Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Elevated NAT10 expression correlates with poor prognosis in HCC patients. (A) Dot blot analyses of total RNA (5 µg) isolated from HCC tissues and adjacent noncancerous liver tissues using an anti‐ac4C antibody, with MB staining as loading control (left panel). Calculation of relative ac4C contents on RNA in HCC tissues and adjacent noncancerous liver tissues (right panel, N = 20). (B) Detection of ac4C levels on mRNA in the same 20 HCC tissues and adjacent noncancerous liver tissues by UPLC‐MS/MS analysis. (C) Large‐scale data mining was used to compare the expression differences in NAT10 mRNA between HCC tissues and adjacent noncancerous liver tissues. (D) Analysis of NAT10 protein levels in HCC and adjacent noncancerous liver tissues ( N = 165) using CPTAC data. (E) NAT10 protein levels in HCC tissues and adjacent noncancerous liver tissues measured by western blotting ( N = 8). (F) Representative IHC images of NAT10 expression in the Tongji cohort tissue microarray (TMA) including HCC tissues and adjacent noncancerous liver tissues (left panel); pie chart showing percentages of cases with differential NAT10 expression profiles compared to adjacent noncancerous liver tissues ( N = 103, right panel, scale bar: 200 µm or 20 µm). (G) Kaplan‐Meier plot correlating NAT10 expression with patient overall and recurrence‐free survival using CPTAC, TCGA, and Tongji datasets. (H) Analysis of NAT10 expression correlation with malignant features of HCC using Tongji TMA cohort and TCGA LIHC datasets. Statistical tests: (A, B, D) paired t test; (G) log‐rank test; (H) Pearson chi‐squared test (2‐sided). Abbreviations: ANL, adjacent noncancerous liver tissue; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; LIHC, Liver hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; CPTAC, Clinical Proteomic Tumor Analysis Consortium; MB, methylene blue; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; mRNA, messenger RNA.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Expressing, Dot Blot, Isolation, Staining, Control, Tandem Mass Spectroscopy, Western Blot, Microarray, Immunohistochemistry

