Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV envelope protein (ZIKV-E, green) and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h post-infection (p.i.) with ZIKV. Scale bar, 50 μm. (B) Quantification of infection efficiency in four GSC and NPC lines 48 h p.i. with ZIKV. (C) Quantification of ZIKV+ cells in a panel of human GSCs and NPCs. (D) Kinetics of viral RNA copies p.i. with ZIKV by measuring viral RNA copies by qRT-PCR in NPC C4–7 and GSC3565. (E) ZIKV infection efficiency of GSCs and NPCs was measured by direct measurement of viral RNA copies. (F) Representative bright-field images 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. Scale bars, 50 μm. (G) Cell viability normalized to day 5 mock, as measured 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. (H) GSCs (GSC3565), differentiated GSCs, NPCs (NPC C4–7), and differentiated NPCs were assayed for cell viability 72 h p.i. with ZIKV. (I) Apoptosis of GSCs (387, 3565) and primary (NPC194, fetal human [fh] NPC) or iPSC-derived NPCs (WT83, C4–7) p.i. with ZIKV was measured by cleaved caspase-3 (CC3) staining. (J) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (K) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 72 h p.i. with ZIKV. Scale bars, 50 μm. (L) Quantification of the percentage of CC3+ cells in DAPI+ cells for GSCs and NPCs 72 h p.i. with ZIKV. (M) Cell viability of patient-derived cultures from GBM (387 and 3565), pontine glioma (3752 and 007), meningioma (CH-157MN, IOMM-LEE), ependymoma (EP1), and medulloblastoma cell lines (DAOY, D283, HDMB03, D341) 72 h after ZIKV infection. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. NS, no significance. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Quantitative RT-PCR, Derivative Assay, Staining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV-E (green), SOX2 (red), and DAPI (blue) of GSCs and forebrain-specific hiPSC-derived NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (B) Quantification of the percentage of SOX2+ cells in DAPI+ cells for GSCs and NPCs 48 h p.i. with ZIKV. (C) Representative immunostaining for ZIKV-E (green), SOX2 or AXL (red), and DAPI (blue) of GSCs (GSC3565) without transduction (shRNA) or transduced with control shRNA (shCONT), AXL shRNA (shAXL.2), or SOX2 shRNA (shSOX2.53) for 72 h and then 48 h with ZIKV infection. Scale bars, 100 μm. (D) Quantification of the percentage of ZIKV+ cells in DAPI+ cells in GSCs 1517 and 3565 under conditions for (C), with a range of ZIKV infection. (E) Viral copy number by qRT-PCR of GSCs (GSC3565 or GSC1517) or NPC C4–7 transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53) for 72 h and then either exposed to mock conditions or infected with ZIKV for another 72 h. All comparisons are versus shCONT. (F) Gene set enrichment (GSE) bubble plots showing pathways positively (top, r > 0.4) or negatively (bottom, r < −0.4) correlated with SOX2 expression in the TCGA GBM HG-U133A microarray dataset. Each circle represents an enriched pathway, with the border color indicating the false discovery rate (FDR)-corrected p value. (G) GSE graph showing the top pathway enrichments positively or negatively correlated with SOX2 as described in (F). (H) Correlation of mRNA levels of SOX2 with IFNAR1, IRF1, promyelocytic leukemia (PML), and IFITIM1 from the TCGA GBM HG-U133A microarray dataset. (I) Correlation between SOX2 with ISGs from the TCGA GBM HG-U133A microarray dataset. The size and color of the dots indicate the degree of correlation (p < 0.001). Blank cells indicate a non-significant correlation. (J) qPCR of ISGs (IFNAR-1, ISH20, IRF1, IFITM1, TLR3, and OAS2) in GSCs (GSC3565) transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53). Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. **p < 0.001, ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Derivative Assay, Transduction, shRNA, Control, Infection, Quantitative RT-PCR, Expressing, Microarray

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative images of mock- or ZIKV-infected BCOs stained with neuronal markers (CTIP2 and NeuN), a neural progenitor cell marker (SOX2), and DAPI. Scale bars, 100 μm. (B) Quantification of BCO size p.i. with ZIKV. Significance was assessed by two-tailed Student’s t test, and experiments were performed in two batches with 12 organoids per group per batch. (C) BCO size fold change of ZIKV- and mock-treated groups over a period of 1 month. (D) Quantification of SOX2+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (E) Quantification of CC3+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (F) Quantification of SATB2+ cells within MAP2+ cells in ZIKV- versus mock-infected groups. **p < 0.01 by two-tailed Student’s t test. (G) Quantification of GFAP+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (H) Quantification of NeuN+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (I) Quantification of CTIP2+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (J) Bright-field images of engraftment of two patient-derived GSCs (387 and 3565) transduced with GFP into human BCOs over a time course. Scale bars, 1 mm. (K) Engrafted GSCs (GFP+) with normal BCO immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 200 μm. (L) Quantification of integrin αvβ5+ cells in normal BCOs or GSC-BCOs. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (M) Representative images of GFP-labeled GSC-BCOs immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (N) Representative images of GFP-labeled GSC-BCOs immunostained for SOX2 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (O) Images of GFP-labeled GSC-GFP BCOs 13 days p.i. with ZIKV. Scale bars, 1 mm. (P) Representative images of residual GSCs (green) and DAPI staining (blue) of GFP-labeled GSC-GFP BCOs cultured under mock conditions or with ZIKV for 2–4 weeks. Scale bars, 200 μm. The percentage of GFP+ cells among DAPI+ cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-way ANOVA. (Q) Representative immunostaining for integrin αvβ5 (red), GFP (green), ZIKV-E (white), and DAPI (blue) of GFP-labeled GSC-GFP BCOs mock- or ZIKV-infected for 2–4 weeks. Scale bars, 200 μm (left) and 100 μm (center). The percentage of ZIKV-E+ cells among integrin αvβ5 cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (R) Representative images of 387 and 3565 GSC-BCOs with or without ZIKV, respectively, stained with SOX2, ZIKV-E, and DAPI. GFP shows the presence of GSCs (scale bars, 50 μm). ZIKV-E+, GFP+, and ZIKV-E+ cells among GFP+ cells were quantified by counting (two GSCs cell lines, two repeats, n = 12 organoids/group); *p < 0.05 by two-tailed Student’s t test. (S) Schematic of the experiment design. (T) Volcano plot showing differences between GSC-BCO ZIKV versus GSC-BCO mock. 113 genes were differentially expressed (greater than 1.5-fold) between these two groups (*p < 0.05). (U) Network analysis of genes differentially expressed upon ZIKV infection, represented as a bubble plot.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Infection, Staining, Marker, Two Tailed Test, Derivative Assay, Transduction, Labeling, Cell Culture, Immunostaining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Immunostaining of the subventricular zone (SVZ) of mice 72 h following ZIKV infection ZIKV-E (green), SOX2 (red, top panels), and integrin αvβ5 (red, bottom panels). Scale bars, 50 μm. (B) Higher magnification of images from (A), demonstrating ZIKV infection of SOX2+ (top panels) and integrin αvβ5+ cells. Scale bars, 10 μm. (C) Survival of ZIKV-infected NSG mice from (A) was plotted by the Kaplan-Meier method. (D) ZIKV-infected brains from the mice in (A) were collected upon death, and histology was assessed by H&E staining. Scale bars, 20 μm. (E) Survival of NSG mice following implantation of GSCs treated with isotype control, P1F6 antibody, ZIKV, combined P1F6 and ZIKV, combined CRISPR knockout (KO) of integrin β5 (sgRNA1 sgRNA2) with ZIKV inoculation, analyzed by log rank test; p < 0.01. (F) H&E staining of tumor-bearing brains from (E). Scale bars, 50 μm. (G) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection (10e3 FFU). Slices then underwent immunofluorescence staining for ZIKV-E (green), integrin αvβ5 (red), and DAPI (blue). Scale bars, 10 μm. (H) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection. Slices then underwent a viral RNA copy assay by qRT-PCR. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Staining, Control, CRISPR, Knock-Out, Incubation, Blocking Assay, Immunofluorescence, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

