braf v600e mutant human melanoma cell line a375  (ATCC)


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    ATCC braf v600e mutant human melanoma cell line a375
    Braf V600e Mutant Human Melanoma Cell Line A375, 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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    adenovirus transfection braf mutant brafv600e melanoma cell line a375  (ATCC)


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    ATCC adenovirus transfection braf mutant brafv600e melanoma cell line a375
    Adenovirus Transfection Braf Mutant Brafv600e Melanoma Cell Line A375, 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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    adenovirus transfection braf mutant brafv600e melanoma cell line a375  (ATCC)


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    ATCC adenovirus transfection braf mutant brafv600e melanoma cell line a375
    Adenovirus Transfection Braf Mutant Brafv600e Melanoma Cell Line A375, 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

    braf mutant melanoma cell line a375  (ATCC)


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    Structured Review

    ATCC braf mutant melanoma cell line a375
    Characteristics of miRNA-mRNA hybrids discovered in melanoma cells. ( A ) Binding characteristics of miRNA–mRNA interactions. Binding region: mRNA part of the hybrids mapped to the ENSEMBL transcript database with regions corresponding to the 5′UTR and 3′UTR, CDS, 5′-UTR-CDS, and CDS-3′UTR. Seed match: nucleotides (nt) 2–7 at 5′ end of miRNA with zero (2–7, 0 mm), 2–8 with zero (2–8, 0 mm), one (2–8, 1 mm), or two mismatches (2–8, 2 mm), and the remaining binding modes were classified as “other”. Seed pairing at nucleotides 2–7 (blue) and 2–8 (red) without mismatches was considered as canonical seed pairing, whereas any additional type of pairing was referred to as non-canonical seed pairing. Strength: “strength” of binding outside the seed region at the 3′ end of the miRNA based on number of bound nt: >8 nt (strong), 5–8 nt (moderate), 1–4 nt (weak), and 0 nt (absent). ( B ) Proportion of miRNA-mRNA hybrids across three biological replicates of the <t>BRAF-mutant</t> <t>melanoma</t> <t>cell</t> <t>line</t> <t>A375</t> (A1, A2, and A3) with miRNA binding sites mapped to the mRNA sequence, and the number of identified hybrid sequences in each replicate. Hybrid mRNAs for which the transcript was not annotated in the corresponding database were excluded. The number above the bars displays the number of hybrids identified in each biological replicate. ( C ) The two types of miRNA-mRNA hybrid molecules that were generated during the intermolecular ligation: 5′ and 3′ miRNA hybrids, in which the miRNA is located at the 5′ end or the 3′ end of the hybrids, respectively. ( D ) M-plots showing the number of miRNA-mRNA hybrids bound at each miRNA nt position ( x -axis, nt 1–22), and showing that most genes are targeted via the seed sequence but also with supplementary sites in the 3′ region of the miRNA. ( E ) The type of seed pairing (5′ miRNA sequence) was divided into classes as shown in ( A ). The proportion of miRNA-mRNA hybrids across the three biological replicates was plotted for the different seed categories for all hybrids as well as for 5′ and 3′ hybrids separately. ( F ) miRNA base-pairing via the 3′ portion of the miRNA (supplementary 3′ or non-seed pairing). The strength of 3′ sequence binding was classified as in ( A ) and was plotted for each seed sequence type across the biological replicates.
    Braf Mutant Melanoma Cell Line A375, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/braf mutant melanoma cell line a375/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    braf mutant melanoma cell line a375 - by Bioz Stars, 2024-05
    86/100 stars

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    1) Product Images from "Cross-Linking Ligation and Sequencing of Hybrids (qCLASH) Reveals an Unpredicted miRNA Targetome in Melanoma Cells"

    Article Title: Cross-Linking Ligation and Sequencing of Hybrids (qCLASH) Reveals an Unpredicted miRNA Targetome in Melanoma Cells

