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Bio-Techne corporation arf6
Fascin distribution affects TMV shedding and clinical outcomes. (A and B) Endogenous fascin localization was determined in prostate cancer cells (A) and breast (B) tumor cell lines during amoeboid cell invasion. (C) Equal amounts of protein (determined using a BCA assay) from unfractionated ovarian ascites fluid and purified TMVs from patient samples were separated by SDS-PAGE and probed for fascin, <t>ARF6,</t> or HE4 content by Western blotting. Note that the protein is equal within but not between patients. Representative data are shown from patients later diagnosed with serous cystadenocarcinoma of the ovary (29), high-grade serous ovarian carcinoma (33), and poorly differentiated ovarian carcinoma (34). (D and E) Overall survival (D) and progression-free survival (E) were compared in patient cohorts (n = 1,657 and n = 1,436, respectively) with high or low fascin expression based on the various quantile expression levels; the two patient cohorts are compared by a Kaplan-Meier survival plot, with the hazard ratio, 95% confidence intervals, and log-rank P value calculated as previously reported (49, 50). (F and G) Overall (F) and progression-free (G) survival measures were also calculated using similar techniques for high fascin values coupled to low Rab35 expression levels.
Arf6, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Cell Signaling Technology Inc 92599 antibodies
Fascin distribution affects TMV shedding and clinical outcomes. (A and B) Endogenous fascin localization was determined in prostate cancer cells (A) and breast (B) tumor cell lines during amoeboid cell invasion. (C) Equal amounts of protein (determined using a BCA assay) from unfractionated ovarian ascites fluid and purified TMVs from patient samples were separated by SDS-PAGE and probed for fascin, <t>ARF6,</t> or HE4 content by Western blotting. Note that the protein is equal within but not between patients. Representative data are shown from patients later diagnosed with serous cystadenocarcinoma of the ovary (29), high-grade serous ovarian carcinoma (33), and poorly differentiated ovarian carcinoma (34). (D and E) Overall survival (D) and progression-free survival (E) were compared in patient cohorts (n = 1,657 and n = 1,436, respectively) with high or low fascin expression based on the various quantile expression levels; the two patient cohorts are compared by a Kaplan-Meier survival plot, with the hazard ratio, 95% confidence intervals, and log-rank P value calculated as previously reported (49, 50). (F and G) Overall (F) and progression-free (G) survival measures were also calculated using similar techniques for high fascin values coupled to low Rab35 expression levels.
92599 Antibodies, supplied by Cell Signaling Technology Inc, 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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Cerestar USA Inc f 92599 rueil malmaison
Fascin distribution affects TMV shedding and clinical outcomes. (A and B) Endogenous fascin localization was determined in prostate cancer cells (A) and breast (B) tumor cell lines during amoeboid cell invasion. (C) Equal amounts of protein (determined using a BCA assay) from unfractionated ovarian ascites fluid and purified TMVs from patient samples were separated by SDS-PAGE and probed for fascin, <t>ARF6,</t> or HE4 content by Western blotting. Note that the protein is equal within but not between patients. Representative data are shown from patients later diagnosed with serous cystadenocarcinoma of the ovary (29), high-grade serous ovarian carcinoma (33), and poorly differentiated ovarian carcinoma (34). (D and E) Overall survival (D) and progression-free survival (E) were compared in patient cohorts (n = 1,657 and n = 1,436, respectively) with high or low fascin expression based on the various quantile expression levels; the two patient cohorts are compared by a Kaplan-Meier survival plot, with the hazard ratio, 95% confidence intervals, and log-rank P value calculated as previously reported (49, 50). (F and G) Overall (F) and progression-free (G) survival measures were also calculated using similar techniques for high fascin values coupled to low Rab35 expression levels.
F 92599 Rueil Malmaison, supplied by Cerestar USA Inc, 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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Fascin distribution affects TMV shedding and clinical outcomes. (A and B) Endogenous fascin localization was determined in prostate cancer cells (A) and breast (B) tumor cell lines during amoeboid cell invasion. (C) Equal amounts of protein (determined using a BCA assay) from unfractionated ovarian ascites fluid and purified TMVs from patient samples were separated by SDS-PAGE and probed for fascin, ARF6, or HE4 content by Western blotting. Note that the protein is equal within but not between patients. Representative data are shown from patients later diagnosed with serous cystadenocarcinoma of the ovary (29), high-grade serous ovarian carcinoma (33), and poorly differentiated ovarian carcinoma (34). (D and E) Overall survival (D) and progression-free survival (E) were compared in patient cohorts (n = 1,657 and n = 1,436, respectively) with high or low fascin expression based on the various quantile expression levels; the two patient cohorts are compared by a Kaplan-Meier survival plot, with the hazard ratio, 95% confidence intervals, and log-rank P value calculated as previously reported (49, 50). (F and G) Overall (F) and progression-free (G) survival measures were also calculated using similar techniques for high fascin values coupled to low Rab35 expression levels.

