mda mb 453 ar v7 geo database gse244283 spatial genomics geo database gse245202 mda mb 453 xenograft geo database gse244283  (ATCC)


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    ATCC mda mb 453 ar v7 geo database gse244283 spatial genomics geo database gse245202 mda mb 453 xenograft geo database gse244283
    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and <t>AR-V7</t> probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).
    Mda Mb 453 Ar V7 Geo Database Gse244283 Spatial Genomics Geo Database Gse245202 Mda Mb 453 Xenograft Geo Database Gse244283, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype"

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    Journal: Cell reports

    doi: 10.1016/j.celrep.2023.113461

    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and AR-V7 probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).
    Figure Legend Snippet: A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and AR-V7 probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Immunohistochemistry, Ligand Binding Assay, Activation Assay, Variant Assay

    TNBC cell lines were stably transfected with AR-V7 using lentivirus. A. BrdU assay of control and AR-V7 lentivirus transfected TNBC cell lines MDA-MB-453 (453), MDA-MB-231 (231), BT549, and MFM223. Cells were plated in growth medium and BrDU assay was performed after 72 hours (n=4/group). B. Scratch assay in MDA-MB-453 and MDA-MB-453-V7. Cells were plated in growth medium and imaged at the start and after 72 hours, and the gap closure measured by imaging software (n=4/group). C. Ki67 staining of TNBC patient specimens (23 (AR-positive (AR+)), and 11 (AR and AR-SV -positive (AR+/AR-SV+)). D and E. RNA sequencing was performed with 453 and 453-V7 cell lines and AR+ and AR+/AR-SV+ patient specimens. Gene set from the Molecular Signatures Database is reported in 453 cells compared to 453-V7 cells and AR+ patient specimens compared to AR+/AR-SV+ patient specimens. Values are expressed as mean -/+ SEM. Experiments in panels A-C were reproduced at least three times and representative experiment is shown. * p<0.05, ** p<0.01, ****p<0.00001 (t-test).
    Figure Legend Snippet: TNBC cell lines were stably transfected with AR-V7 using lentivirus. A. BrdU assay of control and AR-V7 lentivirus transfected TNBC cell lines MDA-MB-453 (453), MDA-MB-231 (231), BT549, and MFM223. Cells were plated in growth medium and BrDU assay was performed after 72 hours (n=4/group). B. Scratch assay in MDA-MB-453 and MDA-MB-453-V7. Cells were plated in growth medium and imaged at the start and after 72 hours, and the gap closure measured by imaging software (n=4/group). C. Ki67 staining of TNBC patient specimens (23 (AR-positive (AR+)), and 11 (AR and AR-SV -positive (AR+/AR-SV+)). D and E. RNA sequencing was performed with 453 and 453-V7 cell lines and AR+ and AR+/AR-SV+ patient specimens. Gene set from the Molecular Signatures Database is reported in 453 cells compared to 453-V7 cells and AR+ patient specimens compared to AR+/AR-SV+ patient specimens. Values are expressed as mean -/+ SEM. Experiments in panels A-C were reproduced at least three times and representative experiment is shown. * p<0.05, ** p<0.01, ****p<0.00001 (t-test).

    Techniques Used: Stable Transfection, Transfection, BrdU Staining, Wound Healing Assay, Imaging, Software, Staining, RNA Sequencing Assay

    A. Structure of UT-105. B. UT-105 inhibits wildtype and mutant AR transactivation. AR or AR F876L (50 ng), 0.25 μg GRE-LUC, and 10 ng CMV-renilla-LUC were transfected into COS7 cells. Cells were treated 24 hours after transfection with a dose response of UT-105 or enzalutamide in the presence of 0.1 nM R1881, and luciferase assay was performed 24 hours after treatment. Firefly luciferase values were normalized to renilla luciferase. Numbers provided in bracket are IC50 values. C. UT-105 degrades AR. LNCaP PCa cells were maintained in charcoal-stripped FBS-containing medium for 2 days before treating with UT-105 in the presence of 0.1 nM R1881. Cells were harvested 24 hours after treatment, and Western blot for AR and GAPDH was performed. D. Top. UT-105 binds to AR-NTD. Recombinant purified AR NTD was incubated with DMSO or 10 μM UT-105 overnight at 4°C. SYPRO orange dye was added to the mixture and a PCR was performed with increasing temperature. SYPRO orange signal was monitored. Bottom. UT-105 stabilized purified AF-1 and AR-V7 recombinant protein. Recombinant purified AF-1 or AR-V7 protein (5 ng) were incubated at room temperature for 4 hours with DMSO or 100 μM of UT-105 or UT-34. Proteins were fractionated on an SDS-PAGE and Western blot was performed with AR antibody (AR-441). E. Illustration depicting the binding regions of UT-105 and enzalutamide. F. UT-105 irreversibly inhibits AR. Transactivation assay was performed with AR with a dose response of R1881 in the presence of 3 and 10 μM UT-105 as indicated in panel B. G. UT-105 inhibits AR-target genes. MDA-MB-453 cells in charcoal stripped serum-containing medium were treated for 16–20 hours. RNA was extracted and expression of FKBP5, TMPRSS2, and STEAP4 was quantified by real-time PCR and normalized to GAPDH (n=4/group). * p<0.05 (one-way ANOVA) H. RNA-seq. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium for 2 days were treated with vehicle or 3 μM UT-105 in the presence of 0.1 nM R1881 for 20 hours (n=3/group). Cells were harvested, RNA extracted, and sequenced. Heatmap of global gene expression changes, top GSEA pathways enriched, and heatmap and bar graphs of AR signaling pathway are represented. I. UT-105 inhibits proliferation of TNBC cells. MDA-MB-453 cells plated in charcoal stripped serum-containing medium and treated for seven days with medium change and retreatment after day 3 (n=3/group). Sulforhodamine B (SRB) colorimetric assay was performed to measure cell viability. J. UT-105 inhibits clonogenicity. TNBC cells stably expressing AR-V7 were plated in 6-well plates and treated with 10 μM of the indicated compounds for two weeks. Colonies were imaged and the number of colonies formed was counted using an imaging software (n=4/group). Panels B-D and F, G, I-J are representatives of at least three independent replicates. * p<0.05, ** p<0.01 (one-way ANOVA). Enza- enzalutamide; V7- AR-V7 splice variant. Values are expressed as mean -/+ SEM.
    Figure Legend Snippet: A. Structure of UT-105. B. UT-105 inhibits wildtype and mutant AR transactivation. AR or AR F876L (50 ng), 0.25 μg GRE-LUC, and 10 ng CMV-renilla-LUC were transfected into COS7 cells. Cells were treated 24 hours after transfection with a dose response of UT-105 or enzalutamide in the presence of 0.1 nM R1881, and luciferase assay was performed 24 hours after treatment. Firefly luciferase values were normalized to renilla luciferase. Numbers provided in bracket are IC50 values. C. UT-105 degrades AR. LNCaP PCa cells were maintained in charcoal-stripped FBS-containing medium for 2 days before treating with UT-105 in the presence of 0.1 nM R1881. Cells were harvested 24 hours after treatment, and Western blot for AR and GAPDH was performed. D. Top. UT-105 binds to AR-NTD. Recombinant purified AR NTD was incubated with DMSO or 10 μM UT-105 overnight at 4°C. SYPRO orange dye was added to the mixture and a PCR was performed with increasing temperature. SYPRO orange signal was monitored. Bottom. UT-105 stabilized purified AF-1 and AR-V7 recombinant protein. Recombinant purified AF-1 or AR-V7 protein (5 ng) were incubated at room temperature for 4 hours with DMSO or 100 μM of UT-105 or UT-34. Proteins were fractionated on an SDS-PAGE and Western blot was performed with AR antibody (AR-441). E. Illustration depicting the binding regions of UT-105 and enzalutamide. F. UT-105 irreversibly inhibits AR. Transactivation assay was performed with AR with a dose response of R1881 in the presence of 3 and 10 μM UT-105 as indicated in panel B. G. UT-105 inhibits AR-target genes. MDA-MB-453 cells in charcoal stripped serum-containing medium were treated for 16–20 hours. RNA was extracted and expression of FKBP5, TMPRSS2, and STEAP4 was quantified by real-time PCR and normalized to GAPDH (n=4/group). * p<0.05 (one-way ANOVA) H. RNA-seq. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium for 2 days were treated with vehicle or 3 μM UT-105 in the presence of 0.1 nM R1881 for 20 hours (n=3/group). Cells were harvested, RNA extracted, and sequenced. Heatmap of global gene expression changes, top GSEA pathways enriched, and heatmap and bar graphs of AR signaling pathway are represented. I. UT-105 inhibits proliferation of TNBC cells. MDA-MB-453 cells plated in charcoal stripped serum-containing medium and treated for seven days with medium change and retreatment after day 3 (n=3/group). Sulforhodamine B (SRB) colorimetric assay was performed to measure cell viability. J. UT-105 inhibits clonogenicity. TNBC cells stably expressing AR-V7 were plated in 6-well plates and treated with 10 μM of the indicated compounds for two weeks. Colonies were imaged and the number of colonies formed was counted using an imaging software (n=4/group). Panels B-D and F, G, I-J are representatives of at least three independent replicates. * p<0.05, ** p<0.01 (one-way ANOVA). Enza- enzalutamide; V7- AR-V7 splice variant. Values are expressed as mean -/+ SEM.

    Techniques Used: Mutagenesis, Transfection, Luciferase, Western Blot, Recombinant, Purification, Incubation, SDS Page, Binding Assay, Transactivation Assay, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Colorimetric Assay, Stable Transfection, Imaging, Software, Variant Assay

    (A-F) RNA was extracted from MDA-MB-453 tumors (shown in Fig. 4B) and sequenced (n = 3–5/group). A. Differentially expressed genes (DEGs) are visualized as a heatmap. The number of significant DEGs in each treatment compared to the vehicle control is shown below. B. GSEA analysis was performed on UT-105-treated tumors. Pathways with an FDR<0.25 are shown with corresponding normalized enrichment scores (NES). C. Enrichment plots are shown for interferon ɤ (top) and interferon α (bottom) Hallmark response pathways. D. Fold change in gene expression from RNA sequencing with MDA-MB-453 tumors for STAT signaling pathway genes IRF1, IRF7, and IRF9 is shown. E. A heatmap of chemokine normalized means from the RNA-seq data performed in MDA-MB-453 xenograft tumors. F. Western immunoblot of phospho-STAT1, phospho-STAT3, and phospho-AKT in representative samples from vehicle and UT-105-treated MDA-MB-453 xenograft tumors (from Fig 4B). G. Effect of UT-105 and ruxolitinib on STAT1 phosphorylation in MDA-MB-453 cells. MDA-MB-453 cells were maintained in csFBS-containing medium for 48 hours. Cells were treated with 3 μM UT-105 or ruxolitinib for 24 hours before the induction of STAT1 phosphorylation with interferon α (1000 units). Cells were harvested 30 minutes after induction, protein was extracted, and Western blot for pSTAT1 and GAPDH was performed. Representative blot is shown. The bands were quantified and fold change from vehicle is provided under the blots. H. Effect of UT-105 and ruxolitinib on cell proliferation. Cells were plated in growth medium and treated with vehicle, UT-105, or JAK-STAT inhibitor ruxolitinib for seven days with medium change and retreatment after day 3 (n=4/group; representative of three replicates; one-way ANOVA). SRB assay was performed. I. AR and AR-SV -positive TNBC specimens are enriched for JAK-STAT pathway. RNA was extracted from TNBC specimens (n=41) shown in Fig. 1C and sequenced. The top pathways enriched in AR and AR-SV compared to AR negative specimens obtained from GSEA (FDR <0.25) are shown as a bar graph. J. Enrichment plots are shown. Rux- ruxolitinib; enza- enzalutamide. Mean -/+ SEM: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.00001.
    Figure Legend Snippet: (A-F) RNA was extracted from MDA-MB-453 tumors (shown in Fig. 4B) and sequenced (n = 3–5/group). A. Differentially expressed genes (DEGs) are visualized as a heatmap. The number of significant DEGs in each treatment compared to the vehicle control is shown below. B. GSEA analysis was performed on UT-105-treated tumors. Pathways with an FDR<0.25 are shown with corresponding normalized enrichment scores (NES). C. Enrichment plots are shown for interferon ɤ (top) and interferon α (bottom) Hallmark response pathways. D. Fold change in gene expression from RNA sequencing with MDA-MB-453 tumors for STAT signaling pathway genes IRF1, IRF7, and IRF9 is shown. E. A heatmap of chemokine normalized means from the RNA-seq data performed in MDA-MB-453 xenograft tumors. F. Western immunoblot of phospho-STAT1, phospho-STAT3, and phospho-AKT in representative samples from vehicle and UT-105-treated MDA-MB-453 xenograft tumors (from Fig 4B). G. Effect of UT-105 and ruxolitinib on STAT1 phosphorylation in MDA-MB-453 cells. MDA-MB-453 cells were maintained in csFBS-containing medium for 48 hours. Cells were treated with 3 μM UT-105 or ruxolitinib for 24 hours before the induction of STAT1 phosphorylation with interferon α (1000 units). Cells were harvested 30 minutes after induction, protein was extracted, and Western blot for pSTAT1 and GAPDH was performed. Representative blot is shown. The bands were quantified and fold change from vehicle is provided under the blots. H. Effect of UT-105 and ruxolitinib on cell proliferation. Cells were plated in growth medium and treated with vehicle, UT-105, or JAK-STAT inhibitor ruxolitinib for seven days with medium change and retreatment after day 3 (n=4/group; representative of three replicates; one-way ANOVA). SRB assay was performed. I. AR and AR-SV -positive TNBC specimens are enriched for JAK-STAT pathway. RNA was extracted from TNBC specimens (n=41) shown in Fig. 1C and sequenced. The top pathways enriched in AR and AR-SV compared to AR negative specimens obtained from GSEA (FDR <0.25) are shown as a bar graph. J. Enrichment plots are shown. Rux- ruxolitinib; enza- enzalutamide. Mean -/+ SEM: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.00001.