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: NAT10 knockdown inhibits HCC progression in vitro and in vivo. (A) Western blot analysis of NAT10 expression post NAT10 knockdown (upper panel). Dot blot analysis of ac4C levels in total RNA and mRNA isolated from control or NAT10‐knockdown HCC cells (lower panel, MHCC‐97H and SNU449), with MB staining as loading control. (B) and (C) CCK8 assay for cell viability of NAT10‐knockdown and control cells at indicated time points. (D) Colony formation assay quantification for indicated cells. (E) Soft agar colony formation assay quantification for indicated cells. (F) Scratch wound healing assays quantification for indicated cells. (G) Cell migration and invasion assays quantification for MHCC‐97H and SNU449 cells. (H) Subcutaneous xenograft models with NAT10‐knockdown and control cells ( n = 5, scale bar: 1 cm). Tumor volume monitored and growth curves generated over 24 days (left panel); tumors weighed (right panel). (I) and (J) Representative images and quantitative data analysis of Ki67‐positive (I) and TUNEL‐positive (J) cells, (scale bar: 20 µm). (K) Representative bioluminescence imaging of liver orthotopic implantation models (left panel) and quantification of intrahepatic metastatic nodules (right panel) ( n = 5). (L) Representative bioluminescence imaging of lung metastasis models (left panel) and quantification of lung metastatic nodules (right panel) ( n = 5). (B‐G) Data shown as mean ± SD, n = 3. Statistical analysis: Unpaired t tests. ** P < 0.01, *** P < 0.001. Abbreviations: MB, methylene blue; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling; CCK‐8, Cell Counting Kit‐8; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Expressing, Dot Blot, Isolation, Control, Staining, CCK-8 Assay, Colony Assay, Soft Agar Assay, Migration, Generated, TUNEL Assay, Imaging, End Labeling, Cell Counting, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Effects of nat10 on ac4c mRNA modification and global mRNA translation. (A) Flow chart depicting acRIP‐seq. (B) Number of ac4C peaks identified in acRIP‐seq in shCtrl and shNAT10 MHCC‐97H cells. (C) Number of ac4C‐modified genes identified in acRIP‐seq. Common ac4C genes have ≥ 1 common ac4C peak, while unique ac4C genes have no common ac4C peaks. (D) Top consensus motif identified by HOMER with acRIP‐seq peaks in MHCC‐97H cells with or without NAT10 knockdown. (E) Normalized distribution of ac4C peaks on mRNA in shCtrl and shNAT10 MHCC‐97H cells. (F) Heatmaps of 125 transcripts displaying reduced ac4C peaks in shNAT10 cells. (G) Flow chart depicting RNA‐seq and Ribo‐seq. (H) Heatmaps of transcript level (RNA‐seq) and RPF abundance (Ribo‐seq). (I) Cumulative distribution of log2‐fold changes of mRNA‐normalized ribosome footprint reads (T.E) for ac4C(−) and ac4C(+) transcripts in shCtrl and shNAT10 MHCC‐97H cells (Kolmogorov‐Smirnov test or two‐tailed t‐test). (J) Polysome profiling of shCtrl and shNAT10 MHCC‐97H cells. (K) Western blot images of SUnSET assays quantifying nascent (puromycin‐labeled) peptides in shCtrl and shNAT10 MHCC‐97H cells. GAPDH used as loading control. Abbreviations: acRIP‐seq, acetylated RNA immunoprecipitation and sequencing; RNA‐seq, RNA sequencing; Ribo‐seq, ribosome profiling analyses; T.E, translation efficiency; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; RPF, ribosome protected fragment; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Modification, Knockdown, RNA Sequencing, Two Tailed Test, Western Blot, Labeling, Control, RNA Immunoprecipitation, Sequencing, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: ac4C modification enhances translation of HMGB2. (A) Schematic diagram of the strategy for pinpointing key NAT10 targets in HCC. (B) Fold changes of transcript level (RNA‐seq) and RPF abundance (Ribo‐seq) of 4 downstream targets. (C) Heatmap of RPF abundance for downstream 4 target genes and rank in ordered NAT10 targets gene list using RPF and T.E fold changes. (D) ac4C (top) and RPF (bottom) abundances on HMGB2 mRNA transcripts in shCtrl and shNAT10 MHCC‐97H cells. (E) acRIP‐qPCR analysis for indicated cells. (F) Western blot of HMGB2 protein expression in NAT10‐deficient cells. (G) HCC cells were treated with 10 µmol/L MG132 for 12 hours. (H) NAT10‐deficient or control cells transfected with pmirGLO‐HMGB2 reporter for 24 h, and HMGB2 translation efficiency defined as reporter protein production (F‐luc/R‐luc) divided by mRNA abundance . (I) Relative mRNA distribution of HMGB2 in ribosome fractions analyzed by qRT‐PCR in shCtrl and shNAT10 MHCC‐97H cells. (J) Subcutaneous xenograft model transplanted with indicated cells ( n = 6, scale bar: 1 cm). Tumor volume monitored and growth curves generated over 24 days (left panel); tumors weighed (right panel). (K) Liver orthotopic implantation models transplanted with indicated cells (scale bar: 1,000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of tumor nodules (right panel) ( n = 5). (L) Lung metastasis model transplanted with indicated cells (scale bar: 1,000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of lung metastatic nodules (right panel) ( n = 5). (M) Representative images of NAT10 and HMGB2 protein expression in HCC tissue arrays ( N = 103) by IHC (upper panel). Pearson correlation analysis between NAT10 and HMGB2 protein expression (lower panel). (N) Pearson correlation analysis between NAT10 and HMGB2 protein expression using CPTAC LIHC dataset. (E) and (H) Data are shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; CPTAC, Clinical Proteomic Tumor Analysis Consortium; qRT‐PCR, quantitative real‐time polymerase chain reaction; RNA‐seq, RNA sequencing; Ribo‐seq, ribosome profiling analyses; T.E, translation efficiency; acRIP, acetylated RNA immunoprecipitation; HMGB2, High Mobility Group Protein B2; IHC, immunohistochemistry; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Modification, RNA Sequencing, Western Blot, Expressing, Control, Transfection, Quantitative RT-PCR, Generated, Imaging, Real-time Polymerase Chain Reaction, RNA Immunoprecipitation, Immunohistochemistry, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: CDS ac4C sites of HMGB2 mRNA enhance binding of eEF2. (A) Schematic of RNA affinity chromatography and MS analysis. (B) Dot blot showing ac4C levels (left) and MB (right, loading controls) in ac4C/C