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: 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 primary antibodies against β‐actin (3700), NAT10 (66548), E‐cadherin (14472), N‐cadherin (13116), Cathepsin D (2284), USP39 (23303), LAMP1 (9091), peroxidase‐labeled antibody to rabbit IgG (7074), peroxidase‐labeled antibody to mouse IgG (7076), anti‐mouse IgG (H + L) F(ab“) 2 Fragment (Alexa Fluor 488 Conjugate) (4408), and anti‐rabbit IgG (H+L) F(ab”) 2 Fragment (Alexa Fluor 594 Conjugate) (8889) were all from Cell Signaling Technology (Boston, MA, USA).

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 primary antibodies against β‐actin (3700), NAT10 (66548), E‐cadherin (14472), N‐cadherin (13116), Cathepsin D (2284), USP39 (23303), LAMP1 (9091), peroxidase‐labeled antibody to rabbit IgG (7074), peroxidase‐labeled antibody to mouse IgG (7076), anti‐mouse IgG (H + L) F(ab“) 2 Fragment (Alexa Fluor 488 Conjugate) (4408), and anti‐rabbit IgG (H+L) F(ab”) 2 Fragment (Alexa Fluor 594 Conjugate) (8889) were all from Cell Signaling Technology (Boston, MA, USA).

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 primary antibodies against β‐actin (3700), NAT10 (66548), E‐cadherin (14472), N‐cadherin (13116), Cathepsin D (2284), USP39 (23303), LAMP1 (9091), peroxidase‐labeled antibody to rabbit IgG (7074), peroxidase‐labeled antibody to mouse IgG (7076), anti‐mouse IgG (H + L) F(ab“) 2 Fragment (Alexa Fluor 488 Conjugate) (4408), and anti‐rabbit IgG (H+L) F(ab”) 2 Fragment (Alexa Fluor 594 Conjugate) (8889) were all from Cell Signaling Technology (Boston, MA, USA).

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 primary antibodies against β‐actin (3700), NAT10 (66548), E‐cadherin (14472), N‐cadherin (13116), Cathepsin D (2284), USP39 (23303), LAMP1 (9091), peroxidase‐labeled antibody to rabbit IgG (7074), peroxidase‐labeled antibody to mouse IgG (7076), anti‐mouse IgG (H + L) F(ab“) 2 Fragment (Alexa Fluor 488 Conjugate) (4408), and anti‐rabbit IgG (H+L) F(ab”) 2 Fragment (Alexa Fluor 594 Conjugate) (8889) were all from Cell Signaling Technology (Boston, MA, USA).

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 primary antibodies against β‐actin (3700), NAT10 (66548), E‐cadherin (14472), N‐cadherin (13116), Cathepsin D (2284), USP39 (23303), LAMP1 (9091), peroxidase‐labeled antibody to rabbit IgG (7074), peroxidase‐labeled antibody to mouse IgG (7076), anti‐mouse IgG (H + L) F(ab“) 2 Fragment (Alexa Fluor 488 Conjugate) (4408), and anti‐rabbit IgG (H+L) F(ab”) 2 Fragment (Alexa Fluor 594 Conjugate) (8889) were all from Cell Signaling Technology (Boston, MA, USA).

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