    Techniques Used: Expressing, RNA Sequencing Assay, Western Blot, Sulforhodamine B Assay

    A. Schematic representation of CDX and PDX experiments (biorender.com). B. Left. MDA-MB-453 orthotopic xenograft was conducted by implanting 5 million cells into the mammary fat pad of female NSG mice. Once the tumors reach 100–300 mm3, the animals (n=8–10/group) were randomized and treated orally with UT-105 (60 mg/kg/day) or vehicle control (DMSO + PEG-300) for 28 days. Tumor volume was measured by digital caliper twice weekly and the percent change in tumor volume is represented in the graph. Right. MDA-MB-453 tumor-bearing female NSG mice (n=8–10/group) were treated with enzalutamide (60 mg/kg/day), bicalutamide (60 mg/kg/day), or vehicle control for 28 days. C. Change in body weight of MDA-MB-453 tumor-bearing mice. D-E. UT-1355 TNBC PDX characterization. Protein was extracted from UT-1355 PDX tumor fragments and Western blot with AR NTD-binding antibody or AR-V7 antibody, and GAPDH antibody was performed. LNCaP and 22RV1 prostate cancer cells were used as control for AR and AR-SV, respectively. Representative blots shown. F. UT-105 completely inhibits UT-1355 PDX tumor growth. UT-1355 PDX tumor fragments (1 mm3) were orthotopically implanted into the mammary fat pad in female NSG mice (n=8–10/group), and a xenograft experiment was performed as indicated above for MDA-MB-453. G-H. Change in body weight of UT-1355-bearing mice and tumor weight. Mean -/+ SEM is shown with One way ANOVA conducted in Graph Pad Prism: * p<0.05, ** p<0.01, *** p<0.001.
    Figure Legend Snippet: A. Schematic representation of CDX and PDX experiments (biorender.com). B. Left. MDA-MB-453 orthotopic xenograft was conducted by implanting 5 million cells into the mammary fat pad of female NSG mice. Once the tumors reach 100–300 mm3, the animals (n=8–10/group) were randomized and treated orally with UT-105 (60 mg/kg/day) or vehicle control (DMSO + PEG-300) for 28 days. Tumor volume was measured by digital caliper twice weekly and the percent change in tumor volume is represented in the graph. Right. MDA-MB-453 tumor-bearing female NSG mice (n=8–10/group) were treated with enzalutamide (60 mg/kg/day), bicalutamide (60 mg/kg/day), or vehicle control for 28 days. C. Change in body weight of MDA-MB-453 tumor-bearing mice. D-E. UT-1355 TNBC PDX characterization. Protein was extracted from UT-1355 PDX tumor fragments and Western blot with AR NTD-binding antibody or AR-V7 antibody, and GAPDH antibody was performed. LNCaP and 22RV1 prostate cancer cells were used as control for AR and AR-SV, respectively. Representative blots shown. F. UT-105 completely inhibits UT-1355 PDX tumor growth. UT-1355 PDX tumor fragments (1 mm3) were orthotopically implanted into the mammary fat pad in female NSG mice (n=8–10/group), and a xenograft experiment was performed as indicated above for MDA-MB-453. G-H. Change in body weight of UT-1355-bearing mice and tumor weight. Mean -/+ SEM is shown with One way ANOVA conducted in Graph Pad Prism: * p<0.05, ** p<0.01, *** p<0.001.

    Techniques Used: Western Blot, Binding Assay

    A. STAT1 increases R1881-induced AR transactivation. AR transactivation was performed in HEK-293 cells with 0.1 μg vector or STAT1. Numbers provided in bracket are R1881’s EC50 values. B. STAT1 and AR interact. AR and STAT1 (5 μg) were transfected into HEK-293 cells. Cells were treated 48 hours after transfection with 2000 IU interferon α and 10 nM R1881. Cells were harvested 4 hours after treatment, immunoprecipitation was performed with IgG or AR antibody, and Western blot was performed for STAT1. C. AR and STAT1 interact in MDA-MB-453 cells. MDA-MB-453 cells were maintained in charcoal-stripped serum-containing medium for two days and treated with interferon α and R1881 for 4 hours. Cells were fixed and immunostained for AR (red) and STAT1 (green), and confocal microscopy was performed. Scale is 2 μm. D. STAT1 is recruited to ARE (on FKBP5 regulatory region). MDA-MB-453 cells were treated as indicated. The cells were crosslinked, sheared, and ChIP PCR was performed with the STAT1 and IgG antibodies. PCR was performed using the primers for the indicated regions. E. AR is recruited to STAT1 responsive gene regulatory region. Data from AR ChIP-seq performed in PCa cells (n=2) were loaded in IGV browser and CXCL8 (IL8) and FKBP5 regulatory regions were scanned to determine the binding of AR. F. UT-105 inhibits STAT1 recruitment to STAT1RE and ARE. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium were treated with interferon α (1000 units), R1881 (1 nM), combination in the presence and absence of UT-105 (10 μM) for 4 hours. ChIP assay was performed with STAT1 antibody and real-time PCR was performed with primers specific for hIL8 STATRE and FKBP5 ARE. Representative experiment is shown in the figure. G. Ruxolitinib inhibits AR-target gene expression. MDA-MB-453 cells maintained in charcoal stripped serum-containing medium were treated with R1881 (0.1 nM) alone or in combination with ruxolitinib (3 μM) for 24 hours. RNA was extracted, and real-time PCR for FKBP5 was performed (n=4/group; one-way ANOVA). H. UT-105 inhibits STAT1-dependent coactivation of AR. AR transactivation was performed in HEK-293 cells in the presence or absence of 0.1 μg STAT1. I. Effect of AR and JAK inhibitors on the proliferation of patient tumors explants growth. Illustration of gelatin sponge culture (Biorender.com). Tumor tissue from two patients (1473 and 1474) were placed on pre-soaked gelatin sponges and treated as indicated for 48 hours. The tissues were fixed and stained with Ki67. The bar graphs show the percentage of cells that stained positively for Ki67. J. Model. A schematic of the proposed mechanism of action of SARDs in TNBC tumors (biorender.com). The model summarizes the choice of drug for inhibiting the growth of LAR TNBC tumors. This would depend on the specific mechanisms involved in AR activation and tumor progression. Enzalutamide may work if AR activity is the primary driver, but its efficacy could be limited by androgen surges. STAT inhibitors like ruxolitinib may not be effective if AR activation is independent of STAT1. Degraders like UT-105 seem promising as they degrade AR directly and could prevent AR or AR splice variant activation, offering a more comprehensive approach to inhibit tumor growth. However, the actual effectiveness of these drugs would need to be studied in clinical settings and may vary from patient to patient. Panels A-H representative of three independent experiments is shown.
    Figure Legend Snippet: A. STAT1 increases R1881-induced AR transactivation. AR transactivation was performed in HEK-293 cells with 0.1 μg vector or STAT1. Numbers provided in bracket are R1881’s EC50 values. B. STAT1 and AR interact. AR and STAT1 (5 μg) were transfected into HEK-293 cells. Cells were treated 48 hours after transfection with 2000 IU interferon α and 10 nM R1881. Cells were harvested 4 hours after treatment, immunoprecipitation was performed with IgG or AR antibody, and Western blot was performed for STAT1. C. AR and STAT1 interact in MDA-MB-453 cells. MDA-MB-453 cells were maintained in charcoal-stripped serum-containing medium for two days and treated with interferon α and R1881 for 4 hours. Cells were fixed and immunostained for AR (red) and STAT1 (green), and confocal microscopy was performed. Scale is 2 μm. D. STAT1 is recruited to ARE (on FKBP5 regulatory region). MDA-MB-453 cells were treated as indicated. The cells were crosslinked, sheared, and ChIP PCR was performed with the STAT1 and IgG antibodies. PCR was performed using the primers for the indicated regions. E. AR is recruited to STAT1 responsive gene regulatory region. Data from AR ChIP-seq performed in PCa cells (n=2) were loaded in IGV browser and CXCL8 (IL8) and FKBP5 regulatory regions were scanned to determine the binding of AR. F. UT-105 inhibits STAT1 recruitment to STAT1RE and ARE. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium were treated with interferon α (1000 units), R1881 (1 nM), combination in the presence and absence of UT-105 (10 μM) for 4 hours. ChIP assay was performed with STAT1 antibody and real-time PCR was performed with primers specific for hIL8 STATRE and FKBP5 ARE. Representative experiment is shown in the figure. G. Ruxolitinib inhibits AR-target gene expression. MDA-MB-453 cells maintained in charcoal stripped serum-containing medium were treated with R1881 (0.1 nM) alone or in combination with ruxolitinib (3 μM) for 24 hours. RNA was extracted, and real-time PCR for FKBP5 was performed (n=4/group; one-way ANOVA). H. UT-105 inhibits STAT1-dependent coactivation of AR. AR transactivation was performed in HEK-293 cells in the presence or absence of 0.1 μg STAT1. I. Effect of AR and JAK inhibitors on the proliferation of patient tumors explants growth. Illustration of gelatin sponge culture (Biorender.com). Tumor tissue from two patients (1473 and 1474) were placed on pre-soaked gelatin sponges and treated as indicated for 48 hours. The tissues were fixed and stained with Ki67. The bar graphs show the percentage of cells that stained positively for Ki67. J. Model. A schematic of the proposed mechanism of action of SARDs in TNBC tumors (biorender.com). The model summarizes the choice of drug for inhibiting the growth of LAR TNBC tumors. This would depend on the specific mechanisms involved in AR activation and tumor progression. Enzalutamide may work if AR activity is the primary driver, but its efficacy could be limited by androgen surges. STAT inhibitors like ruxolitinib may not be effective if AR activation is independent of STAT1. Degraders like UT-105 seem promising as they degrade AR directly and could prevent AR or AR splice variant activation, offering a more comprehensive approach to inhibit tumor growth. However, the actual effectiveness of these drugs would need to be studied in clinical settings and may vary from patient to patient. Panels A-H representative of three independent experiments is shown.

    Techniques Used: Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Confocal Microscopy, ChIP-sequencing, Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Staining, Activation Assay, Activity Assay, Variant Assay

    Key resources table
    Figure Legend Snippet: Key resources table

    Techniques Used: Virus, Plasmid Preparation, Recombinant, RNA Sequencing Assay, Synthesized, Expressing, Construct, Software

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    genome edited mda mb 231  (ATCC)


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    ATCC genome edited mda mb 231
    (A) Growth factor screening. Serum starved <t>MDA-MB-231</t> cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
    Genome Edited Mda Mb 231, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genome edited mda mb 231/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    genome edited mda mb 231 - by Bioz Stars, 2024-06
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    1) Product Images from "Growth factor-induced desialylation for the fast control of endocytosis"

    Article Title: Growth factor-induced desialylation for the fast control of endocytosis

    Journal: bioRxiv

    doi: 10.1101/2023.09.12.557183

    (A) Growth factor screening. Serum starved MDA-MB-231 cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: (A) Growth factor screening. Serum starved MDA-MB-231 cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Incubation, Labeling, Fluorescence, Comparison, Immunolabeling, MANN-WHITNEY