ssRNA probes. (C) GO biological process analysis of proteins identified in quantitative MS via the Database for Annotation, Visualization, and Integrated Discovery. (D) Venn diagram comparing proteins identified in ss‐ac4C and acFUS. (E) Volcano plot of identified proteins. Proteins significantly enriched in ss‐ac4C RNA are shown as red dots. Log2 fold change plotted on x‐axis; ‐log10 P value on y‐axis. (F) Western blot images of endogenous eEF2 and SRP68 proteins pulled down by biotin‐labeled C‐Oligos and ac4C‐Oligos from MHCC‐97H whole cell lysates. (G) RNA pull‐down assays showing dose‐dependent interaction between endogenous eEF2 and biotin‐labeled C‐Oligos (top) or ac4C‐Oligos (below). Gray signal of bands in Western blots (left) quantified by Image Master Total Lab (right). (H) RIP‐qPCR analysis of enrichment of HMGB2 mRNA on eEF2 relative to IgG in shCtrl and shNAT10 MHCC‐97H cells. (I) Western blot analysis of HMGB2 expressions in Huh7 cells co‐transfected with NAT10 CDS plasmid or eEF2 siRNA. (J) Schematic representation of acRIP‐qPCR with fragmented RNA from cells (left). ac4C in HMGB2 mRNA analyzed by acRIP‐qPCR using fragmented RNA in MHCC‐97H cells with shCtrl and shNAT10 (right). (K) Western blot analysis on HA‐HMGB2 in MHCC‐97H cells seeded in 6‐well plates and transfected with either wild‐type HMGB2 CDS or mutant1/2/3 HMGB2 CDS plasmid, along with control or NAT10‐deficient cells. (L) NAT10‐deficient or control cells transfected with either wild‐type HMGB2 CDS or mutant1/2/3 HMGB2 CDS reporter for 24 h, and the quotient of reporter protein production defines translation efficiency of HMGB2. (M) Binding of eEF2 with CDS or 5′ UTR in control or NAT10‐deficient cells analyzed by eEF2 RIP‐qPCR using fragmented RNA. (N) Western blotting showing endogenous eEF2 proteins pulled down by biotin‐labeled exon‐C RNA and exon‐ac4C RNA from MHCC‐97H whole cell lysates. (H, J, L‐N) Data are shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: MS, mass spectrometry; ac4C, N4‐acetylcytidine; MB, methylene blue; GO, Gene ontology analysis; RIP, RNA immunoprecipitates; NAT10, N‐acetyltransferase 10; HMGB2, High Mobility Group Protein B2; eEF2, eukaryotic Elongation Factor 2; CDS, coding sequence; UTR, untranslated regions; acRIP, acetylated RNA immunoprecipitation; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Binding Assay, Affinity Chromatography, Dot Blot, Western Blot, Labeling, Transfection, Plasmid Preparation, Control, Mass Spectrometry, Sequencing, RNA Immunoprecipitation, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Characterization of the NAT10 inhibitor panobinostat. (A) Western blot and dot blot were used to detect HMGB2 expression and ac4C levels after transfection of NAT10 (WT) or G641E NAT10 mutant plasmid, respectively. (B) Flow diagram of NAT10 inhibitor screening. (C) Three‐dimensional (3D) binding model of Panobinostat in NAT10 catalytic pocket. (D) Biacore analysis revealing binding between NAT10 protein and Panobinostat. (E) Western blots for effects of Panobinostat on thermal stabilization of NAT10 protein. CETSA assayed in cell lysates. (F) Identification of direct binding between Panobinostat and NAT10 via DARTS assays. (G) Effects of various concentrations of Panobinostat on global ac4C modification in MHCC‐97H cells. Dot blot assays were conducted with total RNA. (H) Effects of various concentrations of Panobinostat on poly(A)+ RNA ac4C modification in MHCC‐97H cells. (I) Effects of Panobinostat on levels of nascent (puromycin‐labeled) peptides and expression of HMGB2. (J) Effects of Panobinostat on HMGB2 mRNA ac4C level using acRIP‐qPCR. (K) Effects of Panobinostat on HMGB2 mRNA translation efficiency. (L) RIP‐qPCR analysis was performed to determine the enrichment of HMGB2 mRNA on eEF2 relative to IgG in both control and Panobinostat‐treated cells. (J‐L) Data is shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. ** P < 0.01, *** P < 0.001. Abbreviations: WT, wild type; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; HMGB2, High Mobility Group Protein B2; eEF2, eukaryotic Elongation Factor 2; RIP, RNA immunoprecipitates; DARTS, drug affinity responsive targets stability assay; CETSA, cellular thermal shift assay; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Western Blot, Dot Blot, Expressing, Transfection, Mutagenesis, Plasmid Preparation, Binding Assay, Modification, Labeling, Control, Stability Assay, Thermal Shift Assay, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Panobinostat exhibits promising anti‐HCC efficacy in vitro and in vivo. (A) IC50 values of Panobinostat in MHCC‐97H after 48 h of treatment. (B) Effects of various concentrations of Panobinostat on colony formation abilities of MHCC‐97H cells. (C) Effects of various concentrations of Panobinostat on cell migration and invasion abilities of MHCC‐97H cells (scale bars = 100 µm). (D) Schematic diagram of generation and treatment of HCC models in mice. (E) Effects of different concentrations of Panobinostat on tumor volume and weight of subcutaneous xenograft model ( n = 8, scale bar: 1 cm). (F) Effects of various concentrations of Panobinostat on ac4C level in subcutaneous xenograft model. (G) Effects of various concentrations of Panobinostat on HMGB2 expression level in subcutaneous xenograft model. (H) Representative IHC images of HMGB2 expression in subcutaneous xenograft model (scale bar: 20 µm). (I) Liver orthotopic implantation models transplanted with indicated cells (scale bar: 1000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of tumor nodules (right panel) (0 mg, n = 8; 5‐10 mg, n = 10). (J) Lung metastasis model transplanted with indicated cells (scale bar: 1000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of lung metastatic nodules (right panel) ( n = 9). (K) Graphic illustration depicts how NAT10 modulates ac4C‐mediated translation elongation to promote HCC progression. (A‐C) Data is shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. ** P < 0.01, *** P < 0.001. Abbreviations: IC50, a half‐maximal inhibitory concentration; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; HMGB2, High Mobility Group Protein B2; IHC, immunohistochemistry; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: In Vitro, In Vivo, Migration, Expressing, Imaging, Concentration Assay, Immunohistochemistry, Standard Deviation

