transduced cb cd34 Search Results


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  • 99
    Millipore hela cells
    Ataxin-3 regulates starvation-induced autophagy. a-b, <t>HeLa</t> cells stably expressing <t>GFP-LC3</t> were treated with control siRNA or ataxin-3 siRNA and incubated for 1 hr with carrier alone or carrier with 1 µM of PI3P phospholipid. Then, the control cells and the ataxin-3 KD cells with the different treatments were shifted to starvation condition (HBSS, 4 hr) or kept in full-media. a, The number of LC3 dots was analysed for each of the conditions and presented as mean LC3 dots per cell ± s.e.m. S.e.m. was determined from n=5 fields from a single representative experiment out of three independent experiments. Two-way ANOVA (column factor siRNA *** P
    Hela Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 293t cells
    Purification of cTag8-Modified LVs in an Envelope-Independent Manner Thawed NM LVs and cTag8 LVs pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins were incubated with streptavidin Dynabeads for 1 hr at room temperature. Dynabeads were immobilized by magnetic capture, and flow-through fractions were collected. Streptavidin Dynabeads were then washed 4 times with cold PBS and resuspended in cold medium in the same volume as starting viral supernatants. Viral titers (international units per milliliter) of all fractions: (1) crude (Neat), (2) re-suspended magnetic beads (Beads), and (3) post-capture incubation flow-through (Flow-Through) fractions were determined by infectivity assay on <t>293T</t> cells. Data represent the viral recovery of each fraction compared to corresponding total vector input and are plotted ± SD of triplicate determinations; **p ≤ 0.01; ****p ≤ 0.0001.
    293t Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Calithera cb 839
    Combined <t>CB-839/anti-EGFR</t> mAb therapy results in cooperative efficacy in 3D culture in vitro . CRC cell lines were grown in 3D collagen culture. Cells were treated with either cetuximab (black), CB-839 (white), or cetuximab and CB-839 (grey). The number of colonies were counted. Shown is the percentage change in colony number normalized to vehicle control. Combination therapy results in decreased colonies in a 3D culture model compared to single agents (relative to vehicle control). Error bars represent ± SD (n = 2–3 biological replicates and 3 technical replicates).
    Cb 839, supplied by Calithera, 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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    99
    Millipore mda mb 231 cells
    - DHA induced Caspase-1 and Gasdermin D activation and IL-1β secretion in <t>MDA-MB-231.</t> Cells were stimulated at the indicated concentrations of DHA ( A ) or AA ( B ) for 6 h and the amount of active caspase-1 was assessed by labeling cells with FAM-YVAD-FMK FLICA followed by flow cytometry analysis. Histograms are representative of three independent experiments. Cells were stimulated at the indicated concentrations of DHA ( C ) or AA ( D ) for 6 or 18 h and IL-1β secretion was assessed by ELISA. Each bar represents the mean concentration ± SD. *p
    Mda Mb 231 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Ataxin-3 regulates starvation-induced autophagy. a-b, HeLa cells stably expressing GFP-LC3 were treated with control siRNA or ataxin-3 siRNA and incubated for 1 hr with carrier alone or carrier with 1 µM of PI3P phospholipid. Then, the control cells and the ataxin-3 KD cells with the different treatments were shifted to starvation condition (HBSS, 4 hr) or kept in full-media. a, The number of LC3 dots was analysed for each of the conditions and presented as mean LC3 dots per cell ± s.e.m. S.e.m. was determined from n=5 fields from a single representative experiment out of three independent experiments. Two-way ANOVA (column factor siRNA *** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Ataxin-3 regulates starvation-induced autophagy. a-b, HeLa cells stably expressing GFP-LC3 were treated with control siRNA or ataxin-3 siRNA and incubated for 1 hr with carrier alone or carrier with 1 µM of PI3P phospholipid. Then, the control cells and the ataxin-3 KD cells with the different treatments were shifted to starvation condition (HBSS, 4 hr) or kept in full-media. a, The number of LC3 dots was analysed for each of the conditions and presented as mean LC3 dots per cell ± s.e.m. S.e.m. was determined from n=5 fields from a single representative experiment out of three independent experiments. Two-way ANOVA (column factor siRNA *** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Stable Transfection, Expressing, Incubation

    Ataxin-3 contributes to autophagosome formation. a, Primary cultures of mouse cortical neurons were transduced with control or ataxin-3 lentiviral shRNAs and analysed for the levels of LC3-I under starvation condition (HBSS, 4 hr) or together with BafA1 (400 nM, 4 hr). Results are normalised to control cells (HBSS+BafA1). Mean ± s.e.m, n=5 replicates from two independent cultures. Two-way ANOVA (N.S not significant). b, HeLa cells were transfected with different ataxin-3 siRNA and scrambled siRNA used as a control. Ataxin-3 knockdown (KD) efficiency is presented as well as basal LC3-II levels. LC3-II levels in ataxin-3-depleted HeLa cells were normalised to control cells, n=4 independent experiments. One-way ANOVA (** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Ataxin-3 contributes to autophagosome formation. a, Primary cultures of mouse cortical neurons were transduced with control or ataxin-3 lentiviral shRNAs and analysed for the levels of LC3-I under starvation condition (HBSS, 4 hr) or together with BafA1 (400 nM, 4 hr). Results are normalised to control cells (HBSS+BafA1). Mean ± s.e.m, n=5 replicates from two independent cultures. Two-way ANOVA (N.S not significant). b, HeLa cells were transfected with different ataxin-3 siRNA and scrambled siRNA used as a control. Ataxin-3 knockdown (KD) efficiency is presented as well as basal LC3-II levels. LC3-II levels in ataxin-3-depleted HeLa cells were normalised to control cells, n=4 independent experiments. One-way ANOVA (** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Transduction, Transfection

    Expansion of the polyQ domain in ataxin-3 decreased deubiquitinase activity and increased its interaction with beclin 1. a , Beclin 1 was purified from proteasome inhibitor-treated cells that co-expressed HA-Ub and was incubated in-vitro with recombinant ataxin-3 Q22 or ataxin-3 Q80 for 30 min in deubiquitination buffer and samples were analysed for beclin 1 ubiquitination using anti-HA antibodies. b, Number of LC3 dots in ataxin-3 KD HeLa cells that were transfected with GFP ataxin-3 Q28, GFP ataxin-3 Q84 and GFP ataxin-3 ΔQ and starved with HBSS in the last 4 hr. Results are normalised to control siRNA-treated cells from n=4 independent experiments. One-way ANOVA (*** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Expansion of the polyQ domain in ataxin-3 decreased deubiquitinase activity and increased its interaction with beclin 1. a , Beclin 1 was purified from proteasome inhibitor-treated cells that co-expressed HA-Ub and was incubated in-vitro with recombinant ataxin-3 Q22 or ataxin-3 Q80 for 30 min in deubiquitination buffer and samples were analysed for beclin 1 ubiquitination using anti-HA antibodies. b, Number of LC3 dots in ataxin-3 KD HeLa cells that were transfected with GFP ataxin-3 Q28, GFP ataxin-3 Q84 and GFP ataxin-3 ΔQ and starved with HBSS in the last 4 hr. Results are normalised to control siRNA-treated cells from n=4 independent experiments. One-way ANOVA (*** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Activity Assay, Purification, Incubation, In Vitro, Recombinant, Transfection

    Analysis of the interaction of the polyQ domain with beclin 1. a, Empty FLAG, FLAG beclin 1 evolutionary conserved domain (ECD) alone, FLAG beclin 1 ΔECD, FLAG beclin 1 full length and GFP Q35 were transfected in HeLa cells for 24 hr and the cell lysates were immunoprecipitated with anti-FLAG antibody. Immunocomplexes were analysed using anti-GFP antibody. b, Superimposition of human beclin 1 ECD (pdb 4DDP) and Vps30 (pdb 5DFZ), the yeast orthologue of beclin 1. Structures reveal that the N-terminal helix (dark blue helix) of the human structure is displaced, most likely due to protein truncation for crystallographic purposes. The yeast structure suggests that this helix is part of the coiled-coil CC2 of beclin 1 instead of the ECD. c, Two binding-sites in human beclin 1 ECD reveal high docking scores for polyQ 7 (the docking scores for site 1 and site 2 are -10.394 and -10.721, respectively). Sites comprising the region adjacent to the N-terminal helix (dark blue) were not considered for the docking. d-e, Surface charge illustrations of human beclin 1 ECD with the two sites of polyQ interaction. Site 2 is in close proximity to a protruding hydrophobic loop (aromatic finger) composed by Phe359, Phe360 and Trp361 (top right e - cartoon view), which are thought to be implicated in beclin 1 anchorage to lipid membranes.

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Analysis of the interaction of the polyQ domain with beclin 1. a, Empty FLAG, FLAG beclin 1 evolutionary conserved domain (ECD) alone, FLAG beclin 1 ΔECD, FLAG beclin 1 full length and GFP Q35 were transfected in HeLa cells for 24 hr and the cell lysates were immunoprecipitated with anti-FLAG antibody. Immunocomplexes were analysed using anti-GFP antibody. b, Superimposition of human beclin 1 ECD (pdb 4DDP) and Vps30 (pdb 5DFZ), the yeast orthologue of beclin 1. Structures reveal that the N-terminal helix (dark blue helix) of the human structure is displaced, most likely due to protein truncation for crystallographic purposes. The yeast structure suggests that this helix is part of the coiled-coil CC2 of beclin 1 instead of the ECD. c, Two binding-sites in human beclin 1 ECD reveal high docking scores for polyQ 7 (the docking scores for site 1 and site 2 are -10.394 and -10.721, respectively). Sites comprising the region adjacent to the N-terminal helix (dark blue) were not considered for the docking. d-e, Surface charge illustrations of human beclin 1 ECD with the two sites of polyQ interaction. Site 2 is in close proximity to a protruding hydrophobic loop (aromatic finger) composed by Phe359, Phe360 and Trp361 (top right e - cartoon view), which are thought to be implicated in beclin 1 anchorage to lipid membranes.

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Transfection, Immunoprecipitation, Binding Assay

    Impaired starvation-induced autophagy and reduced beclin 1 levels in cells expressing expanded polyQ forms of huntingtin. a, Empty FLAG, FLAG huntingtin (HTT) N-terminal fragment (1-350) Q17, FLAG HTT (1-350) ΔQ and beclin 1 were transfected in HeLa cells for 24 hr and cell lysates were immunoprecipitated with anti-beclin 1 antibody. Immunocomplexes were analysed using anti-FLAG antibody. b , GFP ataxin-3 Q28 and FLAG full-length HTT Q138 were transfected in HeLa cells for 24 hr and endogenous beclin 1 was immunoprecipitated. Immunocomplexes were analysed using anti-ataxin-3 antibody (detect GFP-ataxin-3) and anti-FLAG antibody (detect HTT). The ratio of the bound ataxin-3 to beclin 1 is presented. c, stable-inducible HEK293 cells were switched on for 48 hr with doxycycline (Dox) to express GFP-HTT wild-type exon 1 (GFP-HTT Q23) or mutant GFP HTT exon 1 (GFP-HTT Q74). In the last 4 hr cells were starved (HBSS) and beclin 1 levels were analysed in each cell type. Results are normalised to control HTT Q23 cells no Dox (n=4 independent experiments). Two-way ANOVA (column factor Dox ** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Impaired starvation-induced autophagy and reduced beclin 1 levels in cells expressing expanded polyQ forms of huntingtin. a, Empty FLAG, FLAG huntingtin (HTT) N-terminal fragment (1-350) Q17, FLAG HTT (1-350) ΔQ and beclin 1 were transfected in HeLa cells for 24 hr and cell lysates were immunoprecipitated with anti-beclin 1 antibody. Immunocomplexes were analysed using anti-FLAG antibody. b , GFP ataxin-3 Q28 and FLAG full-length HTT Q138 were transfected in HeLa cells for 24 hr and endogenous beclin 1 was immunoprecipitated. Immunocomplexes were analysed using anti-ataxin-3 antibody (detect GFP-ataxin-3) and anti-FLAG antibody (detect HTT). The ratio of the bound ataxin-3 to beclin 1 is presented. c, stable-inducible HEK293 cells were switched on for 48 hr with doxycycline (Dox) to express GFP-HTT wild-type exon 1 (GFP-HTT Q23) or mutant GFP HTT exon 1 (GFP-HTT Q74). In the last 4 hr cells were starved (HBSS) and beclin 1 levels were analysed in each cell type. Results are normalised to control HTT Q23 cells no Dox (n=4 independent experiments). Two-way ANOVA (column factor Dox ** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Expressing, Transfection, Immunoprecipitation, Mutagenesis

    Expression of polyQ tracts impairs beclin 1-dependent starvation-induced autophagy. a, HeLa cells were transfected with empty GFP or GFP Q35 with or without FLAG ataxin-3 Q22 for 24 hr and were shifted to starvation condition (HBSS) in the last 4 hr. LC3-II and beclin 1 levels were analysed from the cell lysates. Results are mean ± s.e.m normalised to control (empty GFP), n=5 independent experiments, One-way ANOVA (for LC3-II ** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Expression of polyQ tracts impairs beclin 1-dependent starvation-induced autophagy. a, HeLa cells were transfected with empty GFP or GFP Q35 with or without FLAG ataxin-3 Q22 for 24 hr and were shifted to starvation condition (HBSS) in the last 4 hr. LC3-II and beclin 1 levels were analysed from the cell lysates. Results are mean ± s.e.m normalised to control (empty GFP), n=5 independent experiments, One-way ANOVA (for LC3-II ** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Expressing, Transfection

    Effect of different disease proteins with polyQ expansion on beclin 1 ubiquitination, beclin 1 levels and starvation-induced autophagy. a, HeLa cells were transfected with empty vector, GFP atrophin-1 (ATN-1) Q19, GFP ATN-1 Q71 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound ATN-1 using anti-ATN-1 antibody. b, HeLa cells were transfected with empty vector, HA androgen receptor (AR) Q25, HA AR Q120 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound AR using anti-AR antibody. c , Primary fibroblasts derived from healthy controls (n=5) and from HD patients (n=7) were starved with HBSS together with bafA1 (400 nM) for 4 hr and analysed for LC3-II levels. Results are mean ± s.e.m. * P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Effect of different disease proteins with polyQ expansion on beclin 1 ubiquitination, beclin 1 levels and starvation-induced autophagy. a, HeLa cells were transfected with empty vector, GFP atrophin-1 (ATN-1) Q19, GFP ATN-1 Q71 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound ATN-1 using anti-ATN-1 antibody. b, HeLa cells were transfected with empty vector, HA androgen receptor (AR) Q25, HA AR Q120 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound AR using anti-AR antibody. c , Primary fibroblasts derived from healthy controls (n=5) and from HD patients (n=7) were starved with HBSS together with bafA1 (400 nM) for 4 hr and analysed for LC3-II levels. Results are mean ± s.e.m. * P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Derivative Assay

    Purification of cTag8-Modified LVs in an Envelope-Independent Manner Thawed NM LVs and cTag8 LVs pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins were incubated with streptavidin Dynabeads for 1 hr at room temperature. Dynabeads were immobilized by magnetic capture, and flow-through fractions were collected. Streptavidin Dynabeads were then washed 4 times with cold PBS and resuspended in cold medium in the same volume as starting viral supernatants. Viral titers (international units per milliliter) of all fractions: (1) crude (Neat), (2) re-suspended magnetic beads (Beads), and (3) post-capture incubation flow-through (Flow-Through) fractions were determined by infectivity assay on 293T cells. Data represent the viral recovery of each fraction compared to corresponding total vector input and are plotted ± SD of triplicate determinations; **p ≤ 0.01; ****p ≤ 0.0001.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell

    doi: 10.1016/j.omtm.2018.10.008

    Figure Lengend Snippet: Purification of cTag8-Modified LVs in an Envelope-Independent Manner Thawed NM LVs and cTag8 LVs pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins were incubated with streptavidin Dynabeads for 1 hr at room temperature. Dynabeads were immobilized by magnetic capture, and flow-through fractions were collected. Streptavidin Dynabeads were then washed 4 times with cold PBS and resuspended in cold medium in the same volume as starting viral supernatants. Viral titers (international units per milliliter) of all fractions: (1) crude (Neat), (2) re-suspended magnetic beads (Beads), and (3) post-capture incubation flow-through (Flow-Through) fractions were determined by infectivity assay on 293T cells. Data represent the viral recovery of each fraction compared to corresponding total vector input and are plotted ± SD of triplicate determinations; **p ≤ 0.01; ****p ≤ 0.0001.

    Article Snippet: 293T cells were passaged 1:4–1:10 when cell density reached 75%–85% confluence using trypsin-EDTA solution (Sigma).

    Techniques: Purification, Modification, Incubation, Flow Cytometry, Magnetic Beads, Infection, Plasmid Preparation

    Biotin-Mediated Concentration and Purification of cTag8 LVs LVs produced from cTag8 293T cells were incubated with streptavidin Dynabeads for 1 hr at room temperature. Dynabeads were then washed 4 times with PBS and resuspended in plain DMEM and a fraction was collected. Subsequently, Dynabeads were magnetically immobilized and resuspended in Opti-MEM supplemented with 500 μM biotin and 0.5% BSA in 1/50 th of the starting volume and incubated for 1 hr at room temperature. Post-magnetic immobilization of Dynabeads, concentrated eluted LVs (eluate) were collected. (A) Viral titers (IU/mL) of starting (Neat), captured (Beads) and elution (Eluate) fractions were determined by infectivity assay, from which (B) the total transducing units (Total TU) in starting and eluted fractions were calculated. (C) Concentrations of dsDNA were quantified in collected viral fractions by PicoGreen analysis (detection limit

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell

    doi: 10.1016/j.omtm.2018.10.008

    Figure Lengend Snippet: Biotin-Mediated Concentration and Purification of cTag8 LVs LVs produced from cTag8 293T cells were incubated with streptavidin Dynabeads for 1 hr at room temperature. Dynabeads were then washed 4 times with PBS and resuspended in plain DMEM and a fraction was collected. Subsequently, Dynabeads were magnetically immobilized and resuspended in Opti-MEM supplemented with 500 μM biotin and 0.5% BSA in 1/50 th of the starting volume and incubated for 1 hr at room temperature. Post-magnetic immobilization of Dynabeads, concentrated eluted LVs (eluate) were collected. (A) Viral titers (IU/mL) of starting (Neat), captured (Beads) and elution (Eluate) fractions were determined by infectivity assay, from which (B) the total transducing units (Total TU) in starting and eluted fractions were calculated. (C) Concentrations of dsDNA were quantified in collected viral fractions by PicoGreen analysis (detection limit

    Article Snippet: 293T cells were passaged 1:4–1:10 when cell density reached 75%–85% confluence using trypsin-EDTA solution (Sigma).

    Techniques: Concentration Assay, Purification, Produced, Incubation, Infection

    Generation of cTag8-Expressing 293T Cells for Modified Vector Production (A) Both 293T (non-transduced) and 293T cells expressing cTag8 (cTag8 293T) by γ-retroviral transduction with cTag8 co-expressed with EGFP were stained with streptavidin-APC for cTag8 expression analysis by flow cytometry. (B) Surface expression analysis of cTag8 by immunofluorescence staining of cTag8 293T cells with streptavidin-APC. Engineered cells were assessed for LV packaging capacity by the production of LVs from both 293T cells (non-modified, NM LVs) and cTag8 293T cells (cTag8 LVs) in plain DMEM, pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins. (C) Viral supernatants were frozen, viral titers (infectious units (IU)/mL) of stocks were determined by infectivity assay, and mean values are presented ± SD of triplicate determinations. (D) Sucrose-cushion-ultracentrifuge-purified VSV-G pseudotyped NM LVs and cTag8 LVs were negatively stained and analyzed by transmission electron microscopy (TEM). Scale bars, 200 nm.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell

    doi: 10.1016/j.omtm.2018.10.008

    Figure Lengend Snippet: Generation of cTag8-Expressing 293T Cells for Modified Vector Production (A) Both 293T (non-transduced) and 293T cells expressing cTag8 (cTag8 293T) by γ-retroviral transduction with cTag8 co-expressed with EGFP were stained with streptavidin-APC for cTag8 expression analysis by flow cytometry. (B) Surface expression analysis of cTag8 by immunofluorescence staining of cTag8 293T cells with streptavidin-APC. Engineered cells were assessed for LV packaging capacity by the production of LVs from both 293T cells (non-modified, NM LVs) and cTag8 293T cells (cTag8 LVs) in plain DMEM, pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins. (C) Viral supernatants were frozen, viral titers (infectious units (IU)/mL) of stocks were determined by infectivity assay, and mean values are presented ± SD of triplicate determinations. (D) Sucrose-cushion-ultracentrifuge-purified VSV-G pseudotyped NM LVs and cTag8 LVs were negatively stained and analyzed by transmission electron microscopy (TEM). Scale bars, 200 nm.

    Article Snippet: 293T cells were passaged 1:4–1:10 when cell density reached 75%–85% confluence using trypsin-EDTA solution (Sigma).

    Techniques: Expressing, Modification, Plasmid Preparation, Transduction, Staining, Flow Cytometry, Cytometry, Immunofluorescence, Infection, Purification, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    Streptavidin-Mediated Complete Capture of cTag8-Modified LVs Transiently produced LVs from 293T and cTag8 293T cells, termed NM LVs and cTag8 LVs, respectively, were incubated with streptavidin magnetic beads. Dynabeads were immobilized by magnetic capture, and flow-through fractions were collected. Streptavidin Dynabeads were then washed 4 times with cold PBS and resuspended in cold medium, in the same volume as starting viral supernatants. Viral titers (IU/mL) of all fractions: (1) crude (Neat), (2) re-suspended magnetic bead (Beads), and (3) post-capture incubation flow-through (Flow-Through) fractions were determined by infectivity assay on 293T cells. (A) NM LVs and cTag8 LVs were produced in either serum-supplemented IMDM (serum) or serum-free DMEM (plain) medium. Viral supernatants were then subjected to capture methodology for 2 hr at 4°C. (B) Streptavidin Dynabeads were pre-treated either with plain or 15 mM biotin-supplemented PBS for 1 hr at room temperature. After 3 washes with PBS, pre-treated beads were incubated with cTag8 LVs in serum-free media for 1 hr at room temperature. (C) LVs were produced in serum-free media from low (L), medium (M), and high (H) cTag8-expressing 293T cells transiently. Along with control NM LVs, all viral supernatants were incubated with streptavidin Dynabeads for 1 hr at room temperature. All values presented represent viral recovery of each fraction compared to corresponding total vector input. Data are plotted ± SD of triplicate determinations. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns, non-significant.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell

    doi: 10.1016/j.omtm.2018.10.008

    Figure Lengend Snippet: Streptavidin-Mediated Complete Capture of cTag8-Modified LVs Transiently produced LVs from 293T and cTag8 293T cells, termed NM LVs and cTag8 LVs, respectively, were incubated with streptavidin magnetic beads. Dynabeads were immobilized by magnetic capture, and flow-through fractions were collected. Streptavidin Dynabeads were then washed 4 times with cold PBS and resuspended in cold medium, in the same volume as starting viral supernatants. Viral titers (IU/mL) of all fractions: (1) crude (Neat), (2) re-suspended magnetic bead (Beads), and (3) post-capture incubation flow-through (Flow-Through) fractions were determined by infectivity assay on 293T cells. (A) NM LVs and cTag8 LVs were produced in either serum-supplemented IMDM (serum) or serum-free DMEM (plain) medium. Viral supernatants were then subjected to capture methodology for 2 hr at 4°C. (B) Streptavidin Dynabeads were pre-treated either with plain or 15 mM biotin-supplemented PBS for 1 hr at room temperature. After 3 washes with PBS, pre-treated beads were incubated with cTag8 LVs in serum-free media for 1 hr at room temperature. (C) LVs were produced in serum-free media from low (L), medium (M), and high (H) cTag8-expressing 293T cells transiently. Along with control NM LVs, all viral supernatants were incubated with streptavidin Dynabeads for 1 hr at room temperature. All values presented represent viral recovery of each fraction compared to corresponding total vector input. Data are plotted ± SD of triplicate determinations. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns, non-significant.

    Article Snippet: 293T cells were passaged 1:4–1:10 when cell density reached 75%–85% confluence using trypsin-EDTA solution (Sigma).

    Techniques: Modification, Produced, Incubation, Magnetic Beads, Flow Cytometry, Infection, Expressing, Plasmid Preparation

    Reversible Streptavidin Binding of Synthetic Surface Expressing Biotin Mimics (A) Schematic diagrams of the chosen mimics with their reported dissociation constants (K D ). Four surface expression structures were cloned, and their architectures are represented: (1) Flush, consisting of a CD8-derived transmembrane (TM) and endodomain (Endo); (2) GPI, consisting of GPI anchor sequence (25 amino acids [aa]), which leads to the addition of GPI at the anchor sequence, with a serine-glycine linker (6 aa); (3) x2-GPI, consisting of 2 copies of the epitopes’ open reading frame (ORF) separated by a serine-glycine linker with the first 14 aa of the CD8α stalk ectodomain (Ecto; Linker_CD8), on a GPI anchor sequence; (4) CD8α, consisting of the CD8α stalk comprising the ecto-, transmembrane, and endodomains of the human CD8α molecule, with a serine-glycine linker. All peptides were cloned into a retroviral plasmid termed “SFG,” derived from the Moloney murine leukemia virus (MoMLV), upstream of the EGFP marker gene expressed by an internal ribosome entry site (IRES). (B) 293T cells were transiently transfected with all cloned constructs and stained with APC-conjugated streptavidin 48 hr later. The median fluorescence intensity (MedFI) of streptavidin binding of EGFP-positive cells are presented in a graph indicating ±SD of three independent transient expression experiments; ****p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell

    doi: 10.1016/j.omtm.2018.10.008

    Figure Lengend Snippet: Reversible Streptavidin Binding of Synthetic Surface Expressing Biotin Mimics (A) Schematic diagrams of the chosen mimics with their reported dissociation constants (K D ). Four surface expression structures were cloned, and their architectures are represented: (1) Flush, consisting of a CD8-derived transmembrane (TM) and endodomain (Endo); (2) GPI, consisting of GPI anchor sequence (25 amino acids [aa]), which leads to the addition of GPI at the anchor sequence, with a serine-glycine linker (6 aa); (3) x2-GPI, consisting of 2 copies of the epitopes’ open reading frame (ORF) separated by a serine-glycine linker with the first 14 aa of the CD8α stalk ectodomain (Ecto; Linker_CD8), on a GPI anchor sequence; (4) CD8α, consisting of the CD8α stalk comprising the ecto-, transmembrane, and endodomains of the human CD8α molecule, with a serine-glycine linker. All peptides were cloned into a retroviral plasmid termed “SFG,” derived from the Moloney murine leukemia virus (MoMLV), upstream of the EGFP marker gene expressed by an internal ribosome entry site (IRES). (B) 293T cells were transiently transfected with all cloned constructs and stained with APC-conjugated streptavidin 48 hr later. The median fluorescence intensity (MedFI) of streptavidin binding of EGFP-positive cells are presented in a graph indicating ±SD of three independent transient expression experiments; ****p

    Article Snippet: 293T cells were passaged 1:4–1:10 when cell density reached 75%–85% confluence using trypsin-EDTA solution (Sigma).

    Techniques: Binding Assay, Expressing, Clone Assay, Derivative Assay, Sequencing, Plasmid Preparation, Marker, Transfection, Construct, Staining, Fluorescence

    Combined CB-839/anti-EGFR mAb therapy results in cooperative efficacy in 3D culture in vitro . CRC cell lines were grown in 3D collagen culture. Cells were treated with either cetuximab (black), CB-839 (white), or cetuximab and CB-839 (grey). The number of colonies were counted. Shown is the percentage change in colony number normalized to vehicle control. Combination therapy results in decreased colonies in a 3D culture model compared to single agents (relative to vehicle control). Error bars represent ± SD (n = 2–3 biological replicates and 3 technical replicates).

    Journal: Translational Oncology

    Article Title: Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer

    doi: 10.1016/j.tranon.2020.100828

    Figure Lengend Snippet: Combined CB-839/anti-EGFR mAb therapy results in cooperative efficacy in 3D culture in vitro . CRC cell lines were grown in 3D collagen culture. Cells were treated with either cetuximab (black), CB-839 (white), or cetuximab and CB-839 (grey). The number of colonies were counted. Shown is the percentage change in colony number normalized to vehicle control. Combination therapy results in decreased colonies in a 3D culture model compared to single agents (relative to vehicle control). Error bars represent ± SD (n = 2–3 biological replicates and 3 technical replicates).

    Article Snippet: Shown are data for the live cell fluorescence assay with the following treatments: SW48 and HCA-7 cells were exposed to vehicle, 5 μg/mL cetuximab, 250 nM CB-839, or 5 μg/mL cetuximab and 250 nM CB-839; V9P, LoVo, and LS 174 T cells were exposed to vehicle, 10 μg/mL cetuximab, 1 μM CB-839, or 10 μg/mL cetuximab and 1 μM CB-839; and DiFi and LIM1215 cells were exposed to vehicle, 0.01 μg cetuximab, 1 μM CB-839, or 0.01 μg cetuximab and 1 μM CB-839.

    Techniques: In Vitro

    Combined CB-839/anti-EGFR mAb therapy results in cooperative efficacy in 2D culture in vitro . CRC cell lines were propagated in 2D culture. Cells were treated with either cetuximab (black), CB-839 (white), or cetuximab and CB-839 (grey). Cell viability was determined using MultiTox Glo cytotoxicity assay. Shown is the percentage change in live cells from the fluorescence assay normalized to vehicle control. CRC cell lines propagated in 2D culture exhibit reduced viability with combined therapy compared to single agents (relative to vehicle control). Error bars represent ± standard deviation (SD) (n = 3 or 4 technical replicates). Combination indices were determined and are indicated for each cell line by the following symbols: * synergism, ^ additive, and” not available.

    Journal: Translational Oncology

    Article Title: Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer

    doi: 10.1016/j.tranon.2020.100828

    Figure Lengend Snippet: Combined CB-839/anti-EGFR mAb therapy results in cooperative efficacy in 2D culture in vitro . CRC cell lines were propagated in 2D culture. Cells were treated with either cetuximab (black), CB-839 (white), or cetuximab and CB-839 (grey). Cell viability was determined using MultiTox Glo cytotoxicity assay. Shown is the percentage change in live cells from the fluorescence assay normalized to vehicle control. CRC cell lines propagated in 2D culture exhibit reduced viability with combined therapy compared to single agents (relative to vehicle control). Error bars represent ± standard deviation (SD) (n = 3 or 4 technical replicates). Combination indices were determined and are indicated for each cell line by the following symbols: * synergism, ^ additive, and” not available.

    Article Snippet: Shown are data for the live cell fluorescence assay with the following treatments: SW48 and HCA-7 cells were exposed to vehicle, 5 μg/mL cetuximab, 250 nM CB-839, or 5 μg/mL cetuximab and 250 nM CB-839; V9P, LoVo, and LS 174 T cells were exposed to vehicle, 10 μg/mL cetuximab, 1 μM CB-839, or 10 μg/mL cetuximab and 1 μM CB-839; and DiFi and LIM1215 cells were exposed to vehicle, 0.01 μg cetuximab, 1 μM CB-839, or 0.01 μg cetuximab and 1 μM CB-839.

    Techniques: In Vitro, Cytotoxicity Assay, Fluorescence, Standard Deviation

    Combined CB-839/anti-EGFR mAb therapy overcomes intrinsic cetuximab-resistance in SW48 xenografts in vivo . (A) Mice with SW48 cell line xenografts, a model of intrinsic cetuximab resistance, were treated with either vehicle (black), CB-839 (red), cetuximab (blue) or cetuximab and CB-839 (purple) for 30 days and tumor volumes were monitored (n = 5 mice/group). (B) Representative images of mice treated with cetuximab alone (left) or cetuximab in combination with CB-839 (right). Mice progressed on single agent CB-839 or cetuximab yet exhibit reduced tumor volume with combination cetuximab and CB-839. (C) Immunohistochemistry (IHC) was performed on tumors from each of the treatment groups to evaluate Ki67 and pS6 expression. Shown are representative images (40×). The number of positive cells were counted in regions of interest on images from Ki67 IHC (D) and pS6 IHC (E) (n = 3–15 fields of view, at least 3 animals/treatment group). Reduced Ki67 and pS6 were observed when treated with CB-839 in combination with cetuximab compared to cetuximab or CB-839 alone. (F) Representative H E images (0.46×) of tumor tissues from each of the treatment groups. (G) IHC was performed on tumors from each of the treatment groups to evaluate cleaved caspase-3 expression. Shown are representative images (20×). (H) The number of positive cells were counted in regions of interest on images from cleaved caspase-3 IHC (n = 3–15 fields of view, at least 3 animals/treatment group). Error bars represent ± SD. Statistical significance is defined as follows: *p

    Journal: Translational Oncology

    Article Title: Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer

    doi: 10.1016/j.tranon.2020.100828

    Figure Lengend Snippet: Combined CB-839/anti-EGFR mAb therapy overcomes intrinsic cetuximab-resistance in SW48 xenografts in vivo . (A) Mice with SW48 cell line xenografts, a model of intrinsic cetuximab resistance, were treated with either vehicle (black), CB-839 (red), cetuximab (blue) or cetuximab and CB-839 (purple) for 30 days and tumor volumes were monitored (n = 5 mice/group). (B) Representative images of mice treated with cetuximab alone (left) or cetuximab in combination with CB-839 (right). Mice progressed on single agent CB-839 or cetuximab yet exhibit reduced tumor volume with combination cetuximab and CB-839. (C) Immunohistochemistry (IHC) was performed on tumors from each of the treatment groups to evaluate Ki67 and pS6 expression. Shown are representative images (40×). The number of positive cells were counted in regions of interest on images from Ki67 IHC (D) and pS6 IHC (E) (n = 3–15 fields of view, at least 3 animals/treatment group). Reduced Ki67 and pS6 were observed when treated with CB-839 in combination with cetuximab compared to cetuximab or CB-839 alone. (F) Representative H E images (0.46×) of tumor tissues from each of the treatment groups. (G) IHC was performed on tumors from each of the treatment groups to evaluate cleaved caspase-3 expression. Shown are representative images (20×). (H) The number of positive cells were counted in regions of interest on images from cleaved caspase-3 IHC (n = 3–15 fields of view, at least 3 animals/treatment group). Error bars represent ± SD. Statistical significance is defined as follows: *p

    Article Snippet: Shown are data for the live cell fluorescence assay with the following treatments: SW48 and HCA-7 cells were exposed to vehicle, 5 μg/mL cetuximab, 250 nM CB-839, or 5 μg/mL cetuximab and 250 nM CB-839; V9P, LoVo, and LS 174 T cells were exposed to vehicle, 10 μg/mL cetuximab, 1 μM CB-839, or 10 μg/mL cetuximab and 1 μM CB-839; and DiFi and LIM1215 cells were exposed to vehicle, 0.01 μg cetuximab, 1 μM CB-839, or 0.01 μg cetuximab and 1 μM CB-839.

    Techniques: In Vivo, Mouse Assay, Immunohistochemistry, Expressing

    Combined CB-839/anti-EGFR mAb therapy overcomes acquired cetuximab-resistance in HCA-7 CC-CR xenografts in vivo . (A) Mice with HCA-7 CC-CR cell line xenografts, a model of acquired cetuximab-resistance, were treated with either vehicle (black), CB-839 (red), cetuximab (blue) or cetuximab and CB-839 (purple) for 21 days. At this time point, treatment was stopped (dotted line). Tumor volumes were monitored to day 57 (n = 8 mice/group). Mice progressed on single agent CB-839 or cetuximab yet exhibit reduced tumor volume with combination cetuximab and CB-839. (B) IHC was performed on tumors from each of the treatment groups to evaluate Ki67 and pS6 expression. Shown are representative images (40×). The number of positive cells were counted in regions of interest on images from Ki67 IHC (C) and pS6 IHC (D) (n = 3–15 fields of view, at least 3 animals/treatment group). Reduced Ki67 and pS6 were observed when treated with CB-839 in combination with cetuximab compared to cetuximab or CB-839 alone. (E) Representative H E images (0.46×) of tumor tissues from each of the treatment groups. (F) IHC was performed on tumors from each of the treatment groups to evaluate cleaved caspase-3 expression. Shown are representative images (20×). (G) The number of positive cells were counted in regions of interest on images from cleaved caspase-3 IHC (n = 3–15 fields of view, at least 3 animals/treatment group). Error bars represent ± SD. Statistical significance is defined as follows: # p = 0.0574, *p

    Journal: Translational Oncology

    Article Title: Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer

    doi: 10.1016/j.tranon.2020.100828

    Figure Lengend Snippet: Combined CB-839/anti-EGFR mAb therapy overcomes acquired cetuximab-resistance in HCA-7 CC-CR xenografts in vivo . (A) Mice with HCA-7 CC-CR cell line xenografts, a model of acquired cetuximab-resistance, were treated with either vehicle (black), CB-839 (red), cetuximab (blue) or cetuximab and CB-839 (purple) for 21 days. At this time point, treatment was stopped (dotted line). Tumor volumes were monitored to day 57 (n = 8 mice/group). Mice progressed on single agent CB-839 or cetuximab yet exhibit reduced tumor volume with combination cetuximab and CB-839. (B) IHC was performed on tumors from each of the treatment groups to evaluate Ki67 and pS6 expression. Shown are representative images (40×). The number of positive cells were counted in regions of interest on images from Ki67 IHC (C) and pS6 IHC (D) (n = 3–15 fields of view, at least 3 animals/treatment group). Reduced Ki67 and pS6 were observed when treated with CB-839 in combination with cetuximab compared to cetuximab or CB-839 alone. (E) Representative H E images (0.46×) of tumor tissues from each of the treatment groups. (F) IHC was performed on tumors from each of the treatment groups to evaluate cleaved caspase-3 expression. Shown are representative images (20×). (G) The number of positive cells were counted in regions of interest on images from cleaved caspase-3 IHC (n = 3–15 fields of view, at least 3 animals/treatment group). Error bars represent ± SD. Statistical significance is defined as follows: # p = 0.0574, *p

    Article Snippet: Shown are data for the live cell fluorescence assay with the following treatments: SW48 and HCA-7 cells were exposed to vehicle, 5 μg/mL cetuximab, 250 nM CB-839, or 5 μg/mL cetuximab and 250 nM CB-839; V9P, LoVo, and LS 174 T cells were exposed to vehicle, 10 μg/mL cetuximab, 1 μM CB-839, or 10 μg/mL cetuximab and 1 μM CB-839; and DiFi and LIM1215 cells were exposed to vehicle, 0.01 μg cetuximab, 1 μM CB-839, or 0.01 μg cetuximab and 1 μM CB-839.

    Techniques: High Content Screening, In Vivo, Mouse Assay, Immunohistochemistry, Expressing

    Glutamine (Gln) and EGFR cooperate to promote growth and proliferation. Gln “fuels” the citric acid cycle (CAC) as required for signal transduction-mediated growth and proliferation. Glutamine transport is mediated through solute carrier transporters including ASCT2 ( SLC1A5 ), a key Gln transporter in CRC, and xCT ( SLC7A11 ), an exchanger of glutamine-derived glutamate (Glu) and cystine. Intracellular Gln is metabolized by mitochondrial enzymes, glutaminase 1 and 2 (GLS1/2), to Glu, which fuels the CAC and contributes to redox balance via glutathione biosynthesis, a process that requires exchange via xCT ( SLC7A11 ). This work explored the combination of an EGFR neutralizing mAb, cetuximab, with GLS1 inhibition using CB-839 to enhance the anti-tumor efficacy of cetuximab.

    Journal: Translational Oncology

    Article Title: Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer

    doi: 10.1016/j.tranon.2020.100828

    Figure Lengend Snippet: Glutamine (Gln) and EGFR cooperate to promote growth and proliferation. Gln “fuels” the citric acid cycle (CAC) as required for signal transduction-mediated growth and proliferation. Glutamine transport is mediated through solute carrier transporters including ASCT2 ( SLC1A5 ), a key Gln transporter in CRC, and xCT ( SLC7A11 ), an exchanger of glutamine-derived glutamate (Glu) and cystine. Intracellular Gln is metabolized by mitochondrial enzymes, glutaminase 1 and 2 (GLS1/2), to Glu, which fuels the CAC and contributes to redox balance via glutathione biosynthesis, a process that requires exchange via xCT ( SLC7A11 ). This work explored the combination of an EGFR neutralizing mAb, cetuximab, with GLS1 inhibition using CB-839 to enhance the anti-tumor efficacy of cetuximab.

    Article Snippet: Shown are data for the live cell fluorescence assay with the following treatments: SW48 and HCA-7 cells were exposed to vehicle, 5 μg/mL cetuximab, 250 nM CB-839, or 5 μg/mL cetuximab and 250 nM CB-839; V9P, LoVo, and LS 174 T cells were exposed to vehicle, 10 μg/mL cetuximab, 1 μM CB-839, or 10 μg/mL cetuximab and 1 μM CB-839; and DiFi and LIM1215 cells were exposed to vehicle, 0.01 μg cetuximab, 1 μM CB-839, or 0.01 μg cetuximab and 1 μM CB-839.

    Techniques: Transduction, Derivative Assay, Inhibition

    - DHA induced Caspase-1 and Gasdermin D activation and IL-1β secretion in MDA-MB-231. Cells were stimulated at the indicated concentrations of DHA ( A ) or AA ( B ) for 6 h and the amount of active caspase-1 was assessed by labeling cells with FAM-YVAD-FMK FLICA followed by flow cytometry analysis. Histograms are representative of three independent experiments. Cells were stimulated at the indicated concentrations of DHA ( C ) or AA ( D ) for 6 or 18 h and IL-1β secretion was assessed by ELISA. Each bar represents the mean concentration ± SD. *p

    Journal: Scientific Reports

    Article Title: Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells

    doi: 10.1038/s41598-018-20422-0

    Figure Lengend Snippet: - DHA induced Caspase-1 and Gasdermin D activation and IL-1β secretion in MDA-MB-231. Cells were stimulated at the indicated concentrations of DHA ( A ) or AA ( B ) for 6 h and the amount of active caspase-1 was assessed by labeling cells with FAM-YVAD-FMK FLICA followed by flow cytometry analysis. Histograms are representative of three independent experiments. Cells were stimulated at the indicated concentrations of DHA ( C ) or AA ( D ) for 6 or 18 h and IL-1β secretion was assessed by ELISA. Each bar represents the mean concentration ± SD. *p

    Article Snippet: MDA-MB-231 cells were cultivated in L-15 medium (Sigma Chemical Company, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, Gaithersburg, USA) and 1% antibiotic-antimycotic (GIBCO) at 37 °C.

    Techniques: Activation Assay, Multiple Displacement Amplification, Labeling, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay

    - DHA led to cell death in breast cancer cells. MDA-MB-231 ( A ) and 4T1 breast cells were stimulated with different concentrations of DHA for 24 h and cell death was assessed by labeling cells with annexin-V and propidium iodide followed by flow cytometry analysis. The percentage of necrotic, apoptotic or live cells is indicated. Data are representative of five independent experiments. UNS represents unstimulated cells. Each point represents the average percentage ± SD. (n = 5) *p

    Journal: Scientific Reports

    Article Title: Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells

    doi: 10.1038/s41598-018-20422-0

    Figure Lengend Snippet: - DHA led to cell death in breast cancer cells. MDA-MB-231 ( A ) and 4T1 breast cells were stimulated with different concentrations of DHA for 24 h and cell death was assessed by labeling cells with annexin-V and propidium iodide followed by flow cytometry analysis. The percentage of necrotic, apoptotic or live cells is indicated. Data are representative of five independent experiments. UNS represents unstimulated cells. Each point represents the average percentage ± SD. (n = 5) *p

    Article Snippet: MDA-MB-231 cells were cultivated in L-15 medium (Sigma Chemical Company, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, Gaithersburg, USA) and 1% antibiotic-antimycotic (GIBCO) at 37 °C.

    Techniques: Multiple Displacement Amplification, Labeling, Flow Cytometry, Cytometry

    – DHA induced membrane pore formation dependent on caspase-1 activation, lysosomal damage and ROS formation. MDA-MB-231 cells were pre-treated for 1 h with caspase-1 inhibitor Ac-YVAD-cmk 100 μM ( A ) or ROS inhibitor N-acetyl-cysteine NAC 5 mM ( B ), potassium efflux inhibitor glyburide GB 150 µM ( C ) and CA-074, a lysosomal cathepsin B inhibitor 50 µM ( D ). Then, cells were stimulated with 100 µM DHA for 180 minutes and propidium iodide uptake was measured by spectrophotometry. Graphics are representative of three independent experiments (n = 5 each). UNS: Unstimulated cells. RFU: Relative fluorescence units.

    Journal: Scientific Reports

    Article Title: Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells

    doi: 10.1038/s41598-018-20422-0

    Figure Lengend Snippet: – DHA induced membrane pore formation dependent on caspase-1 activation, lysosomal damage and ROS formation. MDA-MB-231 cells were pre-treated for 1 h with caspase-1 inhibitor Ac-YVAD-cmk 100 μM ( A ) or ROS inhibitor N-acetyl-cysteine NAC 5 mM ( B ), potassium efflux inhibitor glyburide GB 150 µM ( C ) and CA-074, a lysosomal cathepsin B inhibitor 50 µM ( D ). Then, cells were stimulated with 100 µM DHA for 180 minutes and propidium iodide uptake was measured by spectrophotometry. Graphics are representative of three independent experiments (n = 5 each). UNS: Unstimulated cells. RFU: Relative fluorescence units.

    Article Snippet: MDA-MB-231 cells were cultivated in L-15 medium (Sigma Chemical Company, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, Gaithersburg, USA) and 1% antibiotic-antimycotic (GIBCO) at 37 °C.

    Techniques: Activation Assay, Multiple Displacement Amplification, Spectrophotometry, Fluorescence

    - DHA triggered priming of inflammasome signaling pathway. Breast cancer MDA-MB-231 cells were stimulated with 50 or 100μM of DHA or AA ( A ) for 18 h and NFκB translocation towards to nucleus was analyzed by confocal microscopy (NF-κB in red and nuclei in blue) compared to unstimulated cells (UNS). MDA-MB-231 cells were also stimulated with 50, 100 or 200μM of DHA ( B ) for 6 h and expression of pro-caspase-1, pro-IL-1β and adaptor protein ASC was analyzed by western blotting and bands densitometry was performed using Image J software ( C ).

    Journal: Scientific Reports

    Article Title: Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells

    doi: 10.1038/s41598-018-20422-0

    Figure Lengend Snippet: - DHA triggered priming of inflammasome signaling pathway. Breast cancer MDA-MB-231 cells were stimulated with 50 or 100μM of DHA or AA ( A ) for 18 h and NFκB translocation towards to nucleus was analyzed by confocal microscopy (NF-κB in red and nuclei in blue) compared to unstimulated cells (UNS). MDA-MB-231 cells were also stimulated with 50, 100 or 200μM of DHA ( B ) for 6 h and expression of pro-caspase-1, pro-IL-1β and adaptor protein ASC was analyzed by western blotting and bands densitometry was performed using Image J software ( C ).

    Article Snippet: MDA-MB-231 cells were cultivated in L-15 medium (Sigma Chemical Company, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, Gaithersburg, USA) and 1% antibiotic-antimycotic (GIBCO) at 37 °C.

    Techniques: Multiple Displacement Amplification, Translocation Assay, Confocal Microscopy, Expressing, Western Blot, Software

    – DHA induced HMGB1 translocation towards the cytoplasm in breast cancer cells MDA-MB-231. MDA-MB-231 cells were stimulated with the indicated concentrations of DHA or AA for 18 hours. Immunofluorescence staining was performed to analyze HMGB1 cellular localization by confocal microscopy. This figure shows fluorescence microscopy analysis with HMGB1 in red and nuclei in blue. UNS: Unstimulated cells.

    Journal: Scientific Reports

    Article Title: Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells

    doi: 10.1038/s41598-018-20422-0

    Figure Lengend Snippet: – DHA induced HMGB1 translocation towards the cytoplasm in breast cancer cells MDA-MB-231. MDA-MB-231 cells were stimulated with the indicated concentrations of DHA or AA for 18 hours. Immunofluorescence staining was performed to analyze HMGB1 cellular localization by confocal microscopy. This figure shows fluorescence microscopy analysis with HMGB1 in red and nuclei in blue. UNS: Unstimulated cells.

    Article Snippet: MDA-MB-231 cells were cultivated in L-15 medium (Sigma Chemical Company, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, Gaithersburg, USA) and 1% antibiotic-antimycotic (GIBCO) at 37 °C.

    Techniques: Translocation Assay, Multiple Displacement Amplification, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Microscopy

    - DHA decreased cell viability of breast cancer cells (MDA-MB-231 and 4T1) but not normal mammary epithelial cells after 24 h of stimulation. MDA-MB-231 ( A , B ) and PBMC ( C , D ) were stimulated with different concentrations of AA or DHA for 24 h (lines with black circle) or 48 h (lines with black square) ( A – D ). Cell viability of the three different cell lines (MDA-MB-231, MCF-10A and 4T1) was also assessed after 24 h of stimulation ( E ) by MTT assay. The data are presented as percentage of cell viability relative to unstimulated cells (0 μM). Each point represents the average percentage ± SD. (n = 5) *p

    Journal: Scientific Reports

    Article Title: Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells

    doi: 10.1038/s41598-018-20422-0

    Figure Lengend Snippet: - DHA decreased cell viability of breast cancer cells (MDA-MB-231 and 4T1) but not normal mammary epithelial cells after 24 h of stimulation. MDA-MB-231 ( A , B ) and PBMC ( C , D ) were stimulated with different concentrations of AA or DHA for 24 h (lines with black circle) or 48 h (lines with black square) ( A – D ). Cell viability of the three different cell lines (MDA-MB-231, MCF-10A and 4T1) was also assessed after 24 h of stimulation ( E ) by MTT assay. The data are presented as percentage of cell viability relative to unstimulated cells (0 μM). Each point represents the average percentage ± SD. (n = 5) *p

    Article Snippet: MDA-MB-231 cells were cultivated in L-15 medium (Sigma Chemical Company, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, Gaithersburg, USA) and 1% antibiotic-antimycotic (GIBCO) at 37 °C.

    Techniques: Multiple Displacement Amplification, MTT Assay

    - DHA triggered membrane pore formation in breast cancer cells MDA-MB-231 and 4T1. MDA-MB-231 and MCF-10A ( A ) or 4T1 ( B ) cells were stimulated at the indicated concentrations of DHA ( A ) for 3 hours and stained with propidium iodide. Data are representative of three independent experiments (n = 5 each). UNS: unstimulated cells.

    Journal: Scientific Reports

    Article Title: Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells

    doi: 10.1038/s41598-018-20422-0

    Figure Lengend Snippet: - DHA triggered membrane pore formation in breast cancer cells MDA-MB-231 and 4T1. MDA-MB-231 and MCF-10A ( A ) or 4T1 ( B ) cells were stimulated at the indicated concentrations of DHA ( A ) for 3 hours and stained with propidium iodide. Data are representative of three independent experiments (n = 5 each). UNS: unstimulated cells.

    Article Snippet: MDA-MB-231 cells were cultivated in L-15 medium (Sigma Chemical Company, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, Gaithersburg, USA) and 1% antibiotic-antimycotic (GIBCO) at 37 °C.

    Techniques: Multiple Displacement Amplification, Staining