    Journal: Cancers

    doi: 10.3390/cancers13051096

    Characteristics of miRNA-mRNA hybrids discovered in melanoma cells. ( A ) Binding characteristics of miRNA–mRNA interactions. Binding region: mRNA part of the hybrids mapped to the ENSEMBL transcript database with regions corresponding to the 5′UTR and 3′UTR, CDS, 5′-UTR-CDS, and CDS-3′UTR. Seed match: nucleotides (nt) 2–7 at 5′ end of miRNA with zero (2–7, 0 mm), 2–8 with zero (2–8, 0 mm), one (2–8, 1 mm), or two mismatches (2–8, 2 mm), and the remaining binding modes were classified as “other”. Seed pairing at nucleotides 2–7 (blue) and 2–8 (red) without mismatches was considered as canonical seed pairing, whereas any additional type of pairing was referred to as non-canonical seed pairing. Strength: “strength” of binding outside the seed region at the 3′ end of the miRNA based on number of bound nt: >8 nt (strong), 5–8 nt (moderate), 1–4 nt (weak), and 0 nt (absent). ( B ) Proportion of miRNA-mRNA hybrids across three biological replicates of the BRAF-mutant melanoma cell line A375 (A1, A2, and A3) with miRNA binding sites mapped to the mRNA sequence, and the number of identified hybrid sequences in each replicate. Hybrid mRNAs for which the transcript was not annotated in the corresponding database were excluded. The number above the bars displays the number of hybrids identified in each biological replicate. ( C ) The two types of miRNA-mRNA hybrid molecules that were generated during the intermolecular ligation: 5′ and 3′ miRNA hybrids, in which the miRNA is located at the 5′ end or the 3′ end of the hybrids, respectively. ( D ) M-plots showing the number of miRNA-mRNA hybrids bound at each miRNA nt position ( x -axis, nt 1–22), and showing that most genes are targeted via the seed sequence but also with supplementary sites in the 3′ region of the miRNA. ( E ) The type of seed pairing (5′ miRNA sequence) was divided into classes as shown in ( A ). The proportion of miRNA-mRNA hybrids across the three biological replicates was plotted for the different seed categories for all hybrids as well as for 5′ and 3′ hybrids separately. ( F ) miRNA base-pairing via the 3′ portion of the miRNA (supplementary 3′ or non-seed pairing). The strength of 3′ sequence binding was classified as in ( A ) and was plotted for each seed sequence type across the biological replicates.
    Figure Legend Snippet: Characteristics of miRNA-mRNA hybrids discovered in melanoma cells. ( A ) Binding characteristics of miRNA–mRNA interactions. Binding region: mRNA part of the hybrids mapped to the ENSEMBL transcript database with regions corresponding to the 5′UTR and 3′UTR, CDS, 5′-UTR-CDS, and CDS-3′UTR. Seed match: nucleotides (nt) 2–7 at 5′ end of miRNA with zero (2–7, 0 mm), 2–8 with zero (2–8, 0 mm), one (2–8, 1 mm), or two mismatches (2–8, 2 mm), and the remaining binding modes were classified as “other”. Seed pairing at nucleotides 2–7 (blue) and 2–8 (red) without mismatches was considered as canonical seed pairing, whereas any additional type of pairing was referred to as non-canonical seed pairing. Strength: “strength” of binding outside the seed region at the 3′ end of the miRNA based on number of bound nt: >8 nt (strong), 5–8 nt (moderate), 1–4 nt (weak), and 0 nt (absent). ( B ) Proportion of miRNA-mRNA hybrids across three biological replicates of the BRAF-mutant melanoma cell line A375 (A1, A2, and A3) with miRNA binding sites mapped to the mRNA sequence, and the number of identified hybrid sequences in each replicate. Hybrid mRNAs for which the transcript was not annotated in the corresponding database were excluded. The number above the bars displays the number of hybrids identified in each biological replicate. ( C ) The two types of miRNA-mRNA hybrid molecules that were generated during the intermolecular ligation: 5′ and 3′ miRNA hybrids, in which the miRNA is located at the 5′ end or the 3′ end of the hybrids, respectively. ( D ) M-plots showing the number of miRNA-mRNA hybrids bound at each miRNA nt position ( x -axis, nt 1–22), and showing that most genes are targeted via the seed sequence but also with supplementary sites in the 3′ region of the miRNA. ( E ) The type of seed pairing (5′ miRNA sequence) was divided into classes as shown in ( A ). The proportion of miRNA-mRNA hybrids across the three biological replicates was plotted for the different seed categories for all hybrids as well as for 5′ and 3′ hybrids separately. ( F ) miRNA base-pairing via the 3′ portion of the miRNA (supplementary 3′ or non-seed pairing). The strength of 3′ sequence binding was classified as in ( A ) and was plotted for each seed sequence type across the biological replicates.

    Techniques Used: Binding Assay, Mutagenesis, Sequencing, Generated, Ligation

    Most commonly occurring hybrids. ( A ) UpSet plot representing the number of distinct miRNA-mRNA hybrids across three biological replicates (horizontal bars) that are unique or common across the replicates (vertical bars). miRNA-mRNA hybrids present in all replicates (A1, A2, and A3) of the BRAF-mutant melanoma cell line A375 were highlighted in red. Top 25 ( B ) miRNAs and ( C ) mRNAs that occur in miRNA-mRNA hybrids showing the number of hybrids detected and the corresponding binding region on the mRNA. As opposed to the data shown in ( A ), the number of hybrids in ( B , C ) was not collapsed for unique miRNA-mRNA pairs; thus, it displays the number of all sequences/hybrids that were identified for that particular miRNA ( B ) or mRNA ( C ).
    Figure Legend Snippet: Most commonly occurring hybrids. ( A ) UpSet plot representing the number of distinct miRNA-mRNA hybrids across three biological replicates (horizontal bars) that are unique or common across the replicates (vertical bars). miRNA-mRNA hybrids present in all replicates (A1, A2, and A3) of the BRAF-mutant melanoma cell line A375 were highlighted in red. Top 25 ( B ) miRNAs and ( C ) mRNAs that occur in miRNA-mRNA hybrids showing the number of hybrids detected and the corresponding binding region on the mRNA. As opposed to the data shown in ( A ), the number of hybrids in ( B , C ) was not collapsed for unique miRNA-mRNA pairs; thus, it displays the number of all sequences/hybrids that were identified for that particular miRNA ( B ) or mRNA ( C ).

    Techniques Used: Mutagenesis, Binding Assay

    Correlation between hybrid number and miRNA or mRNA expression levels. Number of miRNA occurrence frequency in miRNA-mRNA hybrids of the biological replicates (A1, A2, and A3) of the melanoma cell line A375 compared to ( A ) log2 miRNA expression levels in A375 and ( B ) log2 average reads per million (rpm) in melanoma patient samples (TCGA 2015 data). ( C ) Number of mRNA occurrence frequency in miRNA-mRNA hybrids compared to log2 gene expression (mRNA) levels in melanoma patient samples (TCGA2015 data).
    Figure Legend Snippet: Correlation between hybrid number and miRNA or mRNA expression levels. Number of miRNA occurrence frequency in miRNA-mRNA hybrids of the biological replicates (A1, A2, and A3) of the melanoma cell line A375 compared to ( A ) log2 miRNA expression levels in A375 and ( B ) log2 average reads per million (rpm) in melanoma patient samples (TCGA 2015 data). ( C ) Number of mRNA occurrence frequency in miRNA-mRNA hybrids compared to log2 gene expression (mRNA) levels in melanoma patient samples (TCGA2015 data).

    Techniques Used: Expressing

    Experimental validation of miRNA–mRNA interactions. ( A ) Luciferase reporter vectors containing stretches of the target CDS or 3′UTR for selected miRNAs were transfected into A375 cells together with 50 nM miRNA mimic or NCM for 72 h. Illustrated are the mean ± standard deviation (SD) of at least three replicates. Statistical significance was determined by one-way ANOVA, followed by Dunnett’s multiple comparisons test with ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. P1 and P2 represent the parts of the cloned region. ( B ) Protein expression levels of selected miRNA targets upon 50 nM miRNA mimic or NCM treatment for 72 h in three different melanoma cell lines.
    Figure Legend Snippet: Experimental validation of miRNA–mRNA interactions. ( A ) Luciferase reporter vectors containing stretches of the target CDS or 3′UTR for selected miRNAs were transfected into A375 cells together with 50 nM miRNA mimic or NCM for 72 h. Illustrated are the mean ± standard deviation (SD) of at least three replicates. Statistical significance was determined by one-way ANOVA, followed by Dunnett’s multiple comparisons test with ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. P1 and P2 represent the parts of the cloned region. ( B ) Protein expression levels of selected miRNA targets upon 50 nM miRNA mimic or NCM treatment for 72 h in three different melanoma cell lines.

    Techniques Used: Luciferase, Transfection, Standard Deviation, Clone Assay, Expressing

    miRNA-lncRNA hybrids. ( A ) Number of miRNA-lncRNA hybrids in A375 replicates. ( B ) The type of seed pairing (5′ miRNA sequence) was divided and plotted as described in E. The predicted and canonical seed sequence was displayed in blue (2–7 nt no mismatches) and red (2-nt no mismatches). Non-canonical seed pairing was displayed in yellow and green (one or two mismatches within the seed) or in grey for additional types of seed pairing (Other). ( C ) miRNA base-pairing via the 3′ portion of the miRNA (supplementary 3′ or non-seed pairing) as described in F. The “strength” of binding outside the seed region at the 3′ end of the miRNA based on number of bound nt: >8 nt (strong), 5–8 nt (moderate), 1–4 nt (weak), and 0 nt (absent). Top 25 ( D ) miRNAs and ( E ) lncRNAs that occur in miRNA-lncRNA hybrids showing the number of hybrid sequences each RNA species occurs in. ( F ) Number of occurrences of pri-miRNA genes, which were labelled as lncRNAs in miRNA-lncRNA hybrids across three biological A375 replicates (A1, A2, A3).
    Figure Legend Snippet: miRNA-lncRNA hybrids. ( A ) Number of miRNA-lncRNA hybrids in A375 replicates. ( B ) The type of seed pairing (5′ miRNA sequence) was divided and plotted as described in E. The predicted and canonical seed sequence was displayed in blue (2–7 nt no mismatches) and red (2-nt no mismatches). Non-canonical seed pairing was displayed in yellow and green (one or two mismatches within the seed) or in grey for additional types of seed pairing (Other). ( C ) miRNA base-pairing via the 3′ portion of the miRNA (supplementary 3′ or non-seed pairing) as described in F. The “strength” of binding outside the seed region at the 3′ end of the miRNA based on number of bound nt: >8 nt (strong), 5–8 nt (moderate), 1–4 nt (weak), and 0 nt (absent). Top 25 ( D ) miRNAs and ( E ) lncRNAs that occur in miRNA-lncRNA hybrids showing the number of hybrid sequences each RNA species occurs in. ( F ) Number of occurrences of pri-miRNA genes, which were labelled as lncRNAs in miRNA-lncRNA hybrids across three biological A375 replicates (A1, A2, A3).

    Techniques Used: Sequencing, Binding Assay

    braf mutant human melanoma cell lines a375  (ATCC)


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    ATCC braf mutant human melanoma cell lines a375
    Braf Mutant Human Melanoma Cell Lines A375, 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 human melanoma cell lines a375  (ATCC)


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    ATCC braf mutant human melanoma cell lines a375
    miRNAs differentially expressed between A375R and <t>A375</t> cells and KEGG pathway analysis of putative targets. a Heatmap showing miRNA up-regulated (red = expression above the mean) and down-regulated (blue = expression below the mean) in dabrafenib-resistant A375R cells as compared with dabrafenib-sensitive A375 cells (SAM analysis; FC ≥ 2, FDR = 0%). miRNAs are indicated according to annotation provided by Affymetrix. b Putative target genes of differentially expressed miRNAs were obtained from TargetScan and used for KEGG pathway enrichment analysis. Only pathways with an adjusted P value <0.01 were considered and listed according to a decreasing value of the combined score. c, d Expression of miRNAs down-regulated (c) or up-regulated (d) in A375R cells according to microarray results was validated using specific TaqMan® MicroRNA Assays. The data were normalized to the level of RNU44 in each sample and expressed as 2 -ΔCt x10 5 values. Each value represents the arithmetic mean of at least three independent experiments performed with duplicate samples. Bars, standard error of the mean (SEM). ** P <0.01 and * P <0.05, A375R versus ( vs ) A375.
    Braf Mutant Human Melanoma Cell Lines A375, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A"

    Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1238-4

    miRNAs differentially expressed between A375R and A375 cells and KEGG pathway analysis of putative targets. a Heatmap showing miRNA up-regulated (red = expression above the mean) and down-regulated (blue = expression below the mean) in dabrafenib-resistant A375R cells as compared with dabrafenib-sensitive A375 cells (SAM analysis; FC ≥ 2, FDR = 0%). miRNAs are indicated according to annotation provided by Affymetrix. b Putative target genes of differentially expressed miRNAs were obtained from TargetScan and used for KEGG pathway enrichment analysis. Only pathways with an adjusted P value <0.01 were considered and listed according to a decreasing value of the combined score. c, d Expression of miRNAs down-regulated (c) or up-regulated (d) in A375R cells according to microarray results was validated using specific TaqMan® MicroRNA Assays. The data were normalized to the level of RNU44 in each sample and expressed as 2 -ΔCt x10 5 values. Each value represents the arithmetic mean of at least three independent experiments performed with duplicate samples. Bars, standard error of the mean (SEM). ** P <0.01 and * P <0.05, A375R versus ( vs ) A375.
    Figure Legend Snippet: miRNAs differentially expressed between A375R and A375 cells and KEGG pathway analysis of putative targets. a Heatmap showing miRNA up-regulated (red = expression above the mean) and down-regulated (blue = expression below the mean) in dabrafenib-resistant A375R cells as compared with dabrafenib-sensitive A375 cells (SAM analysis; FC ≥ 2, FDR = 0%). miRNAs are indicated according to annotation provided by Affymetrix. b Putative target genes of differentially expressed miRNAs were obtained from TargetScan and used for KEGG pathway enrichment analysis. Only pathways with an adjusted P value <0.01 were considered and listed according to a decreasing value of the combined score. c, d Expression of miRNAs down-regulated (c) or up-regulated (d) in A375R cells according to microarray results was validated using specific TaqMan® MicroRNA Assays. The data were normalized to the level of RNU44 in each sample and expressed as 2 -ΔCt x10 5 values. Each value represents the arithmetic mean of at least three independent experiments performed with duplicate samples. Bars, standard error of the mean (SEM). ** P <0.01 and * P <0.05, A375R versus ( vs ) A375.

    Techniques Used: Expressing, Microarray

    miR-126-3p is up-regulated by dabrafenib only in drug-sensitive cells and inhibits their proliferation. a, b Melanoma cells were incubated with 100 nM dabrafenib (DAB) or with DMSO alone and after 48 h of culture miR-126-3p expression was evaluated by qRT-PCR. 2 -ΔCt x10 5 values calculated relative to RNU44 as the internal reference are shown. Each value represents the arithmetic mean of three independent experiments performed with duplicate samples. Bars, SEM. ** P <0.01 and * P <0.05, DAB vs matched DMSO; ## P <0.01 and # P <0.05, resistant cells/DMSO vs parental cells/DMSO. c A375 and SK-Mel28 were transiently transfected with 50 nM pre-miR-126-3p or pre-miR-CTRL, cultured for six days and then assayed for proliferation by the MTT assay. Data are expressed in terms of absorbance at 595 nM. Each value represents the arithmetic mean of three (A375) or four (SK-Mel28) independent experiments performed with triplicate samples. Bars, SEM. * P <0.05, pre-miR-126-3p vs pre-miR-CTRL.
    Figure Legend Snippet: miR-126-3p is up-regulated by dabrafenib only in drug-sensitive cells and inhibits their proliferation. a, b Melanoma cells were incubated with 100 nM dabrafenib (DAB) or with DMSO alone and after 48 h of culture miR-126-3p expression was evaluated by qRT-PCR. 2 -ΔCt x10 5 values calculated relative to RNU44 as the internal reference are shown. Each value represents the arithmetic mean of three independent experiments performed with duplicate samples. Bars, SEM. ** P <0.01 and * P <0.05, DAB vs matched DMSO; ## P <0.01 and # P <0.05, resistant cells/DMSO vs parental cells/DMSO. c A375 and SK-Mel28 were transiently transfected with 50 nM pre-miR-126-3p or pre-miR-CTRL, cultured for six days and then assayed for proliferation by the MTT assay. Data are expressed in terms of absorbance at 595 nM. Each value represents the arithmetic mean of three (A375) or four (SK-Mel28) independent experiments performed with triplicate samples. Bars, SEM. * P <0.05, pre-miR-126-3p vs pre-miR-CTRL.

    Techniques Used: Incubation, Expressing, Quantitative RT-PCR, Transfection, Cell Culture, MTT Assay

    ADAM9 silencing delays the development of resistance to dabrafenib. A375 cells were seeded into 96-well plates and every eight days transfected with 50 nM siADAM9 or siCTRL and treated with 100 nM dabrafenib (DAB) or DMSO. Cell cultures were photographed and processed for quantitative analysis of proliferation on day 0 (i.e after the first transfection), 8, 16 and 24. Images from a representative experiment are shown. b Quantitative analysis of proliferation of cell cultures described in (a). Crystal violet was solubilized and absorbance was read at 595 nm. Each value represents the arithmetic mean of three independent experiments performed with triplicate cultures. Bars, SEM. ** P <0.01, siADAM9 vs matched siCTRL; §§ P <0.01, siCTRL/DAB/Day 8 vs siCTRL/DMSO/Day 8; †† P <0.01, siADAM9/DAB/Day 8 vs siADAM9/DMSO/Day8; ## P <0.01, siCTRL/DAB/Day 16 vs siCTRL/DAB/Day 8; ⁋⁋ P <0.01, siCTRL/DAB/Day 24 vs siCTRL/DAB/Day 16.
    Figure Legend Snippet: ADAM9 silencing delays the development of resistance to dabrafenib. A375 cells were seeded into 96-well plates and every eight days transfected with 50 nM siADAM9 or siCTRL and treated with 100 nM dabrafenib (DAB) or DMSO. Cell cultures were photographed and processed for quantitative analysis of proliferation on day 0 (i.e after the first transfection), 8, 16 and 24. Images from a representative experiment are shown. b Quantitative analysis of proliferation of cell cultures described in (a). Crystal violet was solubilized and absorbance was read at 595 nm. Each value represents the arithmetic mean of three independent experiments performed with triplicate cultures. Bars, SEM. ** P <0.01, siADAM9 vs matched siCTRL; §§ P <0.01, siCTRL/DAB/Day 8 vs siCTRL/DMSO/Day 8; †† P <0.01, siADAM9/DAB/Day 8 vs siADAM9/DMSO/Day8; ## P <0.01, siCTRL/DAB/Day 16 vs siCTRL/DAB/Day 8; ⁋⁋ P <0.01, siCTRL/DAB/Day 24 vs siCTRL/DAB/Day 16.

    Techniques Used: Transfection

    braf mutant melanoma cell line a375  (ATCC)


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    ATCC braf mutant melanoma cell line a375
    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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    ATCC braf v600e mutant human melanoma cell line a375
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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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    ATCC braf mutant human melanoma cell lines 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.
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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 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