Journal: Cell Death & Disease

Article Title: Impact of N-acetyltransferase 10 on macrophage activation and inflammation-induced cardiac dysfunction

doi: 10.1038/s41419-025-07796-6

Figure Lengend Snippet: A Western blot analysis of NAT10 protein levels in BMDMs treated with PBS, LPS, or IL-4 for 24 h. B Western blot analysis of NAT10 protein levels in RAW264.7 cells treated with PBS, LPS, or IL-4 for 24 h. C Quantitative analysis of NAT10 protein levels in BMDMs and RAW264.7 cells ( n = 6 per group). D Quantitative analysis of NAT10 protein levels in RAW264.7 cells ( n = 4 per group). E Western blot analysis of NAT10 protein levels in BMDMs treated with different doses of LPS. F Dot blot assay showing ac4C modification levels in BMDMs treated with PBS, LPS, or IL-4 for 24 h. G Dot blot assay showing ac4C modification levels in BMDMs treated with different doses of LPS. H Immunofluorescence staining showing the localization of NAT10 in BMDMs (scale bar = 10 μm). Data are shown as mean ± SD. Statistical analyses were performed using the One-way two-sided ANOVA.

Article Snippet: The generation of RAW 264.7 cells with stable Nat10 knockdown, Nat10 overexpression, and Ets2 knockdown was achieved through the use of the CRISPR-Cas9 system, as developed by Cyagen (Suzhou, China).

Techniques: Western Blot, Dot Blot, Modification, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: Impact of N-acetyltransferase 10 on macrophage activation and inflammation-induced cardiac dysfunction

doi: 10.1038/s41419-025-07796-6

Figure Lengend Snippet: A Protein decay assays for NAT10 in LPS-treated BMDMs at different time points following CHX treatment ( n = 3 independent experiments). B Western blot analysis of NAT10 protein expression in BMDMs treated with MG132 (10 μM), NH₄Cl (25 mM), or chloroquine (25 μM) for 6 h. C Ubiquitination plasmids (Ub) were transfected into RAW264.7 cells treated with PBS or LPS, and NAT10 protein was immunoprecipitated. RAW264.7 cells were treated with MG132 (25 μM) for 8 h before sample collection, and NAT10 ubiquitination levels were detected by Western blot. D Western blot analysis of NAT10 protein levels in BMDMs treated with LPS and pan-deubiquitinase inhibitor PR-619 (25 μM) for 8 h. E , F A Venn diagram reveals that USP39 is the only deubiquitinase identified at the intersection of the three IP-MS groups. Potential deubiquitinating enzymes (DUBs) interacting with NAT10 were identified by mass spectrometry in BMDMs. G Western blot analysis of cytoplasmic and nuclear fractions of BMDMs after LPS treatment shows that USP39 is predominantly localized in the nucleus. H RAW264.7 cells were transfected with Myc-tagged USP39 and Flag-tagged NAT10, followed by treatment with PBS or LPS for 24 h. Cell lysates were immunoprecipitated using an anti-Flag antibody, and both the immunoprecipitated proteins and input were analyzed by Western blot to assess protein interactions and expression levels. I Colocalization of USP39 and NAT10 was examined by confocal microscopy (Scale bar = 10 μm). J RAW264.7 cells were transfected with USP39, NAT10, and HA-UB plasmids, and ubiquitinated NAT10 protein was immunoprecipitated. Ubiquitinated RAW264.7 cells were treated with MG132 (25 μM) for 8 h before sample collection, and NAT10 ubiquitination levels were detected by Western blot. K 293 T cells transfected with the indicated constructs were treated with MG132 for 8 h before collection. Whole-cell lysates were subjected to immunoprecipitation with a Flag antibody and Western blot with an anti-HA antibody to detect NAT10 ubiquitination levels. L USP39-mediated deubiquitination of NAT10 was significantly reduced in 293 T cells transfected with NAT10-K195R or NAT10-K426R mutants. M Effect of USP39 and the catalytically inactive USP39-C306A mutant on the ubiquitination of NAT10. N USP39 regulates K48-linked ubiquitination of NAT10. Data are shown as mean ± SD. Statistical analyses were performed using the One-way two-sided ANOVA.

Article Snippet: The generation of RAW 264.7 cells with stable Nat10 knockdown, Nat10 overexpression, and Ets2 knockdown was achieved through the use of the CRISPR-Cas9 system, as developed by Cyagen (Suzhou, China).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Protein-Protein interactions, Mass Spectrometry, Confocal Microscopy, Construct, Mutagenesis

Journal: Cell Death & Disease

Article Title: Impact of N-acetyltransferase 10 on macrophage activation and inflammation-induced cardiac dysfunction

doi: 10.1038/s41419-025-07796-6

Figure Lengend Snippet: A Immunoblot analysis of INOS in whole cell lysates of Flox or Nat10-/- BMDMs stimulated with LPS (100 ng/mL) for 24 h. B Quantitative analysis of INOS protein levels in BMDMs( n = 3 per group). C Heatmap showing the expression levels of inflammation-related genes from RNA sequencing of Flox and NAT10-/- BMDM cells following LPS treatment. D Real-time PCR analysis of mRNA expression of cytokines in Flox or Nat10-/- BMDMs with or without LPS treatment( n = 6 per group). E ELISA analysis of cytokine secretion in the culture medium of Flox or Nat10-/- BMDMs with or without LPS treatment( n = 6 per group). F , G , H Flow cytometry analysis of macrophage surface markers CD80 and CD86 expression in Flox or Nat10-/- BMDMs with or without LPS treatment. I Immunoblot analysis of INOS in whole cell lysates of WT or Nat10 + /- RAW264.7 cells stimulated with LPS (100 ng/mL) for 24 h. J Quantitative analysis of INOS protein levels in RAW264.7 cells ( n = 4 per group). K Real-time PCR analysis of mRNA expression of cytokines in WT or Nat10 + /- RAW264.7 cells with or without LPS treatment. Data are shown as mean ± SD. Statistical analyses were performed using the Two-way two-sided ANOVA.

Article Snippet: The generation of RAW 264.7 cells with stable Nat10 knockdown, Nat10 overexpression, and Ets2 knockdown was achieved through the use of the CRISPR-Cas9 system, as developed by Cyagen (Suzhou, China).

Techniques: Western Blot, Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Impact of N-acetyltransferase 10 on macrophage activation and inflammation-induced cardiac dysfunction

doi: 10.1038/s41419-025-07796-6

Figure Lengend Snippet: A Schematic of the acRIP-seq procedure. B Predominant motif identified within ac4C-seq peaks among WT-CON, WT-LPS, and NAT10-/- -LPS groups. C Visualization of ac4C modification on the target gene kif20a and the non-acetylated gene clec12a using the IGV browser. D Genomic distributions of ac4C peaks in WT-CON, WT-LPS, and NAT10-/- -LPS groups. E Distribution of acetylated positions in mRNA between WT-LPS vs. WT-CON and NAT10-/- -LPS vs. WT-LPS groups. F GO biological process analysis for hyperacetylated and hypoacetylated transcripts between WT-LPS and WT-CON groups. G Normalized exonic reads from ac4C(+) transcripts in WT-LPS vs. WT-CON and NAT10-/- -LPS vs. WT-LPS groups. H Left: CDF plot depicting differential expression of ac4C (−) or ac4C (+) transcripts in WT-LPS vs. WT-CON and NAT10-/- -LPS vs. WT-LPS groups. Right: CDF plot depicting differential expression genes in WT-LPS vs. WT-CON and NAT10-/- -LPS vs. WT-LPS groups for ac4C (−) and ac4C (+) transcripts with peaks occurring within the CDS, 5’UTR, or 3’UTR.

Article Snippet: The generation of RAW 264.7 cells with stable Nat10 knockdown, Nat10 overexpression, and Ets2 knockdown was achieved through the use of the CRISPR-Cas9 system, as developed by Cyagen (Suzhou, China).

Techniques: Modification, Quantitative Proteomics

Journal: Cell Death & Disease

Article Title: Impact of N-acetyltransferase 10 on macrophage activation and inflammation-induced cardiac dysfunction

doi: 10.1038/s41419-025-07796-6

Figure Lengend Snippet: A Venn diagram showing the downstream target genes regulated by NAT10 via ac4C modification. Left: Hypoacetylated genes after NAT10 knockout (acRIP-seq); Right: Downregulated genes after NAT10 knockout (RNA-seq, P < 0.05, log2FoldChange < -1). B Fold changes in transcript levels (RNA-seq) and ac4C modification levels of 44 downstream targets. C Visualization of ac4C peaks in Ets2 across different groups using IGV software. D RT-qPCR detection of the relative enrichment of Ets2 mRNA in acRIP products from BMDMs. E RT-qPCR detection of the relative enrichment of Ets2 mRNA in NAT10 RIP products from BMDMs. F Decay curves for Ets2 mRNA in Flox and Nat10-/- BMDMs. G RT-qPCR detection of Ets2 mRNA expression in BMDMs after Nat10 knockout. H Volcano plot of differentially expressed genes ( | log2FC | > 1 and FDR < 0.05) at the translation level in LPS-treated BMDMs after Nat10 knockout. I Venn diagram showing the overlap of target genes with changes in ac4C acetylation, transcription, and translation levels after NAT10 knockout. J Heatmap of translation efficiency (Ribo-seq) for the overlapping 27 target genes., expressed as Z-score in relation to median. K KEGG analysis of differentially expressed genes (DEGs) in Ribo-seq. L Nat10-depleted or control cells were transfected with lentivirus containing pmirGLO-Ets2 reporter for 24 h, and Ets2 translation efficiency was defined as reporter protein production (F-luc/R-luc) divided by mRNA abundance. M Relative mRNA distribution of Ets2 in ribosome fractions analyzed by qRT-PCR in BMDMs. N Western blot analysis and densitometric quantification of ETS2 protein in Flox and Nat10-/- BMDMs. O Immunofluorescence analysis showing changes in ETS2 localization and expression. (scale bar = 10 um). Data are shown as mean ± SD. Statistical analyses were performed using the student t -test ( E , L ) and two-way two-sided ANOVA ( D , G , N ).

Article Snippet: The generation of RAW 264.7 cells with stable Nat10 knockdown, Nat10 overexpression, and Ets2 knockdown was achieved through the use of the CRISPR-Cas9 system, as developed by Cyagen (Suzhou, China).

Techniques: Modification, Knock-Out, RNA Sequencing, Software, Quantitative RT-PCR, Expressing, Control, Transfection, Western Blot, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Impact of N-acetyltransferase 10 on macrophage activation and inflammation-induced cardiac dysfunction

doi: 10.1038/s41419-025-07796-6

Figure Lengend Snippet: A Western blot detection of iNOS, NAT10, and ETS2 protein levels in BMDMs. Flox and Nat10-/- BMDMs were transfected with or without Ets2 lentivirus. B– D Flow cytometry analysis of macrophage surface markers CD80 and CD86 expression in BMDMs of each group( n = 6 per group). E Real-time PCR analysis of mRNA expression of cytokines in BMDMs of each group( n = 6 per group). F ELISA analysis of cytokine secretion in the culture medium of BMDMs of each group( n = 6 per group). G Western blot detection of iNOS, NAT10, and ETS2 protein levels in RAW264.7 cells. WT and Nat10 stable overexpressing RAW264.7 cells were transfected with or without Ets2 lentivirus. H– J Flow cytometry analysis of macrophage surface markers CD80 and CD86 expression in RAW264.7 cells of each group( n = 6 per group). K Real-time PCR analysis of mRNA expression of cytokines in RAW264.7 cells of each group( n = 6 per group). L ELISA analysis of cytokine secretion in the culture medium of RAW264.7 cells of each group ( n = 6 per group). Data are shown as mean ± SD. Statistical analyses were performed using two-way two-sided ANOVA.