Journal: Molecular and Cellular Biology

Article Title: Coordinated Regulation of Intracellular Fascin Distribution Governs Tumor Microvesicle Release and Invasive Cell Capacity

doi: 10.1128/MCB.00264-18

Figure Lengend Snippet: Fascin distribution affects TMV shedding and clinical outcomes. (A and B) Endogenous fascin localization was determined in prostate cancer cells (A) and breast (B) tumor cell lines during amoeboid cell invasion. (C) Equal amounts of protein (determined using a BCA assay) from unfractionated ovarian ascites fluid and purified TMVs from patient samples were separated by SDS-PAGE and probed for fascin, ARF6, or HE4 content by Western blotting. Note that the protein is equal within but not between patients. Representative data are shown from patients later diagnosed with serous cystadenocarcinoma of the ovary (29), high-grade serous ovarian carcinoma (33), and poorly differentiated ovarian carcinoma (34). (D and E) Overall survival (D) and progression-free survival (E) were compared in patient cohorts (n = 1,657 and n = 1,436, respectively) with high or low fascin expression based on the various quantile expression levels; the two patient cohorts are compared by a Kaplan-Meier survival plot, with the hazard ratio, 95% confidence intervals, and log-rank P value calculated as previously reported (49, 50). (F and G) Overall (F) and progression-free (G) survival measures were also calculated using similar techniques for high fascin values coupled to low Rab35 expression levels.

Article Snippet: Antibody to human PODXL was obtained from BioTechne, and antibodies to ARF6 were as described previously ( 42 ).

Techniques: BIA-KA, Purification, SDS Page, Western Blot, Expressing

Rab35 knockout facilitates amoeboid cell invasion. (A) LOX or LOXRab35KO cells were plated in an in vitro amoeboid cell invasion assay and allowed to invade for 24 h. Invasive cells were fixed, stained as indicated, and examined by confocal microscopy in order to quantify changes in invasive potential. Scale bars, 25 µm. (B) The average displacement of LOX, LOXRab35KO, and LOXRab35KO cells expressing ARF6-T27N was calculated after 8 h of invasion into compliant matrix. Data are presented as means ± the standard deviations (SD; *, P < 0.05). (C) Equal amounts of protein contained in lysates generated from TMVs isolated from LOX or LOXRab35KO cells were separated by SDS-PAGE, and the cargo content was examined as indicated by Western blotting. (D to F) Parental LOX cells alone (D) or pretreated with TMVs isolated from LOX cells (E) or LOXRab35KO cells (F) were plated in an amoeboid invasion assay. After 8 h of invasion, the cells were fixed, and the actin cytoskeleton was stained and imaged. Scale bars, 20 µm. (G) The average displacement per field of cells described for panels D to F was calculated after 8 h of invasion into compliant matrix. Data are presented as means ± the SD (*, P < 0.05; N.S., not statistically significant).

Journal: Molecular and Cellular Biology

Article Title: Coordinated Regulation of Intracellular Fascin Distribution Governs Tumor Microvesicle Release and Invasive Cell Capacity

doi: 10.1128/MCB.00264-18

Figure Lengend Snippet: Rab35 knockout facilitates amoeboid cell invasion. (A) LOX or LOXRab35KO cells were plated in an in vitro amoeboid cell invasion assay and allowed to invade for 24 h. Invasive cells were fixed, stained as indicated, and examined by confocal microscopy in order to quantify changes in invasive potential. Scale bars, 25 µm. (B) The average displacement of LOX, LOXRab35KO, and LOXRab35KO cells expressing ARF6-T27N was calculated after 8 h of invasion into compliant matrix. Data are presented as means ± the standard deviations (SD; *, P < 0.05). (C) Equal amounts of protein contained in lysates generated from TMVs isolated from LOX or LOXRab35KO cells were separated by SDS-PAGE, and the cargo content was examined as indicated by Western blotting. (D to F) Parental LOX cells alone (D) or pretreated with TMVs isolated from LOX cells (E) or LOXRab35KO cells (F) were plated in an amoeboid invasion assay. After 8 h of invasion, the cells were fixed, and the actin cytoskeleton was stained and imaged. Scale bars, 20 µm. (G) The average displacement per field of cells described for panels D to F was calculated after 8 h of invasion into compliant matrix. Data are presented as means ± the SD (*, P < 0.05; N.S., not statistically significant).

Article Snippet: Antibody to human PODXL was obtained from BioTechne, and antibodies to ARF6 were as described previously ( 42 ).

Techniques: Knock-Out, In Vitro, Invasion Assay, Staining, Confocal Microscopy, Expressing, Generated, Isolation, SDS Page, Western Blot

Rab35 knockout uncouples ARF6-regulated TMV shedding. (A) Control and LOXRab35KO cells were lysed and subjected to MT2-based GTP-specific pulldown as described in Materials and Methods. Loss of Rab35 leads to increased levels of ARF6-GTP. Data are presented as means ± the SD (*, P < 0.035). (B) Expression of dominant-negative ARF6-T27N in LOXRab35KO cells in an amoeboid cell invasion assay blocks invasion placing ARF6 activity downstream of Rab35. Scale bars, 25 µm (top panel) or 75 µm (lower panel). The quantification is shown in Fig. 2B. (C) LOXRab35KO cells were infected with retrovirus containing HA-ARF6-T27N, and the TMV levels were measured in conditioned media by nanoparticle tracking. (D) The transduction efficiency of HA-ARF6-T27N retrovirus was determined by Western blotting for the HA tag. (E) Particle concentrations, as measured by NTA, in conditioned media of HA-ARF6-T27N-infected cells. Data are presented as means ± the SEM (*, P < 0.01). (F) Lysates from and LOXRab35KO cells were separated by SDS-PAGE, and the levels of total and phosphorylated ERK were examined by Western blotting. (G) LOXRab35KO cells show sustained and elevated levels of active ERK. *, P < 0.01. (H) LOXRab35KO cells were treated with the small molecule ERK inhibitor U-0126 or vehicle control during amoeboid cell invasion. Scale bars, 25 µm. (I) The average displacement of the cells described for panel H was calculated after 8 h of invasion into compliant matrix. Data are presented as means ± the SD (*, P < 0.001). (J) Nanoparticle tracking analysis of conditioned media from untreated, vehicle control, or U-0126 treated LOXRab35KO cells. Data are presented as means ± the SEM for each particle diameter.

Journal: Molecular and Cellular Biology

Article Title: Coordinated Regulation of Intracellular Fascin Distribution Governs Tumor Microvesicle Release and Invasive Cell Capacity

doi: 10.1128/MCB.00264-18

Figure Lengend Snippet: Rab35 knockout uncouples ARF6-regulated TMV shedding. (A) Control and LOXRab35KO cells were lysed and subjected to MT2-based GTP-specific pulldown as described in Materials and Methods. Loss of Rab35 leads to increased levels of ARF6-GTP. Data are presented as means ± the SD (*, P < 0.035). (B) Expression of dominant-negative ARF6-T27N in LOXRab35KO cells in an amoeboid cell invasion assay blocks invasion placing ARF6 activity downstream of Rab35. Scale bars, 25 µm (top panel) or 75 µm (lower panel). The quantification is shown in Fig. 2B. (C) LOXRab35KO cells were infected with retrovirus containing HA-ARF6-T27N, and the TMV levels were measured in conditioned media by nanoparticle tracking. (D) The transduction efficiency of HA-ARF6-T27N retrovirus was determined by Western blotting for the HA tag. (E) Particle concentrations, as measured by NTA, in conditioned media of HA-ARF6-T27N-infected cells. Data are presented as means ± the SEM (*, P < 0.01). (F) Lysates from and LOXRab35KO cells were separated by SDS-PAGE, and the levels of total and phosphorylated ERK were examined by Western blotting. (G) LOXRab35KO cells show sustained and elevated levels of active ERK. *, P < 0.01. (H) LOXRab35KO cells were treated with the small molecule ERK inhibitor U-0126 or vehicle control during amoeboid cell invasion. Scale bars, 25 µm. (I) The average displacement of the cells described for panel H was calculated after 8 h of invasion into compliant matrix. Data are presented as means ± the SD (*, P < 0.001). (J) Nanoparticle tracking analysis of conditioned media from untreated, vehicle control, or U-0126 treated LOXRab35KO cells. Data are presented as means ± the SEM for each particle diameter.

Article Snippet: Antibody to human PODXL was obtained from BioTechne, and antibodies to ARF6 were as described previously ( 42 ).

Techniques: Knock-Out, Expressing, Dominant Negative Mutation, Invasion Assay, Activity Assay, Infection, Transduction, Western Blot, SDS Page

Loss of Rab35 alters intracellular fascin localization. (A) Parental LOX cells plated on unlabeled gelatin were fixed, stained, and analyzed by confocal microscopy to examine the localization of endogenous fascin at the invasive, ventral surfaces of the cells. (B) Fascin localization and cortactin localization were examined together in cells transiently expressing mEmerald-fascin on unlabeled gelatin. Colocalization of both markers is indicative of localization of both components to invadopodia. (C) Invadopodium-mediated degradation, together with the intracellular distribution of endogenous fascin or cortactin, was examined in mesenchymal LOX cells transfected with myc–Rab35-Q67L. Matrix degradation appears as dark spots on the fluorescent background. (D) LOX cells stably expressing dominant-negative ARF6-T27N were plated in a mesenchymal invasion assay and allowed to degrade matrix overnight before being fixed, stained as indicated, and analyzed by confocal microscopy. (E) Confocal immunofluorescent analysis of fascin distribution and invasive capacity in cells subjected to dominant inhibition of Rab35. (F) LOX cells were transfected with dominant-negative Rab35-S22N and used in an invadopodium invasion assay. Scale bar, 25 µm. (G) LOX and LOXRab35KO cells were plated in an in vitro mesenchymal invasion assay and allowed to degrade matrix overnight before being counterstained for actin and β1-integrin to define cell boundaries; they were then imaged by confocal microscopy. Depletion of Rab35 blocks invadopodium-mediated matrix degradation, which appears as dark spots in the control image. (H) Immunofluorescent staining of endogenous fascin at the invasive surface revealed that the intracellular distribution associated with invadopodium formation is lost with depletion of Rab35 and that fascin has a predominantly peripheral localization. (I) Expression of wild-type Rab35 rescues invadopodium-mediated invasive capacity in LOXRab35KO cells. Unless otherwise indicated, scale bars indicate 20 µm.

Journal: Molecular and Cellular Biology

Article Title: Coordinated Regulation of Intracellular Fascin Distribution Governs Tumor Microvesicle Release and Invasive Cell Capacity

doi: 10.1128/MCB.00264-18

Figure Lengend Snippet: Loss of Rab35 alters intracellular fascin localization. (A) Parental LOX cells plated on unlabeled gelatin were fixed, stained, and analyzed by confocal microscopy to examine the localization of endogenous fascin at the invasive, ventral surfaces of the cells. (B) Fascin localization and cortactin localization were examined together in cells transiently expressing mEmerald-fascin on unlabeled gelatin. Colocalization of both markers is indicative of localization of both components to invadopodia. (C) Invadopodium-mediated degradation, together with the intracellular distribution of endogenous fascin or cortactin, was examined in mesenchymal LOX cells transfected with myc–Rab35-Q67L. Matrix degradation appears as dark spots on the fluorescent background. (D) LOX cells stably expressing dominant-negative ARF6-T27N were plated in a mesenchymal invasion assay and allowed to degrade matrix overnight before being fixed, stained as indicated, and analyzed by confocal microscopy. (E) Confocal immunofluorescent analysis of fascin distribution and invasive capacity in cells subjected to dominant inhibition of Rab35. (F) LOX cells were transfected with dominant-negative Rab35-S22N and used in an invadopodium invasion assay. Scale bar, 25 µm. (G) LOX and LOXRab35KO cells were plated in an in vitro mesenchymal invasion assay and allowed to degrade matrix overnight before being counterstained for actin and β1-integrin to define cell boundaries; they were then imaged by confocal microscopy. Depletion of Rab35 blocks invadopodium-mediated matrix degradation, which appears as dark spots in the control image. (H) Immunofluorescent staining of endogenous fascin at the invasive surface revealed that the intracellular distribution associated with invadopodium formation is lost with depletion of Rab35 and that fascin has a predominantly peripheral localization. (I) Expression of wild-type Rab35 rescues invadopodium-mediated invasive capacity in LOXRab35KO cells. Unless otherwise indicated, scale bars indicate 20 µm.

Article Snippet: Antibody to human PODXL was obtained from BioTechne, and antibodies to ARF6 were as described previously ( 42 ).

Techniques: Staining, Confocal Microscopy, Expressing, Transfection, Stable Transfection, Dominant Negative Mutation, Invasion Assay, Inhibition, In Vitro

Working model outlines Rab35-ARF6-regulated TMV shedding. Working model for the intracellular signaling pathways governing the Rab35- and ARF6-regulated trafficking of fascin to distinct invasive structures. Rab35 inactivation blocks fascin recruitment to invadopodia from endomembrane structures. Instead, peripheral fascin is trapped at sites of TMV shedding, where it is bound in a complex with ezrin and PODXL and readily available to facilitate amoeboid cell invasion.

Journal: Molecular and Cellular Biology

Article Title: Coordinated Regulation of Intracellular Fascin Distribution Governs Tumor Microvesicle Release and Invasive Cell Capacity

doi: 10.1128/MCB.00264-18

Figure Lengend Snippet: Working model outlines Rab35-ARF6-regulated TMV shedding. Working model for the intracellular signaling pathways governing the Rab35- and ARF6-regulated trafficking of fascin to distinct invasive structures. Rab35 inactivation blocks fascin recruitment to invadopodia from endomembrane structures. Instead, peripheral fascin is trapped at sites of TMV shedding, where it is bound in a complex with ezrin and PODXL and readily available to facilitate amoeboid cell invasion.

Article Snippet: Antibody to human PODXL was obtained from BioTechne, and antibodies to ARF6 were as described previously ( 42 ).

Techniques: