) The proposed working model. Under unstressed conditions, MORC2 is acetylated by NAT10 and deacetylated by SIRT2 at K767. DNA damage induced by chemotherapeutic drugs and ionizing radation promotes the translocation of NAT10 from the nucleous to the nucleoplasm, resulting in enhanced interaction between MORC2 and NAT10 and subsequent MORC2 K767Ac. MORC2 K767Ac mediates DNA damage-induced reduction of H3T11P and transcriptional repression of its downstream target genes CDK1 and Cyclin B1 , thus contributing to DNA damage-induced G2 checkpoint activation and facilitating cell survival.

Article Snippet: Myc-DDK-tagged MORC2 (#RC200518) and GFP-tagged NAT10 (#RG207082) cDNAs were obtained from Origene.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining, Translocation Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Sequential hydrolysis of FAD by ecto-5′ nucleotidase CD73 and alkaline phosphatase is required for uptake of vitamin B 2 into cells

doi: 10.1016/j.jbc.2022.102640

Figure Lengend Snippet: Flavin-adenine dinucleotide ( FMN ) hydrolysis by human bone alkaline phosphatase (ALP) and flavin-adenine dinucleotide ( FAD ) hydrolysis by recombinant human CD73 . A , time course of riboflavin (RF) production from FMN by ALP. The RF concentration was measured after incubation of 10 μM FMN with ALP (10 μg/ml). Data represent means ± SD ( n = 3). B , ALP concentration dependence of RF production from FMN. The RF concentration was measured 5 min after incubation of 10 μM FMN with ALP (2.5–10 μg/ml). Data represent means ± SD ( n = 3). C , Michaelis–Menten plot of RF production from FMN by human bone ALP. The RF concentration was measured 5 min after incubation of FMN (0.5–5 μM) with ALP (5 μg/ml). The enzyme activity was expressed in product concentration per minute per mg protein. Data represent means ± SD ( n = 3). D and G , time course of FMN production ( D ) or adenosine production ( G ) from FAD by CD73. The FMN or adenosine concentration was measured after incubation of 10 μM FAD with CD73 (10 ng/ml). Data represent means ± SD ( n = 3). E and H , CD73 concentration dependence of FMN production ( E ) or adenosine production ( H ) from FAD by recombinant CD73. The FMN or adenosine concentration was measured 15 min after incubation of 10 μM FAD with CD73 (5–20 ng/ml). Data represent means ± SD ( n = 3). F and I , Michaelis-Menten plot of FMN production ( F ) or adenosine production ( I ) from FAD by recombinant CD73. The FMN or adenosine concentration was measured 15 min after incubation of 10 μM FAD with CD73 (10 ng/ml). The enzyme activity was expressed in product concentration per minute per μg protein. Data represent means ± SD ( n = 3). When the SD is smaller than the symbols, it is not shown.

Article Snippet: Human bone ALP was purchased from Calzyme.

Techniques: Recombinant, Concentration Assay, Incubation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Sequential hydrolysis of FAD by ecto-5′ nucleotidase CD73 and alkaline phosphatase is required for uptake of vitamin B 2 into cells

doi: 10.1016/j.jbc.2022.102640

Figure Lengend Snippet: Kinetic parameters for hydrolysis reactions catalyzed by human bone ALP and recombinant human CD73

Article Snippet: Human bone ALP was purchased from Calzyme.

Techniques: Recombinant