Article Snippet: The generation of RAW 264.7 cells with stable Nat10 knockdown, Nat10 overexpression, and Ets2 knockdown was achieved through the use of the CRISPR-Cas9 system, as developed by Cyagen (Suzhou, China).

Techniques: Western Blot, Transfection, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Impact of N-acetyltransferase 10 on macrophage activation and inflammation-induced cardiac dysfunction

doi: 10.1038/s41419-025-07796-6

Figure Lengend Snippet: A Kaplan-Meier survival curves of Flox and Nat10-/- mice ( n = 10 male mice per group). B Mean heart rate of LPS-challenged Flox and Nat10-/- mice over 48 h ( n = 10 male mice per group). C , D Representative M-mode echocardiographic images and statistical analysis of Left Ventricular Ejection Fraction, Fractional Shortening, and Stroke Volume in Flox and Nat10-/- mice 12 h after LPS challenge ( n = 6 male mice per group). E Representative photomicrographs of ventricular tissues stained with hematoxylin and eosin (H&E). (scale bar = 100 um). F Serum levels of cardiac troponin-T, troponin-I, and creatine kinase-MB in LPS-challenged mice. G Representative immunohistochemical staining of CD45 in left ventricular myocardium. (scale bar = 100 um). H Representative immunofluorescent staining images of CD68 (green) and iNOS (red) in hearts of Flox and Nat10-/- mice during endotoxemia. (scale bar = 100 um). I Flow cytometry gating strategy and corresponding quantification showing the ratio of CD11B + + F4/80 + + macrophages and F4/80 + + Ly6C+high macrophages in each group ( n = 6). J Heatmap of plasma cytokine changes in Flox and Nat10-/- mice during endotoxemia, expressed as Z-scores relative to the median. K ELISA analysis of cytokine levels in serum. L Real-time PCR analysis of cytokine expression in heart tissue ( n = 6 per group). Data are shown as mean ± SD. Statistical analyses were performed using the Log-rank test ( A ) and two-way two-sided ANOVA ( D , F , H , K , L ).

Article Snippet: The generation of RAW 264.7 cells with stable Nat10 knockdown, Nat10 overexpression, and Ets2 knockdown was achieved through the use of the CRISPR-Cas9 system, as developed by Cyagen (Suzhou, China).

Techniques: Staining, Immunohistochemical staining, Flow Cytometry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing

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: Cancer research

Article Title: Sleeping Beauty insertional mutagenesis in mice identifies drivers of steatosis-associated hepatic tumor

doi: 10.1158/0008-5472.CAN-17-2281

Figure Lengend Snippet: Steatosis-associated CIS list: top 51 CIS genes

Article Snippet: Mouse Nat10 cDNA (#MC202909, Origene) and Prkaca transcript variant 1 (Accession #NM008854) open reading frame with nucleotides 616 and 617 mutated from TT to CG (generating the L206R mutation) with a C-terminal V5-tag were cloned into the previously generated pT2/GD-IRES-GFP( 14 ) Gateway destination SB transposon plasmid co-expressing Fah and GFP using Gateway LR clonase mix (#11791-020, Thermo Fisher Scientific) to generate pT2/GD-Nat10 and pT2/GD/Prkaca L206R . pKT2/GD-GFP and pT2/shp53 plasmids were generated previously. ( 13 ) Twenty μg of each plasmid was delivered by hydrodynamic tail vein injection as described. ( 15 ) Mice were sacrificed at 150–180 days post-injection, livers examined using GFP goggles (#FHS/EF-2G2, BLS-ltd), and GFP-positive tumors counted and collected.

Techniques:

Journal: Cancer research

Article Title: Sleeping Beauty insertional mutagenesis in mice identifies drivers of steatosis-associated hepatic tumor

doi: 10.1158/0008-5472.CAN-17-2281

Figure Lengend Snippet: (A) Overall survival for TCGA HCC cases with and without NAT10 overexpression. (B) NAT10 expression by gene copy number for TCGA HCC cases. (C) NAT10 expression in TCGA no risk HCC and steatosis-associated HCC cases compared to average normal liver. D) Representative immunohistochemistry for NAT10-stained sections of human HCC (top) or matched normal liver (bottom) from tissue microarray (TMA) cases with hepatic steatosis indicated in pathology reports. Scalebars, 100µm. T, tumor. N, non-tumor liver tissue. E) NAT10 immunohistochemistry stain intensity of human HCC and matched normal liver TMA cases stratified by mention of steatosis in pathology report. Yes, steatosis reported. No, no mention of steatosis or steatosis reported absent. Unknown, pathology report not available. *P < .05. **P < .01. ****P < .0001. Error bars, SEM.

Article Snippet: Mouse Nat10 cDNA (#MC202909, Origene) and Prkaca transcript variant 1 (Accession #NM008854) open reading frame with nucleotides 616 and 617 mutated from TT to CG (generating the L206R mutation) with a C-terminal V5-tag were cloned into the previously generated pT2/GD-IRES-GFP( 14 ) Gateway destination SB transposon plasmid co-expressing Fah and GFP using Gateway LR clonase mix (#11791-020, Thermo Fisher Scientific) to generate pT2/GD-Nat10 and pT2/GD/Prkaca L206R . pKT2/GD-GFP and pT2/shp53 plasmids were generated previously. ( 13 ) Twenty μg of each plasmid was delivered by hydrodynamic tail vein injection as described. ( 15 ) Mice were sacrificed at 150–180 days post-injection, livers examined using GFP goggles (#FHS/EF-2G2, BLS-ltd), and GFP-positive tumors counted and collected.

