Journal: Marine Drugs

Article Title: Heparanase and Syndecan-4 Are Involved in Low Molecular Weight Fucoidan-Induced Angiogenesis

doi: 10.3390/md13116588

Figure Lengend Snippet: Effects of Low molecular weight fucoidan (LMWF) on endothelial cell abilities: migration and 2D-angiogenesis. Human vascular endothelial cells (HUVEC) were incubated with 10 µg/mL LMWF for 24 h and the migration ( A ), the lamellipodia formation ( B ) and the capillary tube formation (length and area) ( C ) were determined. ( A ) Migration chamber assay. HUVECs incubated with or without 10 μg/mL LMWF, were allowed to migrate through the porous fibronectin-coated membrane. They were stained with Mayer’s hemalum and counted. The results are expressed as cell number per field; ( B ) Lamellipodia formation. LMWF induced the formation of lamellipodia and ruffles (white arrows indicate lamellipodia/ruffle formation, DAPI-nucleus (blue), Phalloidin-F-actin (red)). Bar = 10 µm; ( C ) Capillary tube formation (2D-angiogenesis assay) on Matrigel. Left and right panels show the length ( left ) and area ( right ) of endothelial capillaries formed by HUVECs treated with or without 10 µg/mL LMWF. Lower right panel shows a representative image of capillary network, as photographed with phase contrast microscopy (magnification ×100). * p < 0.05 versus control untreated (UT) cells. A.U.: arbitrary unit.

Article Snippet: Human umbilical vein endothelial cells (HUVEC, N°CRL-1730, ATCC) were cultured in Endothelial Cell Basal Media 2 (PromoCell, Heidelberg, Germany) supplemented with 10% of fetal calf serum (Lonza, Levallois-Perret, France), and a mix from PromoCell containing EGF (5.0 ng/mL), Hydrocortisone (0.2 μg/mL), VEGF (0.5 ng/mL), bFGF (10 ng/mL), R3 IGF-1 (20 ng/mL), Ascorbic Acid (1 μg/mL), Heparin (22.5 μg/mL), 1% Penicillin Streptomycin (PAA Laboratories, Pasching, Austria).

Techniques: Molecular Weight, Migration, Incubation, Boyden Chamber Assay, Membrane, Staining, Angiogenesis Assay, Microscopy, Control

Journal: Marine Drugs

Article Title: Heparanase and Syndecan-4 Are Involved in Low Molecular Weight Fucoidan-Induced Angiogenesis

doi: 10.3390/md13116588

Figure Lengend Snippet: Effects of LMWF on the SDC expression in HUVECs. SDC-1 and SDC-4 mRNA or protein levels in endothelial cells treated or not with 10 µg/mL LMWF were analyzed respectively by real time RT-PCR ( A ) or western blot ( B , C ). SDC-1 and SDC-4 ectodomains in the supernatant of cells treated with or without 10 µg/mL LMWF were analyzed by dot blot ( D ). * p < 0.05, ** p < 0.005, significantly different to LMWF-untreated cells (UT). A.U.: arbitrary unit.

Article Snippet: Human umbilical vein endothelial cells (HUVEC, N°CRL-1730, ATCC) were cultured in Endothelial Cell Basal Media 2 (PromoCell, Heidelberg, Germany) supplemented with 10% of fetal calf serum (Lonza, Levallois-Perret, France), and a mix from PromoCell containing EGF (5.0 ng/mL), Hydrocortisone (0.2 μg/mL), VEGF (0.5 ng/mL), bFGF (10 ng/mL), R3 IGF-1 (20 ng/mL), Ascorbic Acid (1 μg/mL), Heparin (22.5 μg/mL), 1% Penicillin Streptomycin (PAA Laboratories, Pasching, Austria).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Dot Blot

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: NAT10 enhances the lysosomal acidification through increasing v‐ATPase activity. A) KEGG pathway enrichment analysis was conducted on significantly differentially expressed genes in cells overexpressing NAT10 or control. B,C) LysoTracker‐Green staining was used to evaluate the lysosomal acidification status in KYSE150Luc and KYSE410Luc cells upon NAT10 overexpression or control (B), and KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells with NAT10 knockout (C). Scale bar: 20 µm. D) Expression of Cathepsin D (CTSD) among primary ESCC tumor tissues (Tumor), matched normal tissues (Normal), and lymph node metastatic tissues (Lymphatic metastasis) ( n = 20). E,F) Western blot analysis showing expression of pro‐cathepsin D and mature‐cathepsin D in NAT10‐expressing KYSE150Luc and KYSE410Luc cells or control (E), as well as in NAT10‐knockout KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells (F). G,H) Detection of v‐ATPase activity in KYSE150Luc and KYSE410Luc cells with NAT10 overexpression (G), or KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells with NAT10 knockout (H). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The human ESCC cell lines KYSE150 (DSMZ, ACC 375) and KYSE410 (DSMZ, ACC 381) were purchased from German Center for the Collection of Microbial Species (Braunschweig, Germany) and maintained with RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, Gaithersburg, MD, USA) at 37 °C in a 5% CO 2 environment.

Techniques: Activity Assay, Control, Staining, Over Expression, Knock-Out, Expressing, Western Blot

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: NAT10‐catalyzed ac4C modification increases the translation efficiency of ATP6V0E1 mRNA. A) Diagram showing the strategy to screen target genes via overlapping the v‐ATPase subunits and acRIP‐seq gene lists. B) RIP‐RT‐qPCR was applied to detect the binding between NAT10 and ATP6V0El mRNA using NAT10 antibody or normal mouse IgG control in KYSE150Luc‐LM5 and KYSE410Luc‐13 cells. C,D) acRIP‐RT‐qPCR was performed to detect the ac4C modification level of ATP6V0E1 mRNA in KYSE150Luc and KYSE410Luc cells with NAT10‐expression (C) and KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells with NAT10‐knockout (D). E) Base resolution mapping of ATP6V0E1 in the acPeak region from Sanger sequencing (RedaC:T‐seq). F,G) Western blot analysis was performed to analyze ATP6V0E1 expression as indicated. H) Western blot showing the expression of ATP6V0E1 when transfected with wild‐type (WT) or mutant NAT10 (G641E). I) Mutants design of ATP6V0E1 acPeak. J) Translation efficiency of ATP6V0E1 was detected in ESCC cells co‐transfected with plasmids expressing WT or mutant ATP6V0E1 acPeak and NAT10 using a luciferase reporter assay. K) Dot blot analysis determined the ac4C modification of ATP6V0E1 catalyzed by NAT10 in vitro. L) Western blot analysis assessed the in vitro translation efficiency of unmodified or ac4C‐modified ATP6V0E1 catalyzed by NAT10. M) Representative images and expression patterns of ATP6V0E1 in primary ESCC tumor tissues and adjacent normal tissues (upper panel), as well as in matched primary and metastatic tissues (lower panel). Scale bar: 40 µm. N) Kaplan‐Meier analysis evaluating the overall survival of ESCC patients according to ATP6V0E1 expression. O) Correlation analysis between the expression of NAT10 and ATP6V0E1. Bars, SDs; ns, no significance; ** P < 0.01; *** P < 0.001.

Article Snippet: The human ESCC cell lines KYSE150 (DSMZ, ACC 375) and KYSE410 (DSMZ, ACC 381) were purchased from German Center for the Collection of Microbial Species (Braunschweig, Germany) and maintained with RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, Gaithersburg, MD, USA) at 37 °C in a 5% CO 2 environment.

Techniques: Modification, Quantitative RT-PCR, Binding Assay, Control, Expressing, Knock-Out, Sequencing, Western Blot, Transfection, Mutagenesis, Luciferase, Reporter Assay, Dot Blot, In Vitro

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: ATP6V0E1 promotes lysosomal acidification and cancer metastasis. A) LysoTracker‐Green staining was used to evaluate the lysosomal acidification status in KYSE150Luc and KYSE410Luc cells overexpressing ATP6V0E1 or vector control. Scale bar: 20 µm. B) Western blot revealing the expression levels of pro‐cathepsin D, mature‐cathepsin D, LC3, and p62 in ESCC cells overexpressing ATP6V0E1 or vector control. C) Live imaging of LysoTracker‐Green dye detecting lysosomal acidification in ESCC cells with control or ATP6V0E1 knockout. Scale bar: 20 µm. D) Western blot analysis revealing the expression levels of pro‐cathepsin D, mature‐cathepsin D, LC3, and p62 in ATP6V0E1‐knockout ESCC cells and control. E) Transwell assay comparing cell invasion between ATP6V0E1‐overexpressing and control cells. Scale bar: 200 µm. F) Western blot showing the expression of E‐cadherin and N‐cadherin in ATP6V0E1‐overexpressing or control cells. G,H) The cell invasion and EMT phenotypes were detected when ATP6V0E1 was knocked out. Scale bar: 200 µm. Bars, SDs; ** P < 0.01; *** P < 0.001.

Article Snippet: The human ESCC cell lines KYSE150 (DSMZ, ACC 375) and KYSE410 (DSMZ, ACC 381) were purchased from German Center for the Collection of Microbial Species (Braunschweig, Germany) and maintained with RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, Gaithersburg, MD, USA) at 37 °C in a 5% CO 2 environment.

Techniques: Staining, Plasmid Preparation, Control, Western Blot, Expressing, Imaging, Knock-Out, Transwell Assay

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: NAT10 induces the lysosomal degradation of E‐cadherin to promote cancer metastasis via upregulation of ATP6V0E1. A,B) Immunofluorescence showing the effects of NAT10 on the expression of E‐cadherin. Scale bar: 20 µm. C) Western blot detection of E‐cadherin expression in NAT10‐knockout ESCC cells pretreated with CHX (50 µg mL −1 ) for different durations (0, 3, 6, and 12 h). D) In the presence of Bafilomycin A1 (Baf‐A1, 0.1 µ m ) or CHX (50 µg mL −1 ), the expression of E‐cadherin is shown in NAT10‐overexpressing cells or control. E) The co‐localization of E‐cadherin with LAMP1 was detected by confocal microscopy in NAT10‐overexpressing ESCC cells or control in the absence or presence of Baf‐A1. The white arrows represent the co‐localization of E‐cadherin and LAMP1. Scale bar: 5 µm. F) LysoTracker‐Green staining shows that ATP6V0E1 mediated the effect of NAT10 on lysosomal acidification status in NAT10‐knockout KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells. Scale bar: 20 µm. G,H) Western blot analysis (G) and the Boyden chamber assay (H) indicated that overexpression of ATP6V0E1 attenuated the effect of NAT10 on invasion and EMT phenotypes. Scale bar: 200 µm. I,J) Bioluminescence imaging and quantification show that ATP6V0E1 abolishes the effect of NAT10 on lung metastasis. Scale bar: 400 µm. Bars, SDs; ** P < 0.01; *** P < 0.001.

Article Snippet: The human ESCC cell lines KYSE150 (DSMZ, ACC 375) and KYSE410 (DSMZ, ACC 381) were purchased from German Center for the Collection of Microbial Species (Braunschweig, Germany) and maintained with RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, Gaithersburg, MD, USA) at 37 °C in a 5% CO 2 environment.

Techniques: Immunofluorescence, Expressing, Western Blot, Knock-Out, Control, Confocal Microscopy, Staining, Boyden Chamber Assay, Over Expression, Imaging

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: Identification of G‐749 as a NAT10 inhibitor to enhance ubiquitin‐dependent degradation of NAT10 via interaction with USP39. A) Diagram illustrating the strategy to screen drug candidates. B) Schematic diagram of the molecular structure of G‐749. C) Invasion assay was conducted on KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells after treatment with increasing doses of G‐749. Scale bar: 200 µm. D) Western blot analysis detected the expression of NAT10 protein in ESCC cells upon G‐749 treatment. E) CETSA analysis revealed the binding between the NAT10 protein and G‐749. F) NAT10 were labeled with biotin and pulled down by avidin on magnetic beads after GP treatment. A biotin‐avidin experiment was conducted. G) Molecular docking indicating the specific binding sites between G‐749 and NAT10. H) Immunoblot showing NAT10 expression in KYSE150Luc‐LM5 or KYSE410‐I3 cells pretreated with CHX (50 µg mL −1 ) for different durations (0, 3, 6, and 12 h), with or without G‐749 (5 µ m ) treatment. I) Immunoprecipitation analysis of NAT10‐binding ubiquitin proteins in KYSE150Luc and KYSE410Luc cells following treatment with MG132 (8 µ m ) or G‐749 (0, 1.25, 2.5, and 5 µ m ) for 24 h. J,K) KYSE150Luc and KYSE410Luc cells were co‐transfected with NAT10‐Flag and USP39‐HA plasmids, then subjected to G‐749 (5 µ m ) treatment. The content changes of USP39‐HA or NAT10‐Flag in immunoprecipitates were analyzed. With normal IgG as a negative control. L) GST‐NAT10 pulldown assays were performed in KYSE150Luc or KYSE410Luc cells after G‐749 incubation in vitro. Bars, SDs; ** P < 0.01; *** P < 0.001.

Article Snippet: The human ESCC cell lines KYSE150 (DSMZ, ACC 375) and KYSE410 (DSMZ, ACC 381) were purchased from German Center for the Collection of Microbial Species (Braunschweig, Germany) and maintained with RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, Gaithersburg, MD, USA) at 37 °C in a 5% CO 2 environment.

Techniques: Ubiquitin Proteomics, Invasion Assay, Western Blot, Expressing, Binding Assay, Labeling, Avidin-Biotin Assay, Magnetic Beads, Immunoprecipitation, Transfection, Negative Control, Incubation, In Vitro

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: G‐749 decreases ATP6V0E1 ac4C modification and inhibits lysosomal acidification. A) acRIP‐RT‐qPCR showing the effect of G‐749 on ac4C modification of ATP6V0E1 mRNA. B) Western blot analysis of ATP6V0E1 protein in ESCC cells upon G‐749 treatment. C) V‐ATPase detection showing the effect of G‐749 on the activity of v‐ATPase in ESCC cells. D) LysoTracker‐Green staining evaluating the effects of G‐749 on lysosomal acidification in KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells. Scale bar: 20 µm. E) Western blot revealing that G‐749 resulted in a decrease of the maturation of cathepsin D and an increase of its precursor form in ESCC cells in a dose‐ and time‐dependent manner. F) ESCC cells were transfected with the mCherry‐GFP‐LC3 plasmid and then treated with G‐749 for 24 h. The autophagosome (yellow) or co‐localization with lysosomes forming autolysosomes (red/yellow) was observed using confocal laser microscopy. Scale bar: 5 µm. G) ESCC cells were transfected with the GFP‐LC3 plasmid and then subjected to G‐749 intervention for 24 h. The numbers of green puncta, which represent autophagosomes, were observed using confocal microscopy. Scale bar: 20 µm. H) Western blot analysis of the protein expression of LC3 and p62 in KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells after treatment with G‐749 at the indicated concentrations and time. I) Western blotting detection of E‐cadherin and N‐cadherin expression in KYSE150Luc‐LM5 cells treated with G‐749 at different concentrations. J) Immunofluorescence analysis revealing the expression of E‐cadherin upon G‐749. Scale bar: 20 µm. Bars, SDs; ** P < 0.01; *** P < 0.001.

Article Snippet: The human ESCC cell lines KYSE150 (DSMZ, ACC 375) and KYSE410 (DSMZ, ACC 381) were purchased from German Center for the Collection of Microbial Species (Braunschweig, Germany) and maintained with RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, Gaithersburg, MD, USA) at 37 °C in a 5% CO 2 environment.

Techniques: Modification, Quantitative RT-PCR, Western Blot, Activity Assay, Staining, Transfection, Plasmid Preparation, Microscopy, Confocal Microscopy, Expressing, Immunofluorescence

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: NAT10 is required for the bioactivity of G‐749 in suppressing lysosomal dysregulation and tumor metastasis. A) LysoTracker‐Green staining showing the inhibitory effect of G‐749 on lysosomal acidification, while not in NAT10‐knockout group, and that the suppressive effect of G‐749 was restored when the cells were reconstituted with NAT10. Scale bar: 20 µm. B) Detection of v‐ATPase activity in ESCC cells as indicated. C) Invasion chamber assay comparing the invasive ability of NAT10‐knockout ESCC cells that were further transfected with NAT10 or control in the presence or absence of G‐749. Scale bar: 200 µm. D) Bioluminescence imaging and quantification of lung metastasis showing the inhibitory effect of G‐749 on tumor metastasis as indicated. E) Hematoxylin and eosin (H&E) staining of lung sections as indicated. Scale bar: 400 µm. F) Histological analysis of major organs in the groups. G) The ac4C modification level of ATP6V0E1 mRNA in lung metastatic tumor tissues was assessed by acRIP‐RT‐qPCR. Scale bar: 100 µm. Bars, SDs; ns, no significance; ** P < 0.01; *** P < 0.001.

Article Snippet: The human ESCC cell lines KYSE150 (DSMZ, ACC 375) and KYSE410 (DSMZ, ACC 381) were purchased from German Center for the Collection of Microbial Species (Braunschweig, Germany) and maintained with RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, Gaithersburg, MD, USA) at 37 °C in a 5% CO 2 environment.

Techniques: Staining, Knock-Out, Activity Assay, Invasion Chamber Assay, Transfection, Control, Imaging, Modification, Quantitative RT-PCR

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: NAT10 enhances the lysosomal acidification through increasing v‐ATPase activity. A) KEGG pathway enrichment analysis was conducted on significantly differentially expressed genes in cells overexpressing NAT10 or control. B,C) LysoTracker‐Green staining was used to evaluate the lysosomal acidification status in KYSE150Luc and KYSE410Luc cells upon NAT10 overexpression or control (B), and KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells with NAT10 knockout (C). Scale bar: 20 µm. D) Expression of Cathepsin D (CTSD) among primary ESCC tumor tissues (Tumor), matched normal tissues (Normal), and lymph node metastatic tissues (Lymphatic metastasis) ( n = 20). E,F) Western blot analysis showing expression of pro‐cathepsin D and mature‐cathepsin D in NAT10‐expressing KYSE150Luc and KYSE410Luc cells or control (E), as well as in NAT10‐knockout KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells (F). G,H) Detection of v‐ATPase activity in KYSE150Luc and KYSE410Luc cells with NAT10 overexpression (G), or KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells with NAT10 knockout (H). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The coding sequences of NAT10 and ATP6V0E1 were cloned into pcDNA3.1, and the NAT10 and ATP6V0E1 sgRNAs were inserted into the lentiCRISPRV2 vector (52961, Addgene).

Techniques: Activity Assay, Control, Staining, Over Expression, Knock-Out, Expressing, Western Blot

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: NAT10‐catalyzed ac4C modification increases the translation efficiency of ATP6V0E1 mRNA. A) Diagram showing the strategy to screen target genes via overlapping the v‐ATPase subunits and acRIP‐seq gene lists. B) RIP‐RT‐qPCR was applied to detect the binding between NAT10 and ATP6V0El mRNA using NAT10 antibody or normal mouse IgG control in KYSE150Luc‐LM5 and KYSE410Luc‐13 cells. C,D) acRIP‐RT‐qPCR was performed to detect the ac4C modification level of ATP6V0E1 mRNA in KYSE150Luc and KYSE410Luc cells with NAT10‐expression (C) and KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells with NAT10‐knockout (D). E) Base resolution mapping of ATP6V0E1 in the acPeak region from Sanger sequencing (RedaC:T‐seq). F,G) Western blot analysis was performed to analyze ATP6V0E1 expression as indicated. H) Western blot showing the expression of ATP6V0E1 when transfected with wild‐type (WT) or mutant NAT10 (G641E). I) Mutants design of ATP6V0E1 acPeak. J) Translation efficiency of ATP6V0E1 was detected in ESCC cells co‐transfected with plasmids expressing WT or mutant ATP6V0E1 acPeak and NAT10 using a luciferase reporter assay. K) Dot blot analysis determined the ac4C modification of ATP6V0E1 catalyzed by NAT10 in vitro. L) Western blot analysis assessed the in vitro translation efficiency of unmodified or ac4C‐modified ATP6V0E1 catalyzed by NAT10. M) Representative images and expression patterns of ATP6V0E1 in primary ESCC tumor tissues and adjacent normal tissues (upper panel), as well as in matched primary and metastatic tissues (lower panel). Scale bar: 40 µm. N) Kaplan‐Meier analysis evaluating the overall survival of ESCC patients according to ATP6V0E1 expression. O) Correlation analysis between the expression of NAT10 and ATP6V0E1. Bars, SDs; ns, no significance; ** P < 0.01; *** P < 0.001.

Article Snippet: The coding sequences of NAT10 and ATP6V0E1 were cloned into pcDNA3.1, and the NAT10 and ATP6V0E1 sgRNAs were inserted into the lentiCRISPRV2 vector (52961, Addgene).

Techniques: Modification, Quantitative RT-PCR, Binding Assay, Control, Expressing, Knock-Out, Sequencing, Western Blot, Transfection, Mutagenesis, Luciferase, Reporter Assay, Dot Blot, In Vitro

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: NAT10 induces the lysosomal degradation of E‐cadherin to promote cancer metastasis via upregulation of ATP6V0E1. A,B) Immunofluorescence showing the effects of NAT10 on the expression of E‐cadherin. Scale bar: 20 µm. C) Western blot detection of E‐cadherin expression in NAT10‐knockout ESCC cells pretreated with CHX (50 µg mL −1 ) for different durations (0, 3, 6, and 12 h). D) In the presence of Bafilomycin A1 (Baf‐A1, 0.1 µ m ) or CHX (50 µg mL −1 ), the expression of E‐cadherin is shown in NAT10‐overexpressing cells or control. E) The co‐localization of E‐cadherin with LAMP1 was detected by confocal microscopy in NAT10‐overexpressing ESCC cells or control in the absence or presence of Baf‐A1. The white arrows represent the co‐localization of E‐cadherin and LAMP1. Scale bar: 5 µm. F) LysoTracker‐Green staining shows that ATP6V0E1 mediated the effect of NAT10 on lysosomal acidification status in NAT10‐knockout KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells. Scale bar: 20 µm. G,H) Western blot analysis (G) and the Boyden chamber assay (H) indicated that overexpression of ATP6V0E1 attenuated the effect of NAT10 on invasion and EMT phenotypes. Scale bar: 200 µm. I,J) Bioluminescence imaging and quantification show that ATP6V0E1 abolishes the effect of NAT10 on lung metastasis. Scale bar: 400 µm. Bars, SDs; ** P < 0.01; *** P < 0.001.

Article Snippet: The coding sequences of NAT10 and ATP6V0E1 were cloned into pcDNA3.1, and the NAT10 and ATP6V0E1 sgRNAs were inserted into the lentiCRISPRV2 vector (52961, Addgene).

Techniques: Immunofluorescence, Expressing, Western Blot, Knock-Out, Control, Confocal Microscopy, Staining, Boyden Chamber Assay, Over Expression, Imaging

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: Identification of G‐749 as a NAT10 inhibitor to enhance ubiquitin‐dependent degradation of NAT10 via interaction with USP39. A) Diagram illustrating the strategy to screen drug candidates. B) Schematic diagram of the molecular structure of G‐749. C) Invasion assay was conducted on KYSE150Luc‐LM5 and KYSE410Luc‐I3 cells after treatment with increasing doses of G‐749. Scale bar: 200 µm. D) Western blot analysis detected the expression of NAT10 protein in ESCC cells upon G‐749 treatment. E) CETSA analysis revealed the binding between the NAT10 protein and G‐749. F) NAT10 were labeled with biotin and pulled down by avidin on magnetic beads after GP treatment. A biotin‐avidin experiment was conducted. G) Molecular docking indicating the specific binding sites between G‐749 and NAT10. H) Immunoblot showing NAT10 expression in KYSE150Luc‐LM5 or KYSE410‐I3 cells pretreated with CHX (50 µg mL −1 ) for different durations (0, 3, 6, and 12 h), with or without G‐749 (5 µ m ) treatment. I) Immunoprecipitation analysis of NAT10‐binding ubiquitin proteins in KYSE150Luc and KYSE410Luc cells following treatment with MG132 (8 µ m ) or G‐749 (0, 1.25, 2.5, and 5 µ m ) for 24 h. J,K) KYSE150Luc and KYSE410Luc cells were co‐transfected with NAT10‐Flag and USP39‐HA plasmids, then subjected to G‐749 (5 µ m ) treatment. The content changes of USP39‐HA or NAT10‐Flag in immunoprecipitates were analyzed. With normal IgG as a negative control. L) GST‐NAT10 pulldown assays were performed in KYSE150Luc or KYSE410Luc cells after G‐749 incubation in vitro. Bars, SDs; ** P < 0.01; *** P < 0.001.

Article Snippet: The coding sequences of NAT10 and ATP6V0E1 were cloned into pcDNA3.1, and the NAT10 and ATP6V0E1 sgRNAs were inserted into the lentiCRISPRV2 vector (52961, Addgene).

Techniques: Ubiquitin Proteomics, Invasion Assay, Western Blot, Expressing, Binding Assay, Labeling, Avidin-Biotin Assay, Magnetic Beads, Immunoprecipitation, Transfection, Negative Control, Incubation, In Vitro

Journal: Advanced Science

Article Title: NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA

doi: 10.1002/advs.202502931

Figure Lengend Snippet: NAT10 is required for the bioactivity of G‐749 in suppressing lysosomal dysregulation and tumor metastasis. A) LysoTracker‐Green staining showing the inhibitory effect of G‐749 on lysosomal acidification, while not in NAT10‐knockout group, and that the suppressive effect of G‐749 was restored when the cells were reconstituted with NAT10. Scale bar: 20 µm. B) Detection of v‐ATPase activity in ESCC cells as indicated. C) Invasion chamber assay comparing the invasive ability of NAT10‐knockout ESCC cells that were further transfected with NAT10 or control in the presence or absence of G‐749. Scale bar: 200 µm. D) Bioluminescence imaging and quantification of lung metastasis showing the inhibitory effect of G‐749 on tumor metastasis as indicated. E) Hematoxylin and eosin (H&E) staining of lung sections as indicated. Scale bar: 400 µm. F) Histological analysis of major organs in the groups. G) The ac4C modification level of ATP6V0E1 mRNA in lung metastatic tumor tissues was assessed by acRIP‐RT‐qPCR. Scale bar: 100 µm. Bars, SDs; ns, no significance; ** P < 0.01; *** P < 0.001.

Article Snippet: The coding sequences of NAT10 and ATP6V0E1 were cloned into pcDNA3.1, and the NAT10 and ATP6V0E1 sgRNAs were inserted into the lentiCRISPRV2 vector (52961, Addgene).

Techniques: Staining, Knock-Out, Activity Assay, Invasion Chamber Assay, Transfection, Control, Imaging, Modification, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Syntaxin 18 regulates the DNA damage response and epithelial-to-mesenchymal transition to promote radiation resistance of lung cancer

doi: 10.1038/s41419-022-04978-4

Figure Lengend Snippet: a A549 and H460 cells were transduced with a shRNA targeting STX18 and its expression was quantified by RT-qPCR. n = 3–4. The insert shows respective immunoblotting analyses of STX18 expression. Here, quantification was achieved after normalization to the loading control (Actin) and the shScr sample. n = 3. b Proliferation of unirradiated cells was measured by proliferation/cell viability assay. n = 3. c Quantification of sub-G1 fraction by flow cytometry after irradiation. Cells were irradiated and cell cycle distribution was quantified by PI staining after 72 h. n = 3–5. d Colony formation ability was assessed after irradiation. Cells were irradiated and colonies were counted after 10 days. n = 3.

Article Snippet: The following antibodies were used for immunoblotting: beta-Actin (MP Biomedicals, Santa Ana, CA, USA, #691002), STX18, p53 (Santa Cruz Biotechnology, Dallas, TX, USA, #293067, #71817, respectively), Frizzled 5, E-cadherin, ZO-1, Vimentin, Zeb1 (all from Cell Signaling Technologies, #5266, #3195, #5406, #5741, #3396), Vinculin (1:2000, Abcam, #129002), phospho-ATR, ATR, phospho-Chk1, Chk1, phospho-ATM, ATM, phospho-Chk2, Chk2 (all from Cell Signaling Technologies, #58014, #13934, #2348, #2360, #13050, #2873, #2669, #2662), ATR (Cell Signaling Technologies, #13934).

Techniques: Transduction, shRNA, Expressing, Quantitative RT-PCR, Western Blot, Viability Assay, Flow Cytometry, Irradiation, Staining

Journal: Cell Death & Disease

Article Title: Syntaxin 18 regulates the DNA damage response and epithelial-to-mesenchymal transition to promote radiation resistance of lung cancer

doi: 10.1038/s41419-022-04978-4

Figure Lengend Snippet: For the representation of STX18 knockdown, A549-shSTX18 K3 cells were used. a Immunoblotting analysis of proteins involved in cell cycle checkpoints after 10 Gy. Vinculin was used as control. After normalization to the loading control, the samples were compared to the shScr-0.5 h sample. b Detection by flow cytometry of Annexin V positive cells following irradiation with 10 Gy and/or pre-treatment with 1 µM berzosertib or Chir-124. Cells were incubated for 72 h then stained with Annexin V. c Quantification of mitotic index after irradiation with 2 Gy by quantification of cells positive for phosphorylation of Histone H3 by flow cytometry. d Quantification of fragmented nuclei and nuclei with abnormal shape in the cell population 72 h after irradiation. e Cell cycle analysis by flow cytometry after irradiation. Cells were irradiated and cell cycle distribution was analyzed by PI staining after 72 h. n = 3 for all experiments.

Article Snippet: The following antibodies were used for immunoblotting: beta-Actin (MP Biomedicals, Santa Ana, CA, USA, #691002), STX18, p53 (Santa Cruz Biotechnology, Dallas, TX, USA, #293067, #71817, respectively), Frizzled 5, E-cadherin, ZO-1, Vimentin, Zeb1 (all from Cell Signaling Technologies, #5266, #3195, #5406, #5741, #3396), Vinculin (1:2000, Abcam, #129002), phospho-ATR, ATR, phospho-Chk1, Chk1, phospho-ATM, ATM, phospho-Chk2, Chk2 (all from Cell Signaling Technologies, #58014, #13934, #2348, #2360, #13050, #2873, #2669, #2662), ATR (Cell Signaling Technologies, #13934).

Techniques: Western Blot, Flow Cytometry, Irradiation, Incubation, Staining, Cell Cycle Assay

Journal: Cell Death & Disease

Article Title: Syntaxin 18 regulates the DNA damage response and epithelial-to-mesenchymal transition to promote radiation resistance of lung cancer

doi: 10.1038/s41419-022-04978-4

Figure Lengend Snippet: a Immunoblotting analysis of E-cadherin, vimentin, ZO-1 and Zeb1 expression. Vinculin and actin were used as controls. After normalization to the loading control, the samples were compared to the shScr sample (set to 1). b RT-qPCR analysis of MMP9 mRNA expression. c Detection by flow cytometry of Annexin V positive cells. Cells were incubated for 72 h on poly(2-hydroxyethyl methacrylate)-coated (Poly-Hema) plates and stained with Annexin V. d Boyden chamber assay for A549 migration and invasion. Cells were seeded on the transwell plate after overnight serum starvation and incubated for 24 h. Migrated and invaded cells were then stained with crystal violet. e Representative pictures from Boyden chamber assay results shown in ( d ). Scale bar, 500 µm. n = 3 for all experiments.

Article Snippet: The following antibodies were used for immunoblotting: beta-Actin (MP Biomedicals, Santa Ana, CA, USA, #691002), STX18, p53 (Santa Cruz Biotechnology, Dallas, TX, USA, #293067, #71817, respectively), Frizzled 5, E-cadherin, ZO-1, Vimentin, Zeb1 (all from Cell Signaling Technologies, #5266, #3195, #5406, #5741, #3396), Vinculin (1:2000, Abcam, #129002), phospho-ATR, ATR, phospho-Chk1, Chk1, phospho-ATM, ATM, phospho-Chk2, Chk2 (all from Cell Signaling Technologies, #58014, #13934, #2348, #2360, #13050, #2873, #2669, #2662), ATR (Cell Signaling Technologies, #13934).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Flow Cytometry, Incubation, Staining, Boyden Chamber Assay, Migration

Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A Transcriptional expression pattern of seven APOBEC3 family members in LUAD (green) and normal lung tissues (red), which was visualized using ridgeline plot generated by TNMplot. B Protein expression levels of A3C in LUAD or normal lung tissue based on the CPTAC proteomics datasets. The Mann-Whitney U test was used to compare the mean of two groups (** P < 0.01). C Genetic dependency scores of seven APOBEC3 family members across hundreds to thousands of cancer cell lines using genome-wide CRISPR-Cas9 screening data in the Broad DepMap database. A dependency score < 0 indicates that loss of the gene impairs cell viability, with more negative scores reflecting higher survival essentiality. D Differential expression of seven APOBEC3 family members according to TP53 mutation status in cancer cell lines (wildtype, n = 536; mutant, n = 910) in the Broad DepMap database. The Mann-Whitney U test was used to compare the mean of two groups. *** P < 0.001 for A3A, A3C, A3D, A3F and A3H, ** P = 0.0054 for A3G, P = 0.1740 for A3B (N.S. not significant). E ChIP-seq analysis from multiple datasets ( GSE46641 , GSE56674 , ENCODE) revealed p53 binding enrichment at seven APOBEC3 family genes across various cell types and stress conditions. F Relative mRNA expression levels of the seven APOBEC3 family members in A549 (TP53 wild-type) and H1299 (TP53-null) cells using quantitative RT-PCR (normalized to GAPDH, fold matched A3A, representative of n = 3). G Relative mRNA expression levels of A3C in A549 cells transfected p53-targeting siRNAs (normalized to GAPDH, fold matched siControl, representative of n = 3, * P = 0.0364). H Relative mRNA expression levels of A3C in H1299 cells transfected with empty vector (pcDNA3.1), wild-type or mutant p53 (R175H, R248W, R273H) expression vectors (normalized to GAPDH, fold matched null control, representative of n = 3, * P = 0.0240). The non-parametric Kruskal-Wallis test was used to compare differences among groups for quantitative RT-PCR analysis. I Immunohistochemical staining of A3C in three NSCLC patient samples. A3C expression was detected in bronchial epithelium (Patient #1), alveolar walls including a mitotic cell (Patient #2), and scattered atypical cells resembling tumor cells in the interstitium (Patient #3), with nuclear and cytoplasmic localization. J Patients were stratified into high and low A3C expression groups using the median expression value as the cutoff. Survival analysis, including overall survival (OS) and first progression (FP) was performed by Kaplan–Meier plotter website. K Genes were ranked by their correlation with A3C expression in LUAD and enriched pathways were identified using corGSEA tool offered by Correlation AnalyzeR website. L Predicted gemcitabine response in LUAD patients was assessed using the OncoPredict R package. Patients were stratified into A3C high and low expression groups based on the median expression level, and differences in predicted response were analyzed using the non-parametric Kruskal-Wallis test ( P = 6.3e-18). M Pearson correlation analysis was conducted between A3C mRNA expression levels derived from the Broad DepMap database and gemcitabine sensitivity of matched cancer cell lines obtained from the GDSC database.

Article Snippet: All the mutants were generated and fully sequenced by GENEWIZ, Inc. CDA (RNA accession: NM_001785 , cat. no. RC208922), A3C (RNA accession: NM_014508 , cat. no. RC203971), human Myc-DDK-tagged ORF clone and corresponding pCMV6-Entry (cat. no. PS100001 ) vectors were purchased from OriGene Technologies, Inc. C97S/C100S mutants of A3C were generated via overlapping polymerase chain reactions (PCRs) using the specific mutation-containing primers and fully sequenced by GENEWIZ.

Techniques: Expressing, Generated, MANN-WHITNEY, Genome Wide, CRISPR, Quantitative Proteomics, Mutagenesis, ChIP-sequencing, Binding Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Control, Immunohistochemical staining, Staining, Derivative Assay

Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A H1299 cells were stably transfected with empty vector pCMV6, CDA or A3C expression vectors. NC stands for negative control; OE stands for overexpression. Cells were challenged with 0.01, 0.1, 1, 10, 100 μM gemcitabine (GEM) for 24, 48 and 72 h. The cell viability was determined by MTS assay and IC 50 values were calculated. Data are presented as the mean ± SD of three biologically independent experiments, one-way ANOVA does not reach statistical significance. B H1299 cells stably expressing A3C (A3C OE ) or negative control cells (A3C NC ) were initially cultured at a density as low as 500 cells/well in a 96-well plate. Cell growth was monitored using the method of MTS. The absorbance over time at OD490 values was measured every day. Cell expansion over time periods between two groups were compared by two-way repeated-measures ANOVA with the Greenhouse-Geisser correction and post-hoc analyses were performed by Šidák- adjusted multiple comparison tests. *** P < 0.001, compared with NC group. C For xenograft mouse model, H1299 cells stably transfected with empty vector pCMV6 (A3C NC ) or A3C expression vector (A3C OE ) were inoculated subcutaneously into the right flank of nude mice. After developing tumors, the mice were divided into four cohorts (n = 6) and treated with either (1) vehicle (normal saline, NS); (2) gemcitabine (100 mg/kg, i.p, Q3Dx5 or every third day for five doses). Tumor size was recorded using caliper measurements. Tumor growth over time periods among four groups were compared by two-way repeated-measures ANOVA with the Greenhouse-Geisser correction and post-hoc analyses were performed by Šidák- adjusted multiple comparison tests. ** P = 0.0046 compared between gemcitabine-treated A3C-overexpressing xenografts and control xenografts, P = 0.2032 compared between A3C-overexpressing xenografts and control xenografts. D Daily mean body weights were determined and recorded. E H1299 cells were stably transfected with CDA, A3C or empty vector, the intracellular pharmacokinetics of gemcitabine was determined using UHPLC-MS/MS. Intracellular gemcitabine concentrations were normalized by protein concentration of whole-cell lysates. F H1299 cells expressing A3C, CDA or control vector were challenged with 1 μM gemcitabine for 6 h. Whole cell lysates were harvested for immunoblot of phospho-Chk1 (Ser 345), Chk1 and FLAG. GAPDH was used as an internal control. G H1299 cells expressing wild-type A3C or enzymatic mutant A3C were challenged with 1 μM gemcitabine for 6 h. Whole cell lysates were harvested for immunoblot of phospho-Chk1 (Ser 345), Chk1, A3C and FLAG. GAPDH was used as an internal control. H A3C expression was suppressed by shRNA-mediated knockdown in A3C-overexpressing H1299 cells. Cells were challenged with 1 μM gemcitabine for 6 h. Whole cell lysates were harvested for immunoblot of phospho-Chk1 (Ser 345), Chk1, A3C and FLAG. GAPDH was used as an internal control. shCon, non-targeting control shRNA. I A3C-overexpressing H1299 cells with A3C knockdown were processed for DNA fiber analysis. A total of 150 forks was scored for replication fork speed (kb/min) per group, representing the aggregate of two biologically replicates. Each biological replicate included three technical replicates (three microscope slides). Top: Schematic of the single-molecule DNA fiber tract labeling used to evaluate fork progression. Representative DNA fibers featured by the replication fork speed were shown. Scale bar: 5 µm. Bottom: Distribution of fork speed in H1299-A3C cells after transduction with control shRNA or shRNA targeting A3C. The horizontal line indicates the median fork speed. Statistical significance was determined by one-way ANOVA using Dunnett’s multiple comparisons test. *** P < 0.001 compared with shCon. J A3C-overexpressing H1299 cells with A3C knockdown were processed for gemcitabine treatment and MST assay. Downward sigmoidal dose-response curves were plotted by GraphPad Prism 9.3.1 software.

Article Snippet: All the mutants were generated and fully sequenced by GENEWIZ, Inc. CDA (RNA accession: NM_001785 , cat. no. RC208922), A3C (RNA accession: NM_014508 , cat. no. RC203971), human Myc-DDK-tagged ORF clone and corresponding pCMV6-Entry (cat. no. PS100001 ) vectors were purchased from OriGene Technologies, Inc. C97S/C100S mutants of A3C were generated via overlapping polymerase chain reactions (PCRs) using the specific mutation-containing primers and fully sequenced by GENEWIZ.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Negative Control, Over Expression, MTS Assay, Cell Culture, Comparison, Saline, Control, Drug discovery, Tandem Mass Spectroscopy, Protein Concentration, Western Blot, Mutagenesis, shRNA, Knockdown, Microscopy, Labeling, Transduction, Software

Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A A3C-overexpressing (A3C OE ) or empty vector control (A3C NC ) H1299 cells were incubated with Chk1 inhibitors (AZD-7762 or AZD, MK8776 or MK, x-axis) and gemcitabine (y-axis) in an 8 × 8 concentration checkerboard format for 72 h. Cell viability was determined by MTS assay. The experiment data on top panel (values were relative cell viability compared with control) were analyzed and calculated with Combenefit software. The predicted data were subtracted from the experimental data, yielding a final difference value for each combination. Data are expressed as mean values of three biologically independent experiments. The greater the difference value, the more synergistic that particular combination was. Three mathematical models (Loewe additivity, Bliss independence and Highest single agent) were used to robustly identify synergistic combinations. B Synergistic effect was also evaluated by CompuSyn based on the median-effect principle of Chou and Talalay. Combination index (CI) is widely accepted as an indicative of the degree of synergistic interaction. CI values less than 1.0 (horizontal line) correspond to a synergistic interaction. CI values below 0.3 indicate strong synergy. The data are presented as the mean ± SD by three biologically independent experiments. One-way ANOVA followed by post-hoc Tukey’s analysis was performed. ** P = 0.0011, *** P < 0.001. C The cytotoxic effects of two Chk1 inhibitors on A3C-overexpressing (A3C OE ) or empty vector control (A3C NC ) H1299 cells were measured by MTS assay. D The effect of A3C on the colony-forming ability of H1299 cells in the presence of gemcitabine or/and two Chk1 inhibitors. The data are presented as the mean ± SD by three biologically independent experiments. One-way ANOVA followed by post-hoc Tukey’s analysis was performed. * P = 0.0217 between A3C NC and A3C OE H1299 cells co-treated with gemcitabine and MK-8776. E The effect of A3C on the DNA replication and cell cycle distribution of H1299 cells in the presence of gemcitabine or/and two Chk1 inhibitors for 24 h, which was assessed using a click reaction with Alexa Fluor 647 azide and Flow Cytometry. The percentages of a cell population in the different phases were marked on flow cytometry diagram. The percentage of cells at S-phase are presented as the mean ± SD by three biologically independent experiments. One-way ANOVA followed by post-hoc Tukey’s analysis was performed (*** P < 0.001).

Article Snippet: All the mutants were generated and fully sequenced by GENEWIZ, Inc. CDA (RNA accession: NM_001785 , cat. no. RC208922), A3C (RNA accession: NM_014508 , cat. no. RC203971), human Myc-DDK-tagged ORF clone and corresponding pCMV6-Entry (cat. no. PS100001 ) vectors were purchased from OriGene Technologies, Inc. C97S/C100S mutants of A3C were generated via overlapping polymerase chain reactions (PCRs) using the specific mutation-containing primers and fully sequenced by GENEWIZ.

Techniques: Plasmid Preparation, Control, Incubation, Concentration Assay, MTS Assay, Software, Flow Cytometry

Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A Representative images of comet assay measuring the amount of DNA damage in A3C-over expressing (A3C OE ) or control (A3C NC ) H1299 cells treated with gemcitabine or/and two Chk1 inhibitors for 24 h. A total of 50 comets was analyzed per group, representing the aggregate of two biologically independent replicates. Each biological replicate included three technical replicates (three replicate wells for one comet slide). Data are mean ± SD of tail moments (tail DNA% × length of tail). Scale bar, 200 μm. Scatter plots show all points, medians, and lower and upper quantiles. One-way ANOVA followed by post-hoc Tukey’s analysis was performed. ** P = 0.0027 between A3C OE and A3C NC H1299 cells treated with GEM, ** P = 0.0014 between A3C OE and A3C NC H1299 cells treated with AZD, *** P < 0.001). B Representative images of chromosome spreading assay to measure the chromosomal aberrations in A3C (A3C OE ) or control (A3C NC ) H1299 cells treated with gemcitabine or/and two Chk1 inhibitors for 24 h. A total of 20 metaphase spreads was analyzed per group, representing the aggregate of two biologically replicates. Each biological replicate included three technical replicates (three microscope slides). Data represent the percentage (%) of abnormal metaphase spreads (unrepaired chromatid breaks or chromosome pulverization) in a total of 20 metaphase spreads. Scale bar, 50 μm. C Representative images of pan-nuclear 53BP1 foci formation in A3C (A3C OE ) or control (A3C NC ) H1299 cells treated with gemcitabine or/and two Chk1 inhibitors for 6 h. Confocal images of indicated treatments with antibodies against 53BP1 (green). Nuclei were stained with DAPI (blue). 630× magnification images, scale bar represents 5 μm. Quantification of the number of 53BP1 foci per nucleus and the percentage of nucleus displaying more than 10 foci is plotted. Each bar represents the mean percentage (%) ± SD by three biologically triplicate experiments and at least 40 cells per group were analyzed for each experiment. Comparisons test was performed by one-way ANOVA followed by post-hoc Tukey’s test. * P = 0.0280, *** P < 0.001. ( D ) H1299 cells were transfected with A3C (A3C OE ) or empty vector (A3C NC ) for 48 h, and further challenged with gemcitabine (1 μM) in the presence or absence of Chk1 inhibitors (AZD, 1 μM; MK, 10 μM) for another 6 h. Whole cell lysates were harvested for detecting the levels of phosphorylated RPA32 (Ser8), phosphorylated Chk1 (Ser345), phosphorylated histone H2AX on serine 139 (γH2AX), total RPA32 and Chk1. GAPDH was used as loading control. E The effect of A3C on DNA repair activity using GFP-reporter assay and quantified by Flow Cytometry. Schematic representation of the HR reporter substrate DR-GFP and NHEJ reporter substrate EJ5-GFP. HEK-293T cells grown on 6-well plates were used as transfection efficiency control by transiently transfected with 1 μg/well pCBASceI and 1 μg/well DR-GFP/EJ5-GFP for 48 h and harvested for Flow Cytometry. H1299 control cells and A3C-overexpressing cells grown on 6-well plates were transiently transfected with 1 μg/well pCBASceI, 1 μg/well DR-GFP/EJ5-GFP for 48 h and harvested for Flow Cytometry. A3C-overexpressing cells and A3C knockdown cells established from A3C-overexpressing cells grown on 6-well plates were transiently transfected with 1 μg/well pCBASceI, 1 μg/well DR-GFP/EJ5-GFP for 48 h and harvested for Flow Cytometry. Data are expressed as the mean ± SD by three biologically triplicate values. Comparisons test was performed by unpaired t -test or one-way ANOVA (*** P < 0.001).

Article Snippet: All the mutants were generated and fully sequenced by GENEWIZ, Inc. CDA (RNA accession: NM_001785 , cat. no. RC208922), A3C (RNA accession: NM_014508 , cat. no. RC203971), human Myc-DDK-tagged ORF clone and corresponding pCMV6-Entry (cat. no. PS100001 ) vectors were purchased from OriGene Technologies, Inc. C97S/C100S mutants of A3C were generated via overlapping polymerase chain reactions (PCRs) using the specific mutation-containing primers and fully sequenced by GENEWIZ.

Techniques: Single Cell Gel Electrophoresis, Expressing, Control, Microscopy, Staining, Transfection, Plasmid Preparation, Activity Assay, Reporter Assay, Flow Cytometry, Knockdown

Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A A3C pulldown and immunoblot analysis of H1299 cell lysates with or without gemcitabine treatment. * Shorter exposure time. B Venn diagram illustrated the intersection of A3C interactome with or without gemcitabine treatment. C Enrichment analysis of pathways based on Gene Ontology (GO). D A3C interactome was screened by mass spectrometry-based proteomics. The table showed the representative highest scored peptides that physically bind to A3C. Our AP-MS dataset was verified by published proteomic data (Cheung group, 2018) from RNA/DNA hybrids immunoprecipitation (DRIP) located at the BAMBI promoter and DPP9 3’ UTR. E A Venn diagram illustrated the intersection of A3C interactome (291, after removed keratins and other contaminants) with 803 proteins that interact with RNA/DNA hybrids published by Cheung group. Molecular Complex Detection (MCODE) was executed to identify key modules in the protein-protein interaction (PPI) network of A3C-hybrids binding. DDX5, DDX3X and DHX9 were highlighted in the highly interconnected clusters. F R-loop-dependent A3C-DDX5 interaction. H1299 cells grown on 10 cm dishes were co-transfected with 5 μg FLAG-tagged A3C and 5 μg HA-tagged DDX5, along with either 5 μg wild-type RNase H1 (ppyCAG_RNaseH1), the catalytically inactive D210N mutant, or an empty vector (pcDNA3.1). Cell lysates were analyzed by immunoprecipitation using anti-FLAG antibodies followed by immunoblots with the indicated antibodies. Input indicates 20% of pre-immunoprecipitated samples. G The full-length (FL), N-terminal (ΔΝ), C-terminal (ΔC) or both (ΔΝC) truncated forms of DDX5. H HEK-H293T cells were transfected with full-length (FL) HA-DDX5, or the DDX5 truncation mutants for 24 h. Lysates were incubated with immobilized HA. Affinity precipitated exogenous DDX5 was detected with anti-HA antibody, with the relative levels in the lysates also shown. I Display of the Predicted Aligned Error (PAE) for the AlphaFold-predicted A3C/DDX5 complex. DDX5 and A3C chains are labeled respectively in cyan and orange. A dark green tile designates a good prediction, and the region of DDX5 that is expected to be in proximity to A3C includes residues 50-100, while for A3C, the residue range is 30-180. Structure-based protein interaction interface analysis between A3C and DDX5. Image represents the predicted A3C-DDX5 complex, where interaction interface and hotspot residues are labeled. J Structure-based protein interaction interface between A3C (PDB ID: 3vow) and DDX5 (PDB ID: 4a4d) where interaction region and two hotspot residues (Tyr59/Tyr97) are labeled.

Article Snippet: All the mutants were generated and fully sequenced by GENEWIZ, Inc. CDA (RNA accession: NM_001785 , cat. no. RC208922), A3C (RNA accession: NM_014508 , cat. no. RC203971), human Myc-DDK-tagged ORF clone and corresponding pCMV6-Entry (cat. no. PS100001 ) vectors were purchased from OriGene Technologies, Inc. C97S/C100S mutants of A3C were generated via overlapping polymerase chain reactions (PCRs) using the specific mutation-containing primers and fully sequenced by GENEWIZ.

Techniques: Western Blot, Mass Spectrometry, Protein-Protein interactions, Immunoprecipitation, Binding Assay, Transfection, Mutagenesis, Plasmid Preparation, Incubation, Labeling, Residue

Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A Immunofluorescent staining with the S9.6 antibody showed the R-loop formation (green foci) in response to gemcitabine or/and Chk1 inhibitors in H1299 cells expressing A3C, DDX5 or their control counterparts (pCMV6). Nuclei were counterstained with DAPI (blue). Scale bar represents 50 μm. Quantification of the number of S9.6 foci per nucleus and the percentage of nucleus displaying more than 3 foci is plotted. Each bar represents the mean percentage (%) ± SD by three biologically triplicate experiments and at least 40 cells per group were analyzed for each experiment. Comparisons test was performed by one-way ANOVA followed by post-hoc Tukey’s test. * P = 0.0112, *** P < 0.001. B H1299 cells were treated with gemcitabine or/and Chk1 inhibitors for 6 and genomic DNA in a dilution of 400 ng/μL was extracted for assessing R-loop levels. Nuclear samples digested with or without RNase H (0.5 U) were loaded onto nylon membranes (2.5 μL per dot) and probed with S9.6 antibody. C Dot-blot analysis for A3C or DDX5-overexpressing cells treated with gemcitabine or/and Chk1 inhibitors. D H1299 cells were transfected with DDX5 (DDX5 OE ) or empty vector (DDX5 NC ) for 48 h, and further challenged with gemcitabine (1 μM) in the presence or absence of Chk1 inhibitors (AZD, 1 μM; MK, 10 μM) for another 6 h. Whole cell lysates were harvested for detecting the levels of phosphorylated RPA32 (Ser8), phosphorylated Chk1 (Ser345), phosphorylated histone H2AX on serine 139 (γH2AX), total RPA32 and Chk1. GAPDH was used as loading control. E A3C-overexpressing H1299 cells were transfected with DDX5 (DDX5 OE ) or empty vector (DDX5 NC ) for 48 h and challenged with gemcitabine (1 μM) for 6 h. Whole cell lysates were harvested for detecting the levels of phosphorylated Chk1 (Ser345) and Chk1. F EdU incorporation assay was performed in A3C OE /DDX5 NC and A3C OE /DDX5 OE H1299 cells. All the cells were stimulated with or without gemcitabine for 24 h. Data are expressed as the mean ± SD by triplicate assays. One-way ANOVA followed by post-hoc Tukey’s analysis was performed. ** P = 0.0098.

Article Snippet: All the mutants were generated and fully sequenced by GENEWIZ, Inc. CDA (RNA accession: NM_001785 , cat. no. RC208922), A3C (RNA accession: NM_014508 , cat. no. RC203971), human Myc-DDK-tagged ORF clone and corresponding pCMV6-Entry (cat. no. PS100001 ) vectors were purchased from OriGene Technologies, Inc. C97S/C100S mutants of A3C were generated via overlapping polymerase chain reactions (PCRs) using the specific mutation-containing primers and fully sequenced by GENEWIZ.

Techniques: Staining, Expressing, Control, Dot Blot, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells

doi: 10.1038/s41419-025-08215-6

Figure Lengend Snippet: A Further modulation of DDX5 expression in A3C-overexpressing H1299 cells for evaluating the functional interplay between A3C and DDX5 (overexpression, OE; knockdown, KD; NC, negative control). B A3C OE /DDX5 NC and A3C OE /DDX5 KD cells were co-treated with gemcitabine or/and Chk1 inhibitors for 6 h, and dot-blot analysis was performed for detecting R-loop levels using S9.6 antibody. Methylene blue was a loading control. C Drug sensitivity interaction between gemcitabine and two Chk1 inhibitors were evaluated in A3C OE /DDX5 NC and A3C OE /DDX5 KD H1299 cells using Combenefit and CompuSyn programs. * P = 0.0359, ** P = 0.0085, *** P < 0.001. D Comet assay in A3C OE /DDX5 NC and A3C OE /DDX5 KD H1299 cells. Data are expressed as the mean ± SD. One-way ANOVA followed by post-hoc Tukey’s analysis was performed (* P = 0.0279, *** P < 0.001). Scatter plots show all points, medians, and lower and upper quantiles. E For xenograft mouse model, A3C OE /DDX5 NC and A3C OE /DDX5 KD cells were inoculated subcutaneously into the right flank of nude mice. After developing tumors, the mice were divided into four cohorts (n = 6) and treated with either (1) vehicle (normal saline); (2) gemcitabine (100 mg/kg, i.p., Q3Dx5 or every third day for five doses). F Tumor size was recorded using caliper measurements. Tumor growth over time periods among four groups were compared by two-way repeated-measures ANOVA with the Greenhouse-Geisser correction and post-hoc analyses were performed by Šidák- adjusted multiple comparison tests. ** P = 0.0016 between gemcitabine-treated A3C OE /DDX5 KD xenografts and control xenografts. G Daily mean body weights were determined for all groups. H Representative fluorescence images of DDX5 (red) and S9.6 (green foci) staining positivity from xenograft tissues at 400× magnification. Nuclei were stained with DAPI. Scale bar represents 50 μm.

Article Snippet: All the mutants were generated and fully sequenced by GENEWIZ, Inc. CDA (RNA accession: NM_001785 , cat. no. RC208922), A3C (RNA accession: NM_014508 , cat. no. RC203971), human Myc-DDK-tagged ORF clone and corresponding pCMV6-Entry (cat. no. PS100001 ) vectors were purchased from OriGene Technologies, Inc. C97S/C100S mutants of A3C were generated via overlapping polymerase chain reactions (PCRs) using the specific mutation-containing primers and fully sequenced by GENEWIZ.

Techniques: Expressing, Functional Assay, Over Expression, Knockdown, Negative Control, Dot Blot, Control, Single Cell Gel Electrophoresis, Saline, Comparison, Fluorescence, Staining