Journal: PLoS ONE

Article Title: The Response of the Prostate to Circulating Cholesterol: Activating Transcription Factor 3 (ATF3) as a Prominent Node in a Cholesterol-Sensing Network

doi: 10.1371/journal.pone.0039448

Figure Lengend Snippet: ( A ) A provisional network was generated from integration of two microarray data sets. Node color represents increases (red), no significant changes (yellow), and decreases (green) in gene expression in murine prostate tissue after cholesterol alteration as ascertained by cDNA microarray. Changes in RNA expression levels of the corresponding nodes in LNCaP cells are shown as colored node boundaries (donut shape) and the color represents increases (red), no significant change (yellow), and decreases (green) in gene expression under CDM conditions compared to control. Arrows indicate direct activation, T-shaped lines direct repression, dashed arrows indirect activation, and lines physical interaction. ( B ) Gene expression under Normo and Hyper conditions ( in vivo ). To verify in vivo microarray data obtained from SCID experiments, mRNA levels of the indicated genes were determined. GAPDH expression was used to normalize gene expression. Error bars represent SD (n = 3). ( C ) Gene expression under Control and Cholesterol-depleted conditions ( in vitro ). LNCaP cells were incubated in CDM for 0, 3 or 16 h, and mRNA levels of the indicated genes were measured by RT-PCR analysis to validate cDNA microarray data. Error bars represent SD (n = 3). * p <0.05 (Student’s t-test).

Article Snippet: LNCaP human prostate tumor cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C under 5% CO 2 .

Techniques: Generated, Microarray, Gene Expression, RNA Expression, Control, Activation Assay, In Vivo, Expressing, In Vitro, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: The Response of the Prostate to Circulating Cholesterol: Activating Transcription Factor 3 (ATF3) as a Prominent Node in a Cholesterol-Sensing Network

doi: 10.1371/journal.pone.0039448

Figure Lengend Snippet: ( A ) RT-PCR analysis in vivo . ATF3 levels are reduced in all prostatic lobes from Hyper mice, compared to those from the Normo group (AP = anterior prostate; VP = ventral prostate; DLP = dorsal prostate). ( B ) Immunoblot analysis. Immunoblot of PrEC lysates showed induction of ATF3 protein by CDM (left panel) and by β-cyclodextrin (right panel). MG132, a proteasome inhibitor, also increased ATF3 expression. ( C ) Immunofluorescence analysis. Induction of ATF3 protein by CDM in LNCaP cells as shown by IF. LNCaP cells were treated with CDM for 18 h, stained with anti-ATF3 antibody and nuclei were counterstained with DAPI (left panel: ATF3; middle panel: DAPI; right panel: overlay). ( D ) RT-PCR analysis. ATF3 mRNA levels in LNCaP cells treated with CDM were normalized to levels of GAPDH. RT-PCR analysis shows induction of ATF3 mRNA levels by CDM. ( E–F ) Promoter reporter analysis. A full-length ATF3 promoter was cloned into a luciferase reporter vector and transfected into LNCaP (D) or PrEC (E). Cells were then incubated in Control and CDM medium. ATF3 promoter activity was plotted as arbitrary units (± SD) after normalization with total protein concentration.

Article Snippet: LNCaP human prostate tumor cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C under 5% CO 2 .

Techniques: Reverse Transcription Polymerase Chain Reaction, In Vivo, Western Blot, Expressing, Immunofluorescence, Staining, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Control, Activity Assay, Protein Concentration

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 1. Mitotic spindle checkpoint genes are broadly overexpressed in human urothelial carcinoma. A, human samples of normal urothelium (N ¼ 10) and urothelial carcinoma of the bladder (N ¼ 8) were subjected to RNA microarray. A subset of 13 gene transcripts related to the mitotic spindle checkpoint, including Aurora A and B, were upregulated at least 5-fold in the urothelial carcinoma (UCC) compared with the normal urothelium. B, upregulation of these genes was validated by 2-step quantitative real-time PCR on a separate set of human samples of urothelial carcinoma (N ¼ 3) and normal urothelium (N ¼ 3). Ten of 13 genes (asterisked) showed statistical significance (t test; P < 0.05) for differential expression in urothelial carcinoma compared with normal urothelium.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Quantitative Proteomics

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 2. MLN8237 (MLN) induces cell-cycle arrest and aneuploidy of bladder cancer cell lines. A, MLN8237 inhibited expression of phospho-Aurora A-T288 at mitotic spindles. B, MLN8237 showed specificity for inhibiting Aurora A, as expression of histone-H3 and phospho-histone-H3, markers of Aurora B function, was maintained. C, PI staining with flow cytometry analysis was conducted to assess cell-cycle changes. T24, UM-UC- 3, and RT4 cells were treated with 10 nmol/L to 1 mmol/L MLN8237 for 48 hours. All 3 cell lines showed dramatic cell-cycle arrest and increase in the 4N cell population in a dose-dependent manner. T24 and UM-UC-3 cells also showed a considerable increase in aneuploidy, whereas RT4 cells did not.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Expressing, Staining, Cytometry

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 3. Cellular phenotypes of T24 and RT4 cells differ after MLN8237 (MLN) treatment. A, MLN8237 induces a dramatic increase in cell size in T24 cells but not RT4 cells. B, immunocytochemistry and fluorescence microscopy of T24 and RT4 cells revealed the formation of aberrant spindle figures upon MLN8237 treatment, with multipolar spindle apparatuses and failure of localization of chromatids to a single metaphase plate. C, T24 cells show a phenotype of increased cell size and ploidy, whereas RT4 cells do not. D, T24 cells also exhibit a subpopulation of cells exhibiting marked cytoplasmic Aurora A expression, whereas RT4 cells lacked cytoplasmic Aurora A expression. E, real-time imaging of T24 and RT4 cells treated with MLN8237 was conducted over 48 hours. T24 cells exhibited dramatic increases in cell size as a result of repeated cell-cycle progressions without separation of daughter cells. RT4 cells seemed to become arrested after one failed mitotic attempt, preventing repeated cell-cycle progressions that could otherwise result in increased ploidy.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Immunocytochemistry, Microscopy, Expressing, Imaging