    (A) Schematics of interaction between ⍰2-6 Sia-modified glycans and Gal3 or SNA. (B) Identification of Gal3 interactors. MDA-MB-231 cells were incubated for 1 h on ice with Gal3-His. Pulldowns were subjected to quantitative mass spectrometry analysis. Red quadrant shows significantly enriched Gal3 binders. (C) Identification of Gal3 binders that are also SNA interactors. MDA-MB-231 cells, either untreated or treated with STI, were incubated for 1 h on ice with SNA-biotin. Pulldowns were analyzed by quantitative mass spectrometry. 574 proteins from 5 independent experiments were common to Gal3 (from B) and SNA (from C) data sets. 93 of these were enriched with high confidence for both (yellow quadrant). (D) Volcano plot of these 93 high confidence interactors as to their propensity to undergo EGF-induced desialylation (experiment as in (C) in the presence of 100 ng/mL of EGF). 30 responders are highlighted in the green quadrant. Note the presence of ⍰3β1 integrin and CD97 amongst the hits. (E) Cluster analysis of the summarized list of “biological processes” gene ontology terms associated with the 30 EGF-sensitive Gal3 and SNA interactors. (F) Validation of proteomics experiments by SNA pulldown. SNA-biotin pulldown was performed as in (B) in the presence or absence of STI or EGF. Samples were analyzed by Western blotting against α3 integrin. Quantifications from 3 independent experiments. One-way ANOVA with Tukey’s multiple comparison test. (G) Validation of proteomics experiments by α3 integrin immunoprecipitation. MDA-MB-231 cells were incubated for 1 h on ice anti-α3 integrin antibodies in the presence or absence of EGF. After pulldown, samples were analyzed by Western blotting using SNA-biotin. Quantifications from 3 independent experiments. Two tailed unpaired t-test. In (F,G): ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: (A) Schematics of interaction between ⍰2-6 Sia-modified glycans and Gal3 or SNA. (B) Identification of Gal3 interactors. MDA-MB-231 cells were incubated for 1 h on ice with Gal3-His. Pulldowns were subjected to quantitative mass spectrometry analysis. Red quadrant shows significantly enriched Gal3 binders. (C) Identification of Gal3 binders that are also SNA interactors. MDA-MB-231 cells, either untreated or treated with STI, were incubated for 1 h on ice with SNA-biotin. Pulldowns were analyzed by quantitative mass spectrometry. 574 proteins from 5 independent experiments were common to Gal3 (from B) and SNA (from C) data sets. 93 of these were enriched with high confidence for both (yellow quadrant). (D) Volcano plot of these 93 high confidence interactors as to their propensity to undergo EGF-induced desialylation (experiment as in (C) in the presence of 100 ng/mL of EGF). 30 responders are highlighted in the green quadrant. Note the presence of ⍰3β1 integrin and CD97 amongst the hits. (E) Cluster analysis of the summarized list of “biological processes” gene ontology terms associated with the 30 EGF-sensitive Gal3 and SNA interactors. (F) Validation of proteomics experiments by SNA pulldown. SNA-biotin pulldown was performed as in (B) in the presence or absence of STI or EGF. Samples were analyzed by Western blotting against α3 integrin. Quantifications from 3 independent experiments. One-way ANOVA with Tukey’s multiple comparison test. (G) Validation of proteomics experiments by α3 integrin immunoprecipitation. MDA-MB-231 cells were incubated for 1 h on ice anti-α3 integrin antibodies in the presence or absence of EGF. After pulldown, samples were analyzed by Western blotting using SNA-biotin. Quantifications from 3 independent experiments. Two tailed unpaired t-test. In (F,G): ** p < 0.01, *** p < 0.001.

    Techniques Used: Modification, Incubation, Mass Spectrometry, Western Blot, Comparison, Immunoprecipitation, Two Tailed Test

    Neuraminidases and NHE1
    Figure Legend Snippet: Neuraminidases and NHE1

    Techniques Used:

    (A) Neuraminidases. Serum starved wildtype MDA-MB-231 cells or CRISPR-based knockouts of NEU1 , NEU3 or NEU1/3 were incubated for 1 h on ice with Alexa-488 coupled Gal3 in the presence or absence of EGF. Fluorescence signals were quantified from 3 independent experiments (≈ 75 cells per condition). One-way ANOVA with Tukey’s multiple comparison test. (B) Experiment as in (A) with patient skin fibroblasts harboring inactivating mutations in the indicated enzymes. 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Tukey’s multiple comparison test. (C) Schematics of N-glycosylation pathway. (D) pH quenching effect. Serum starved MDA-MB-231 cells were incubated for 1 h on ice at pH 7.4 or pH 8.4 in the presence or absence of EGF, fixed, and incubated at neutral pH with Alexa-488 coupled Gal3. Quantifications as in (A) on 3 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (E) Cells were treated as in (D), lysed after EGF stimulation, and analyzed by Western blotting against EGFR Y1068. 3 independent experiments. Unpaired t-test. (F) pH triggering effect. Serum starved MDA-MB-231 cells were incubated for 1 h on ice in pH 5.5 or pH 7.4 buffers with or without EGF and/or DANA, and then for 1 h on ice at neutral pH with Alexa-488 coupled Gal3. Quantifications as in (A) on 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (G) Role of NHE1, CRISPR. Serum starved wild-type MDA-MB-468 cells, CRISPR-based knockouts of NHE1, and NHE1-GFP rescue cells were incubated and quantified as in (A) on 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (H) pH 5.5 buffer can rescue NHE knockout cells. Serum starved MDA-MB-231 wildtype and NHE1 knockout cells were incubated for 1 h on ice in pH 5.5 or pH 7.4 buffers with or without EGF. Quantifications as in (A) on 4 independent experiments and normalized to controls (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (I) Role of NHE1, inhibitors. MDA-MB-231 cells were pretreated for 30 min at 37 °C with the indicated inhibitors or DMSO (CTRL) in serum free media before incubation in the continued presence of the inhibitors for 1 h on ice with Alexa-488 coupled Gal3 in the presence or absence of EGF. Quantifications as in (A) on 5 independent experiments (≈150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. Means ± SEM are shown in this figure. Except for (E): Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
    Figure Legend Snippet: (A) Neuraminidases. Serum starved wildtype MDA-MB-231 cells or CRISPR-based knockouts of NEU1 , NEU3 or NEU1/3 were incubated for 1 h on ice with Alexa-488 coupled Gal3 in the presence or absence of EGF. Fluorescence signals were quantified from 3 independent experiments (≈ 75 cells per condition). One-way ANOVA with Tukey’s multiple comparison test. (B) Experiment as in (A) with patient skin fibroblasts harboring inactivating mutations in the indicated enzymes. 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Tukey’s multiple comparison test. (C) Schematics of N-glycosylation pathway. (D) pH quenching effect. Serum starved MDA-MB-231 cells were incubated for 1 h on ice at pH 7.4 or pH 8.4 in the presence or absence of EGF, fixed, and incubated at neutral pH with Alexa-488 coupled Gal3. Quantifications as in (A) on 3 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (E) Cells were treated as in (D), lysed after EGF stimulation, and analyzed by Western blotting against EGFR Y1068. 3 independent experiments. Unpaired t-test. (F) pH triggering effect. Serum starved MDA-MB-231 cells were incubated for 1 h on ice in pH 5.5 or pH 7.4 buffers with or without EGF and/or DANA, and then for 1 h on ice at neutral pH with Alexa-488 coupled Gal3. Quantifications as in (A) on 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (G) Role of NHE1, CRISPR. Serum starved wild-type MDA-MB-468 cells, CRISPR-based knockouts of NHE1, and NHE1-GFP rescue cells were incubated and quantified as in (A) on 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (H) pH 5.5 buffer can rescue NHE knockout cells. Serum starved MDA-MB-231 wildtype and NHE1 knockout cells were incubated for 1 h on ice in pH 5.5 or pH 7.4 buffers with or without EGF. Quantifications as in (A) on 4 independent experiments and normalized to controls (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (I) Role of NHE1, inhibitors. MDA-MB-231 cells were pretreated for 30 min at 37 °C with the indicated inhibitors or DMSO (CTRL) in serum free media before incubation in the continued presence of the inhibitors for 1 h on ice with Alexa-488 coupled Gal3 in the presence or absence of EGF. Quantifications as in (A) on 5 independent experiments (≈150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. Means ± SEM are shown in this figure. Except for (E): Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

    Techniques Used: CRISPR, Incubation, Fluorescence, Comparison, Western Blot, Knock-Out

    (A) ⍰3 integrin uptake. Serum starved MDA-MB-231 cells were continuously incubated for 10 min at 37 °C in the presence or absence of EGF with Atto647N-labeled anti-⍰3 integrin antibody and Alexa488-transferrin (Tf). Note that ⍰3 integrin uptake was stimulated in the presence of EGF. Scale bar = 10 µm. (B) Quantification of fluorescence from 3 independent experiments (≈ 100 cells per condition) as in (A) in which cells were incubated in the presence or absence of EGF and/or the Gal3 inhibitor I3. One-way ANOVA with Dunnett’s multiple comparison test. (C-F) β1 integrin and CD97 uptake. Experiments as in (A) in which anti-β1 integrin or anti-CD47 antibodies were used in the presence or absence of the indicated inhibitors and growth factors. 3-6 independent experiments (≈ 200 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. In (D): Scale bar = 10 µm. (G) pH imaging. MDA-MB-231 cells were incubated continuously for 4 min at 37 °C with AcidiFluor-coupled anti-α3 integrin antibody, and then imaged for 10 min sequentially in TIRF and epifluorescence modalities. EGF was added after 18 sec. Note that increased fluorescence in the TIRF field (red) indicates acidification. Arrows indicate 1 representative event. Scale bar = 10 µm. (H) Dynamic pH monitoring. Acquisition of images started immediately after incubation with antibody in sequential TIRF and epifluorescence modes at 37 °C. pH 4 buffer was added at the indicated time point to document how the setup reacted. Total fluorescence (mean) and Emax are shown for a representative cell. (I) Intensity traces for the event shown by arrows in (G). * indicates where vesicle leaves frame. TIRF in red, widefield in green. (J,K) Quantification of numbers per cell (I) or of maximum intensity values (Emax) (J) for tracks that undergo a pH change as shown in (G). 3 independent experiments (≈ 15 cells counted per condition). Holm-Šídák’s multiple comparisons test. Means ± SEM are shown in this figure. In (B,C,E,F), means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: (A) ⍰3 integrin uptake. Serum starved MDA-MB-231 cells were continuously incubated for 10 min at 37 °C in the presence or absence of EGF with Atto647N-labeled anti-⍰3 integrin antibody and Alexa488-transferrin (Tf). Note that ⍰3 integrin uptake was stimulated in the presence of EGF. Scale bar = 10 µm. (B) Quantification of fluorescence from 3 independent experiments (≈ 100 cells per condition) as in (A) in which cells were incubated in the presence or absence of EGF and/or the Gal3 inhibitor I3. One-way ANOVA with Dunnett’s multiple comparison test. (C-F) β1 integrin and CD97 uptake. Experiments as in (A) in which anti-β1 integrin or anti-CD47 antibodies were used in the presence or absence of the indicated inhibitors and growth factors. 3-6 independent experiments (≈ 200 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. In (D): Scale bar = 10 µm. (G) pH imaging. MDA-MB-231 cells were incubated continuously for 4 min at 37 °C with AcidiFluor-coupled anti-α3 integrin antibody, and then imaged for 10 min sequentially in TIRF and epifluorescence modalities. EGF was added after 18 sec. Note that increased fluorescence in the TIRF field (red) indicates acidification. Arrows indicate 1 representative event. Scale bar = 10 µm. (H) Dynamic pH monitoring. Acquisition of images started immediately after incubation with antibody in sequential TIRF and epifluorescence modes at 37 °C. pH 4 buffer was added at the indicated time point to document how the setup reacted. Total fluorescence (mean) and Emax are shown for a representative cell. (I) Intensity traces for the event shown by arrows in (G). * indicates where vesicle leaves frame. TIRF in red, widefield in green. (J,K) Quantification of numbers per cell (I) or of maximum intensity values (Emax) (J) for tracks that undergo a pH change as shown in (G). 3 independent experiments (≈ 15 cells counted per condition). Holm-Šídák’s multiple comparisons test. Means ± SEM are shown in this figure. In (B,C,E,F), means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Incubation, Labeling, Fluorescence, Comparison, Imaging