Techniques: Over Expression, Expressing, Immunohistochemistry, Staining, Microarray

Journal: Cancer research

Article Title: Sleeping Beauty insertional mutagenesis in mice identifies drivers of steatosis-associated hepatic tumor

doi: 10.1158/0008-5472.CAN-17-2281

Figure Lengend Snippet: (A) Transposons for tumor induction. Red triangles, SB inverted repeat/ direct repeat sequences. Caggs, Caggs promoter. Gene-of-interest, mouse cDNA sequence for either Nat10 or PrkacaL206R with V5 tag. IRES, internal ribosomal entry site. F. luc, firefly luciferase gene sequence. pA, polyadenylation signal. PGK, PGK promoter. Fah, mouse Fah cDNA. GFP, GFP cDNA sequence. GOI, Gene of interest refers to Nat10 or PrkacaL206R. (B) Treatment plan. EtOH, 5% ethanol CDD, choline-deficient diet. d, days. mo, months. (C) Immunohistochemistry for NAT10, FAH, and ALB-stained liver sections from eCDD-treated mice injected with GFP/shp53 (top) or Nat10/shp53 (middle and bottom). Scalebars, 100µm. T, tumor. N, non-tumor liver. (D) Nat10 expression measured by qRT-PCR from non-tumor liver tissue (L) or liver tumor (T) from Nat10/shp53 or GFP/shp53-injected eCDD-treated mice, normalized to Actb and to wild-type mouse liver Nat10 (n = 5 each). (E) NAT10 immunohistochemical stain intensity of non-tumor liver tissue (L) or liver tumors (T) from Nat10/shp53 (n = 11 L; n = 17 T) or GFP/shp53-injected mice (n = 6 L) from both diets combined. (F) Box-and-whisker plot of tumor burden or (G) tumor penetrance for Nat10/shp53-injected mice treated with eCDD (n = 24) or ND (n = 25) or GFP/shp53-injected mice treated with eCDD (n = 36) or ND (n = 42). *P < .05. **P < .01. ***P < .001. ****P < .0001. Error bars, SEM.

Article Snippet: Mouse Nat10 cDNA (#MC202909, Origene) and Prkaca transcript variant 1 (Accession #NM008854) open reading frame with nucleotides 616 and 617 mutated from TT to CG (generating the L206R mutation) with a C-terminal V5-tag were cloned into the previously generated pT2/GD-IRES-GFP( 14 ) Gateway destination SB transposon plasmid co-expressing Fah and GFP using Gateway LR clonase mix (#11791-020, Thermo Fisher Scientific) to generate pT2/GD-Nat10 and pT2/GD/Prkaca L206R . pKT2/GD-GFP and pT2/shp53 plasmids were generated previously. ( 13 ) Twenty μg of each plasmid was delivered by hydrodynamic tail vein injection as described. ( 15 ) Mice were sacrificed at 150–180 days post-injection, livers examined using GFP goggles (#FHS/EF-2G2, BLS-ltd), and GFP-positive tumors counted and collected.

Techniques: Sequencing, Luciferase, Immunohistochemistry, Staining, Injection, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Whisker Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Helicobacter pylori-induced NAT10 stabilizes MDM2 mRNA via RNA acetylation to facilitate gastric cancer progression.

doi: 10.1186/s13046-022-02586-w

Figure Lengend Snippet: Fig. 5 NAT10 overexpression correlates with high levels of MDM2 ac4C modification and MDM2 expression in gastric cancer specimens. A and B The ac4C levels of the MDM2 transcript were measured by acRIP-qPCR analysis (A), and MDM2 mRNA levels were tested by qRT–PCR (B) in 20 GC and paired normal gastric mucosal tissues. The differences were determined with a two-tailed t-test. C ac4C levels of MDM2 mRNA were positively correlated with MDM2 and NAT10 expression in GC specimens. D The graph shows a significant correlation of NAT10 mRNA with MDM2 expression in GCs. E The TCGA and GEO datasets shows that NAT10 and MDM2 levels were correlated in GC tissues. r and P values were determined by Pearson correlation test (C-E). F–H Representative images of IHC staining of NAT10 and MDM2 in normal gastric tissues and two GC samples with high or low expression of both proteins are shown (F). Scale bar, 150 μm. NAT10 expression was positively correlated with MDM2 expression (G). r and P values were calculated using the Pearson correlation test. Kaplan–Meier analysis indicates the correlation between the combination of high expression of NAT10 and MDM2 and poorer OS (H). Error bars, SD

Article Snippet: Briefly, the small guide RNA (sgRNA) targeting the genomic locus of NAT10 was synthesized and cloned into the lentiCRISPRv2 vector (52,961, Addgene).

Techniques: Over Expression, Modification, Expressing, Quantitative RT-PCR, Two Tailed Test, Immunohistochemistry

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Helicobacter pylori-induced NAT10 stabilizes MDM2 mRNA via RNA acetylation to facilitate gastric cancer progression.

doi: 10.1186/s13046-022-02586-w

Figure Lengend Snippet: Fig. 6 Hp infection enhances NAT10 expression and regulates p53 stability. A Western blot analysis of p53, NAT10 and MDM2 following coculture of GES1 and AGS cells with Hp. B The stability of p53 protein was determined in GES1 cells cocultured with Hp SS1 using the cycloheximide chase method. C qRT–PCR was performed to analyze NAT10, MDM2 and CDKN1A expression in the indicated cells. D and E qRT–PCR analysis of NAT10, MDM2 and CDKN1A expression (D) and Western blot analysis of NAT10, MDM2 and p53 (E) in gastric tissues from mice challenged with Hp SS1 or Brucella broth (Control) for 3 weeks. F The global ac4C acetylation levels in mRNA and total RNA from GES1 and AGS cells cultured in the presence or absence of Hp. G The ac4C levels of MDM2 mRNA in cells treated as described in H. H The stability of MDM2 mRNA was assessed in the indicated cells treated with 5 μg/mL ACD. The MDM2 mRNA abundance relative to that of GAPDH as quantified by qRT–PCR. I Protein levels were tested in NAT10-knockout and control cells following coculture with Hp. SS1, Hp strain SS1; 43,504, Hp strain ATCC43504. Error bars, SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test)

Article Snippet: Briefly, the small guide RNA (sgRNA) targeting the genomic locus of NAT10 was synthesized and cloned into the lentiCRISPRv2 vector (52,961, Addgene).

Techniques: Infection, Expressing, Western Blot, Quantitative RT-PCR, Control, Cell Culture, Knock-Out, Two Tailed Test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Helicobacter pylori-induced NAT10 stabilizes MDM2 mRNA via RNA acetylation to facilitate gastric cancer progression.

doi: 10.1186/s13046-022-02586-w

Figure Lengend Snippet: Fig. 8 Schematic model for the Hp-NAT10-MDM2-p53 axis in promoting the development of GC

Article Snippet: Briefly, the small guide RNA (sgRNA) targeting the genomic locus of NAT10 was synthesized and cloned into the lentiCRISPRv2 vector (52,961, Addgene).

Techniques:

Journal: Cell reports

Article Title: NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer.

doi: 10.1016/j.celrep.2023.112810

Figure Lengend Snippet: Figure 1. NAT10 is upregulated in ESCA and associated with poor ESCA prognosis

Article Snippet: The NAT10 overexpression plasmid pICE-FLAG-NAT10-siR-WT (oeWT), and the functional mutation overexpression plasmid pICE-FLAG-NAT10-siR-G641E (oeMUT) were purchased from Addgene (https://www.addgene.org/ ).

Techniques:

Journal: Cell reports

Article Title: NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer.

doi: 10.1016/j.celrep.2023.112810

Figure Lengend Snippet: Figure 2. NAT10 promotes ESCA progression in vitro and in cKI mice

Article Snippet: The NAT10 overexpression plasmid pICE-FLAG-NAT10-siR-WT (oeWT), and the functional mutation overexpression plasmid pICE-FLAG-NAT10-siR-G641E (oeMUT) were purchased from Addgene (https://www.addgene.org/ ).

Techniques: In Vitro

Journal: Cell reports

Article Title: NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer.

doi: 10.1016/j.celrep.2023.112810

Figure Lengend Snippet: Figure 4. cKO and chemical inhibition of Nat10 inhibit ESCA progression in vitro and in vivo

Article Snippet: The NAT10 overexpression plasmid pICE-FLAG-NAT10-siR-WT (oeWT), and the functional mutation overexpression plasmid pICE-FLAG-NAT10-siR-G641E (oeMUT) were purchased from Addgene (https://www.addgene.org/ ).

Techniques: Inhibition, In Vitro, In Vivo

Journal: Cell reports

Article Title: NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer.

doi: 10.1016/j.celrep.2023.112810

Figure Lengend Snippet: Figure 5. NAT10 catalyzes tRNA ac4C modifications, stabilizes tRNA expression, and promotes mRNA translation

Article Snippet: The NAT10 overexpression plasmid pICE-FLAG-NAT10-siR-WT (oeWT), and the functional mutation overexpression plasmid pICE-FLAG-NAT10-siR-G641E (oeMUT) were purchased from Addgene (https://www.addgene.org/ ).

Techniques: Expressing

Journal: Cell reports

Article Title: NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer.

doi: 10.1016/j.celrep.2023.112810

Figure Lengend Snippet: Figure 6. EGFR is a key downstream target of NAT10 that mediates its function in gefitinib resistance

Article Snippet: The NAT10 overexpression plasmid pICE-FLAG-NAT10-siR-WT (oeWT), and the functional mutation overexpression plasmid pICE-FLAG-NAT10-siR-G641E (oeMUT) were purchased from Addgene (https://www.addgene.org/ ).

Techniques:

Journal: Cell reports

Article Title: NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer.

doi: 10.1016/j.celrep.2023.112810

Figure Lengend Snippet: Figure 7. Combination of NAT10 depletion and gefitinib treatment synergistically inhibits ESCA progression

Article Snippet: The NAT10 overexpression plasmid pICE-FLAG-NAT10-siR-WT (oeWT), and the functional mutation overexpression plasmid pICE-FLAG-NAT10-siR-G641E (oeMUT) were purchased from Addgene (https://www.addgene.org/ ).

Techniques: