crl 1619ig 2 luc2  (ATCC)


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    ATCC crl 1619ig 2 luc2
    Crl 1619ig 2 Luc2, 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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    braf mutant melanoma cell line a375  (ATCC)


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    ATCC braf mutant melanoma cell line a375
    A WB analysis of changes in CD44, CXCR2, CXCR4, CXCR7, CD74, MIF, pAKT, AKT, pERK1/2, and ERK1/2 expressions in response to IFN-γ (0–500 IU/mL) in <t>A375,</t> SB2, SK-MEL-2, and MeWo. Actin, AKT, and ERK1/2 were used as loading controls. B Release of sCD74 in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 3). C Release of MIF in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 4). D WB analysis of sCD74 in supernatants of A375, SB2, SK-MEL-2, MeWo, and THP-1 MΦ with or without 500 IU/mL IFN-γ stimulation. E WB analysis of sCD74 in the sera of 2 melanoma patients and 2 NHDs. Supernatant of THP-1 MΦ after 500 IU/mL IFN-γ stimulation was used as a reference control. F WB analysis of CD74 in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74. Parental cells with or without 100 IU/mL IFN-γ stimulation were used as controls. G Release of sCD74 in supernatants under basal conditions in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74 measured by ELISA ( n = 3), and the fold-change relative to sCD74 levels in supernatants of SC cells is shown as bar graphs. H WB analysis of sCD74 in supernatants of A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33, and p35 CD74. Parental cells under 500 IU/mL IFN-γ stimulation were used as a control. I WB analysis of sCD74 in supernatants of A375, SK-MEL-2, and THP-1 MΦ with or without deglycosylation treatment under 500 IU/mL IFN-γ stimulatory conditions. Serum of a melanoma patient was also deglycosylated. J Schematic illustration of deglycosylated full-length p33 CD74 1-216 . Considering that MW of deglycosylated sCD74 was approximately 16 KDa, sCD74 was equivalent to a part of full-length CD74 (red box). Graph values represent mean ± SD. CLIP class-II-associated invariant chain peptide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MW molecular weight, MΦ macrophage, NHD normal healthy donor , N.D. not detectable, SC scramble, SD standard deviation, TM transmembrane, WB Western blot.
    Braf Mutant Melanoma Cell Line A375, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma"

    Article Title: Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04552-y

    A WB analysis of changes in CD44, CXCR2, CXCR4, CXCR7, CD74, MIF, pAKT, AKT, pERK1/2, and ERK1/2 expressions in response to IFN-γ (0–500 IU/mL) in A375, SB2, SK-MEL-2, and MeWo. Actin, AKT, and ERK1/2 were used as loading controls. B Release of sCD74 in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 3). C Release of MIF in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 4). D WB analysis of sCD74 in supernatants of A375, SB2, SK-MEL-2, MeWo, and THP-1 MΦ with or without 500 IU/mL IFN-γ stimulation. E WB analysis of sCD74 in the sera of 2 melanoma patients and 2 NHDs. Supernatant of THP-1 MΦ after 500 IU/mL IFN-γ stimulation was used as a reference control. F WB analysis of CD74 in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74. Parental cells with or without 100 IU/mL IFN-γ stimulation were used as controls. G Release of sCD74 in supernatants under basal conditions in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74 measured by ELISA ( n = 3), and the fold-change relative to sCD74 levels in supernatants of SC cells is shown as bar graphs. H WB analysis of sCD74 in supernatants of A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33, and p35 CD74. Parental cells under 500 IU/mL IFN-γ stimulation were used as a control. I WB analysis of sCD74 in supernatants of A375, SK-MEL-2, and THP-1 MΦ with or without deglycosylation treatment under 500 IU/mL IFN-γ stimulatory conditions. Serum of a melanoma patient was also deglycosylated. J Schematic illustration of deglycosylated full-length p33 CD74 1-216 . Considering that MW of deglycosylated sCD74 was approximately 16 KDa, sCD74 was equivalent to a part of full-length CD74 (red box). Graph values represent mean ± SD. CLIP class-II-associated invariant chain peptide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MW molecular weight, MΦ macrophage, NHD normal healthy donor , N.D. not detectable, SC scramble, SD standard deviation, TM transmembrane, WB Western blot.
    Figure Legend Snippet: A WB analysis of changes in CD44, CXCR2, CXCR4, CXCR7, CD74, MIF, pAKT, AKT, pERK1/2, and ERK1/2 expressions in response to IFN-γ (0–500 IU/mL) in A375, SB2, SK-MEL-2, and MeWo. Actin, AKT, and ERK1/2 were used as loading controls. B Release of sCD74 in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 3). C Release of MIF in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 4). D WB analysis of sCD74 in supernatants of A375, SB2, SK-MEL-2, MeWo, and THP-1 MΦ with or without 500 IU/mL IFN-γ stimulation. E WB analysis of sCD74 in the sera of 2 melanoma patients and 2 NHDs. Supernatant of THP-1 MΦ after 500 IU/mL IFN-γ stimulation was used as a reference control. F WB analysis of CD74 in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74. Parental cells with or without 100 IU/mL IFN-γ stimulation were used as controls. G Release of sCD74 in supernatants under basal conditions in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74 measured by ELISA ( n = 3), and the fold-change relative to sCD74 levels in supernatants of SC cells is shown as bar graphs. H WB analysis of sCD74 in supernatants of A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33, and p35 CD74. Parental cells under 500 IU/mL IFN-γ stimulation were used as a control. I WB analysis of sCD74 in supernatants of A375, SK-MEL-2, and THP-1 MΦ with or without deglycosylation treatment under 500 IU/mL IFN-γ stimulatory conditions. Serum of a melanoma patient was also deglycosylated. J Schematic illustration of deglycosylated full-length p33 CD74 1-216 . Considering that MW of deglycosylated sCD74 was approximately 16 KDa, sCD74 was equivalent to a part of full-length CD74 (red box). Graph values represent mean ± SD. CLIP class-II-associated invariant chain peptide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MW molecular weight, MΦ macrophage, NHD normal healthy donor , N.D. not detectable, SC scramble, SD standard deviation, TM transmembrane, WB Western blot.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection, Expressing, Peptide ELISA, Molecular Weight, Standard Deviation, Western Blot

    A , B A375, SK-MEL-2, and THP-1 MΦ were treated with broad protease inhibitors ( A ), including GM6001 (MMP and ADAM inhibitor), GM1489 (MMP inhibitor), E-64 (cysteine inhibitor), leupeptin (serine, cysteine, and threonine inhibitor), 3,4-DCI (serine inhibitor), and β-secretase inhibitor IV (BACE inhibitor) or selective inhibitors ( B ), including GI254023X (ADAM10 inhibitor), TAPI-1 (ADAM17 inhibitor), and LY3000328 (cathepsin-S inhibitor) under 500 IU/mL IFN-γ stimulatory conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3), and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. C Representative images of immunocytochemical staining of cell-surface CD74 in SK-MEL-2 and THP-1 MΦ treated with GI254023X and TAPI-1 under 500 IU/ml IFN-γ stimulation. Two cell lines were immunostained with CD74 (green) and DAPI (blue). Scale bar = 20 μm. D , E Efficacies of two individual siRNAs in knocking down ADAM10 expression ( D ) and ADAM17 expression ( E ) were analyzed by WB in SK-MEL-2 and THP-1 MΦ. SC siRNA was used as a control. F Release of sCD74 in supernatants was measured by ELISA in SK-MEL-2 and THP-1 MΦ transfected with SC siRNA, ADAM10 RNAi-1 and -2, and ADAM17 RNAi-1 and -2 under 500 IU/mL IFN-γ stimulatory conditions. Bar graphs show the fold change relative to sCD74 levels in supernatants of cells transfected with SC siRNA ( n = 3). G A375 and SK-MEL-2 infected with lentivirus-expressing p33 CD74 were treated with GI254023X and TAPI-1 under basal conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3), and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. H WB analysis of CD74 expression in A375, SK-MEL-2, and THP-1 MΦ with or without 100 IU/mL IFN-γ stimulation after a short exposure (upper) and a long exposure (lower). Cell lysates precipitated with acetone were subjected to WB analysis, and actin was used as a loading control. Arrow indicates 25-KDa bands. I A375, SK-MEL-2, and THP-1 MΦ were treated with 100 nM BFA for 24 h under 500 IU/mL IFN-γ-stimulated conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3) and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. Graph values represent mean ± SD. ADAM a disintegrin and metalloproteinase, BFA brefeldin A, DAPI 4′,6-diamidino-2-phenylindole, DMSO dimethyl sulfoxide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MMP matrix metalloproteinase, MΦ macrophage, SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot, 3,4-DCI 3,4-dichloroisocoumarin.
    Figure Legend Snippet: A , B A375, SK-MEL-2, and THP-1 MΦ were treated with broad protease inhibitors ( A ), including GM6001 (MMP and ADAM inhibitor), GM1489 (MMP inhibitor), E-64 (cysteine inhibitor), leupeptin (serine, cysteine, and threonine inhibitor), 3,4-DCI (serine inhibitor), and β-secretase inhibitor IV (BACE inhibitor) or selective inhibitors ( B ), including GI254023X (ADAM10 inhibitor), TAPI-1 (ADAM17 inhibitor), and LY3000328 (cathepsin-S inhibitor) under 500 IU/mL IFN-γ stimulatory conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3), and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. C Representative images of immunocytochemical staining of cell-surface CD74 in SK-MEL-2 and THP-1 MΦ treated with GI254023X and TAPI-1 under 500 IU/ml IFN-γ stimulation. Two cell lines were immunostained with CD74 (green) and DAPI (blue). Scale bar = 20 μm. D , E Efficacies of two individual siRNAs in knocking down ADAM10 expression ( D ) and ADAM17 expression ( E ) were analyzed by WB in SK-MEL-2 and THP-1 MΦ. SC siRNA was used as a control. F Release of sCD74 in supernatants was measured by ELISA in SK-MEL-2 and THP-1 MΦ transfected with SC siRNA, ADAM10 RNAi-1 and -2, and ADAM17 RNAi-1 and -2 under 500 IU/mL IFN-γ stimulatory conditions. Bar graphs show the fold change relative to sCD74 levels in supernatants of cells transfected with SC siRNA ( n = 3). G A375 and SK-MEL-2 infected with lentivirus-expressing p33 CD74 were treated with GI254023X and TAPI-1 under basal conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3), and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. H WB analysis of CD74 expression in A375, SK-MEL-2, and THP-1 MΦ with or without 100 IU/mL IFN-γ stimulation after a short exposure (upper) and a long exposure (lower). Cell lysates precipitated with acetone were subjected to WB analysis, and actin was used as a loading control. Arrow indicates 25-KDa bands. I A375, SK-MEL-2, and THP-1 MΦ were treated with 100 nM BFA for 24 h under 500 IU/mL IFN-γ-stimulated conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3) and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. Graph values represent mean ± SD. ADAM a disintegrin and metalloproteinase, BFA brefeldin A, DAPI 4′,6-diamidino-2-phenylindole, DMSO dimethyl sulfoxide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MMP matrix metalloproteinase, MΦ macrophage, SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot, 3,4-DCI 3,4-dichloroisocoumarin.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Staining, Expressing, Transfection, Infection, Standard Deviation, Western Blot

    A , B Cell-proliferation assay in A375, SB2, and MeWo. Cells were treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) for 72 h under basal conditions ( A ) or under 100 IU/mL IFN-γ stimulatory conditions ( B ). Results represent the fold change relative to the O.D. value of each cell line treated with 0 µg/mL rhCD74 ( n = 6). C Efficacies of two individual siRNAs in knocking down MIF were analyzed by WB in A375 and SB2. MIF siRNAs did not change CD74 expression. SC siRNA was used as a reference control. D Efficacies of two individual siRNAs in knocking down CD74 were analyzed by WB in A375 and SB2. CD74 siRNAs did not change MIF expression. SC siRNA was used as a reference control. E , F Cell-proliferation assay in A375 ( E ) and SB2 ( F ) transfected with SC siRNA, MIF RNAi-1 and -2, and CD74 RNAi-1 and -2. Cells were treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) for 72 h under 100 IU/mL IFN-γ stimulatory conditions. Results represent the fold change relative to the O.D. value of each transfected cell treated with 0 µg/mL rhCD74 ( n = 6). Cell-growth inhibitory effect of 5 µg/mL rhCD74 was significantly diminished in A375 and SB2 transfected with MIF RNAi-1 and -2, and CD74 RNAi-1 and -2, compared with those transfected with SC siRNA. G WB analysis of pAKT in A375, SB2, and MeWo treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) without IFN-γ stimulation (upper) or with 100 IU/mL IFN-γ stimulation (lower). AKT was used as a loading control. H Schematic illustration of transwell coculture system. I WB analysis of CD74 and MIF in cell lysate of THP-1 MΦ transfected with SC siRNA or CD74 RNAi-1. J sCD74 and MIF levels in supernatants of THP-1 MΦ transfected with SC siRNA or CD74 RNAi-1. K Cell-proliferation assay in A375, SB2, and MeWo 48 h after coculture with THP-1 MΦ. High and low concentrations of sCD74 in medium were obtained by transfecting SC siRNA or CD74 RNAi-1 to THP-1 MΦ, respectively, in the presence of 100 IU/mL IFN-γ. Results represent the fold change relative to the O.D. value of each cell line cultured in low sCD74-containing medium ( n = 4). L WB analysis of pAKT in A375, SB2, and MeWo in low and high sCD74-containing medium. AKT was used as a loading control. Graph values represent mean ± SD. Significance in difference between two groups was tested by Student t -test. ** p < 0.01. IFN-γ interferon-γ, MIF macrophage-migration inhibitory factor, MΦ macrophage, rh recombinant human , SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot.
    Figure Legend Snippet: A , B Cell-proliferation assay in A375, SB2, and MeWo. Cells were treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) for 72 h under basal conditions ( A ) or under 100 IU/mL IFN-γ stimulatory conditions ( B ). Results represent the fold change relative to the O.D. value of each cell line treated with 0 µg/mL rhCD74 ( n = 6). C Efficacies of two individual siRNAs in knocking down MIF were analyzed by WB in A375 and SB2. MIF siRNAs did not change CD74 expression. SC siRNA was used as a reference control. D Efficacies of two individual siRNAs in knocking down CD74 were analyzed by WB in A375 and SB2. CD74 siRNAs did not change MIF expression. SC siRNA was used as a reference control. E , F Cell-proliferation assay in A375 ( E ) and SB2 ( F ) transfected with SC siRNA, MIF RNAi-1 and -2, and CD74 RNAi-1 and -2. Cells were treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) for 72 h under 100 IU/mL IFN-γ stimulatory conditions. Results represent the fold change relative to the O.D. value of each transfected cell treated with 0 µg/mL rhCD74 ( n = 6). Cell-growth inhibitory effect of 5 µg/mL rhCD74 was significantly diminished in A375 and SB2 transfected with MIF RNAi-1 and -2, and CD74 RNAi-1 and -2, compared with those transfected with SC siRNA. G WB analysis of pAKT in A375, SB2, and MeWo treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) without IFN-γ stimulation (upper) or with 100 IU/mL IFN-γ stimulation (lower). AKT was used as a loading control. H Schematic illustration of transwell coculture system. I WB analysis of CD74 and MIF in cell lysate of THP-1 MΦ transfected with SC siRNA or CD74 RNAi-1. J sCD74 and MIF levels in supernatants of THP-1 MΦ transfected with SC siRNA or CD74 RNAi-1. K Cell-proliferation assay in A375, SB2, and MeWo 48 h after coculture with THP-1 MΦ. High and low concentrations of sCD74 in medium were obtained by transfecting SC siRNA or CD74 RNAi-1 to THP-1 MΦ, respectively, in the presence of 100 IU/mL IFN-γ. Results represent the fold change relative to the O.D. value of each cell line cultured in low sCD74-containing medium ( n = 4). L WB analysis of pAKT in A375, SB2, and MeWo in low and high sCD74-containing medium. AKT was used as a loading control. Graph values represent mean ± SD. Significance in difference between two groups was tested by Student t -test. ** p < 0.01. IFN-γ interferon-γ, MIF macrophage-migration inhibitory factor, MΦ macrophage, rh recombinant human , SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot.

    Techniques Used: Proliferation Assay, Expressing, Transfection, Cell Culture, Migration, Recombinant, Standard Deviation, Western Blot

    A Representative flow-cytometry plots show annexin V–FITC ( x axis) and PI ( y axis) in A375. B The rate of apoptotic cells in A375, SB2, and MeWo quantified by flow cytometry ( n = 3). C , D The rate of apoptotic cells in A375 ( C ) and SB2 ( D ) transfected with SC siRNA, MIF RNAi-1, or CD74 RNAi-1 quantified by flow cytometry ( n = 3). Flow cytometry ( A – D ) was performed 72 h after the administration of 0 or 5 µg/mL rhCD74 under 100 IU/mL IFN-γ stimulation. E Representative flow-cytometry plots show annexin V–FITC ( x axis) and PI ( y axis) in A375, SB2, and MeWo. F Rate of apoptotic cells in A375, SB2, and MeWo ( n = 3). Flow cytometry ( E , F ) was performed 48 h after coculture with THP-1 MΦ. High and low concentrations of sCD74 in medium were obtained by transfecting SC siRNA or CD74 RNAi-1 to THP-1 MΦ, respectively, in the presence of 100 U/mL IFN-γ. G WB analysis of BCL-2, pBAD, BAD, and CASPASE-9 in A375, SB2, and MeWo 72 h after treatment with different concentrations of rhCD74 (0, 1, and 5 µg/mL) under 100 IU/mL IFN-γ stimulation. Actin and BAD were used as loading controls. H WB analysis of BCL-2, pBAD, BAD, and CASPASE-9 in A375, SB2, and MeWo 48 h after coculture with THP-1 MΦ. Actin and BAD were used as loading controls. Graph values represent mean ± SD. Significance in difference between two groups was tested by Student t -test. ** p < 0.01. IFN-γ interferon-γ, MIF macrophage-migration inhibitory factor, MΦ macrophage, PI propidium iodide, rh recombinant human , SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot.
    Figure Legend Snippet: A Representative flow-cytometry plots show annexin V–FITC ( x axis) and PI ( y axis) in A375. B The rate of apoptotic cells in A375, SB2, and MeWo quantified by flow cytometry ( n = 3). C , D The rate of apoptotic cells in A375 ( C ) and SB2 ( D ) transfected with SC siRNA, MIF RNAi-1, or CD74 RNAi-1 quantified by flow cytometry ( n = 3). Flow cytometry ( A – D ) was performed 72 h after the administration of 0 or 5 µg/mL rhCD74 under 100 IU/mL IFN-γ stimulation. E Representative flow-cytometry plots show annexin V–FITC ( x axis) and PI ( y axis) in A375, SB2, and MeWo. F Rate of apoptotic cells in A375, SB2, and MeWo ( n = 3). Flow cytometry ( E , F ) was performed 48 h after coculture with THP-1 MΦ. High and low concentrations of sCD74 in medium were obtained by transfecting SC siRNA or CD74 RNAi-1 to THP-1 MΦ, respectively, in the presence of 100 U/mL IFN-γ. G WB analysis of BCL-2, pBAD, BAD, and CASPASE-9 in A375, SB2, and MeWo 72 h after treatment with different concentrations of rhCD74 (0, 1, and 5 µg/mL) under 100 IU/mL IFN-γ stimulation. Actin and BAD were used as loading controls. H WB analysis of BCL-2, pBAD, BAD, and CASPASE-9 in A375, SB2, and MeWo 48 h after coculture with THP-1 MΦ. Actin and BAD were used as loading controls. Graph values represent mean ± SD. Significance in difference between two groups was tested by Student t -test. ** p < 0.01. IFN-γ interferon-γ, MIF macrophage-migration inhibitory factor, MΦ macrophage, PI propidium iodide, rh recombinant human , SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot.

    Techniques Used: Flow Cytometry, Transfection, Migration, Recombinant, Standard Deviation, Western Blot

    nras mutant a375 luc2  (ATCC)


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    Structured Review

    ATCC nras mutant a375 luc2
    D 2 O-induced apoptosis in a panel of human malignant melanoma cells <t>(A375</t> <t>V600E</t> , A375 <t>NRAS</t> , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).
    Nras Mutant A375 Luc2, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nras mutant a375 luc2/product/ATCC
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    1) Product Images from "Deuterium Oxide (D 2 O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma"

    Article Title: Deuterium Oxide (D 2 O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

    Journal: Cancers

    doi: 10.3390/cancers13040605

    D 2 O-induced apoptosis in a panel of human malignant melanoma cells (A375 V600E , A375 NRAS , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).
    Figure Legend Snippet: D 2 O-induced apoptosis in a panel of human malignant melanoma cells (A375 V600E , A375 NRAS , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).

    Techniques Used: Staining, Transformation Assay, Flow Cytometry, Western Blot

    D 2 O-induced early stress response gene expression comparing melanoma (A375) and pancreatic ductal adenocarcinoma (PANC-1) cells. ( A ) Time-course analysis of cell viability impairment assessed by flow cytometry (performed as in A) in A375 and PANC-1 cells cultured in 90% D 2 O (≤24 h; n = 3; p * < 0.05; p ** < 0.01). ( B ) Volcano plot depicting differential gene expression (untreated versus D 2 O-exposed (90%, 6 h)) as identified by the Human Stress and Toxicity PathwayFinder TM PCR Array technology (cut off criteria: expression differential >2; p -value ≤ 0.05; n = 3; A375 (black diamond); PANC-1 (empty diamond). ( C ) Comparative gene expression array analysis in Venn diagram depiction; in the overlapping region, single arrow indicates congruent up- or downregulation, and double arrows indicate opposing expression changes between cell lines. ( D ) Comparative gene expression array analysis with total number of genes per group as summarized numerically (A375 vs. PANC-1; D 2 O exposed as in panel B).
    Figure Legend Snippet: D 2 O-induced early stress response gene expression comparing melanoma (A375) and pancreatic ductal adenocarcinoma (PANC-1) cells. ( A ) Time-course analysis of cell viability impairment assessed by flow cytometry (performed as in A) in A375 and PANC-1 cells cultured in 90% D 2 O (≤24 h; n = 3; p * < 0.05; p ** < 0.01). ( B ) Volcano plot depicting differential gene expression (untreated versus D 2 O-exposed (90%, 6 h)) as identified by the Human Stress and Toxicity PathwayFinder TM PCR Array technology (cut off criteria: expression differential >2; p -value ≤ 0.05; n = 3; A375 (black diamond); PANC-1 (empty diamond). ( C ) Comparative gene expression array analysis in Venn diagram depiction; in the overlapping region, single arrow indicates congruent up- or downregulation, and double arrows indicate opposing expression changes between cell lines. ( D ) Comparative gene expression array analysis with total number of genes per group as summarized numerically (A375 vs. PANC-1; D 2 O exposed as in panel B).

    Techniques Used: Expressing, Flow Cytometry, Cell Culture

    D 2 O-induced stress response gene expression and rapid onset of modulated phospho-protein signaling in A375 melanoma cells. ( A ) RT-qPCR assessment of gene expression; left row, dose response (≤90 % D 2 O); right row, time course (≤6 h). ( B ) Stress response protein phosphorylation in response to acute D 2 O exposure as profiled by immunoblot analysis: time course (90% D 2 O; ≤24 h). Bar graphs summarize quantitative analysis by densitometry (mean ± SD). ( C ) For γH2AX detection, flow cytometry was performed; bar graphs summarize quantitative analysis (mean ± SD; n = 3; p *** < 0.001).
    Figure Legend Snippet: D 2 O-induced stress response gene expression and rapid onset of modulated phospho-protein signaling in A375 melanoma cells. ( A ) RT-qPCR assessment of gene expression; left row, dose response (≤90 % D 2 O); right row, time course (≤6 h). ( B ) Stress response protein phosphorylation in response to acute D 2 O exposure as profiled by immunoblot analysis: time course (90% D 2 O; ≤24 h). Bar graphs summarize quantitative analysis by densitometry (mean ± SD). ( C ) For γH2AX detection, flow cytometry was performed; bar graphs summarize quantitative analysis (mean ± SD; n = 3; p *** < 0.001).

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry

    Systemic administration of D 2 O attenuates A375 melanoma cell invasiveness in vitro, while impairing metastasis and tumor growth in SCID mouse models of human malignant melanoma. ( A , B ) A375-Luc2 melanoma cells were tail vein injected ( n = 8 per group) followed by bioluminescent image analysis of lung metastasis (14 and 28 days later). Starting at time of cell injection until day 14, mice received H 2 O-based or D 2 O-supplemented (30% v / v in H 2 O) drinking water, followed by 14 days H 2 O in both groups. A, injection scheme; B, bioluminescent imaging (day 28) with bar graph depicting numeric image analysis of bioluminescent signal ( p *** < 0.001). (For bioluminescent imaging on day 14, see ). ( C ) Invasion through Matrigel-coated Boyden chamber (H 2 O-based versus D 2 O-supplemented (27%) medium). Bar graphs with representative images (10× magnification) after crystal violet staining of inserts ( n = 3; p * < 0.05). ( D – G ) A375 melanoma cells were injected subcutaneously ( n = 10 per group); after pair-matching, tumor growth was monitored over a 24-day period; starting at time of pair matching (day 0) until end of experiment (day 24), mice received H 2 O-based or D 2 O-supplemented (30% v / v in H 2 O) drinking water. ( D ) Experimental scheme. ( E ) Kaplan–Meier analysis of mouse survival as a function of treatment groups; numbers indicate survivors per group. ( F ) Tumor burden in treatment groups as a function of time; graph (right panel), individual tumor size at termination. ( G ) At the end of the experiment, tumors were processed for IHC (left panels; 20× magnification); bar graph: tissue H-scores per antigen (right panel; n = 3; p * < 0.05).
    Figure Legend Snippet: Systemic administration of D 2 O attenuates A375 melanoma cell invasiveness in vitro, while impairing metastasis and tumor growth in SCID mouse models of human malignant melanoma. ( A , B ) A375-Luc2 melanoma cells were tail vein injected ( n = 8 per group) followed by bioluminescent image analysis of lung metastasis (14 and 28 days later). Starting at time of cell injection until day 14, mice received H 2 O-based or D 2 O-supplemented (30% v / v in H 2 O) drinking water, followed by 14 days H 2 O in both groups. A, injection scheme; B, bioluminescent imaging (day 28) with bar graph depicting numeric image analysis of bioluminescent signal ( p *** < 0.001). (For bioluminescent imaging on day 14, see ). ( C ) Invasion through Matrigel-coated Boyden chamber (H 2 O-based versus D 2 O-supplemented (27%) medium). Bar graphs with representative images (10× magnification) after crystal violet staining of inserts ( n = 3; p * < 0.05). ( D – G ) A375 melanoma cells were injected subcutaneously ( n = 10 per group); after pair-matching, tumor growth was monitored over a 24-day period; starting at time of pair matching (day 0) until end of experiment (day 24), mice received H 2 O-based or D 2 O-supplemented (30% v / v in H 2 O) drinking water. ( D ) Experimental scheme. ( E ) Kaplan–Meier analysis of mouse survival as a function of treatment groups; numbers indicate survivors per group. ( F ) Tumor burden in treatment groups as a function of time; graph (right panel), individual tumor size at termination. ( G ) At the end of the experiment, tumors were processed for IHC (left panels; 20× magnification); bar graph: tissue H-scores per antigen (right panel; n = 3; p * < 0.05).

    Techniques Used: In Vitro, Injection, Imaging, Staining

    a375 malignant melanoma cells  (ATCC)


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    a 375 human malignant melanoma cells  (ATCC)


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    A WB analysis of changes in CD44, CXCR2, CXCR4, CXCR7, CD74, MIF, pAKT, AKT, pERK1/2, and ERK1/2 expressions in response to IFN-γ (0–500 IU/mL) in <t>A375,</t> SB2, SK-MEL-2, and MeWo. Actin, AKT, and ERK1/2 were used as loading controls. B Release of sCD74 in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 3). C Release of MIF in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 4). D WB analysis of sCD74 in supernatants of A375, SB2, SK-MEL-2, MeWo, and THP-1 MΦ with or without 500 IU/mL IFN-γ stimulation. E WB analysis of sCD74 in the sera of 2 melanoma patients and 2 NHDs. Supernatant of THP-1 MΦ after 500 IU/mL IFN-γ stimulation was used as a reference control. F WB analysis of CD74 in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74. Parental cells with or without 100 IU/mL IFN-γ stimulation were used as controls. G Release of sCD74 in supernatants under basal conditions in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74 measured by ELISA ( n = 3), and the fold-change relative to sCD74 levels in supernatants of SC cells is shown as bar graphs. H WB analysis of sCD74 in supernatants of A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33, and p35 CD74. Parental cells under 500 IU/mL IFN-γ stimulation were used as a control. I WB analysis of sCD74 in supernatants of A375, SK-MEL-2, and THP-1 MΦ with or without deglycosylation treatment under 500 IU/mL IFN-γ stimulatory conditions. Serum of a melanoma patient was also deglycosylated. J Schematic illustration of deglycosylated full-length p33 CD74 1-216 . Considering that MW of deglycosylated sCD74 was approximately 16 KDa, sCD74 was equivalent to a part of full-length CD74 (red box). Graph values represent mean ± SD. CLIP class-II-associated invariant chain peptide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MW molecular weight, MΦ macrophage, NHD normal healthy donor , N.D. not detectable, SC scramble, SD standard deviation, TM transmembrane, WB Western blot.
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    D 2 O-induced apoptosis in a panel of human malignant melanoma cells <t>(A375</t> <t>V600E</t> , A375 <t>NRAS</t> , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).
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    D 2 O-induced apoptosis in a panel of human malignant melanoma cells <t>(A375</t> <t>V600E</t> , A375 <t>NRAS</t> , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).
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    D 2 O-induced apoptosis in a panel of human malignant melanoma cells <t>(A375</t> <t>V600E</t> , A375 <t>NRAS</t> , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).
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    ATCC human pleural malignant mesothelioma
    D 2 O-induced apoptosis in a panel of human malignant melanoma cells <t>(A375</t> <t>V600E</t> , A375 <t>NRAS</t> , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).
    Human Pleural Malignant Mesothelioma, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC a 375 human malignant melanoma cells
    D 2 O-induced apoptosis in a panel of human malignant melanoma cells <t>(A375</t> <t>V600E</t> , A375 <t>NRAS</t> , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).
    A 375 Human Malignant Melanoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A WB analysis of changes in CD44, CXCR2, CXCR4, CXCR7, CD74, MIF, pAKT, AKT, pERK1/2, and ERK1/2 expressions in response to IFN-γ (0–500 IU/mL) in A375, SB2, SK-MEL-2, and MeWo. Actin, AKT, and ERK1/2 were used as loading controls. B Release of sCD74 in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 3). C Release of MIF in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 4). D WB analysis of sCD74 in supernatants of A375, SB2, SK-MEL-2, MeWo, and THP-1 MΦ with or without 500 IU/mL IFN-γ stimulation. E WB analysis of sCD74 in the sera of 2 melanoma patients and 2 NHDs. Supernatant of THP-1 MΦ after 500 IU/mL IFN-γ stimulation was used as a reference control. F WB analysis of CD74 in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74. Parental cells with or without 100 IU/mL IFN-γ stimulation were used as controls. G Release of sCD74 in supernatants under basal conditions in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74 measured by ELISA ( n = 3), and the fold-change relative to sCD74 levels in supernatants of SC cells is shown as bar graphs. H WB analysis of sCD74 in supernatants of A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33, and p35 CD74. Parental cells under 500 IU/mL IFN-γ stimulation were used as a control. I WB analysis of sCD74 in supernatants of A375, SK-MEL-2, and THP-1 MΦ with or without deglycosylation treatment under 500 IU/mL IFN-γ stimulatory conditions. Serum of a melanoma patient was also deglycosylated. J Schematic illustration of deglycosylated full-length p33 CD74 1-216 . Considering that MW of deglycosylated sCD74 was approximately 16 KDa, sCD74 was equivalent to a part of full-length CD74 (red box). Graph values represent mean ± SD. CLIP class-II-associated invariant chain peptide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MW molecular weight, MΦ macrophage, NHD normal healthy donor , N.D. not detectable, SC scramble, SD standard deviation, TM transmembrane, WB Western blot.

    Journal: Cell Death & Disease

    Article Title: Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma

    doi: 10.1038/s41419-022-04552-y

    Figure Lengend Snippet: A WB analysis of changes in CD44, CXCR2, CXCR4, CXCR7, CD74, MIF, pAKT, AKT, pERK1/2, and ERK1/2 expressions in response to IFN-γ (0–500 IU/mL) in A375, SB2, SK-MEL-2, and MeWo. Actin, AKT, and ERK1/2 were used as loading controls. B Release of sCD74 in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 3). C Release of MIF in supernatants 24 h after IFN-γ stimulation (0–500 IU/mL) in A375, SB2, SK-MEL-2, MeWo, THP-1 MΦ, and primary MΦ measured by ELISA ( n = 4). D WB analysis of sCD74 in supernatants of A375, SB2, SK-MEL-2, MeWo, and THP-1 MΦ with or without 500 IU/mL IFN-γ stimulation. E WB analysis of sCD74 in the sera of 2 melanoma patients and 2 NHDs. Supernatant of THP-1 MΦ after 500 IU/mL IFN-γ stimulation was used as a reference control. F WB analysis of CD74 in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74. Parental cells with or without 100 IU/mL IFN-γ stimulation were used as controls. G Release of sCD74 in supernatants under basal conditions in A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33 CD74, and p35 CD74 measured by ELISA ( n = 3), and the fold-change relative to sCD74 levels in supernatants of SC cells is shown as bar graphs. H WB analysis of sCD74 in supernatants of A375 and SK-MEL-2 infected with lentivirus-expressing SC, p33, and p35 CD74. Parental cells under 500 IU/mL IFN-γ stimulation were used as a control. I WB analysis of sCD74 in supernatants of A375, SK-MEL-2, and THP-1 MΦ with or without deglycosylation treatment under 500 IU/mL IFN-γ stimulatory conditions. Serum of a melanoma patient was also deglycosylated. J Schematic illustration of deglycosylated full-length p33 CD74 1-216 . Considering that MW of deglycosylated sCD74 was approximately 16 KDa, sCD74 was equivalent to a part of full-length CD74 (red box). Graph values represent mean ± SD. CLIP class-II-associated invariant chain peptide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MW molecular weight, MΦ macrophage, NHD normal healthy donor , N.D. not detectable, SC scramble, SD standard deviation, TM transmembrane, WB Western blot.

    Article Snippet: BRAF-mutant melanoma cell line A375 (CVCL_0132), NRAS-mutant melanoma line SK-MEL-2 (CVCL_0069), BRAF/NRAS wild-type melanoma line MeWo (CVCL_0445), and monocyte-like cell line THP-1 (CVCL_0006) were purchased from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Infection, Expressing, Peptide ELISA, Molecular Weight, Standard Deviation, Western Blot

    A , B A375, SK-MEL-2, and THP-1 MΦ were treated with broad protease inhibitors ( A ), including GM6001 (MMP and ADAM inhibitor), GM1489 (MMP inhibitor), E-64 (cysteine inhibitor), leupeptin (serine, cysteine, and threonine inhibitor), 3,4-DCI (serine inhibitor), and β-secretase inhibitor IV (BACE inhibitor) or selective inhibitors ( B ), including GI254023X (ADAM10 inhibitor), TAPI-1 (ADAM17 inhibitor), and LY3000328 (cathepsin-S inhibitor) under 500 IU/mL IFN-γ stimulatory conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3), and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. C Representative images of immunocytochemical staining of cell-surface CD74 in SK-MEL-2 and THP-1 MΦ treated with GI254023X and TAPI-1 under 500 IU/ml IFN-γ stimulation. Two cell lines were immunostained with CD74 (green) and DAPI (blue). Scale bar = 20 μm. D , E Efficacies of two individual siRNAs in knocking down ADAM10 expression ( D ) and ADAM17 expression ( E ) were analyzed by WB in SK-MEL-2 and THP-1 MΦ. SC siRNA was used as a control. F Release of sCD74 in supernatants was measured by ELISA in SK-MEL-2 and THP-1 MΦ transfected with SC siRNA, ADAM10 RNAi-1 and -2, and ADAM17 RNAi-1 and -2 under 500 IU/mL IFN-γ stimulatory conditions. Bar graphs show the fold change relative to sCD74 levels in supernatants of cells transfected with SC siRNA ( n = 3). G A375 and SK-MEL-2 infected with lentivirus-expressing p33 CD74 were treated with GI254023X and TAPI-1 under basal conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3), and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. H WB analysis of CD74 expression in A375, SK-MEL-2, and THP-1 MΦ with or without 100 IU/mL IFN-γ stimulation after a short exposure (upper) and a long exposure (lower). Cell lysates precipitated with acetone were subjected to WB analysis, and actin was used as a loading control. Arrow indicates 25-KDa bands. I A375, SK-MEL-2, and THP-1 MΦ were treated with 100 nM BFA for 24 h under 500 IU/mL IFN-γ-stimulated conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3) and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. Graph values represent mean ± SD. ADAM a disintegrin and metalloproteinase, BFA brefeldin A, DAPI 4′,6-diamidino-2-phenylindole, DMSO dimethyl sulfoxide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MMP matrix metalloproteinase, MΦ macrophage, SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot, 3,4-DCI 3,4-dichloroisocoumarin.

    Journal: Cell Death & Disease

    Article Title: Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma

    doi: 10.1038/s41419-022-04552-y

    Figure Lengend Snippet: A , B A375, SK-MEL-2, and THP-1 MΦ were treated with broad protease inhibitors ( A ), including GM6001 (MMP and ADAM inhibitor), GM1489 (MMP inhibitor), E-64 (cysteine inhibitor), leupeptin (serine, cysteine, and threonine inhibitor), 3,4-DCI (serine inhibitor), and β-secretase inhibitor IV (BACE inhibitor) or selective inhibitors ( B ), including GI254023X (ADAM10 inhibitor), TAPI-1 (ADAM17 inhibitor), and LY3000328 (cathepsin-S inhibitor) under 500 IU/mL IFN-γ stimulatory conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3), and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. C Representative images of immunocytochemical staining of cell-surface CD74 in SK-MEL-2 and THP-1 MΦ treated with GI254023X and TAPI-1 under 500 IU/ml IFN-γ stimulation. Two cell lines were immunostained with CD74 (green) and DAPI (blue). Scale bar = 20 μm. D , E Efficacies of two individual siRNAs in knocking down ADAM10 expression ( D ) and ADAM17 expression ( E ) were analyzed by WB in SK-MEL-2 and THP-1 MΦ. SC siRNA was used as a control. F Release of sCD74 in supernatants was measured by ELISA in SK-MEL-2 and THP-1 MΦ transfected with SC siRNA, ADAM10 RNAi-1 and -2, and ADAM17 RNAi-1 and -2 under 500 IU/mL IFN-γ stimulatory conditions. Bar graphs show the fold change relative to sCD74 levels in supernatants of cells transfected with SC siRNA ( n = 3). G A375 and SK-MEL-2 infected with lentivirus-expressing p33 CD74 were treated with GI254023X and TAPI-1 under basal conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3), and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. H WB analysis of CD74 expression in A375, SK-MEL-2, and THP-1 MΦ with or without 100 IU/mL IFN-γ stimulation after a short exposure (upper) and a long exposure (lower). Cell lysates precipitated with acetone were subjected to WB analysis, and actin was used as a loading control. Arrow indicates 25-KDa bands. I A375, SK-MEL-2, and THP-1 MΦ were treated with 100 nM BFA for 24 h under 500 IU/mL IFN-γ-stimulated conditions. Release of sCD74 in supernatants was measured by ELISA ( n = 3) and the fold change relative to sCD74 levels in supernatants of cells treated with 0.5% DMSO is shown as bar graphs. Graph values represent mean ± SD. ADAM a disintegrin and metalloproteinase, BFA brefeldin A, DAPI 4′,6-diamidino-2-phenylindole, DMSO dimethyl sulfoxide, ELISA enzyme-linked immunosorbent assay, IFN-γ interferon-γ, MMP matrix metalloproteinase, MΦ macrophage, SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot, 3,4-DCI 3,4-dichloroisocoumarin.

    Article Snippet: BRAF-mutant melanoma cell line A375 (CVCL_0132), NRAS-mutant melanoma line SK-MEL-2 (CVCL_0069), BRAF/NRAS wild-type melanoma line MeWo (CVCL_0445), and monocyte-like cell line THP-1 (CVCL_0006) were purchased from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Staining, Expressing, Transfection, Infection, Standard Deviation, Western Blot

    A , B Cell-proliferation assay in A375, SB2, and MeWo. Cells were treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) for 72 h under basal conditions ( A ) or under 100 IU/mL IFN-γ stimulatory conditions ( B ). Results represent the fold change relative to the O.D. value of each cell line treated with 0 µg/mL rhCD74 ( n = 6). C Efficacies of two individual siRNAs in knocking down MIF were analyzed by WB in A375 and SB2. MIF siRNAs did not change CD74 expression. SC siRNA was used as a reference control. D Efficacies of two individual siRNAs in knocking down CD74 were analyzed by WB in A375 and SB2. CD74 siRNAs did not change MIF expression. SC siRNA was used as a reference control. E , F Cell-proliferation assay in A375 ( E ) and SB2 ( F ) transfected with SC siRNA, MIF RNAi-1 and -2, and CD74 RNAi-1 and -2. Cells were treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) for 72 h under 100 IU/mL IFN-γ stimulatory conditions. Results represent the fold change relative to the O.D. value of each transfected cell treated with 0 µg/mL rhCD74 ( n = 6). Cell-growth inhibitory effect of 5 µg/mL rhCD74 was significantly diminished in A375 and SB2 transfected with MIF RNAi-1 and -2, and CD74 RNAi-1 and -2, compared with those transfected with SC siRNA. G WB analysis of pAKT in A375, SB2, and MeWo treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) without IFN-γ stimulation (upper) or with 100 IU/mL IFN-γ stimulation (lower). AKT was used as a loading control. H Schematic illustration of transwell coculture system. I WB analysis of CD74 and MIF in cell lysate of THP-1 MΦ transfected with SC siRNA or CD74 RNAi-1. J sCD74 and MIF levels in supernatants of THP-1 MΦ transfected with SC siRNA or CD74 RNAi-1. K Cell-proliferation assay in A375, SB2, and MeWo 48 h after coculture with THP-1 MΦ. High and low concentrations of sCD74 in medium were obtained by transfecting SC siRNA or CD74 RNAi-1 to THP-1 MΦ, respectively, in the presence of 100 IU/mL IFN-γ. Results represent the fold change relative to the O.D. value of each cell line cultured in low sCD74-containing medium ( n = 4). L WB analysis of pAKT in A375, SB2, and MeWo in low and high sCD74-containing medium. AKT was used as a loading control. Graph values represent mean ± SD. Significance in difference between two groups was tested by Student t -test. ** p < 0.01. IFN-γ interferon-γ, MIF macrophage-migration inhibitory factor, MΦ macrophage, rh recombinant human , SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot.

    Journal: Cell Death & Disease

    Article Title: Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma

    doi: 10.1038/s41419-022-04552-y

    Figure Lengend Snippet: A , B Cell-proliferation assay in A375, SB2, and MeWo. Cells were treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) for 72 h under basal conditions ( A ) or under 100 IU/mL IFN-γ stimulatory conditions ( B ). Results represent the fold change relative to the O.D. value of each cell line treated with 0 µg/mL rhCD74 ( n = 6). C Efficacies of two individual siRNAs in knocking down MIF were analyzed by WB in A375 and SB2. MIF siRNAs did not change CD74 expression. SC siRNA was used as a reference control. D Efficacies of two individual siRNAs in knocking down CD74 were analyzed by WB in A375 and SB2. CD74 siRNAs did not change MIF expression. SC siRNA was used as a reference control. E , F Cell-proliferation assay in A375 ( E ) and SB2 ( F ) transfected with SC siRNA, MIF RNAi-1 and -2, and CD74 RNAi-1 and -2. Cells were treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) for 72 h under 100 IU/mL IFN-γ stimulatory conditions. Results represent the fold change relative to the O.D. value of each transfected cell treated with 0 µg/mL rhCD74 ( n = 6). Cell-growth inhibitory effect of 5 µg/mL rhCD74 was significantly diminished in A375 and SB2 transfected with MIF RNAi-1 and -2, and CD74 RNAi-1 and -2, compared with those transfected with SC siRNA. G WB analysis of pAKT in A375, SB2, and MeWo treated with different concentrations of rhCD74 (0, 1, and 5 µg/mL) without IFN-γ stimulation (upper) or with 100 IU/mL IFN-γ stimulation (lower). AKT was used as a loading control. H Schematic illustration of transwell coculture system. I WB analysis of CD74 and MIF in cell lysate of THP-1 MΦ transfected with SC siRNA or CD74 RNAi-1. J sCD74 and MIF levels in supernatants of THP-1 MΦ transfected with SC siRNA or CD74 RNAi-1. K Cell-proliferation assay in A375, SB2, and MeWo 48 h after coculture with THP-1 MΦ. High and low concentrations of sCD74 in medium were obtained by transfecting SC siRNA or CD74 RNAi-1 to THP-1 MΦ, respectively, in the presence of 100 IU/mL IFN-γ. Results represent the fold change relative to the O.D. value of each cell line cultured in low sCD74-containing medium ( n = 4). L WB analysis of pAKT in A375, SB2, and MeWo in low and high sCD74-containing medium. AKT was used as a loading control. Graph values represent mean ± SD. Significance in difference between two groups was tested by Student t -test. ** p < 0.01. IFN-γ interferon-γ, MIF macrophage-migration inhibitory factor, MΦ macrophage, rh recombinant human , SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot.

    Article Snippet: BRAF-mutant melanoma cell line A375 (CVCL_0132), NRAS-mutant melanoma line SK-MEL-2 (CVCL_0069), BRAF/NRAS wild-type melanoma line MeWo (CVCL_0445), and monocyte-like cell line THP-1 (CVCL_0006) were purchased from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Proliferation Assay, Expressing, Transfection, Cell Culture, Migration, Recombinant, Standard Deviation, Western Blot

    A Representative flow-cytometry plots show annexin V–FITC ( x axis) and PI ( y axis) in A375. B The rate of apoptotic cells in A375, SB2, and MeWo quantified by flow cytometry ( n = 3). C , D The rate of apoptotic cells in A375 ( C ) and SB2 ( D ) transfected with SC siRNA, MIF RNAi-1, or CD74 RNAi-1 quantified by flow cytometry ( n = 3). Flow cytometry ( A – D ) was performed 72 h after the administration of 0 or 5 µg/mL rhCD74 under 100 IU/mL IFN-γ stimulation. E Representative flow-cytometry plots show annexin V–FITC ( x axis) and PI ( y axis) in A375, SB2, and MeWo. F Rate of apoptotic cells in A375, SB2, and MeWo ( n = 3). Flow cytometry ( E , F ) was performed 48 h after coculture with THP-1 MΦ. High and low concentrations of sCD74 in medium were obtained by transfecting SC siRNA or CD74 RNAi-1 to THP-1 MΦ, respectively, in the presence of 100 U/mL IFN-γ. G WB analysis of BCL-2, pBAD, BAD, and CASPASE-9 in A375, SB2, and MeWo 72 h after treatment with different concentrations of rhCD74 (0, 1, and 5 µg/mL) under 100 IU/mL IFN-γ stimulation. Actin and BAD were used as loading controls. H WB analysis of BCL-2, pBAD, BAD, and CASPASE-9 in A375, SB2, and MeWo 48 h after coculture with THP-1 MΦ. Actin and BAD were used as loading controls. Graph values represent mean ± SD. Significance in difference between two groups was tested by Student t -test. ** p < 0.01. IFN-γ interferon-γ, MIF macrophage-migration inhibitory factor, MΦ macrophage, PI propidium iodide, rh recombinant human , SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot.

    Journal: Cell Death & Disease

    Article Title: Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma

    doi: 10.1038/s41419-022-04552-y

    Figure Lengend Snippet: A Representative flow-cytometry plots show annexin V–FITC ( x axis) and PI ( y axis) in A375. B The rate of apoptotic cells in A375, SB2, and MeWo quantified by flow cytometry ( n = 3). C , D The rate of apoptotic cells in A375 ( C ) and SB2 ( D ) transfected with SC siRNA, MIF RNAi-1, or CD74 RNAi-1 quantified by flow cytometry ( n = 3). Flow cytometry ( A – D ) was performed 72 h after the administration of 0 or 5 µg/mL rhCD74 under 100 IU/mL IFN-γ stimulation. E Representative flow-cytometry plots show annexin V–FITC ( x axis) and PI ( y axis) in A375, SB2, and MeWo. F Rate of apoptotic cells in A375, SB2, and MeWo ( n = 3). Flow cytometry ( E , F ) was performed 48 h after coculture with THP-1 MΦ. High and low concentrations of sCD74 in medium were obtained by transfecting SC siRNA or CD74 RNAi-1 to THP-1 MΦ, respectively, in the presence of 100 U/mL IFN-γ. G WB analysis of BCL-2, pBAD, BAD, and CASPASE-9 in A375, SB2, and MeWo 72 h after treatment with different concentrations of rhCD74 (0, 1, and 5 µg/mL) under 100 IU/mL IFN-γ stimulation. Actin and BAD were used as loading controls. H WB analysis of BCL-2, pBAD, BAD, and CASPASE-9 in A375, SB2, and MeWo 48 h after coculture with THP-1 MΦ. Actin and BAD were used as loading controls. Graph values represent mean ± SD. Significance in difference between two groups was tested by Student t -test. ** p < 0.01. IFN-γ interferon-γ, MIF macrophage-migration inhibitory factor, MΦ macrophage, PI propidium iodide, rh recombinant human , SC scramble, SD standard deviation, siRNA short-interference RNA, WB Western blot.

    Article Snippet: BRAF-mutant melanoma cell line A375 (CVCL_0132), NRAS-mutant melanoma line SK-MEL-2 (CVCL_0069), BRAF/NRAS wild-type melanoma line MeWo (CVCL_0445), and monocyte-like cell line THP-1 (CVCL_0006) were purchased from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Flow Cytometry, Transfection, Migration, Recombinant, Standard Deviation, Western Blot

    D 2 O-induced apoptosis in a panel of human malignant melanoma cells (A375 V600E , A375 NRAS , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).

    Journal: Cancers

    Article Title: Deuterium Oxide (D 2 O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

    doi: 10.3390/cancers13040605

    Figure Lengend Snippet: D 2 O-induced apoptosis in a panel of human malignant melanoma cells (A375 V600E , A375 NRAS , LOX-IMVI, and G361). ( A ) Impairment of cellular viability in response to culture in D 2 O (90%, 24 h) was monitored using flow cytometric analysis (annexin V-PI staining). Numbers in quadrants indicate percentage of viable cells (AV-negative, PI-negative) from a total of gated cells (mean ± SD, n = 3). Bar graph (right panel) indicates dose response of impaired cell viability (D 2 O (≤90%, 24 h)) ( n = 3). Human Hs27 dermal fibroblasts exposed to D 2 O served as non-transformed, non-malignant controls. ( B ) Impairment of cellular viability in response to long term exposure to D 2 O (27%, ≤6 days). Bar graph depicts dose response and time course ((≤6 days); ( n = 3)). ( C ) Impairment of cellular proliferation (A375) in response to culture in D 2 O (≤27%, 3 days). ( D ) M-phase depletion as a function of culture in D 2 O (27%, 3 days) as assessed by phospho-histone H3 flow cytometry: individual histograms representative of three repeats (left panel); right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( E ) D 2 O-induced (90%, 24 h) cell death (A375 V600E versus A375 NRAS ) in the absence or presence of zVAD-fmk (40 µM). ( F ) D 2 O-induced (90%, 24 h) induction of pro-caspase 3 cleavage as examined in A375 V600E cells by flow cytometry (left panel: representative histograms; right panel: bar graph depiction of numerical analysis ( n = 3; p *** < 0.001). ( G ) Time course of PARP-1 cleavage in response to D 2 O-exposure (90%, ≤24 h) in A375 cells (bottom panel: immunoblot; top panel: bar graph depiction of numerical analysis ( n = 3).

    Article Snippet: The following cell lines were purchased from ATCC (Manassas, VA, USA): human malignant melanoma cells (A375 (CRL-1619), A375-Luc2 (containing BRAF V600E mutation; CRL-1619-LUC2), NRAS-mutant-A375-Luc2 (isogenic variant containing both BRAF V600E and NRAS Q61K mutations; CRL-1619IG-2-LUC2), and G361 (CRL-1424)), human pancreatic ductal adenocarcinoma cells (PANC-1-Luc2 (CRL-1469-LUC2), MIA PaCa-2 (CRL-1420), and Capan-2 (HTB-80)), and normal skin fibroblasts (Hs27 (CRL-1634)), cultured under standard conditions as specified by the manufacturer [ , , ].

    Techniques: Staining, Transformation Assay, Flow Cytometry, Western Blot

    D 2 O-induced early stress response gene expression comparing melanoma (A375) and pancreatic ductal adenocarcinoma (PANC-1) cells. ( A ) Time-course analysis of cell viability impairment assessed by flow cytometry (performed as in A) in A375 and PANC-1 cells cultured in 90% D 2 O (≤24 h; n = 3; p * < 0.05; p ** < 0.01). ( B ) Volcano plot depicting differential gene expression (untreated versus D 2 O-exposed (90%, 6 h)) as identified by the Human Stress and Toxicity PathwayFinder TM PCR Array technology (cut off criteria: expression differential >2; p -value ≤ 0.05; n = 3; A375 (black diamond); PANC-1 (empty diamond). ( C ) Comparative gene expression array analysis in Venn diagram depiction; in the overlapping region, single arrow indicates congruent up- or downregulation, and double arrows indicate opposing expression changes between cell lines. ( D ) Comparative gene expression array analysis with total number of genes per group as summarized numerically (A375 vs. PANC-1; D 2 O exposed as in panel B).

    Journal: Cancers

    Article Title: Deuterium Oxide (D 2 O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

    doi: 10.3390/cancers13040605

    Figure Lengend Snippet: D 2 O-induced early stress response gene expression comparing melanoma (A375) and pancreatic ductal adenocarcinoma (PANC-1) cells. ( A ) Time-course analysis of cell viability impairment assessed by flow cytometry (performed as in A) in A375 and PANC-1 cells cultured in 90% D 2 O (≤24 h; n = 3; p * < 0.05; p ** < 0.01). ( B ) Volcano plot depicting differential gene expression (untreated versus D 2 O-exposed (90%, 6 h)) as identified by the Human Stress and Toxicity PathwayFinder TM PCR Array technology (cut off criteria: expression differential >2; p -value ≤ 0.05; n = 3; A375 (black diamond); PANC-1 (empty diamond). ( C ) Comparative gene expression array analysis in Venn diagram depiction; in the overlapping region, single arrow indicates congruent up- or downregulation, and double arrows indicate opposing expression changes between cell lines. ( D ) Comparative gene expression array analysis with total number of genes per group as summarized numerically (A375 vs. PANC-1; D 2 O exposed as in panel B).

    Article Snippet: The following cell lines were purchased from ATCC (Manassas, VA, USA): human malignant melanoma cells (A375 (CRL-1619), A375-Luc2 (containing BRAF V600E mutation; CRL-1619-LUC2), NRAS-mutant-A375-Luc2 (isogenic variant containing both BRAF V600E and NRAS Q61K mutations; CRL-1619IG-2-LUC2), and G361 (CRL-1424)), human pancreatic ductal adenocarcinoma cells (PANC-1-Luc2 (CRL-1469-LUC2), MIA PaCa-2 (CRL-1420), and Capan-2 (HTB-80)), and normal skin fibroblasts (Hs27 (CRL-1634)), cultured under standard conditions as specified by the manufacturer [ , , ].

    Techniques: Expressing, Flow Cytometry, Cell Culture

    D 2 O-induced stress response gene expression and rapid onset of modulated phospho-protein signaling in A375 melanoma cells. ( A ) RT-qPCR assessment of gene expression; left row, dose response (≤90 % D 2 O); right row, time course (≤6 h). ( B ) Stress response protein phosphorylation in response to acute D 2 O exposure as profiled by immunoblot analysis: time course (90% D 2 O; ≤24 h). Bar graphs summarize quantitative analysis by densitometry (mean ± SD). ( C ) For γH2AX detection, flow cytometry was performed; bar graphs summarize quantitative analysis (mean ± SD; n = 3; p *** < 0.001).

    Journal: Cancers

    Article Title: Deuterium Oxide (D 2 O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

    doi: 10.3390/cancers13040605

    Figure Lengend Snippet: D 2 O-induced stress response gene expression and rapid onset of modulated phospho-protein signaling in A375 melanoma cells. ( A ) RT-qPCR assessment of gene expression; left row, dose response (≤90 % D 2 O); right row, time course (≤6 h). ( B ) Stress response protein phosphorylation in response to acute D 2 O exposure as profiled by immunoblot analysis: time course (90% D 2 O; ≤24 h). Bar graphs summarize quantitative analysis by densitometry (mean ± SD). ( C ) For γH2AX detection, flow cytometry was performed; bar graphs summarize quantitative analysis (mean ± SD; n = 3; p *** < 0.001).

    Article Snippet: The following cell lines were purchased from ATCC (Manassas, VA, USA): human malignant melanoma cells (A375 (CRL-1619), A375-Luc2 (containing BRAF V600E mutation; CRL-1619-LUC2), NRAS-mutant-A375-Luc2 (isogenic variant containing both BRAF V600E and NRAS Q61K mutations; CRL-1619IG-2-LUC2), and G361 (CRL-1424)), human pancreatic ductal adenocarcinoma cells (PANC-1-Luc2 (CRL-1469-LUC2), MIA PaCa-2 (CRL-1420), and Capan-2 (HTB-80)), and normal skin fibroblasts (Hs27 (CRL-1634)), cultured under standard conditions as specified by the manufacturer [ , , ].

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry

    Systemic administration of D 2 O attenuates A375 melanoma cell invasiveness in vitro, while impairing metastasis and tumor growth in SCID mouse models of human malignant melanoma. ( A , B ) A375-Luc2 melanoma cells were tail vein injected ( n = 8 per group) followed by bioluminescent image analysis of lung metastasis (14 and 28 days later). Starting at time of cell injection until day 14, mice received H 2 O-based or D 2 O-supplemented (30% v / v in H 2 O) drinking water, followed by 14 days H 2 O in both groups. A, injection scheme; B, bioluminescent imaging (day 28) with bar graph depicting numeric image analysis of bioluminescent signal ( p *** < 0.001). (For bioluminescent imaging on day 14, see ). ( C ) Invasion through Matrigel-coated Boyden chamber (H 2 O-based versus D 2 O-supplemented (27%) medium). Bar graphs with representative images (10× magnification) after crystal violet staining of inserts ( n = 3; p * < 0.05). ( D – G ) A375 melanoma cells were injected subcutaneously ( n = 10 per group); after pair-matching, tumor growth was monitored over a 24-day period; starting at time of pair matching (day 0) until end of experiment (day 24), mice received H 2 O-based or D 2 O-supplemented (30% v / v in H 2 O) drinking water. ( D ) Experimental scheme. ( E ) Kaplan–Meier analysis of mouse survival as a function of treatment groups; numbers indicate survivors per group. ( F ) Tumor burden in treatment groups as a function of time; graph (right panel), individual tumor size at termination. ( G ) At the end of the experiment, tumors were processed for IHC (left panels; 20× magnification); bar graph: tissue H-scores per antigen (right panel; n = 3; p * < 0.05).

    Journal: Cancers

    Article Title: Deuterium Oxide (D 2 O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

    doi: 10.3390/cancers13040605

    Figure Lengend Snippet: Systemic administration of D 2 O attenuates A375 melanoma cell invasiveness in vitro, while impairing metastasis and tumor growth in SCID mouse models of human malignant melanoma. ( A , B ) A375-Luc2 melanoma cells were tail vein injected ( n = 8 per group) followed by bioluminescent image analysis of lung metastasis (14 and 28 days later). Starting at time of cell injection until day 14, mice received H 2 O-based or D 2 O-supplemented (30% v / v in H 2 O) drinking water, followed by 14 days H 2 O in both groups. A, injection scheme; B, bioluminescent imaging (day 28) with bar graph depicting numeric image analysis of bioluminescent signal ( p *** < 0.001). (For bioluminescent imaging on day 14, see ). ( C ) Invasion through Matrigel-coated Boyden chamber (H 2 O-based versus D 2 O-supplemented (27%) medium). Bar graphs with representative images (10× magnification) after crystal violet staining of inserts ( n = 3; p * < 0.05). ( D – G ) A375 melanoma cells were injected subcutaneously ( n = 10 per group); after pair-matching, tumor growth was monitored over a 24-day period; starting at time of pair matching (day 0) until end of experiment (day 24), mice received H 2 O-based or D 2 O-supplemented (30% v / v in H 2 O) drinking water. ( D ) Experimental scheme. ( E ) Kaplan–Meier analysis of mouse survival as a function of treatment groups; numbers indicate survivors per group. ( F ) Tumor burden in treatment groups as a function of time; graph (right panel), individual tumor size at termination. ( G ) At the end of the experiment, tumors were processed for IHC (left panels; 20× magnification); bar graph: tissue H-scores per antigen (right panel; n = 3; p * < 0.05).

    Article Snippet: The following cell lines were purchased from ATCC (Manassas, VA, USA): human malignant melanoma cells (A375 (CRL-1619), A375-Luc2 (containing BRAF V600E mutation; CRL-1619-LUC2), NRAS-mutant-A375-Luc2 (isogenic variant containing both BRAF V600E and NRAS Q61K mutations; CRL-1619IG-2-LUC2), and G361 (CRL-1424)), human pancreatic ductal adenocarcinoma cells (PANC-1-Luc2 (CRL-1469-LUC2), MIA PaCa-2 (CRL-1420), and Capan-2 (HTB-80)), and normal skin fibroblasts (Hs27 (CRL-1634)), cultured under standard conditions as specified by the manufacturer [ , , ].

    Techniques: In Vitro, Injection, Imaging, Staining