    (A) Electron microcopy. Left: Representative overview electron micrographs of MDA-MB-231 cells that have been incubated with HRP-labeled Gal3 for 6 min at 37 °C in the presence or absence of EGF. Right: Zoomed views of CLICs from EGF-treated MDA-MB-231 cells for which the following HRP-labeled ligands were present during incubations: (i) Gal3, (ii) anti-α3 integrin antibodies, or (iii) anti-β1 integrin antibodies. Scale bars = 10 µm (images on left) and 200 nm (images on right). (B) Quantification of electron micrographs as in (A). HRP-positive structures from 2 independent experiments were counted for the indicated conditions (≈ 10 cells per condition) in a single slice per cell and categorized according to morphology. (C) Lattice light sheet microscopy. Representative maximum intensity projections of AP2-eGFP (green) expressing SUM159 cells in indicated conditions of incubation with anti-β1 integrin-Cy3 antibodies or Tf-Cy3 (magenta) in the presence or absence of EGF. Scale bars = 10 µm. (D) Central section of a SUM159 cell at 1 min 15 s into acquisition, showing an AP2-negative β1 integrin uptake event (in dotted box). Scale bar = 5 µm. (E) High magnifications panels of single frames from boxed area in (D). The AP2-negative uptake event is tracked by white arrowheads. (F) Percentage of β1 integrin and transferrin (Tf) uptake tracks from 3 independent experiments that were AP2-negative as in (C-E). For β1 integrin: 1802 tracks from 16 control cells, and 2157 tracks from 18 EGF-treated cells. For Tf: 1145 tracks from 11 control cells, and 1422 tracks from 12 EGF-treated cells. Note that the percentage of AP2-negative tracks increased upon EGF treatment only for β1 integrin uptake. Means ± SEM, unpaired t-test. (G) Average normalized intensity traces and classifications for β1 integrin and Tf uptake events from experiments in (F). Lifetime cohorts of endocytic trajectories show “dome”-shaped intensity profiles for cargoes, and co-tracking or not with AP2. Dotted lines represent background levels for fluorescence signals. (H) Selected β1 integrin tracks in EGF-treated cells. Axes show X,Y,Z positions of uptake carriers in cells, spot colors depicts AP2 intensity, line color distance to plasma membrane, and X indicates track starting points. Means ± SEM are shown in this Figure. ns = p > 0.05, ** p < 0.01.
    Figure Legend Snippet: (A) Electron microcopy. Left: Representative overview electron micrographs of MDA-MB-231 cells that have been incubated with HRP-labeled Gal3 for 6 min at 37 °C in the presence or absence of EGF. Right: Zoomed views of CLICs from EGF-treated MDA-MB-231 cells for which the following HRP-labeled ligands were present during incubations: (i) Gal3, (ii) anti-α3 integrin antibodies, or (iii) anti-β1 integrin antibodies. Scale bars = 10 µm (images on left) and 200 nm (images on right). (B) Quantification of electron micrographs as in (A). HRP-positive structures from 2 independent experiments were counted for the indicated conditions (≈ 10 cells per condition) in a single slice per cell and categorized according to morphology. (C) Lattice light sheet microscopy. Representative maximum intensity projections of AP2-eGFP (green) expressing SUM159 cells in indicated conditions of incubation with anti-β1 integrin-Cy3 antibodies or Tf-Cy3 (magenta) in the presence or absence of EGF. Scale bars = 10 µm. (D) Central section of a SUM159 cell at 1 min 15 s into acquisition, showing an AP2-negative β1 integrin uptake event (in dotted box). Scale bar = 5 µm. (E) High magnifications panels of single frames from boxed area in (D). The AP2-negative uptake event is tracked by white arrowheads. (F) Percentage of β1 integrin and transferrin (Tf) uptake tracks from 3 independent experiments that were AP2-negative as in (C-E). For β1 integrin: 1802 tracks from 16 control cells, and 2157 tracks from 18 EGF-treated cells. For Tf: 1145 tracks from 11 control cells, and 1422 tracks from 12 EGF-treated cells. Note that the percentage of AP2-negative tracks increased upon EGF treatment only for β1 integrin uptake. Means ± SEM, unpaired t-test. (G) Average normalized intensity traces and classifications for β1 integrin and Tf uptake events from experiments in (F). Lifetime cohorts of endocytic trajectories show “dome”-shaped intensity profiles for cargoes, and co-tracking or not with AP2. Dotted lines represent background levels for fluorescence signals. (H) Selected β1 integrin tracks in EGF-treated cells. Axes show X,Y,Z positions of uptake carriers in cells, spot colors depicts AP2 intensity, line color distance to plasma membrane, and X indicates track starting points. Means ± SEM are shown in this Figure. ns = p > 0.05, ** p < 0.01.

    Techniques Used: Incubation, Labeling, Microscopy, Expressing, Fluorescence, Membrane

    (A) Schematics of retrograde transport assay. Endocytic ligands (including antibodies against cell surface proteins) can either be directly coupled to benzylguanine (BG), as shown in (i), or the cell surface proteome can be modified with cell impermeable BG-NHS (ii). Proteins that undergo retrograde transport are captured in the Golgi by a covalent reaction with a GFP-tagged SNAPtag fusion protein that has been localized there. (B) HeLa cells stably expressing GalT-GFP-SNAP were continuously incubated for 4 h at 37 °C with BG-modified Gal3 before lysis and pulldown using GFP-trap beads. Western blot quantification from 3 independent experiments of Gal3-SNAP conjugate in pulldowns. Means ± SEM, unpaired t-test, *** p < 0.001. (C) Gal3 interaction proteomics. HeLa cells were incubated for 1 h on ice with Gal3-His in the presence or absence of EGF, followed by lysis, pulldown on cobalt-agarose beads, and quantitative mass spectrometry. Correlation plot of proteins. Blue quadrants indicate specific interactor of Gal3 in all conditions from 5 independent experiments. (D) Volcano blot of Gal3 interactors (blue quadrants from (C)) that are significantly (i.e., with 3 peptides and an adjusted p-value ≤ 0.05) enriched after EGF stimulation (red quadrant). (E) Retrograde proteomics. HeLa cells stably expressing GalT-GFP-SNAP were cell-surface modified on ice with NHS-PEG9-BG, followed by 16 h incubation at 37 °C, pulldown with GFP-trap beads, and quantitative mass spectrometry. High confidence retrograde cargoes are shown in the blue quadrant. (F) Comparison of Gal3 interactors and retrograde proteome. The yellow quadrant indicates high confidence Gal3 interactors that were enriched upon EGF stimulation and that also undergo retrograde transport. Note the presence of α3β1integrin and CD97 in this list. (G) 4 independent experiments as in (B) with BG-modified anti-β1 integrin antibodies. Quantification of anti-β1 integrin antibody-SNAP conjugates (IB anti-β1-SNAP) in GFP-trap pulldowns. Means ± SEM, unpaired t-test. (H) Resialylation analysis. MDA-MB-231 cells were incubated for 1 h on ice in the presence or absence of EGF, shifted in all conditions to 37 °C without EGF, and lysed at the indicated timepoints. ⍰3 integrin signal in SNA pulldowns (PD) was revealed by Western blotting. Note that ⍰3 integrin was still desialylated after 1 h of chase, and then became resialylated after 6 h of chase in a protein neosynthesis-independent manner (presence of cycloheximide (CHX). (I) Quantification of ⍰3 integrin signals from 3 independent experiments as in (H). Left, input; right, SNA PD. Means ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, ns = p > 0.05, ** p < 0.01.
    Figure Legend Snippet: (A) Schematics of retrograde transport assay. Endocytic ligands (including antibodies against cell surface proteins) can either be directly coupled to benzylguanine (BG), as shown in (i), or the cell surface proteome can be modified with cell impermeable BG-NHS (ii). Proteins that undergo retrograde transport are captured in the Golgi by a covalent reaction with a GFP-tagged SNAPtag fusion protein that has been localized there. (B) HeLa cells stably expressing GalT-GFP-SNAP were continuously incubated for 4 h at 37 °C with BG-modified Gal3 before lysis and pulldown using GFP-trap beads. Western blot quantification from 3 independent experiments of Gal3-SNAP conjugate in pulldowns. Means ± SEM, unpaired t-test, *** p < 0.001. (C) Gal3 interaction proteomics. HeLa cells were incubated for 1 h on ice with Gal3-His in the presence or absence of EGF, followed by lysis, pulldown on cobalt-agarose beads, and quantitative mass spectrometry. Correlation plot of proteins. Blue quadrants indicate specific interactor of Gal3 in all conditions from 5 independent experiments. (D) Volcano blot of Gal3 interactors (blue quadrants from (C)) that are significantly (i.e., with 3 peptides and an adjusted p-value ≤ 0.05) enriched after EGF stimulation (red quadrant). (E) Retrograde proteomics. HeLa cells stably expressing GalT-GFP-SNAP were cell-surface modified on ice with NHS-PEG9-BG, followed by 16 h incubation at 37 °C, pulldown with GFP-trap beads, and quantitative mass spectrometry. High confidence retrograde cargoes are shown in the blue quadrant. (F) Comparison of Gal3 interactors and retrograde proteome. The yellow quadrant indicates high confidence Gal3 interactors that were enriched upon EGF stimulation and that also undergo retrograde transport. Note the presence of α3β1integrin and CD97 in this list. (G) 4 independent experiments as in (B) with BG-modified anti-β1 integrin antibodies. Quantification of anti-β1 integrin antibody-SNAP conjugates (IB anti-β1-SNAP) in GFP-trap pulldowns. Means ± SEM, unpaired t-test. (H) Resialylation analysis. MDA-MB-231 cells were incubated for 1 h on ice in the presence or absence of EGF, shifted in all conditions to 37 °C without EGF, and lysed at the indicated timepoints. ⍰3 integrin signal in SNA pulldowns (PD) was revealed by Western blotting. Note that ⍰3 integrin was still desialylated after 1 h of chase, and then became resialylated after 6 h of chase in a protein neosynthesis-independent manner (presence of cycloheximide (CHX). (I) Quantification of ⍰3 integrin signals from 3 independent experiments as in (H). Left, input; right, SNA PD. Means ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, ns = p > 0.05, ** p < 0.01.

    Techniques Used: Transport Assay, Modification, Stable Transfection, Expressing, Incubation, Lysis, Western Blot, Mass Spectrometry, Comparison

    (A) Cell migration. MDA-MB-231 cells were seeded under the indicated conditions on cell derived matrices and incubated for 16 h incubations at 37 °C in 1 % FCS. Individual tracks are represented. (B) The Euclidean distance of migration was determined as in (A) in 3 independent experiments in the absence (DMSO) or presence of the Gal3 inhibitor I3 and of EGF. (C) Experiment as in (B) in the absence (DMSO) or presence of the indicated drugs. (D) Inverted invasion assays. MDA-MB-231 cells were attached to fibronectin-spiked collagen plugs and allowed to invade against gravity towards an upper reservoir with or without EGF. Invasion was considered beyond the indicated red line. Knockout of the indicated neuraminidases inhibited invasion. (E-F) Quantification of invasion from 3 independent experiments as in (D) with the indicated inhibitors and KO cells. (G) Schematics depicting the desialylation glycoswitch model. See text for details. Means ± SEM are shown in this figure. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. One-way ANOVA with Dunnett’s multiple comparison test, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: (A) Cell migration. MDA-MB-231 cells were seeded under the indicated conditions on cell derived matrices and incubated for 16 h incubations at 37 °C in 1 % FCS. Individual tracks are represented. (B) The Euclidean distance of migration was determined as in (A) in 3 independent experiments in the absence (DMSO) or presence of the Gal3 inhibitor I3 and of EGF. (C) Experiment as in (B) in the absence (DMSO) or presence of the indicated drugs. (D) Inverted invasion assays. MDA-MB-231 cells were attached to fibronectin-spiked collagen plugs and allowed to invade against gravity towards an upper reservoir with or without EGF. Invasion was considered beyond the indicated red line. Knockout of the indicated neuraminidases inhibited invasion. (E-F) Quantification of invasion from 3 independent experiments as in (D) with the indicated inhibitors and KO cells. (G) Schematics depicting the desialylation glycoswitch model. See text for details. Means ± SEM are shown in this figure. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. One-way ANOVA with Dunnett’s multiple comparison test, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Migration, Derivative Assay, Incubation, Knock-Out, Comparison

    horizon genomics hzghc001844c001 mda mb 231 gift  (ATCC)


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    ATCC horizon genomics hzghc001844c001 mda mb 231 gift
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    ATCC mda mb 453 ar v7 geo database gse244283 spatial genomics geo database gse245202 mda mb 453 xenograft geo database gse244283
    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and <t>AR-V7</t> probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).
    Mda Mb 453 Ar V7 Geo Database Gse244283 Spatial Genomics Geo Database Gse245202 Mda Mb 453 Xenograft Geo Database Gse244283, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC genome length 1 9 mb
    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and <t>AR-V7</t> probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).
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    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and <t>AR-V7</t> probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).
    Mb Genome, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and <t>AR-V7</t> probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).
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    ATCC genome edited mda mb 231
    (A) Growth factor screening. Serum starved <t>MDA-MB-231</t> cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
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    (A) Growth factor screening. Serum starved <t>MDA-MB-231</t> cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
    Horizon Genomics Hzghc001844c001 Mda Mb 231 Gift, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Growth factor screening. Serum starved <t>MDA-MB-231</t> cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
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    (A) Growth factor screening. Serum starved <t>MDA-MB-231</t> cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
    Genome Analysis Organism Strain Isolation Source Genome Size Mb Cds Trna Rrna Genome Reference, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Growth factor screening. Serum starved <t>MDA-MB-231</t> cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
    Mb Whole Genome Sequence, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and AR-V7 probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and AR-V7 probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Immunohistochemistry, Ligand Binding Assay, Activation Assay, Variant Assay

    TNBC cell lines were stably transfected with AR-V7 using lentivirus. A. BrdU assay of control and AR-V7 lentivirus transfected TNBC cell lines MDA-MB-453 (453), MDA-MB-231 (231), BT549, and MFM223. Cells were plated in growth medium and BrDU assay was performed after 72 hours (n=4/group). B. Scratch assay in MDA-MB-453 and MDA-MB-453-V7. Cells were plated in growth medium and imaged at the start and after 72 hours, and the gap closure measured by imaging software (n=4/group). C. Ki67 staining of TNBC patient specimens (23 (AR-positive (AR+)), and 11 (AR and AR-SV -positive (AR+/AR-SV+)). D and E. RNA sequencing was performed with 453 and 453-V7 cell lines and AR+ and AR+/AR-SV+ patient specimens. Gene set from the Molecular Signatures Database is reported in 453 cells compared to 453-V7 cells and AR+ patient specimens compared to AR+/AR-SV+ patient specimens. Values are expressed as mean -/+ SEM. Experiments in panels A-C were reproduced at least three times and representative experiment is shown. * p<0.05, ** p<0.01, ****p<0.00001 (t-test).

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: TNBC cell lines were stably transfected with AR-V7 using lentivirus. A. BrdU assay of control and AR-V7 lentivirus transfected TNBC cell lines MDA-MB-453 (453), MDA-MB-231 (231), BT549, and MFM223. Cells were plated in growth medium and BrDU assay was performed after 72 hours (n=4/group). B. Scratch assay in MDA-MB-453 and MDA-MB-453-V7. Cells were plated in growth medium and imaged at the start and after 72 hours, and the gap closure measured by imaging software (n=4/group). C. Ki67 staining of TNBC patient specimens (23 (AR-positive (AR+)), and 11 (AR and AR-SV -positive (AR+/AR-SV+)). D and E. RNA sequencing was performed with 453 and 453-V7 cell lines and AR+ and AR+/AR-SV+ patient specimens. Gene set from the Molecular Signatures Database is reported in 453 cells compared to 453-V7 cells and AR+ patient specimens compared to AR+/AR-SV+ patient specimens. Values are expressed as mean -/+ SEM. Experiments in panels A-C were reproduced at least three times and representative experiment is shown. * p<0.05, ** p<0.01, ****p<0.00001 (t-test).

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Stable Transfection, Transfection, BrdU Staining, Wound Healing Assay, Imaging, Software, Staining, RNA Sequencing Assay

    A. Structure of UT-105. B. UT-105 inhibits wildtype and mutant AR transactivation. AR or AR F876L (50 ng), 0.25 μg GRE-LUC, and 10 ng CMV-renilla-LUC were transfected into COS7 cells. Cells were treated 24 hours after transfection with a dose response of UT-105 or enzalutamide in the presence of 0.1 nM R1881, and luciferase assay was performed 24 hours after treatment. Firefly luciferase values were normalized to renilla luciferase. Numbers provided in bracket are IC50 values. C. UT-105 degrades AR. LNCaP PCa cells were maintained in charcoal-stripped FBS-containing medium for 2 days before treating with UT-105 in the presence of 0.1 nM R1881. Cells were harvested 24 hours after treatment, and Western blot for AR and GAPDH was performed. D. Top. UT-105 binds to AR-NTD. Recombinant purified AR NTD was incubated with DMSO or 10 μM UT-105 overnight at 4°C. SYPRO orange dye was added to the mixture and a PCR was performed with increasing temperature. SYPRO orange signal was monitored. Bottom. UT-105 stabilized purified AF-1 and AR-V7 recombinant protein. Recombinant purified AF-1 or AR-V7 protein (5 ng) were incubated at room temperature for 4 hours with DMSO or 100 μM of UT-105 or UT-34. Proteins were fractionated on an SDS-PAGE and Western blot was performed with AR antibody (AR-441). E. Illustration depicting the binding regions of UT-105 and enzalutamide. F. UT-105 irreversibly inhibits AR. Transactivation assay was performed with AR with a dose response of R1881 in the presence of 3 and 10 μM UT-105 as indicated in panel B. G. UT-105 inhibits AR-target genes. MDA-MB-453 cells in charcoal stripped serum-containing medium were treated for 16–20 hours. RNA was extracted and expression of FKBP5, TMPRSS2, and STEAP4 was quantified by real-time PCR and normalized to GAPDH (n=4/group). * p<0.05 (one-way ANOVA) H. RNA-seq. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium for 2 days were treated with vehicle or 3 μM UT-105 in the presence of 0.1 nM R1881 for 20 hours (n=3/group). Cells were harvested, RNA extracted, and sequenced. Heatmap of global gene expression changes, top GSEA pathways enriched, and heatmap and bar graphs of AR signaling pathway are represented. I. UT-105 inhibits proliferation of TNBC cells. MDA-MB-453 cells plated in charcoal stripped serum-containing medium and treated for seven days with medium change and retreatment after day 3 (n=3/group). Sulforhodamine B (SRB) colorimetric assay was performed to measure cell viability. J. UT-105 inhibits clonogenicity. TNBC cells stably expressing AR-V7 were plated in 6-well plates and treated with 10 μM of the indicated compounds for two weeks. Colonies were imaged and the number of colonies formed was counted using an imaging software (n=4/group). Panels B-D and F, G, I-J are representatives of at least three independent replicates. * p<0.05, ** p<0.01 (one-way ANOVA). Enza- enzalutamide; V7- AR-V7 splice variant. Values are expressed as mean -/+ SEM.

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: A. Structure of UT-105. B. UT-105 inhibits wildtype and mutant AR transactivation. AR or AR F876L (50 ng), 0.25 μg GRE-LUC, and 10 ng CMV-renilla-LUC were transfected into COS7 cells. Cells were treated 24 hours after transfection with a dose response of UT-105 or enzalutamide in the presence of 0.1 nM R1881, and luciferase assay was performed 24 hours after treatment. Firefly luciferase values were normalized to renilla luciferase. Numbers provided in bracket are IC50 values. C. UT-105 degrades AR. LNCaP PCa cells were maintained in charcoal-stripped FBS-containing medium for 2 days before treating with UT-105 in the presence of 0.1 nM R1881. Cells were harvested 24 hours after treatment, and Western blot for AR and GAPDH was performed. D. Top. UT-105 binds to AR-NTD. Recombinant purified AR NTD was incubated with DMSO or 10 μM UT-105 overnight at 4°C. SYPRO orange dye was added to the mixture and a PCR was performed with increasing temperature. SYPRO orange signal was monitored. Bottom. UT-105 stabilized purified AF-1 and AR-V7 recombinant protein. Recombinant purified AF-1 or AR-V7 protein (5 ng) were incubated at room temperature for 4 hours with DMSO or 100 μM of UT-105 or UT-34. Proteins were fractionated on an SDS-PAGE and Western blot was performed with AR antibody (AR-441). E. Illustration depicting the binding regions of UT-105 and enzalutamide. F. UT-105 irreversibly inhibits AR. Transactivation assay was performed with AR with a dose response of R1881 in the presence of 3 and 10 μM UT-105 as indicated in panel B. G. UT-105 inhibits AR-target genes. MDA-MB-453 cells in charcoal stripped serum-containing medium were treated for 16–20 hours. RNA was extracted and expression of FKBP5, TMPRSS2, and STEAP4 was quantified by real-time PCR and normalized to GAPDH (n=4/group). * p<0.05 (one-way ANOVA) H. RNA-seq. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium for 2 days were treated with vehicle or 3 μM UT-105 in the presence of 0.1 nM R1881 for 20 hours (n=3/group). Cells were harvested, RNA extracted, and sequenced. Heatmap of global gene expression changes, top GSEA pathways enriched, and heatmap and bar graphs of AR signaling pathway are represented. I. UT-105 inhibits proliferation of TNBC cells. MDA-MB-453 cells plated in charcoal stripped serum-containing medium and treated for seven days with medium change and retreatment after day 3 (n=3/group). Sulforhodamine B (SRB) colorimetric assay was performed to measure cell viability. J. UT-105 inhibits clonogenicity. TNBC cells stably expressing AR-V7 were plated in 6-well plates and treated with 10 μM of the indicated compounds for two weeks. Colonies were imaged and the number of colonies formed was counted using an imaging software (n=4/group). Panels B-D and F, G, I-J are representatives of at least three independent replicates. * p<0.05, ** p<0.01 (one-way ANOVA). Enza- enzalutamide; V7- AR-V7 splice variant. Values are expressed as mean -/+ SEM.

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Mutagenesis, Transfection, Luciferase, Western Blot, Recombinant, Purification, Incubation, SDS Page, Binding Assay, Transactivation Assay, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Colorimetric Assay, Stable Transfection, Imaging, Software, Variant Assay

    (A-F) RNA was extracted from MDA-MB-453 tumors (shown in Fig. 4B) and sequenced (n = 3–5/group). A. Differentially expressed genes (DEGs) are visualized as a heatmap. The number of significant DEGs in each treatment compared to the vehicle control is shown below. B. GSEA analysis was performed on UT-105-treated tumors. Pathways with an FDR<0.25 are shown with corresponding normalized enrichment scores (NES). C. Enrichment plots are shown for interferon ɤ (top) and interferon α (bottom) Hallmark response pathways. D. Fold change in gene expression from RNA sequencing with MDA-MB-453 tumors for STAT signaling pathway genes IRF1, IRF7, and IRF9 is shown. E. A heatmap of chemokine normalized means from the RNA-seq data performed in MDA-MB-453 xenograft tumors. F. Western immunoblot of phospho-STAT1, phospho-STAT3, and phospho-AKT in representative samples from vehicle and UT-105-treated MDA-MB-453 xenograft tumors (from Fig 4B). G. Effect of UT-105 and ruxolitinib on STAT1 phosphorylation in MDA-MB-453 cells. MDA-MB-453 cells were maintained in csFBS-containing medium for 48 hours. Cells were treated with 3 μM UT-105 or ruxolitinib for 24 hours before the induction of STAT1 phosphorylation with interferon α (1000 units). Cells were harvested 30 minutes after induction, protein was extracted, and Western blot for pSTAT1 and GAPDH was performed. Representative blot is shown. The bands were quantified and fold change from vehicle is provided under the blots. H. Effect of UT-105 and ruxolitinib on cell proliferation. Cells were plated in growth medium and treated with vehicle, UT-105, or JAK-STAT inhibitor ruxolitinib for seven days with medium change and retreatment after day 3 (n=4/group; representative of three replicates; one-way ANOVA). SRB assay was performed. I. AR and AR-SV -positive TNBC specimens are enriched for JAK-STAT pathway. RNA was extracted from TNBC specimens (n=41) shown in Fig. 1C and sequenced. The top pathways enriched in AR and AR-SV compared to AR negative specimens obtained from GSEA (FDR <0.25) are shown as a bar graph. J. Enrichment plots are shown. Rux- ruxolitinib; enza- enzalutamide. Mean -/+ SEM: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.00001.

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: (A-F) RNA was extracted from MDA-MB-453 tumors (shown in Fig. 4B) and sequenced (n = 3–5/group). A. Differentially expressed genes (DEGs) are visualized as a heatmap. The number of significant DEGs in each treatment compared to the vehicle control is shown below. B. GSEA analysis was performed on UT-105-treated tumors. Pathways with an FDR<0.25 are shown with corresponding normalized enrichment scores (NES). C. Enrichment plots are shown for interferon ɤ (top) and interferon α (bottom) Hallmark response pathways. D. Fold change in gene expression from RNA sequencing with MDA-MB-453 tumors for STAT signaling pathway genes IRF1, IRF7, and IRF9 is shown. E. A heatmap of chemokine normalized means from the RNA-seq data performed in MDA-MB-453 xenograft tumors. F. Western immunoblot of phospho-STAT1, phospho-STAT3, and phospho-AKT in representative samples from vehicle and UT-105-treated MDA-MB-453 xenograft tumors (from Fig 4B). G. Effect of UT-105 and ruxolitinib on STAT1 phosphorylation in MDA-MB-453 cells. MDA-MB-453 cells were maintained in csFBS-containing medium for 48 hours. Cells were treated with 3 μM UT-105 or ruxolitinib for 24 hours before the induction of STAT1 phosphorylation with interferon α (1000 units). Cells were harvested 30 minutes after induction, protein was extracted, and Western blot for pSTAT1 and GAPDH was performed. Representative blot is shown. The bands were quantified and fold change from vehicle is provided under the blots. H. Effect of UT-105 and ruxolitinib on cell proliferation. Cells were plated in growth medium and treated with vehicle, UT-105, or JAK-STAT inhibitor ruxolitinib for seven days with medium change and retreatment after day 3 (n=4/group; representative of three replicates; one-way ANOVA). SRB assay was performed. I. AR and AR-SV -positive TNBC specimens are enriched for JAK-STAT pathway. RNA was extracted from TNBC specimens (n=41) shown in Fig. 1C and sequenced. The top pathways enriched in AR and AR-SV compared to AR negative specimens obtained from GSEA (FDR <0.25) are shown as a bar graph. J. Enrichment plots are shown. Rux- ruxolitinib; enza- enzalutamide. Mean -/+ SEM: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.00001.

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Expressing, RNA Sequencing Assay, Western Blot, Sulforhodamine B Assay

    A. Schematic representation of CDX and PDX experiments (biorender.com). B. Left. MDA-MB-453 orthotopic xenograft was conducted by implanting 5 million cells into the mammary fat pad of female NSG mice. Once the tumors reach 100–300 mm3, the animals (n=8–10/group) were randomized and treated orally with UT-105 (60 mg/kg/day) or vehicle control (DMSO + PEG-300) for 28 days. Tumor volume was measured by digital caliper twice weekly and the percent change in tumor volume is represented in the graph. Right. MDA-MB-453 tumor-bearing female NSG mice (n=8–10/group) were treated with enzalutamide (60 mg/kg/day), bicalutamide (60 mg/kg/day), or vehicle control for 28 days. C. Change in body weight of MDA-MB-453 tumor-bearing mice. D-E. UT-1355 TNBC PDX characterization. Protein was extracted from UT-1355 PDX tumor fragments and Western blot with AR NTD-binding antibody or AR-V7 antibody, and GAPDH antibody was performed. LNCaP and 22RV1 prostate cancer cells were used as control for AR and AR-SV, respectively. Representative blots shown. F. UT-105 completely inhibits UT-1355 PDX tumor growth. UT-1355 PDX tumor fragments (1 mm3) were orthotopically implanted into the mammary fat pad in female NSG mice (n=8–10/group), and a xenograft experiment was performed as indicated above for MDA-MB-453. G-H. Change in body weight of UT-1355-bearing mice and tumor weight. Mean -/+ SEM is shown with One way ANOVA conducted in Graph Pad Prism: * p<0.05, ** p<0.01, *** p<0.001.

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: A. Schematic representation of CDX and PDX experiments (biorender.com). B. Left. MDA-MB-453 orthotopic xenograft was conducted by implanting 5 million cells into the mammary fat pad of female NSG mice. Once the tumors reach 100–300 mm3, the animals (n=8–10/group) were randomized and treated orally with UT-105 (60 mg/kg/day) or vehicle control (DMSO + PEG-300) for 28 days. Tumor volume was measured by digital caliper twice weekly and the percent change in tumor volume is represented in the graph. Right. MDA-MB-453 tumor-bearing female NSG mice (n=8–10/group) were treated with enzalutamide (60 mg/kg/day), bicalutamide (60 mg/kg/day), or vehicle control for 28 days. C. Change in body weight of MDA-MB-453 tumor-bearing mice. D-E. UT-1355 TNBC PDX characterization. Protein was extracted from UT-1355 PDX tumor fragments and Western blot with AR NTD-binding antibody or AR-V7 antibody, and GAPDH antibody was performed. LNCaP and 22RV1 prostate cancer cells were used as control for AR and AR-SV, respectively. Representative blots shown. F. UT-105 completely inhibits UT-1355 PDX tumor growth. UT-1355 PDX tumor fragments (1 mm3) were orthotopically implanted into the mammary fat pad in female NSG mice (n=8–10/group), and a xenograft experiment was performed as indicated above for MDA-MB-453. G-H. Change in body weight of UT-1355-bearing mice and tumor weight. Mean -/+ SEM is shown with One way ANOVA conducted in Graph Pad Prism: * p<0.05, ** p<0.01, *** p<0.001.

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Western Blot, Binding Assay

    A. STAT1 increases R1881-induced AR transactivation. AR transactivation was performed in HEK-293 cells with 0.1 μg vector or STAT1. Numbers provided in bracket are R1881’s EC50 values. B. STAT1 and AR interact. AR and STAT1 (5 μg) were transfected into HEK-293 cells. Cells were treated 48 hours after transfection with 2000 IU interferon α and 10 nM R1881. Cells were harvested 4 hours after treatment, immunoprecipitation was performed with IgG or AR antibody, and Western blot was performed for STAT1. C. AR and STAT1 interact in MDA-MB-453 cells. MDA-MB-453 cells were maintained in charcoal-stripped serum-containing medium for two days and treated with interferon α and R1881 for 4 hours. Cells were fixed and immunostained for AR (red) and STAT1 (green), and confocal microscopy was performed. Scale is 2 μm. D. STAT1 is recruited to ARE (on FKBP5 regulatory region). MDA-MB-453 cells were treated as indicated. The cells were crosslinked, sheared, and ChIP PCR was performed with the STAT1 and IgG antibodies. PCR was performed using the primers for the indicated regions. E. AR is recruited to STAT1 responsive gene regulatory region. Data from AR ChIP-seq performed in PCa cells (n=2) were loaded in IGV browser and CXCL8 (IL8) and FKBP5 regulatory regions were scanned to determine the binding of AR. F. UT-105 inhibits STAT1 recruitment to STAT1RE and ARE. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium were treated with interferon α (1000 units), R1881 (1 nM), combination in the presence and absence of UT-105 (10 μM) for 4 hours. ChIP assay was performed with STAT1 antibody and real-time PCR was performed with primers specific for hIL8 STATRE and FKBP5 ARE. Representative experiment is shown in the figure. G. Ruxolitinib inhibits AR-target gene expression. MDA-MB-453 cells maintained in charcoal stripped serum-containing medium were treated with R1881 (0.1 nM) alone or in combination with ruxolitinib (3 μM) for 24 hours. RNA was extracted, and real-time PCR for FKBP5 was performed (n=4/group; one-way ANOVA). H. UT-105 inhibits STAT1-dependent coactivation of AR. AR transactivation was performed in HEK-293 cells in the presence or absence of 0.1 μg STAT1. I. Effect of AR and JAK inhibitors on the proliferation of patient tumors explants growth. Illustration of gelatin sponge culture (Biorender.com). Tumor tissue from two patients (1473 and 1474) were placed on pre-soaked gelatin sponges and treated as indicated for 48 hours. The tissues were fixed and stained with Ki67. The bar graphs show the percentage of cells that stained positively for Ki67. J. Model. A schematic of the proposed mechanism of action of SARDs in TNBC tumors (biorender.com). The model summarizes the choice of drug for inhibiting the growth of LAR TNBC tumors. This would depend on the specific mechanisms involved in AR activation and tumor progression. Enzalutamide may work if AR activity is the primary driver, but its efficacy could be limited by androgen surges. STAT inhibitors like ruxolitinib may not be effective if AR activation is independent of STAT1. Degraders like UT-105 seem promising as they degrade AR directly and could prevent AR or AR splice variant activation, offering a more comprehensive approach to inhibit tumor growth. However, the actual effectiveness of these drugs would need to be studied in clinical settings and may vary from patient to patient. Panels A-H representative of three independent experiments is shown.

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: A. STAT1 increases R1881-induced AR transactivation. AR transactivation was performed in HEK-293 cells with 0.1 μg vector or STAT1. Numbers provided in bracket are R1881’s EC50 values. B. STAT1 and AR interact. AR and STAT1 (5 μg) were transfected into HEK-293 cells. Cells were treated 48 hours after transfection with 2000 IU interferon α and 10 nM R1881. Cells were harvested 4 hours after treatment, immunoprecipitation was performed with IgG or AR antibody, and Western blot was performed for STAT1. C. AR and STAT1 interact in MDA-MB-453 cells. MDA-MB-453 cells were maintained in charcoal-stripped serum-containing medium for two days and treated with interferon α and R1881 for 4 hours. Cells were fixed and immunostained for AR (red) and STAT1 (green), and confocal microscopy was performed. Scale is 2 μm. D. STAT1 is recruited to ARE (on FKBP5 regulatory region). MDA-MB-453 cells were treated as indicated. The cells were crosslinked, sheared, and ChIP PCR was performed with the STAT1 and IgG antibodies. PCR was performed using the primers for the indicated regions. E. AR is recruited to STAT1 responsive gene regulatory region. Data from AR ChIP-seq performed in PCa cells (n=2) were loaded in IGV browser and CXCL8 (IL8) and FKBP5 regulatory regions were scanned to determine the binding of AR. F. UT-105 inhibits STAT1 recruitment to STAT1RE and ARE. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium were treated with interferon α (1000 units), R1881 (1 nM), combination in the presence and absence of UT-105 (10 μM) for 4 hours. ChIP assay was performed with STAT1 antibody and real-time PCR was performed with primers specific for hIL8 STATRE and FKBP5 ARE. Representative experiment is shown in the figure. G. Ruxolitinib inhibits AR-target gene expression. MDA-MB-453 cells maintained in charcoal stripped serum-containing medium were treated with R1881 (0.1 nM) alone or in combination with ruxolitinib (3 μM) for 24 hours. RNA was extracted, and real-time PCR for FKBP5 was performed (n=4/group; one-way ANOVA). H. UT-105 inhibits STAT1-dependent coactivation of AR. AR transactivation was performed in HEK-293 cells in the presence or absence of 0.1 μg STAT1. I. Effect of AR and JAK inhibitors on the proliferation of patient tumors explants growth. Illustration of gelatin sponge culture (Biorender.com). Tumor tissue from two patients (1473 and 1474) were placed on pre-soaked gelatin sponges and treated as indicated for 48 hours. The tissues were fixed and stained with Ki67. The bar graphs show the percentage of cells that stained positively for Ki67. J. Model. A schematic of the proposed mechanism of action of SARDs in TNBC tumors (biorender.com). The model summarizes the choice of drug for inhibiting the growth of LAR TNBC tumors. This would depend on the specific mechanisms involved in AR activation and tumor progression. Enzalutamide may work if AR activity is the primary driver, but its efficacy could be limited by androgen surges. STAT inhibitors like ruxolitinib may not be effective if AR activation is independent of STAT1. Degraders like UT-105 seem promising as they degrade AR directly and could prevent AR or AR splice variant activation, offering a more comprehensive approach to inhibit tumor growth. However, the actual effectiveness of these drugs would need to be studied in clinical settings and may vary from patient to patient. Panels A-H representative of three independent experiments is shown.

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Confocal Microscopy, ChIP-sequencing, Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Staining, Activation Assay, Activity Assay, Variant Assay

    Key resources table

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: Key resources table

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Virus, Plasmid Preparation, Recombinant, RNA Sequencing Assay, Synthesized, Expressing, Construct, Software

    (A) Growth factor screening. Serum starved MDA-MB-231 cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

    Journal: bioRxiv

    Article Title: Growth factor-induced desialylation for the fast control of endocytosis

    doi: 10.1101/2023.09.12.557183

    Figure Lengend Snippet: (A) Growth factor screening. Serum starved MDA-MB-231 cells were incubated for 1 h on ice with Alexa488-labeled Gal3 in the presence or absence of 100 ng/mL of the indicated growth factors. (B) Fluorescence intensity was determined for 4 independent experiments as in (A) (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (C) Experiment as in (A) with EGF under conditions of afatinib (AFT) or DANA treatment. 4 independent experiments (≈ 150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (D) Simplified scheme of the epithelial architecture of the mouse tongue. (E) EGFR (red) immunolabeling on tissue section of the ventral part of the mouse tongue. (F) Gal3 localization on sections of tongues from mice that had been treated or not with AFT. The mean intensity of Gal3 labeling was analyzed at the level of the basal layer on 6 mice per condition. Mann-Whitney test. Scale bars = 10 µm (A) or 45 µm (E,F). In (A,E,F): Nuclei in blue. In (B,C,F): Means ± SEM. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: MDA-MB-231 (ATCC HTB-26), genome edited MDA-MB-231 (NEU1-/-, NEU3-/-, NHE1-/-), MDA-MB-468 NHE1-/-, MDA-MB-468 NHE1-/- rescued with NHE1-mEmerald, HN12, MEF (mouse), HeLa, SUM159-AP2-eGFP and NR6 (mouse) cells were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine and penicillin/streptomycin (Thermo Fisher Scientific).

    Techniques: Incubation, Labeling, Fluorescence, Comparison, Immunolabeling, MANN-WHITNEY

    (A) Schematics of interaction between ⍰2-6 Sia-modified glycans and Gal3 or SNA. (B) Identification of Gal3 interactors. MDA-MB-231 cells were incubated for 1 h on ice with Gal3-His. Pulldowns were subjected to quantitative mass spectrometry analysis. Red quadrant shows significantly enriched Gal3 binders. (C) Identification of Gal3 binders that are also SNA interactors. MDA-MB-231 cells, either untreated or treated with STI, were incubated for 1 h on ice with SNA-biotin. Pulldowns were analyzed by quantitative mass spectrometry. 574 proteins from 5 independent experiments were common to Gal3 (from B) and SNA (from C) data sets. 93 of these were enriched with high confidence for both (yellow quadrant). (D) Volcano plot of these 93 high confidence interactors as to their propensity to undergo EGF-induced desialylation (experiment as in (C) in the presence of 100 ng/mL of EGF). 30 responders are highlighted in the green quadrant. Note the presence of ⍰3β1 integrin and CD97 amongst the hits. (E) Cluster analysis of the summarized list of “biological processes” gene ontology terms associated with the 30 EGF-sensitive Gal3 and SNA interactors. (F) Validation of proteomics experiments by SNA pulldown. SNA-biotin pulldown was performed as in (B) in the presence or absence of STI or EGF. Samples were analyzed by Western blotting against α3 integrin. Quantifications from 3 independent experiments. One-way ANOVA with Tukey’s multiple comparison test. (G) Validation of proteomics experiments by α3 integrin immunoprecipitation. MDA-MB-231 cells were incubated for 1 h on ice anti-α3 integrin antibodies in the presence or absence of EGF. After pulldown, samples were analyzed by Western blotting using SNA-biotin. Quantifications from 3 independent experiments. Two tailed unpaired t-test. In (F,G): ** p < 0.01, *** p < 0.001.

    Journal: bioRxiv

    Article Title: Growth factor-induced desialylation for the fast control of endocytosis

    doi: 10.1101/2023.09.12.557183

    Figure Lengend Snippet: (A) Schematics of interaction between ⍰2-6 Sia-modified glycans and Gal3 or SNA. (B) Identification of Gal3 interactors. MDA-MB-231 cells were incubated for 1 h on ice with Gal3-His. Pulldowns were subjected to quantitative mass spectrometry analysis. Red quadrant shows significantly enriched Gal3 binders. (C) Identification of Gal3 binders that are also SNA interactors. MDA-MB-231 cells, either untreated or treated with STI, were incubated for 1 h on ice with SNA-biotin. Pulldowns were analyzed by quantitative mass spectrometry. 574 proteins from 5 independent experiments were common to Gal3 (from B) and SNA (from C) data sets. 93 of these were enriched with high confidence for both (yellow quadrant). (D) Volcano plot of these 93 high confidence interactors as to their propensity to undergo EGF-induced desialylation (experiment as in (C) in the presence of 100 ng/mL of EGF). 30 responders are highlighted in the green quadrant. Note the presence of ⍰3β1 integrin and CD97 amongst the hits. (E) Cluster analysis of the summarized list of “biological processes” gene ontology terms associated with the 30 EGF-sensitive Gal3 and SNA interactors. (F) Validation of proteomics experiments by SNA pulldown. SNA-biotin pulldown was performed as in (B) in the presence or absence of STI or EGF. Samples were analyzed by Western blotting against α3 integrin. Quantifications from 3 independent experiments. One-way ANOVA with Tukey’s multiple comparison test. (G) Validation of proteomics experiments by α3 integrin immunoprecipitation. MDA-MB-231 cells were incubated for 1 h on ice anti-α3 integrin antibodies in the presence or absence of EGF. After pulldown, samples were analyzed by Western blotting using SNA-biotin. Quantifications from 3 independent experiments. Two tailed unpaired t-test. In (F,G): ** p < 0.01, *** p < 0.001.

    Article Snippet: MDA-MB-231 (ATCC HTB-26), genome edited MDA-MB-231 (NEU1-/-, NEU3-/-, NHE1-/-), MDA-MB-468 NHE1-/-, MDA-MB-468 NHE1-/- rescued with NHE1-mEmerald, HN12, MEF (mouse), HeLa, SUM159-AP2-eGFP and NR6 (mouse) cells were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine and penicillin/streptomycin (Thermo Fisher Scientific).

    Techniques: Modification, Incubation, Mass Spectrometry, Western Blot, Comparison, Immunoprecipitation, Two Tailed Test

    Neuraminidases and NHE1

    Journal: bioRxiv

    Article Title: Growth factor-induced desialylation for the fast control of endocytosis

    doi: 10.1101/2023.09.12.557183

    Figure Lengend Snippet: Neuraminidases and NHE1

    Article Snippet: MDA-MB-231 (ATCC HTB-26), genome edited MDA-MB-231 (NEU1-/-, NEU3-/-, NHE1-/-), MDA-MB-468 NHE1-/-, MDA-MB-468 NHE1-/- rescued with NHE1-mEmerald, HN12, MEF (mouse), HeLa, SUM159-AP2-eGFP and NR6 (mouse) cells were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine and penicillin/streptomycin (Thermo Fisher Scientific).

    Techniques:

    (A) Neuraminidases. Serum starved wildtype MDA-MB-231 cells or CRISPR-based knockouts of NEU1 , NEU3 or NEU1/3 were incubated for 1 h on ice with Alexa-488 coupled Gal3 in the presence or absence of EGF. Fluorescence signals were quantified from 3 independent experiments (≈ 75 cells per condition). One-way ANOVA with Tukey’s multiple comparison test. (B) Experiment as in (A) with patient skin fibroblasts harboring inactivating mutations in the indicated enzymes. 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Tukey’s multiple comparison test. (C) Schematics of N-glycosylation pathway. (D) pH quenching effect. Serum starved MDA-MB-231 cells were incubated for 1 h on ice at pH 7.4 or pH 8.4 in the presence or absence of EGF, fixed, and incubated at neutral pH with Alexa-488 coupled Gal3. Quantifications as in (A) on 3 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (E) Cells were treated as in (D), lysed after EGF stimulation, and analyzed by Western blotting against EGFR Y1068. 3 independent experiments. Unpaired t-test. (F) pH triggering effect. Serum starved MDA-MB-231 cells were incubated for 1 h on ice in pH 5.5 or pH 7.4 buffers with or without EGF and/or DANA, and then for 1 h on ice at neutral pH with Alexa-488 coupled Gal3. Quantifications as in (A) on 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (G) Role of NHE1, CRISPR. Serum starved wild-type MDA-MB-468 cells, CRISPR-based knockouts of NHE1, and NHE1-GFP rescue cells were incubated and quantified as in (A) on 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (H) pH 5.5 buffer can rescue NHE knockout cells. Serum starved MDA-MB-231 wildtype and NHE1 knockout cells were incubated for 1 h on ice in pH 5.5 or pH 7.4 buffers with or without EGF. Quantifications as in (A) on 4 independent experiments and normalized to controls (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (I) Role of NHE1, inhibitors. MDA-MB-231 cells were pretreated for 30 min at 37 °C with the indicated inhibitors or DMSO (CTRL) in serum free media before incubation in the continued presence of the inhibitors for 1 h on ice with Alexa-488 coupled Gal3 in the presence or absence of EGF. Quantifications as in (A) on 5 independent experiments (≈150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. Means ± SEM are shown in this figure. Except for (E): Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

    Journal: bioRxiv

    Article Title: Growth factor-induced desialylation for the fast control of endocytosis

    doi: 10.1101/2023.09.12.557183

    Figure Lengend Snippet: (A) Neuraminidases. Serum starved wildtype MDA-MB-231 cells or CRISPR-based knockouts of NEU1 , NEU3 or NEU1/3 were incubated for 1 h on ice with Alexa-488 coupled Gal3 in the presence or absence of EGF. Fluorescence signals were quantified from 3 independent experiments (≈ 75 cells per condition). One-way ANOVA with Tukey’s multiple comparison test. (B) Experiment as in (A) with patient skin fibroblasts harboring inactivating mutations in the indicated enzymes. 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Tukey’s multiple comparison test. (C) Schematics of N-glycosylation pathway. (D) pH quenching effect. Serum starved MDA-MB-231 cells were incubated for 1 h on ice at pH 7.4 or pH 8.4 in the presence or absence of EGF, fixed, and incubated at neutral pH with Alexa-488 coupled Gal3. Quantifications as in (A) on 3 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (E) Cells were treated as in (D), lysed after EGF stimulation, and analyzed by Western blotting against EGFR Y1068. 3 independent experiments. Unpaired t-test. (F) pH triggering effect. Serum starved MDA-MB-231 cells were incubated for 1 h on ice in pH 5.5 or pH 7.4 buffers with or without EGF and/or DANA, and then for 1 h on ice at neutral pH with Alexa-488 coupled Gal3. Quantifications as in (A) on 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (G) Role of NHE1, CRISPR. Serum starved wild-type MDA-MB-468 cells, CRISPR-based knockouts of NHE1, and NHE1-GFP rescue cells were incubated and quantified as in (A) on 4 independent experiments (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (H) pH 5.5 buffer can rescue NHE knockout cells. Serum starved MDA-MB-231 wildtype and NHE1 knockout cells were incubated for 1 h on ice in pH 5.5 or pH 7.4 buffers with or without EGF. Quantifications as in (A) on 4 independent experiments and normalized to controls (≈ 100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. (I) Role of NHE1, inhibitors. MDA-MB-231 cells were pretreated for 30 min at 37 °C with the indicated inhibitors or DMSO (CTRL) in serum free media before incubation in the continued presence of the inhibitors for 1 h on ice with Alexa-488 coupled Gal3 in the presence or absence of EGF. Quantifications as in (A) on 5 independent experiments (≈150 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. Means ± SEM are shown in this figure. Except for (E): Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

    Article Snippet: MDA-MB-231 (ATCC HTB-26), genome edited MDA-MB-231 (NEU1-/-, NEU3-/-, NHE1-/-), MDA-MB-468 NHE1-/-, MDA-MB-468 NHE1-/- rescued with NHE1-mEmerald, HN12, MEF (mouse), HeLa, SUM159-AP2-eGFP and NR6 (mouse) cells were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine and penicillin/streptomycin (Thermo Fisher Scientific).

    Techniques: CRISPR, Incubation, Fluorescence, Comparison, Western Blot, Knock-Out

    (A) ⍰3 integrin uptake. Serum starved MDA-MB-231 cells were continuously incubated for 10 min at 37 °C in the presence or absence of EGF with Atto647N-labeled anti-⍰3 integrin antibody and Alexa488-transferrin (Tf). Note that ⍰3 integrin uptake was stimulated in the presence of EGF. Scale bar = 10 µm. (B) Quantification of fluorescence from 3 independent experiments (≈ 100 cells per condition) as in (A) in which cells were incubated in the presence or absence of EGF and/or the Gal3 inhibitor I3. One-way ANOVA with Dunnett’s multiple comparison test. (C-F) β1 integrin and CD97 uptake. Experiments as in (A) in which anti-β1 integrin or anti-CD47 antibodies were used in the presence or absence of the indicated inhibitors and growth factors. 3-6 independent experiments (≈ 200 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. In (D): Scale bar = 10 µm. (G) pH imaging. MDA-MB-231 cells were incubated continuously for 4 min at 37 °C with AcidiFluor-coupled anti-α3 integrin antibody, and then imaged for 10 min sequentially in TIRF and epifluorescence modalities. EGF was added after 18 sec. Note that increased fluorescence in the TIRF field (red) indicates acidification. Arrows indicate 1 representative event. Scale bar = 10 µm. (H) Dynamic pH monitoring. Acquisition of images started immediately after incubation with antibody in sequential TIRF and epifluorescence modes at 37 °C. pH 4 buffer was added at the indicated time point to document how the setup reacted. Total fluorescence (mean) and Emax are shown for a representative cell. (I) Intensity traces for the event shown by arrows in (G). * indicates where vesicle leaves frame. TIRF in red, widefield in green. (J,K) Quantification of numbers per cell (I) or of maximum intensity values (Emax) (J) for tracks that undergo a pH change as shown in (G). 3 independent experiments (≈ 15 cells counted per condition). Holm-Šídák’s multiple comparisons test. Means ± SEM are shown in this figure. In (B,C,E,F), means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

    Journal: bioRxiv

    Article Title: Growth factor-induced desialylation for the fast control of endocytosis

    doi: 10.1101/2023.09.12.557183

    Figure Lengend Snippet: (A) ⍰3 integrin uptake. Serum starved MDA-MB-231 cells were continuously incubated for 10 min at 37 °C in the presence or absence of EGF with Atto647N-labeled anti-⍰3 integrin antibody and Alexa488-transferrin (Tf). Note that ⍰3 integrin uptake was stimulated in the presence of EGF. Scale bar = 10 µm. (B) Quantification of fluorescence from 3 independent experiments (≈ 100 cells per condition) as in (A) in which cells were incubated in the presence or absence of EGF and/or the Gal3 inhibitor I3. One-way ANOVA with Dunnett’s multiple comparison test. (C-F) β1 integrin and CD97 uptake. Experiments as in (A) in which anti-β1 integrin or anti-CD47 antibodies were used in the presence or absence of the indicated inhibitors and growth factors. 3-6 independent experiments (≈ 200 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. In (D): Scale bar = 10 µm. (G) pH imaging. MDA-MB-231 cells were incubated continuously for 4 min at 37 °C with AcidiFluor-coupled anti-α3 integrin antibody, and then imaged for 10 min sequentially in TIRF and epifluorescence modalities. EGF was added after 18 sec. Note that increased fluorescence in the TIRF field (red) indicates acidification. Arrows indicate 1 representative event. Scale bar = 10 µm. (H) Dynamic pH monitoring. Acquisition of images started immediately after incubation with antibody in sequential TIRF and epifluorescence modes at 37 °C. pH 4 buffer was added at the indicated time point to document how the setup reacted. Total fluorescence (mean) and Emax are shown for a representative cell. (I) Intensity traces for the event shown by arrows in (G). * indicates where vesicle leaves frame. TIRF in red, widefield in green. (J,K) Quantification of numbers per cell (I) or of maximum intensity values (Emax) (J) for tracks that undergo a pH change as shown in (G). 3 independent experiments (≈ 15 cells counted per condition). Holm-Šídák’s multiple comparisons test. Means ± SEM are shown in this figure. In (B,C,E,F), means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: MDA-MB-231 (ATCC HTB-26), genome edited MDA-MB-231 (NEU1-/-, NEU3-/-, NHE1-/-), MDA-MB-468 NHE1-/-, MDA-MB-468 NHE1-/- rescued with NHE1-mEmerald, HN12, MEF (mouse), HeLa, SUM159-AP2-eGFP and NR6 (mouse) cells were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine and penicillin/streptomycin (Thermo Fisher Scientific).