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 4. MLN8237 (MLN) induces cytotoxicity and differential apoptotic processes. A, MTS assay was used to calculate IC50 values for each cell line following treatment over a range of MLN8237 concentrations for 48 hours. MLN8237 exhibited highest potency in T24 and UM-UC-3 cells (IC50 of 31 and 45 nmol/L, respectively) and lowest potency in RT4cells (IC50 of 120 nmol/L). B, Western blot analysis of T24 and RT4 cells for apoptotic markers revealed induction of p53 expression in RT4 cells, and induction of p73, but not p53, expression in T24 cells. Both cell lines showed increased expression of the apoptotic marker cleaved PARP starting 24 hours after initiation of treatment. C, annexin V staining with flow cytometry analysis of T24 and RT4 cells revealed an increased apoptotic cell fraction at 48 and 72 hours after initiation of MLN8237 treatment. D, clonogenic assays of T24 and RT4 cells showed 90% inhibition of long-term clone forming capability at 100 nmol/L MLN8237.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: MTS Assay, Western Blot, Expressing, Marker, Staining, Cytometry, Inhibition

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 6. Interactions of MLN8237 with paclitaxel and gemcitabine in vitro are schedule-dependent. MLN8237 (MLN) was combined with either paclitaxel (PTX) or gemcitabine (Gem) in T24 cells. Drugs were administered either simultaneously for 48 hours (left), or sequentially, with one drug for 48 hours, followed by washout and the other drug for 48 hours (middle and right). MTS assay was used to quantify the effect on cell viability of these combination treatments. MLN8237 showed synergistic effects with paclitaxel and gemcitabine when dosed sequentially (middle and right), and antagonistic effects when dosed simultaneously (left).

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: In Vitro, MTS Assay

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Phosphorylation of Trop-2. A, FLAG-tagged Trop-2 was immunoprecipitated (IP) from lysates of HCT116/M (M) and HCT116/WT (WT: #1) cells and subjected to SDS-PAGE, followed by immunoblotting (IB). B, FLAG-tagged Trop-2 immunoprecipitated from a lysate of HCT116/WT (#1) cells with anti-FLAG magnetic beads was treated with or without N-glycanase and then subjected to SDS-PAGE, followed by immunoblotting. C, after N-glycanase treatment, FLAG-tagged Trop-2 obtained from a lysate of HCT116/WT (#1) cells as described above was subjected to SDS-PAGE using a Phos-tag–containing gel (Phos-tag SDS-PAGE), followed by immunoblotting. The top and bottom bands represent phosphorylated and unphosphorylated FLAG-tagged Trop-2, respectively. D, schematic model of WT and mutated FLAG-tagged Trop-2. ED, ectodomain; TM, transmembrane domain; CD, cytoplasmic domain. E, after N-glycanase treatment, FLAG-tagged Trop-2 immunoprecipitated from lysates of HCT116/WT (#1 and #2), HCT116/S303A (S303A: #1 and #2), and HCT116/S322A (S322A: #1 and #2) cells as described above was subjected to Phos-tag (top) and ordinary (bottom) SDS-PAGE, followed by immunoblotting. The arrowhead indicates phosphorylated FLAG-tagged Trop-2. F, intensities of the bands in the Phos-tag gel panel in E were measured, and then the ratio of phosphorylated to total FLAG-tagged Trop-2 was calculated (means ± S.E. (error bars), n = 4).

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Phospho-proteomics, Immunoprecipitation, SDS Page, Western Blot, Magnetic Beads

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Interaction of Trop-2 with claudin-7. A, lysates (left) of HCT116/M (M) and HCT116/WT (WT: #1) cells and the immunoprecipitates (IP) (right) obtained from the lysates with anti-FLAG magnetic beads were subjected to SDS-PAGE, followed by immunoblotting (IB), and detection of claudin-7, occludin, E-cadherin, and FLAG-tagged Trop-2. β-Actin served as a loading control. B and C, HCT116 cells were stably transfected with both a Tet-ON regulator plasmid and a response plasmid–inserted FLAG-tagged Trop-2 gene. Lysates of the cells treated with or without 10 μg/ml doxycycline (Dox) for 1–6 days were subjected to SDS-PAGE, followed by immunoblotting. β-Actin served as a loading control. The intensity of the claudin-7 and β-actin bands in B was measured, and the ratio of claudin-7 to β-actin in each Dox-treated cell is indicated, with that in Dox-nontreated cells being taken as 1 (means ± S.E. (error bars), n = 3; *, p < 0.05; **, p < 0.01). D and E, immunoprecipitates from the lysates of HCT116/WT (#1), HCT116/S322A (SA: #1), and HCT116/S322E (SE: #1) cells with anti-claudin-7 antibodies were subjected to SDS-PAGE, followed by immunoblotting. Histograms show the intensity of the FLAG-tagged Trop-2 bands, each density being normalized as to that of claudin-7, and the value for HCT116/WT (#1) cells was taken as 1 (means ± S.E., n = 3; *, p < 0.05; **, p < 0.01). F, after treatment of HCT116/WT (#1) cells with PMA or 4α-PMA for 2 h, their lysates (left) and claudin-7 coimmunoprecipitated with anti-FLAG magnetic beads (right) were subjected to SDS-PAGE, followed by immunoblotting, and detection with anti-claudin-7 and anti-FLAG antibodies. β-Actin served as a loading control.

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Magnetic Beads, SDS Page, Western Blot, Control, Stable Transfection, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Expression and distribution of Trop-2 and claudin-7 in various types of Trop-2–expressing cells and in cancer tissues. A, levels of claudin-7 mRNA in HCT116/M (M), HCT116/WT (WT: #1), HCT116/S322A (SA: #1), and HCT116/S322E (SE: #1) cells were determined by DNA microarray analysis, and its level in HCT116/M cells was taken as 1. B, expression of claudin-7 protein in the four cell types described above was analyzed by SDS-PAGE, followed by immunoblotting, and that in HCT116/M cells was normalized as to β-actin, and the value for HCT116/M cells was taken as 1. β-Actin served as a loading control (means ± S.E. (error bars), n = 4; *, p < 0.05; **, p < 0.01). C, distribution of claudin-7 (green) and FLAG-tagged Trop-2 (magenta) in HCT116/M, HCT116/WT (#1), HCT116/S322A (#1), and HCT116/S322E (#1) cells was observed immunochemically. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. D, distribution of claudin-7 and Trop-2 was observed immunohistochemically in colorectal cancer tissues. Nuclei were stained with hematoxylin. Scale bars, 100 μm.

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Expressing, Microarray, SDS Page, Western Blot, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Phosphorylation of Trop-2 by PKCα and PKCδ. A, expression of PKC and PKD isoforms in HCT116/WT (WT: #1) cells was examined by DNA microarray analysis. B and C, HCT116/WT (#1) cells were treated with or without a PKC or PKD inhibitor for 1 h and subsequently with PMA or 4α-PMA for 2 h. Each cell lysate was subjected to SDS-PAGE, followed by immunoblotting. The intensities of the bands in B were determined. Histograms show the inhibitory effect of each inhibitor, the levels of phosphorylated Trop-2 being represented as the ratio of phosphorylated to total FLAG-tagged Trop-2 (p-Trop-2/FLAG), and the ratio obtained for 4α-PMA–treated cells was taken as 1 (means ± S.E. (error bars), n = 3; **, p < 0.01; NS, not significant). D and E, HCT116/WT (#1) cells were transiently transfected with PKCα siRNA (#1), PKCδ siRNA (#1), both PKCα and PKCδ siRNAs (#1), or a control siRNA and then treated with PMA or 4α-PMA for 2 h. A lysate of each cell type was subjected to SDS-PAGE, followed by immunoblotting. β-Actin served as a loading control. The intensities of the bands in D were determined, and the relative value (p-Trop-2/FLAG) was calculated as described in B and C. Histograms show the p-Trop-2/FLAG ratio in PMA-treated cells and that in 4α-PMA–treated cells (mean ± S.E., n = 3; **, p < 0.01; NS, not significant).

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Phospho-proteomics, Expressing, Microarray, SDS Page, Western Blot, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Effects of Trop-2 phosphorylation on motility of WT and mutated Trop-2–expressing HCT116 cells. A and B, migration of HCT116/M (M, n = 7), HCT116/WT (WT: #1, n = 4), HCT116/S322A (SA: #1, n = 4), and HCT116/S322E (SE: #1, n = 3) cells was evaluated by means of Transwell assays. A representative photograph of each migrated cell type is shown in A. Scale bars, 100 μm. The number of migrated cells per field was determined and is shown as a histogram in B (means ± S.E. (error bars); **, p < 0.01). C, proliferation of HCT116/M (n = 7), HCT116/WT (#1: n = 4), HCT116/S322A (#1: n = 4), and HCT116/S322E (#1: n = 3) cells was analyzed using MTT assays (means ± S.E.). D and E, migration of HCT116/WT (#1) cells treated with or without PKC inhibitors was measured as described in A and B. A representative photograph of each migrated cell is shown in D. Scale bars, 100 μm. The number of migrated cells per field was determined as described in A and B and is shown as a histogram in E (means ± S.E., n = 3; **, p < 0.01; NS, not significant). F, Proliferation of cells was examined using MTT assays as described in C (means ± S.E., n = 3). G and H, migration of HCT116/WT (#1) cells treated with both PKCα and PKCδ siRNAs (#1) or a control siRNA and then with PMA or 4α-PMA was analyzed by using Transwells. A representative photograph of each migrated cell type is shown in G. Scale bars, 100 μm. The number of migrated cells per field was determined as described in A and B and is shown as a histogram in H (means ± S.E., n = 3; **, p < 0.01; NS, not significant). I, proliferation of cells was analyzed using MTT assays as described in C (means ± S.E., n = 3).

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Phospho-proteomics, Expressing, Migration, Control

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited the proliferation and induced the apoptosis of PCa cells. The IC50 of mannose in ( a ) DU145 and ( b ) PC3 cells was determined using a CCK-8 assay. ( c ) Intracellular mannose concentration in PCa cells. Cell proliferation of ( d ) DU145 and ( e ) PC3 was assessed using growth curves, respectively. ( f ) Colony formation assays were performed and ( g ) colony numbers were counted in PCa cells. ( h ) Flow cytometric analysis was used to assess ( i ) the apoptosis rate of PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; IC50: the half-maximal inhibitory concentration; CCK-8: Cell Counting Kit-8; AAD: Aminoactinomycin D; APC: Allophycocyanin.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: CCK-8 Assay, Concentration Assay, Cell Culture, Cell Counting

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited tumor growth in a PCa xenograft model without affecting mice health. ( a ) Subcutaneous tumors from the xenograft model with DU145 cells. ( b ) Tumor growth was monitored for 30 days after mannose treatment. ( c ) The volume of tumor growth. ( d ) The weight of the tumors. ( e ) Intratumoral mannose concentration and ( f ) ATP content in subcutaneous tumors. The weights of ( g ) mice and ( h ) major metabolic organs. ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Concentration Assay, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted mitochondrial function, led to ROS overproduction, and activated Bax/Bak in PCa cells. ( a ) JC-1 staining and ( b ) rhodamine 123 staining were used to assess the MMP in DU145 and PC3 cells. ( c ) The ATP content in cells. ( d ) Mitochondrial ROS and ( e ) cellular ROS levels in cells. ( f and g ) The protein expression of Bax and Bak in cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine; Bax: BCL2-associated X; Bak: BCL2-antagonist/killer 1; ROS: reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine triphosphate; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Expressing, Cell Culture, Membrane

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted the balance of mitochondrial dynamics in PCa cells. ( a ) Mitochondria stained with MitoTracker Red were observed by confocal microscopy. ( b ) Mitochondrial structure under a transmission electron microscopy, and the mitochondrial cross-sectional area was quantified. ( c ) The protein expression of FIS1 in cells. Upregulated ( d ) FIS1 and ( e ) increased ATP content in PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. ATP: adenosine triphosphate; FIS1: fission, mitochondrial 1; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Expressing, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Downregulation of MPI expression enhances the anticancer effect of mannose. The expression of MPI in human prostate cancer tissues and its prognostic value. ( a and b ) The expression of MPI was silenced by siRNA-MPI and verified by western blotting. siRNA-MPI with different base sequences including si-1, si-2 and si-3 were used for downregulating the MPI expression in PCa cells. According to the degree of down-regulation of MPI protein, si-3 and si-2 with the best interference effect were applied to DU145 and PC3 cells, respectively. ( c ) Intracellular mannose concentration and ( d ) ATP content in cells. ( e ) Growth curves and ( f ) colony formation assays of cells. ( g ) The IHC scores for MPI expression in PCa tissues. ( h ) Kaplan–Meier curves of BCR-free survival and ( i ) overall survival for the low and high MPI expression groups of patients in the TCGA-PRAD dataset. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. MPI: mannose phosphate isomerase; si: siRNA, small interfering RNA; IHC: immunohistochemistry; PCa: prostate cancer; BCR: biochemical recurrence; TCGA-PRAD: The Cancer Genome Atlas-Prostate Adenocarcinoma; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TMA: tissue microarray; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Expressing, Western Blot, Concentration Assay, Cell Culture, Small Interfering RNA, Immunohistochemistry, Microarray

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Effects of hypoxia on cell markers, morphology, and migration (A) Expression in breast cancer MCF7 and MDA-MB-231 cells of E-cadherin (epithelial marker), fibronectin (epithelial marker), HIF-1α (hypoxia marker), β-catenin, and GLUT1. Cells were cultured at 37°C in 5% CO 2 atmosphere for normoxic treatment, and in 1% O 2 / 5% CO 2 / 94% N 2 atmosphere for hypoxic treatment. Cells were harvested, lysed in T-PER Reagent, and protein content was determined by BCA assay. Western blotting was performed as described in M&M. (B) Morphological changes under normoxic and hypoxic conditions. Cells (2 × 10 5 per well) were grown in 6-well plates for 24 h under the two conditions. Photos were taken by phase-contrast microscopy at 200× magnification. (C) Cell migration assessed by wound assay. Cell monolayers under the two conditions were scratched with pipette tip. Cells were washed with ice-cold 1× PBS and cultured in serum-free medium. Pictures of wounds were taken at 0 and 24 h by phase-contrast microscopy (100× magnification).

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration, Expressing, Marker, Cell Culture, BIA-KA, Western Blot, Microscopy, Transferring

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MALDI-TOF-MS spectra of N-glycans from MCF7 cells MCF7 cells were cultured in 10-cm dishes under normoxic and hypoxic conditions, and N-glycans were separated and desalted as described in M&M. Lyophilized N-glycans were dissolved in MW, and an aliquot of mixture with DHB solution was spotted on MTP AnchorChip sample target and air-dried. MALTI-TOF-MS was performed in positive-ion mode. Experiments were performed in biological triplicate, and representative N-glycan spectra are shown. Peaks (signal-to-noise ratio > 5) were selected for relative proportion analysis. Detailed structures were analyzed using the GlycoWorkbench program. Proposed structures are indicated by m/z value.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Cell Culture, Glycoproteomics

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Relative proportions of various types of N-glycans in MCF7 and MDA-MB-231 cells under normoxia and hypoxia.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques:

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Variation of fine glycan structures detected by lectin microarray analysis (A) Variation of levels of glycans from MCF7 (upper) and MDA-MB-231 (lower) cells, detected by 37 lectins, is presented as a heatmap. Lectin microarray analysis was performed as described as M&M. Red: fluorescence signal activation. Green: signal inhibition. Black: missing data. (B) Altered glycan levels evaluated by lectin histochemistry. Four lectins (Con A, MAL-I, LCA, PHA-E) were applied, and lectin histochemistry was performed as described in M&M. Signals are shown from merge images of Cy3-conjugated lectins and DAPI staining of nuclei in MCF7 (left) and MDA-MB-231 (right) under normoxic and hypoxic conditions (60× magnification). (C) Expression in MCF7 and MDA-MB-231 cells of HIF-1α, MGAT3, and tubulin.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Glycoproteomics, Microarray, Fluorescence, Activation Assay, Inhibition, Staining, Expressing

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Differential glycopatterns in normoxia- vs. hypoxia-treated MCF7 cells revealed by lectin microarray analysis.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Microarray

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MGAT3 overexpression suppresses hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-transfected MCF7 cells. Cells were stably transduced with a GFP-marked lentivirus carrying mock gene or MGAT3 gene, harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M using anti-MGAT3 and anti-GFP antibody. (B) Levels of bisecting GlcNAc structures in mock- and MGAT3-transfectants. Whole cell lysates of the two transfectants were subjected to PHA-E lectin blotting as described in M&M. (C) Proliferation of transfectant cells. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (E) Colony formation ability. The two transfectants (2500 cells each) were cultured in 6-cm dishes for 1–2 week, fixed, stained with crystal violet solution, and photographed. Acetic acid was added to dissolve crystal violet, and OD 595 was determined (D) . * p < 0.05; *** p < 0.001. (F) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (H) . * p < 0.05. (G) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure , harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Expressing, Transfection, Stable Transfection, Transduction, Western Blot, Cell Culture, MTS Assay, Staining, Migration, BIA-KA

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MGAT3 knockdown promotes hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-shRNA-transfected MCF7 cells. Cells were stably transduced with lentivirus carrying anti-MGAT3 shRNAs (MCF7/shMGAT3-1/2) or shNC (MCF7/mock), harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M. (B) Cell proliferation. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (C) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (E) . * p < 0.05; ** p < 0.01. (D) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, β-catenin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure , harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Knockdown, Expressing, shRNA, Transfection, Stable Transfection, Transduction, Western Blot, Cell Culture, MTS Assay, Migration, BIA-KA

Journal: Nature communications

Article Title: RNF208, an estrogen-inducible E3 ligase, targets soluble Vimentin to suppress metastasis in triple-negative breast cancers.

doi: 10.1038/s41467-019-13852-5

Figure Lengend Snippet: Fig. 1 RNF208 is significantly underexpressed in highly metastatic breast cancer cells. a Volcano plot of RNF208 expression in luminal subtype (MCF-7, T47D, and ZR-75B) and TNBC (MDA-MB-231 and Hs578T) cells using RNA sequencing (GSE100878). b RT-PCR and immunoblot analysis of RNF208 expression in breast cancer cells. c Scatter plots of RNF208 expression in 52 breast cancer cells from public microarray datasets (GSE41313) (Luminal N = 29 and TNBC N = 23 cells). d, e Heat map and box plots showing the expression levels of RNF208, ESR1, and ERBB2 across the breast cancer subtypes using Genomic Data Commons (GDC). (Luminal A N = 335, Luminal B N = 88, HER2 N = 23 and TNBC N = 55 patients). f Comparison of RNF208 expression in breast cancer subtypes using published microarray datasets (GSE2034) (Luminal N = 154 and TNBC N = 55 patients). g Representative immunohistochemical (IHC) staining of RNF208 protein expression classified in luminal and basal breast cancer patient tissues. Original magnification ×100. Scale bar, 50 μm. h Real-time quantitative RT-PCR of RNF208 expression in luminal subtype tissues (N = 6) and TNBC tissues (N = 6); 18S rRNA was used as an internal control. i Kaplan−Meier analysis showing relapse-free survival depending on RNF208 expression levels from public meta-analysis data (N = 3951). P values were calculated using a log-rank test. All P values were calculated by unpaired two-tailed Student’s t tests (c, e, f, and h). These data represent the mean ± S.D. Source data for (c, f, h) is available in Source Data file. Unprocessed original scans of blots and gels in (b) are shown in Supplementary Fig. 13.

Article Snippet: The human breast cancer cell lines MCF-7, T47D, Hs578T, and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC); ZR-75B, cloned from the ZR-75-1 cell lines26, was obtained from the National Cancer Institute (NCI)/NIH.

Techniques: Expressing, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Microarray, Comparison, Immunohistochemical staining, Immunohistochemistry, Quantitative RT-PCR, Control, Two Tailed Test

Journal: Nature communications

Article Title: RNF208, an estrogen-inducible E3 ligase, targets soluble Vimentin to suppress metastasis in triple-negative breast cancers.

doi: 10.1038/s41467-019-13852-5

Figure Lengend Snippet: Fig. 2 RNF208 expression was transcriptionally activated by ERα in luminal breast cancer cells. a Comparison of RNF208 expression between ERα- positive and -negative breast cancer samples in published microarray datasets (GSE2034 and GSE5460). b RT-PCR (top) and immunoblot (bottom) analysis of ERα target genes (FOXOM1, GREB1, ESR1) and RNF208 expression in EtOH- or E2-treated ERα-positive breast cancer cells. 10 nM or 20 nM of E2 was treated in MCF-7 or T47D cell, respectively and E2 was treated for 24 h for RT-PCR and 48 h for immunoblot analysis. GAPDH and β-actin were used as internal controls. c Real-time quantitative RT-PCR (qRT-PCR) of RNF208 expression in ESR1-knockdown T47D cells upon E2 treatment. d Illustration of luciferase reporters including ERα-binding sites in the RNF208 promoter sequences. e T47D cells were transfected with various deletion constructs of the RNF208 promoter and then treated with or without E2 for 24 h. After E2 treatment, cells were assayed for luciferase activity. f ChIP analysis showing the recruitment of ERα to the human RNF208 promoter in E2-treated T47D cells. Precipitation was conducted with antinormal IgG or anti-ERα antibodies. g T47D cells were transfected with pGL3 control, RNF208 promoter, or its mutant (mtR4) plasmids containing mutated estrogen-responsive element site (CACC sequence replaced by GAAA) and then subjected to luciferase assays. h RT-PCR and immunoblot analysis showing RNF208 expression upon E2 treatment for 24 h (mRNA) or 48 h (protein), respectively, in control or ERE knockout MCF-7 cells. i Immunoblot analysis of ERα with 5-aza-dC treatment in TNBC cells. Hs578T and MDA-MB-231 cells were treated with 10 μM 5-aza-dC for 96 h and β-actin was used for normalization. j TNBC cells were transiently transfected with 20 nM of control siRNA or ESR1 siRNA and then treated with 10 μM 5-aza-dC for 96 h. Cell lysates were immunoblotted with the indicated antibodies. All P values were calculated by unpaired two-tailed Student’s t tests (e, g). These data represent the mean ± SD of three independent experiments. Source data for (c, e, g, h) are available in Source Data file. Unprocessed original scans of blots and gels in (b, f, h−j) are shown in Supplementary Fig. 13.

Article Snippet: The human breast cancer cell lines MCF-7, T47D, Hs578T, and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC); ZR-75B, cloned from the ZR-75-1 cell lines26, was obtained from the National Cancer Institute (NCI)/NIH.

Techniques: Expressing, Comparison, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Knockdown, Luciferase, Binding Assay, Transfection, Construct, Activity Assay, Control, Mutagenesis, Sequencing, Knock-Out, Two Tailed Test

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 2. Loss of ARG1 and AGMAT enhances liver tumor formation (A) Immunoblots of arginine-to-polyamine-converting enzymes (ARG1 and AGMAT) and polyamine metabolism enzymes (ODC, SRM, SMS, SAT1, PAOX, and SMOX) in Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control (same samples were used as in Figure 1E). n = 4 (Ctrl), n = 8 (L-dKO). (B) Total polyamine content in Ctrl liver and L-dKO tumor tissues. n = 6. (C) Relative 3H-putrescine uptake into Ctrl liver and L-dKO tumor tissues. n = 8. (D) Immunohistochemistry of Ctrl and L-dKO liver tissues stained for ARG1 or AGMAT. NT, adjacent non-tumor tissue; T, tumor. (E) Representative images of livers from L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. (F) Number of macroscopic tumors per liver of L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 9–10. (G) Arginine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 4–10. *p < 0.05, **p < 0.01. ***p < 0.001, ****p < 0.0001 by unpaired t test (B and C) and one-way ANOVA (F and G).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER L-Arginine-coupled agarose beads gbiosciences Cat#GENO786-1361 L-leucine-coupled agarose beads gbiosciences Cat#GENO786-1370 Critical commercial assays Total Polyamine Assay Kit BioVision Cat#K475 L-arginine ELISA kit MyBiosource Cat#MBS728648-96 Nano-Glo dual luciferase reporter assay kit Promega Cat#N1610 Deposited data HCC patients’ RNA sequencing data Ng et al., 202250 EGAS00001005073, EGAS00001005074 HCC patients’ proteome data Ng et al., 202250 PRIDE (PXD025705, PXD025836) Mouse RNA sequencing data Dimitrakopoulos et al., 202125 SRP156216 Mouse proteome data Dimitrakopoulos et al., 202125 https://github.com/cbgethz/netics/tree/ master/mouse_data Mouse and HCC patients’ metabolomics This paper MASSIVE: MSV000092406 RNA sequencing data of ARG1/AGMAT-expressing (and control) and RBM39-depleted (and control) SNU-449 cells This paper GEO: PRJNA940402 Mouse, HCC patients, and SNU-449 proteome data of pulldowns using Leu- and Arg-coupled agarose beads This paper MASSIVE: MSV000091516 Experimental models: Cell lines SNU-182 Diego Calvisi (University of Sassari, Italy) N/A SNU-449 Diego Calvisi (University of Sassari, Italy) N/A HLE Diego Calvisi (University of Sassari, Italy) N/A PLC Diego Calvisi (University of Sassari, Italy) N/A Hep40 Diego Calvisi (University of Sassari, Italy) N/A Hep3B Diego Calvisi (University of Sassari, Italy) N/A Huh6 Diego Calvisi (University of Sassari, Italy) N/A Huh7 Diego Calvisi (University of Sassari, Italy) N/A HepG2 Diego Calvisi (University of Sassari, Italy) N/A SNU-475 Gerhard Christofori (University of Basel, Switzerland) N/A SNU-423 Gerhard Christofori (University of Basel, Switzerland) N/A Huh1 Gerhard Christofori (University of Basel, Switzerland) N/A HEK293 ATCC CRL-1573 HEK239T ATCC CRL-3216 SNU-449 control This paper N/A SNU-449 ARG1 This paper N/A SNU-449 AGMAT This paper N/A SNU-449 ARG1/AGMAT This paper N/A SNU-449 ASS1-FLAG This paper N/A SNU-449 ARG1/AGMAT +ASS1 This paper N/A SNU-449 ARG1/AGMAT +3xHA-ASS1 This paper N/A Huh7 sgCtrl This paper N/A Huh7 sgARG1 This paper N/A Huh7 sgAGMAT This paper N/A SNU-449 ARG1/AGMAT +control (for ASNS) This paper N/A (Continued on next page) ll OPEN ACCESS Cell 186, 5068–5083.e1–e12, November 9, 2023 e2 Article

Techniques: Western Blot, Control, Immunohistochemistry, Staining, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 3. ARG1/AGMAT determine metabolic gene expression via arginine (A) Immunoblots of SNU-449 cells upon stable expression of ARG1 and/or AGMAT. Actin serves as loading control. (B) Representative clonogenic growth assay of control, ARG1-, and/or AGMAT-expressing SNU-449 cells grown in arginine-restricted medium. (C) Relative clonogenic growth of control, ARG1-, and/or AGMAT- expressing SNU-449 cells. N = 6. (D) Arginine content of control, ARG1-, and/or AGMAT-expressing SNU-449 cells. N = 4. (E) PCA analysis of RNA-seq data of control and ARG1/AGMAT-expressing SNU-449 cells. (F) Heatmap of a subset of differentially expressed metabolic genes in ARG1/AGMAT-expressing compared to control SNU-449 cells (log2 fold-change). (G) mRNA levels of ASNS, PSAT1, PSPH, GLSK, GLUT3, HK2, NNMT, and AOC3 in control and ARG1/AGMAT-expressing SNU-449 cells. N = 5–7. (H) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ARG1/AGMAT-expressing SNU-449 cells. Calnexin serves as loading control. (I) Immunoblots of ASNS, PSAT, PSPH, and NNMT of Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control. n = 4 (Ctrl), n = 8 (L-dKO). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (C and D) and unpaired t test (G).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER L-Arginine-coupled agarose beads gbiosciences Cat#GENO786-1361 L-leucine-coupled agarose beads gbiosciences Cat#GENO786-1370 Critical commercial assays Total Polyamine Assay Kit BioVision Cat#K475 L-arginine ELISA kit MyBiosource Cat#MBS728648-96 Nano-Glo dual luciferase reporter assay kit Promega Cat#N1610 Deposited data HCC patients’ RNA sequencing data Ng et al., 202250 EGAS00001005073, EGAS00001005074 HCC patients’ proteome data Ng et al., 202250 PRIDE (PXD025705, PXD025836) Mouse RNA sequencing data Dimitrakopoulos et al., 202125 SRP156216 Mouse proteome data Dimitrakopoulos et al., 202125 https://github.com/cbgethz/netics/tree/ master/mouse_data Mouse and HCC patients’ metabolomics This paper MASSIVE: MSV000092406 RNA sequencing data of ARG1/AGMAT-expressing (and control) and RBM39-depleted (and control) SNU-449 cells This paper GEO: PRJNA940402 Mouse, HCC patients, and SNU-449 proteome data of pulldowns using Leu- and Arg-coupled agarose beads This paper MASSIVE: MSV000091516 Experimental models: Cell lines SNU-182 Diego Calvisi (University of Sassari, Italy) N/A SNU-449 Diego Calvisi (University of Sassari, Italy) N/A HLE Diego Calvisi (University of Sassari, Italy) N/A PLC Diego Calvisi (University of Sassari, Italy) N/A Hep40 Diego Calvisi (University of Sassari, Italy) N/A Hep3B Diego Calvisi (University of Sassari, Italy) N/A Huh6 Diego Calvisi (University of Sassari, Italy) N/A Huh7 Diego Calvisi (University of Sassari, Italy) N/A HepG2 Diego Calvisi (University of Sassari, Italy) N/A SNU-475 Gerhard Christofori (University of Basel, Switzerland) N/A SNU-423 Gerhard Christofori (University of Basel, Switzerland) N/A Huh1 Gerhard Christofori (University of Basel, Switzerland) N/A HEK293 ATCC CRL-1573 HEK239T ATCC CRL-3216 SNU-449 control This paper N/A SNU-449 ARG1 This paper N/A SNU-449 AGMAT This paper N/A SNU-449 ARG1/AGMAT This paper N/A SNU-449 ASS1-FLAG This paper N/A SNU-449 ARG1/AGMAT +ASS1 This paper N/A SNU-449 ARG1/AGMAT +3xHA-ASS1 This paper N/A Huh7 sgCtrl This paper N/A Huh7 sgARG1 This paper N/A Huh7 sgAGMAT This paper N/A SNU-449 ARG1/AGMAT +control (for ASNS) This paper N/A (Continued on next page) ll OPEN ACCESS Cell 186, 5068–5083.e1–e12, November 9, 2023 e2 Article

Techniques: Gene Expression, Western Blot, Expressing, Control, Growth Assay, RNA Sequencing

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 4. ASNS promotes arginine uptake in liver cancer (A) Relative 3H-arginine uptake in control and ARG1/AGMAT-expressing SNU-449 cells with or without pre-loading with asparagine (Asn) or glutamine (Gln). N = 5–6. (B) Immunoblots of ARG1/AGMAT-expressing SNU-449 cells upon stable expression of ASNS or control. Calnexin serves as loading control. (C) Relative 3H-arginine uptake in control and ASNS-expressing SNU-449 ARG1/AGMAT-ex- pressing cells. N = 5. (D) Representative clonogenic growth assay of control and ASNS-expressing SNU-449 ARG1/ AGMAT-expressing cells grown in arginine- restricted medium. (E) mRNA levels of PSAT1, PSPH, GLSK, GLUT3, HK2, NNMT, and AOC3 in control and ASNS- expressing SNU-449 ARG1/AGMAT-expressing cells. N = 6–8. (F) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ASNS-expressing SNU-449 ARG1/ AGMAT-expressing cells. Calnexin serves as loading control. (G) mRNA levels of Asns in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV- shCtrl or AAV-shAsns. n = 6–7. (H) Number of macroscopic tumors per liver in L-dKO mice injected with AAV-shCtrl or AAV- shAsns. n = 7. (I) Arginine content in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-shCtrl or AAV-shAsns. n = 4–6. n.s. = not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t test (A, C, E, G, and H) and one-way ANOVA (I).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER L-Arginine-coupled agarose beads gbiosciences Cat#GENO786-1361 L-leucine-coupled agarose beads gbiosciences Cat#GENO786-1370 Critical commercial assays Total Polyamine Assay Kit BioVision Cat#K475 L-arginine ELISA kit MyBiosource Cat#MBS728648-96 Nano-Glo dual luciferase reporter assay kit Promega Cat#N1610 Deposited data HCC patients’ RNA sequencing data Ng et al., 202250 EGAS00001005073, EGAS00001005074 HCC patients’ proteome data Ng et al., 202250 PRIDE (PXD025705, PXD025836) Mouse RNA sequencing data Dimitrakopoulos et al., 202125 SRP156216 Mouse proteome data Dimitrakopoulos et al., 202125 https://github.com/cbgethz/netics/tree/ master/mouse_data Mouse and HCC patients’ metabolomics This paper MASSIVE: MSV000092406 RNA sequencing data of ARG1/AGMAT-expressing (and control) and RBM39-depleted (and control) SNU-449 cells This paper GEO: PRJNA940402 Mouse, HCC patients, and SNU-449 proteome data of pulldowns using Leu- and Arg-coupled agarose beads This paper MASSIVE: MSV000091516 Experimental models: Cell lines SNU-182 Diego Calvisi (University of Sassari, Italy) N/A SNU-449 Diego Calvisi (University of Sassari, Italy) N/A HLE Diego Calvisi (University of Sassari, Italy) N/A PLC Diego Calvisi (University of Sassari, Italy) N/A Hep40 Diego Calvisi (University of Sassari, Italy) N/A Hep3B Diego Calvisi (University of Sassari, Italy) N/A Huh6 Diego Calvisi (University of Sassari, Italy) N/A Huh7 Diego Calvisi (University of Sassari, Italy) N/A HepG2 Diego Calvisi (University of Sassari, Italy) N/A SNU-475 Gerhard Christofori (University of Basel, Switzerland) N/A SNU-423 Gerhard Christofori (University of Basel, Switzerland) N/A Huh1 Gerhard Christofori (University of Basel, Switzerland) N/A HEK293 ATCC CRL-1573 HEK239T ATCC CRL-3216 SNU-449 control This paper N/A SNU-449 ARG1 This paper N/A SNU-449 AGMAT This paper N/A SNU-449 ARG1/AGMAT This paper N/A SNU-449 ASS1-FLAG This paper N/A SNU-449 ARG1/AGMAT +ASS1 This paper N/A SNU-449 ARG1/AGMAT +3xHA-ASS1 This paper N/A Huh7 sgCtrl This paper N/A Huh7 sgARG1 This paper N/A Huh7 sgAGMAT This paper N/A SNU-449 ARG1/AGMAT +control (for ASNS) This paper N/A (Continued on next page) ll OPEN ACCESS Cell 186, 5068–5083.e1–e12, November 9, 2023 e2 Article

Techniques: Control, Expressing, Western Blot, Growth Assay, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 7. ARG1, AGMAT, arginine, and RBM39 in human HCC patients (A) Schematic representation of arginine and polyamine metabolism in HCC patients. Boxes below enzymes indicate changes in mRNA (left box) and protein (right box) levels in human HCC tumors (T) compared to paired non-tumor (NT) biopsies, respectively. Color coding according to level of log2 fold-change as indicated. ‘‘?’’ indicates unknown identity. Tumor aggressiveness is indicated by Edmondson-Steiner grade low (Edm. low, grade I and II) and high (Edm. high, grade III and IV). n = 73 (Edm. low) and n = 49 (Edm. high) for mRNA; n = 30 (Edm. low) and n = 21 (Edm. high) for protein. (B) Immunoblots of ARG1, AGMAT, RBM39, and ASNS in paired non-tumor (NT) and tumor (T) tissues of five HCC patients. Calnexin serves as loading control. (C) Tissue microarray for ARG1 and AGMAT. ARG1, normal liver n = 58, HCC n = 160; AGMAT, normal liver n = 49, HCC n = 142. (D) Representative IHC of ARG1 and AGMAT of an HCC patient (from C). Non-tumor, NT; tumor, T.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER L-Arginine-coupled agarose beads gbiosciences Cat#GENO786-1361 L-leucine-coupled agarose beads gbiosciences Cat#GENO786-1370 Critical commercial assays Total Polyamine Assay Kit BioVision Cat#K475 L-arginine ELISA kit MyBiosource Cat#MBS728648-96 Nano-Glo dual luciferase reporter assay kit Promega Cat#N1610 Deposited data HCC patients’ RNA sequencing data Ng et al., 202250 EGAS00001005073, EGAS00001005074 HCC patients’ proteome data Ng et al., 202250 PRIDE (PXD025705, PXD025836) Mouse RNA sequencing data Dimitrakopoulos et al., 202125 SRP156216 Mouse proteome data Dimitrakopoulos et al., 202125 https://github.com/cbgethz/netics/tree/ master/mouse_data Mouse and HCC patients’ metabolomics This paper MASSIVE: MSV000092406 RNA sequencing data of ARG1/AGMAT-expressing (and control) and RBM39-depleted (and control) SNU-449 cells This paper GEO: PRJNA940402 Mouse, HCC patients, and SNU-449 proteome data of pulldowns using Leu- and Arg-coupled agarose beads This paper MASSIVE: MSV000091516 Experimental models: Cell lines SNU-182 Diego Calvisi (University of Sassari, Italy) N/A SNU-449 Diego Calvisi (University of Sassari, Italy) N/A HLE Diego Calvisi (University of Sassari, Italy) N/A PLC Diego Calvisi (University of Sassari, Italy) N/A Hep40 Diego Calvisi (University of Sassari, Italy) N/A Hep3B Diego Calvisi (University of Sassari, Italy) N/A Huh6 Diego Calvisi (University of Sassari, Italy) N/A Huh7 Diego Calvisi (University of Sassari, Italy) N/A HepG2 Diego Calvisi (University of Sassari, Italy) N/A SNU-475 Gerhard Christofori (University of Basel, Switzerland) N/A SNU-423 Gerhard Christofori (University of Basel, Switzerland) N/A Huh1 Gerhard Christofori (University of Basel, Switzerland) N/A HEK293 ATCC CRL-1573 HEK239T ATCC CRL-3216 SNU-449 control This paper N/A SNU-449 ARG1 This paper N/A SNU-449 AGMAT This paper N/A SNU-449 ARG1/AGMAT This paper N/A SNU-449 ASS1-FLAG This paper N/A SNU-449 ARG1/AGMAT +ASS1 This paper N/A SNU-449 ARG1/AGMAT +3xHA-ASS1 This paper N/A Huh7 sgCtrl This paper N/A Huh7 sgARG1 This paper N/A Huh7 sgAGMAT This paper N/A SNU-449 ARG1/AGMAT +control (for ASNS) This paper N/A (Continued on next page) ll OPEN ACCESS Cell 186, 5068–5083.e1–e12, November 9, 2023 e2 Article

Techniques: Western Blot, Control, Microarray

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy.

doi: 10.1073/pnas.0911220107

Figure Lengend Snippet: Fig. 1. LRP6 expression is frequently up-regulated in a subset of human breast cancer tissues and cell lines. (A) Breast cancer TissueScan Real-Time qPCR array, containing seven normal/Stage 0 cDNAs and 41 human breast cancer cDNAs, was analyzed for LRP6 expression by real-time PCR. Averages of relative LRP6 expression from three independent plates are plotted with clinical status indicated. LRP6 mRNA levels are markedly up-regulated in a subset of human breast cancer tissues. #Samples with elevated HER2 tran- scripts. (B and C) Breast cancer tissue microarray was used for IHC staining of LRP6. (B) Representatives of LRP6 staining in normal and malignant breast tissue are shown. LRP6 antibody (C-term T1546, Abgent), which specifically recognizes human LRP6, was used for IHC staining. (C) The quantification of LRP6 IHC staining was determined from three independent experiments. Staining intensity was scored as absent (0), weak (1), moderate (2), or strong (3). Four observations were made on each slide by independent inves- tigators, and a mean score was recorded. (D) Expression of LRP6 in human mammary epithelial cell (MCF-10A) and indicated breast cancer cell lines analyzed by Western blot analysis. *P < 0.05; **P < 0.01.

Article Snippet: MDA-MB-231, MDA-MB-157, SKBR3, MCF-7, MDA-MB-435s, MDA-MB-361, HCC1187, HCC1143, HCC1806, HCC38, HCC1937, HCC1395, T-47D, and CAMA1 breast cancer cell lines and MCF-10A nontransformed cells were all from the American Type Culture Collection (ATCC) and grown according to ATCC recommendations.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Microarray, Immunohistochemistry, Staining, Western Blot