    Techniques: Incubation, Labeling, Fluorescence, Comparison, Imaging

    (A) Electron microcopy. Left: Representative overview electron micrographs of MDA-MB-231 cells that have been incubated with HRP-labeled Gal3 for 6 min at 37 °C in the presence or absence of EGF. Right: Zoomed views of CLICs from EGF-treated MDA-MB-231 cells for which the following HRP-labeled ligands were present during incubations: (i) Gal3, (ii) anti-α3 integrin antibodies, or (iii) anti-β1 integrin antibodies. Scale bars = 10 µm (images on left) and 200 nm (images on right). (B) Quantification of electron micrographs as in (A). HRP-positive structures from 2 independent experiments were counted for the indicated conditions (≈ 10 cells per condition) in a single slice per cell and categorized according to morphology. (C) Lattice light sheet microscopy. Representative maximum intensity projections of AP2-eGFP (green) expressing SUM159 cells in indicated conditions of incubation with anti-β1 integrin-Cy3 antibodies or Tf-Cy3 (magenta) in the presence or absence of EGF. Scale bars = 10 µm. (D) Central section of a SUM159 cell at 1 min 15 s into acquisition, showing an AP2-negative β1 integrin uptake event (in dotted box). Scale bar = 5 µm. (E) High magnifications panels of single frames from boxed area in (D). The AP2-negative uptake event is tracked by white arrowheads. (F) Percentage of β1 integrin and transferrin (Tf) uptake tracks from 3 independent experiments that were AP2-negative as in (C-E). For β1 integrin: 1802 tracks from 16 control cells, and 2157 tracks from 18 EGF-treated cells. For Tf: 1145 tracks from 11 control cells, and 1422 tracks from 12 EGF-treated cells. Note that the percentage of AP2-negative tracks increased upon EGF treatment only for β1 integrin uptake. Means ± SEM, unpaired t-test. (G) Average normalized intensity traces and classifications for β1 integrin and Tf uptake events from experiments in (F). Lifetime cohorts of endocytic trajectories show “dome”-shaped intensity profiles for cargoes, and co-tracking or not with AP2. Dotted lines represent background levels for fluorescence signals. (H) Selected β1 integrin tracks in EGF-treated cells. Axes show X,Y,Z positions of uptake carriers in cells, spot colors depicts AP2 intensity, line color distance to plasma membrane, and X indicates track starting points. Means ± SEM are shown in this Figure. ns = p > 0.05, ** p < 0.01.

    Journal: bioRxiv

    Article Title: Growth factor-induced desialylation for the fast control of endocytosis

    doi: 10.1101/2023.09.12.557183

    Figure Lengend Snippet: (A) Electron microcopy. Left: Representative overview electron micrographs of MDA-MB-231 cells that have been incubated with HRP-labeled Gal3 for 6 min at 37 °C in the presence or absence of EGF. Right: Zoomed views of CLICs from EGF-treated MDA-MB-231 cells for which the following HRP-labeled ligands were present during incubations: (i) Gal3, (ii) anti-α3 integrin antibodies, or (iii) anti-β1 integrin antibodies. Scale bars = 10 µm (images on left) and 200 nm (images on right). (B) Quantification of electron micrographs as in (A). HRP-positive structures from 2 independent experiments were counted for the indicated conditions (≈ 10 cells per condition) in a single slice per cell and categorized according to morphology. (C) Lattice light sheet microscopy. Representative maximum intensity projections of AP2-eGFP (green) expressing SUM159 cells in indicated conditions of incubation with anti-β1 integrin-Cy3 antibodies or Tf-Cy3 (magenta) in the presence or absence of EGF. Scale bars = 10 µm. (D) Central section of a SUM159 cell at 1 min 15 s into acquisition, showing an AP2-negative β1 integrin uptake event (in dotted box). Scale bar = 5 µm. (E) High magnifications panels of single frames from boxed area in (D). The AP2-negative uptake event is tracked by white arrowheads. (F) Percentage of β1 integrin and transferrin (Tf) uptake tracks from 3 independent experiments that were AP2-negative as in (C-E). For β1 integrin: 1802 tracks from 16 control cells, and 2157 tracks from 18 EGF-treated cells. For Tf: 1145 tracks from 11 control cells, and 1422 tracks from 12 EGF-treated cells. Note that the percentage of AP2-negative tracks increased upon EGF treatment only for β1 integrin uptake. Means ± SEM, unpaired t-test. (G) Average normalized intensity traces and classifications for β1 integrin and Tf uptake events from experiments in (F). Lifetime cohorts of endocytic trajectories show “dome”-shaped intensity profiles for cargoes, and co-tracking or not with AP2. Dotted lines represent background levels for fluorescence signals. (H) Selected β1 integrin tracks in EGF-treated cells. Axes show X,Y,Z positions of uptake carriers in cells, spot colors depicts AP2 intensity, line color distance to plasma membrane, and X indicates track starting points. Means ± SEM are shown in this Figure. ns = p > 0.05, ** p < 0.01.

    Article Snippet: MDA-MB-231 (ATCC HTB-26), genome edited MDA-MB-231 (NEU1-/-, NEU3-/-, NHE1-/-), MDA-MB-468 NHE1-/-, MDA-MB-468 NHE1-/- rescued with NHE1-mEmerald, HN12, MEF (mouse), HeLa, SUM159-AP2-eGFP and NR6 (mouse) cells were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine and penicillin/streptomycin (Thermo Fisher Scientific).

    Techniques: Incubation, Labeling, Microscopy, Expressing, Fluorescence, Membrane

    (A) Schematics of retrograde transport assay. Endocytic ligands (including antibodies against cell surface proteins) can either be directly coupled to benzylguanine (BG), as shown in (i), or the cell surface proteome can be modified with cell impermeable BG-NHS (ii). Proteins that undergo retrograde transport are captured in the Golgi by a covalent reaction with a GFP-tagged SNAPtag fusion protein that has been localized there. (B) HeLa cells stably expressing GalT-GFP-SNAP were continuously incubated for 4 h at 37 °C with BG-modified Gal3 before lysis and pulldown using GFP-trap beads. Western blot quantification from 3 independent experiments of Gal3-SNAP conjugate in pulldowns. Means ± SEM, unpaired t-test, *** p < 0.001. (C) Gal3 interaction proteomics. HeLa cells were incubated for 1 h on ice with Gal3-His in the presence or absence of EGF, followed by lysis, pulldown on cobalt-agarose beads, and quantitative mass spectrometry. Correlation plot of proteins. Blue quadrants indicate specific interactor of Gal3 in all conditions from 5 independent experiments. (D) Volcano blot of Gal3 interactors (blue quadrants from (C)) that are significantly (i.e., with 3 peptides and an adjusted p-value ≤ 0.05) enriched after EGF stimulation (red quadrant). (E) Retrograde proteomics. HeLa cells stably expressing GalT-GFP-SNAP were cell-surface modified on ice with NHS-PEG9-BG, followed by 16 h incubation at 37 °C, pulldown with GFP-trap beads, and quantitative mass spectrometry. High confidence retrograde cargoes are shown in the blue quadrant. (F) Comparison of Gal3 interactors and retrograde proteome. The yellow quadrant indicates high confidence Gal3 interactors that were enriched upon EGF stimulation and that also undergo retrograde transport. Note the presence of α3β1integrin and CD97 in this list. (G) 4 independent experiments as in (B) with BG-modified anti-β1 integrin antibodies. Quantification of anti-β1 integrin antibody-SNAP conjugates (IB anti-β1-SNAP) in GFP-trap pulldowns. Means ± SEM, unpaired t-test. (H) Resialylation analysis. MDA-MB-231 cells were incubated for 1 h on ice in the presence or absence of EGF, shifted in all conditions to 37 °C without EGF, and lysed at the indicated timepoints. ⍰3 integrin signal in SNA pulldowns (PD) was revealed by Western blotting. Note that ⍰3 integrin was still desialylated after 1 h of chase, and then became resialylated after 6 h of chase in a protein neosynthesis-independent manner (presence of cycloheximide (CHX). (I) Quantification of ⍰3 integrin signals from 3 independent experiments as in (H). Left, input; right, SNA PD. Means ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, ns = p > 0.05, ** p < 0.01.

    Journal: bioRxiv

    Article Title: Growth factor-induced desialylation for the fast control of endocytosis

    doi: 10.1101/2023.09.12.557183

    Figure Lengend Snippet: (A) Schematics of retrograde transport assay. Endocytic ligands (including antibodies against cell surface proteins) can either be directly coupled to benzylguanine (BG), as shown in (i), or the cell surface proteome can be modified with cell impermeable BG-NHS (ii). Proteins that undergo retrograde transport are captured in the Golgi by a covalent reaction with a GFP-tagged SNAPtag fusion protein that has been localized there. (B) HeLa cells stably expressing GalT-GFP-SNAP were continuously incubated for 4 h at 37 °C with BG-modified Gal3 before lysis and pulldown using GFP-trap beads. Western blot quantification from 3 independent experiments of Gal3-SNAP conjugate in pulldowns. Means ± SEM, unpaired t-test, *** p < 0.001. (C) Gal3 interaction proteomics. HeLa cells were incubated for 1 h on ice with Gal3-His in the presence or absence of EGF, followed by lysis, pulldown on cobalt-agarose beads, and quantitative mass spectrometry. Correlation plot of proteins. Blue quadrants indicate specific interactor of Gal3 in all conditions from 5 independent experiments. (D) Volcano blot of Gal3 interactors (blue quadrants from (C)) that are significantly (i.e., with 3 peptides and an adjusted p-value ≤ 0.05) enriched after EGF stimulation (red quadrant). (E) Retrograde proteomics. HeLa cells stably expressing GalT-GFP-SNAP were cell-surface modified on ice with NHS-PEG9-BG, followed by 16 h incubation at 37 °C, pulldown with GFP-trap beads, and quantitative mass spectrometry. High confidence retrograde cargoes are shown in the blue quadrant. (F) Comparison of Gal3 interactors and retrograde proteome. The yellow quadrant indicates high confidence Gal3 interactors that were enriched upon EGF stimulation and that also undergo retrograde transport. Note the presence of α3β1integrin and CD97 in this list. (G) 4 independent experiments as in (B) with BG-modified anti-β1 integrin antibodies. Quantification of anti-β1 integrin antibody-SNAP conjugates (IB anti-β1-SNAP) in GFP-trap pulldowns. Means ± SEM, unpaired t-test. (H) Resialylation analysis. MDA-MB-231 cells were incubated for 1 h on ice in the presence or absence of EGF, shifted in all conditions to 37 °C without EGF, and lysed at the indicated timepoints. ⍰3 integrin signal in SNA pulldowns (PD) was revealed by Western blotting. Note that ⍰3 integrin was still desialylated after 1 h of chase, and then became resialylated after 6 h of chase in a protein neosynthesis-independent manner (presence of cycloheximide (CHX). (I) Quantification of ⍰3 integrin signals from 3 independent experiments as in (H). Left, input; right, SNA PD. Means ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, ns = p > 0.05, ** p < 0.01.

    Article Snippet: MDA-MB-231 (ATCC HTB-26), genome edited MDA-MB-231 (NEU1-/-, NEU3-/-, NHE1-/-), MDA-MB-468 NHE1-/-, MDA-MB-468 NHE1-/- rescued with NHE1-mEmerald, HN12, MEF (mouse), HeLa, SUM159-AP2-eGFP and NR6 (mouse) cells were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine and penicillin/streptomycin (Thermo Fisher Scientific).

    Techniques: Transport Assay, Modification, Stable Transfection, Expressing, Incubation, Lysis, Western Blot, Mass Spectrometry, Comparison

    (A) Cell migration. MDA-MB-231 cells were seeded under the indicated conditions on cell derived matrices and incubated for 16 h incubations at 37 °C in 1 % FCS. Individual tracks are represented. (B) The Euclidean distance of migration was determined as in (A) in 3 independent experiments in the absence (DMSO) or presence of the Gal3 inhibitor I3 and of EGF. (C) Experiment as in (B) in the absence (DMSO) or presence of the indicated drugs. (D) Inverted invasion assays. MDA-MB-231 cells were attached to fibronectin-spiked collagen plugs and allowed to invade against gravity towards an upper reservoir with or without EGF. Invasion was considered beyond the indicated red line. Knockout of the indicated neuraminidases inhibited invasion. (E-F) Quantification of invasion from 3 independent experiments as in (D) with the indicated inhibitors and KO cells. (G) Schematics depicting the desialylation glycoswitch model. See text for details. Means ± SEM are shown in this figure. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. One-way ANOVA with Dunnett’s multiple comparison test, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

    Journal: bioRxiv

    Article Title: Growth factor-induced desialylation for the fast control of endocytosis

    doi: 10.1101/2023.09.12.557183

    Figure Lengend Snippet: (A) Cell migration. MDA-MB-231 cells were seeded under the indicated conditions on cell derived matrices and incubated for 16 h incubations at 37 °C in 1 % FCS. Individual tracks are represented. (B) The Euclidean distance of migration was determined as in (A) in 3 independent experiments in the absence (DMSO) or presence of the Gal3 inhibitor I3 and of EGF. (C) Experiment as in (B) in the absence (DMSO) or presence of the indicated drugs. (D) Inverted invasion assays. MDA-MB-231 cells were attached to fibronectin-spiked collagen plugs and allowed to invade against gravity towards an upper reservoir with or without EGF. Invasion was considered beyond the indicated red line. Knockout of the indicated neuraminidases inhibited invasion. (E-F) Quantification of invasion from 3 independent experiments as in (D) with the indicated inhibitors and KO cells. (G) Schematics depicting the desialylation glycoswitch model. See text for details. Means ± SEM are shown in this figure. Means from separate experiments are indicated by solid dots, and measurements of individual cells have different colored symbols for each experiment. One-way ANOVA with Dunnett’s multiple comparison test, * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: MDA-MB-231 (ATCC HTB-26), genome edited MDA-MB-231 (NEU1-/-, NEU3-/-, NHE1-/-), MDA-MB-468 NHE1-/-, MDA-MB-468 NHE1-/- rescued with NHE1-mEmerald, HN12, MEF (mouse), HeLa, SUM159-AP2-eGFP and NR6 (mouse) cells were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine and penicillin/streptomycin (Thermo Fisher Scientific).

    Techniques: Migration, Derivative Assay, Incubation, Knock-Out, Comparison