isogenic mutants  (ATCC)


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    ATCC isogenic mutants
    Isogenic Mutants, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nras mutant a 375 isogenic cell line  (ATCC)


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    ATCC nras mutant a 375 isogenic cell line
    Nras Mutant A 375 Isogenic Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a375 nras q61k  (ATCC)


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    ATCC a375 nras q61k
    CRISPR/Cas9 engineering of isogenic <t>A375</t> models of drug-resistant melanoma. ( A ) Schematic diagram of the CRISPR/Cas9 editing strategy used for the introduction of point mutations associated with BRAF and MEK inhibitor resistance into A375 melanoma cells. Two guide RNAs in complex with Cas9 (sg1 and sg2, scissors) were used to create double-stranded breaks in the intronic regions (black lines) to either side of the target exon (black box). A donor plasmid containing a copy of the target exon with the desired point mutation (star) and flanking intronic sequences was used as a repair template. This strategy ensures that any indels resulting from imperfect sequence repair at the Cas9 cut sites (red lines) are spliced out during mRNA processing and do not affect the resulting cellular protein; ( B ) Sanger sequencing of genomic DNA from the resulting KRAS G13D heterozygous (left), <t>NRAS</t> <t>Q61K</t> heterozygous (middle), and MEK1 Q56P homozygous (right) A375 isogenic lines.
    A375 Nras Q61k, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy"

    Article Title: CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy

    Journal: Cancers

    doi: 10.3390/cancers14215449

    CRISPR/Cas9 engineering of isogenic A375 models of drug-resistant melanoma. ( A ) Schematic diagram of the CRISPR/Cas9 editing strategy used for the introduction of point mutations associated with BRAF and MEK inhibitor resistance into A375 melanoma cells. Two guide RNAs in complex with Cas9 (sg1 and sg2, scissors) were used to create double-stranded breaks in the intronic regions (black lines) to either side of the target exon (black box). A donor plasmid containing a copy of the target exon with the desired point mutation (star) and flanking intronic sequences was used as a repair template. This strategy ensures that any indels resulting from imperfect sequence repair at the Cas9 cut sites (red lines) are spliced out during mRNA processing and do not affect the resulting cellular protein; ( B ) Sanger sequencing of genomic DNA from the resulting KRAS G13D heterozygous (left), NRAS Q61K heterozygous (middle), and MEK1 Q56P homozygous (right) A375 isogenic lines.
    Figure Legend Snippet: CRISPR/Cas9 engineering of isogenic A375 models of drug-resistant melanoma. ( A ) Schematic diagram of the CRISPR/Cas9 editing strategy used for the introduction of point mutations associated with BRAF and MEK inhibitor resistance into A375 melanoma cells. Two guide RNAs in complex with Cas9 (sg1 and sg2, scissors) were used to create double-stranded breaks in the intronic regions (black lines) to either side of the target exon (black box). A donor plasmid containing a copy of the target exon with the desired point mutation (star) and flanking intronic sequences was used as a repair template. This strategy ensures that any indels resulting from imperfect sequence repair at the Cas9 cut sites (red lines) are spliced out during mRNA processing and do not affect the resulting cellular protein; ( B ) Sanger sequencing of genomic DNA from the resulting KRAS G13D heterozygous (left), NRAS Q61K heterozygous (middle), and MEK1 Q56P homozygous (right) A375 isogenic lines.

    Techniques Used: CRISPR, Plasmid Preparation, Mutagenesis, Sequencing

    Isogenic melanoma models are resistant to BRAF inhibitors but not to BRAF non-specific chemotherapeutics in 2D tissue culture. ( A ) Dabrafenib resistance of A375 melanoma models in 2D tissue culture; ( B ) Vemurafenib resistance of A375 melanoma models in 2D tissue culture; ( C ) No resistance to the BRAF non-specific chemotherapeutic doxorubicin in 2D tissue culture; ( D ) Immunoblot demonstrating BRAF inhibitor resistance in EGFR pathway signaling in NRAS Q61K and KRAS G13D A375 melanoma models. Cells were treated with 1.0 μM of the indicated drug for 90 min prior to harvesting protein; ( E ) Dose-response curve for the MEK inhibitor trametinib in the MEK1 Q56P melanoma model in 2D tissue culture; ( F ) The same curve as in ( E ) for the MEK inhibitor binimetinib.
    Figure Legend Snippet: Isogenic melanoma models are resistant to BRAF inhibitors but not to BRAF non-specific chemotherapeutics in 2D tissue culture. ( A ) Dabrafenib resistance of A375 melanoma models in 2D tissue culture; ( B ) Vemurafenib resistance of A375 melanoma models in 2D tissue culture; ( C ) No resistance to the BRAF non-specific chemotherapeutic doxorubicin in 2D tissue culture; ( D ) Immunoblot demonstrating BRAF inhibitor resistance in EGFR pathway signaling in NRAS Q61K and KRAS G13D A375 melanoma models. Cells were treated with 1.0 μM of the indicated drug for 90 min prior to harvesting protein; ( E ) Dose-response curve for the MEK inhibitor trametinib in the MEK1 Q56P melanoma model in 2D tissue culture; ( F ) The same curve as in ( E ) for the MEK inhibitor binimetinib.

    Techniques Used: Western Blot

    Effect of tissue culture format on EGFR pathway signaling in drug-resistant melanoma models. ( A ) A375 WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P cells growing in a 2D monolayer (top) and as 3D spheroids (bottom). For spheroid formation, 500 cells from each line were seeded in each well of a 96-well ultra-low attachment spheroid microplate and grown for five days before imaging; ( B ) Immunoblots tracking EGFR pathway signaling in engineered A375 melanoma model cells in 2D and 3D tissue culture. Protein was harvested from each cell line growing in 2D culture, and cellular protein from 3D spheroids was collected from spheroids seeded at 500 cells per 96-well and grown for seven days. Total protein (20 μg) was loaded in each lane and samples were blotted for total EGFR, total MEK1/2, phospho-MEK1/2 (Ser217/221), total ERK1/2 (p44/42 MAPK), phospho-ERK1/2 (Thr202/Tyr204), AKT, phospho-AKT (Ser473), and GAPDH.
    Figure Legend Snippet: Effect of tissue culture format on EGFR pathway signaling in drug-resistant melanoma models. ( A ) A375 WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P cells growing in a 2D monolayer (top) and as 3D spheroids (bottom). For spheroid formation, 500 cells from each line were seeded in each well of a 96-well ultra-low attachment spheroid microplate and grown for five days before imaging; ( B ) Immunoblots tracking EGFR pathway signaling in engineered A375 melanoma model cells in 2D and 3D tissue culture. Protein was harvested from each cell line growing in 2D culture, and cellular protein from 3D spheroids was collected from spheroids seeded at 500 cells per 96-well and grown for seven days. Total protein (20 μg) was loaded in each lane and samples were blotted for total EGFR, total MEK1/2, phospho-MEK1/2 (Ser217/221), total ERK1/2 (p44/42 MAPK), phospho-ERK1/2 (Thr202/Tyr204), AKT, phospho-AKT (Ser473), and GAPDH.

    Techniques Used: Imaging, Western Blot

    A375 Ras mutant melanoma models are resistant to BRAF inhibitors in 3D tissue culture and A375 MEK1 melanoma model is resistant to both MEK and BRAF inhibitors in 3D tissue culture. ( A ) BRAF inhibitor resistance in A375 RAS mutant melanoma models in 3D tissue culture. For each indicated cell type, 500 cells were seeded in each well of a ULA spheroid microplate and grown for three days in the absence of drug. After 72 h the indicated BRAF-specific inhibitor or the BRAF-nonspecific chemotherapeutic agent doxorubicin was added (Db = dabrafenib 25 nM, Vb = vemurafenib 50 nM, Dx = doxorubicin 100 nM) and the spheroids were grown for an additional three days. Spheroids were then stained with 2 μM Calcein AM green and NucBlue live-cell nuclear marker for 90 min and then imaged; ( B ) Average spheroid size of RAS mutant melanoma models following drug treatment relative to the un-drugged condition. Statistical analysis was performed using two-way ANOVA with multiple comparisons, each condition represents at least n = 3 spheroids; ( C ) MEK and BRAF inhibitor resistance in A375 MEKQ56P spheroid melanoma model. Drugged spheroids were handled as in ( A ) and treated with 25 nM dabrafenib, 50 nM vemurafenib, and 50 nM of the MEK inhibitors trametinib (Tb) and binimetinib (Bb), respectively, or 100 nM doxorubicin; ( D ) Average numbers of nuclei per spheroid were calculated relative to the un-drugged condition. Results from at least n = 3 spheroids for each condition were averaged and plotted, statistical analysis was performed using two-way ANOVA with multiple comparisons. ****, p < 0.0001.
    Figure Legend Snippet: A375 Ras mutant melanoma models are resistant to BRAF inhibitors in 3D tissue culture and A375 MEK1 melanoma model is resistant to both MEK and BRAF inhibitors in 3D tissue culture. ( A ) BRAF inhibitor resistance in A375 RAS mutant melanoma models in 3D tissue culture. For each indicated cell type, 500 cells were seeded in each well of a ULA spheroid microplate and grown for three days in the absence of drug. After 72 h the indicated BRAF-specific inhibitor or the BRAF-nonspecific chemotherapeutic agent doxorubicin was added (Db = dabrafenib 25 nM, Vb = vemurafenib 50 nM, Dx = doxorubicin 100 nM) and the spheroids were grown for an additional three days. Spheroids were then stained with 2 μM Calcein AM green and NucBlue live-cell nuclear marker for 90 min and then imaged; ( B ) Average spheroid size of RAS mutant melanoma models following drug treatment relative to the un-drugged condition. Statistical analysis was performed using two-way ANOVA with multiple comparisons, each condition represents at least n = 3 spheroids; ( C ) MEK and BRAF inhibitor resistance in A375 MEKQ56P spheroid melanoma model. Drugged spheroids were handled as in ( A ) and treated with 25 nM dabrafenib, 50 nM vemurafenib, and 50 nM of the MEK inhibitors trametinib (Tb) and binimetinib (Bb), respectively, or 100 nM doxorubicin; ( D ) Average numbers of nuclei per spheroid were calculated relative to the un-drugged condition. Results from at least n = 3 spheroids for each condition were averaged and plotted, statistical analysis was performed using two-way ANOVA with multiple comparisons. ****, p < 0.0001.

    Techniques Used: Mutagenesis, Staining, Marker

    MEK1 Q56P melanoma model is sensitive to combination BRAF/MEK inhibitor treatment in both 2D and 3D tissue culture. ( A ) Dose-response curves for A375 WT cells in 2D tissue culture with dabrafenib (grey line), trametinib (black line), or a combination of dabrafenib and trametinib (red line, molarity indicates total drug concentration); ( B ) Dose-response curves for NRAS Q61K cells in 2D tissue culture with dabrafenib, trametinib, or a combination of dabrafenib and trametinib; ( C ) Dose-response curves for MEK1 Q56P cells in 2D tissue culture with dabrafenib, trametinib, or combination; ( D ) Dose-response curves for MEK1 Q56P cells in 2D tissue culture with dabrafenib, trametinib, or combination. Lower survival with combination indicates synergistic drug killing in this line; ( E ) Immunoblot demonstrating synergistic inhibition of the MEK/ERK signaling pathway in MEK1 Q56P melanoma model cells in 2D tissue culture. Cells were treated with either 1.0 μM of the indicated inhibitor compound or with 0.5 μM each of each indicated drug for 90 min prior to harvesting protein (Db = dabrafenib 1.0 μM, Vb = vemurafenib 1.0 μM, Tb = trametinib 1.0 μM, Bb = binimetinib 1.0 μM, Db/Tb = dabrafenib 0.5 μM + trametinib 0.5 μM); ( F ) Model of synergistic inhibition of the RAS/RAF/MEK/ERK pathway by combination MEK and BRAF inhibitor treatment. Orange star indicates the primary BRAF V600E mutation which drives cell proliferation in the absence of BRAF inhibitor. Treatment with BRAF inhibitor results in secondary MEK1 Q65P mutation (black outlined orange star). In the presence of these two mutations, upstream pathway inhibition with BRAF inhibitor in combination with downstream pathway inhibition with MEK inhibitor (red arrows) leads to less cell survival and proliferation than is observed when each drug is used alone; ( G ) Susceptibility of MEK1 Q56P cells grown in 3D tissue culture to dabrafenib, trametinib, and combination drug treatment. For each indicated cell type, 500 cells were seeded in each well of a ULA spheroid microplate and grown for three days in the absence of drug. The spheroids were then treated with 12 nM dabrafenib, 4 nM trametinib, a combination of 6 nM dabrafenib and 2 nM trametinib, or vehicle control for an additional three days. Spheroids were then stained with 2 μM Calcein AM green and NucBlue live-cell nuclear marker for 90 min and then imaged; ( H ) Spheroid sizes were calculated relative to the un-drugged condition. Results from at least n = 3 spheroids for each condition were averaged and plotted, statistical analysis was performed using two-way ANOVA with multiple comparisons. ***, p < 0.001.
    Figure Legend Snippet: MEK1 Q56P melanoma model is sensitive to combination BRAF/MEK inhibitor treatment in both 2D and 3D tissue culture. ( A ) Dose-response curves for A375 WT cells in 2D tissue culture with dabrafenib (grey line), trametinib (black line), or a combination of dabrafenib and trametinib (red line, molarity indicates total drug concentration); ( B ) Dose-response curves for NRAS Q61K cells in 2D tissue culture with dabrafenib, trametinib, or a combination of dabrafenib and trametinib; ( C ) Dose-response curves for MEK1 Q56P cells in 2D tissue culture with dabrafenib, trametinib, or combination; ( D ) Dose-response curves for MEK1 Q56P cells in 2D tissue culture with dabrafenib, trametinib, or combination. Lower survival with combination indicates synergistic drug killing in this line; ( E ) Immunoblot demonstrating synergistic inhibition of the MEK/ERK signaling pathway in MEK1 Q56P melanoma model cells in 2D tissue culture. Cells were treated with either 1.0 μM of the indicated inhibitor compound or with 0.5 μM each of each indicated drug for 90 min prior to harvesting protein (Db = dabrafenib 1.0 μM, Vb = vemurafenib 1.0 μM, Tb = trametinib 1.0 μM, Bb = binimetinib 1.0 μM, Db/Tb = dabrafenib 0.5 μM + trametinib 0.5 μM); ( F ) Model of synergistic inhibition of the RAS/RAF/MEK/ERK pathway by combination MEK and BRAF inhibitor treatment. Orange star indicates the primary BRAF V600E mutation which drives cell proliferation in the absence of BRAF inhibitor. Treatment with BRAF inhibitor results in secondary MEK1 Q65P mutation (black outlined orange star). In the presence of these two mutations, upstream pathway inhibition with BRAF inhibitor in combination with downstream pathway inhibition with MEK inhibitor (red arrows) leads to less cell survival and proliferation than is observed when each drug is used alone; ( G ) Susceptibility of MEK1 Q56P cells grown in 3D tissue culture to dabrafenib, trametinib, and combination drug treatment. For each indicated cell type, 500 cells were seeded in each well of a ULA spheroid microplate and grown for three days in the absence of drug. The spheroids were then treated with 12 nM dabrafenib, 4 nM trametinib, a combination of 6 nM dabrafenib and 2 nM trametinib, or vehicle control for an additional three days. Spheroids were then stained with 2 μM Calcein AM green and NucBlue live-cell nuclear marker for 90 min and then imaged; ( H ) Spheroid sizes were calculated relative to the un-drugged condition. Results from at least n = 3 spheroids for each condition were averaged and plotted, statistical analysis was performed using two-way ANOVA with multiple comparisons. ***, p < 0.001.

    Techniques Used: Concentration Assay, Western Blot, Inhibition, Mutagenesis, Staining, Marker

    PD-L1 is constitutively expressed in KRAS G13D, but not in A375 WT, NRAS Q61K, or MEK1 Q56P melanoma models. ( A ) Flow cytometry analysis of cell surface PD-L1 expression in A375 WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P melanoma models grown in 2D tissue culture. Cells were treated overnight with 200 ng/µL interferon gamma (red), or mock treated (blue). The following day the cells were stained with either anti-PD-L1 or isotype control (grey); ( B ) PD-L1 immunoblot of total cellular protein from A375 WT, NRAS Q61K, and KRAS G13D cells grown in either 2D or 3D tissue culture; ( C ) Indirect immunofluorescence staining of PD-L1 in A375 WT, NRAS Q61K and KRAS G13D melanoma model lines.
    Figure Legend Snippet: PD-L1 is constitutively expressed in KRAS G13D, but not in A375 WT, NRAS Q61K, or MEK1 Q56P melanoma models. ( A ) Flow cytometry analysis of cell surface PD-L1 expression in A375 WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P melanoma models grown in 2D tissue culture. Cells were treated overnight with 200 ng/µL interferon gamma (red), or mock treated (blue). The following day the cells were stained with either anti-PD-L1 or isotype control (grey); ( B ) PD-L1 immunoblot of total cellular protein from A375 WT, NRAS Q61K, and KRAS G13D cells grown in either 2D or 3D tissue culture; ( C ) Indirect immunofluorescence staining of PD-L1 in A375 WT, NRAS Q61K and KRAS G13D melanoma model lines.

    Techniques Used: Flow Cytometry, Expressing, Staining, Western Blot, Immunofluorescence

    crl 1619ig 2  (ATCC)


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    ATCC crl 1619ig 2
    KEY RESOURCES TABLE
    Crl 1619ig 2, 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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    crl 1619ig 2 - by Bioz Stars, 2024-05
    86/100 stars

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    1) Product Images from "AP-1 transcription factor network explains diverse patterns of cellular plasticity in melanoma cells"

    Article Title: AP-1 transcription factor network explains diverse patterns of cellular plasticity in melanoma cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2022.111147

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Blocking Assay, Expressing, Activity Assay, Software

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Blocking Assay, Expressing, Activity Assay, Software

    crl 1619ig 2  (ATCC)


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    ATCC crl 1619ig 2
    KEY RESOURCES TABLE
    Crl 1619ig 2, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    crl 1619ig 2 - by Bioz Stars, 2024-05
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    1) Product Images from "AP-1 transcription factor network explains diverse patterns of cellular plasticity in melanoma cells"

    Article Title: AP-1 transcription factor network explains diverse patterns of cellular plasticity in melanoma cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2022.111147

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Blocking Assay, Expressing, Activity Assay, Software

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Blocking Assay, Expressing, Activity Assay, Software

    crl 1619ig  (ATCC)


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    ATCC crl 1619ig
    Crl 1619ig, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human a375 metastatic melanoma cells  (ATCC)


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    ATCC human a375 metastatic melanoma cells
    In vitro screening of novel NL compounds: ( A ) hit compounds were screened at 10 micromolar concentrations for 24 h. All but one compound demonstrated significant activity and were selected for a dose response analysis; ( B ) compounds were screened at increasing concentrations to assay for activity. NL221-75 and NL350-02 were as potent as FDA-approved controls, demonstrating low nanomolar range activity; ( C ) MTT <t>A375</t> cells were treated with increasing concentrations of test items and cell proliferation was determined at 24 h using the MTT method. All compounds demonstrated dose-dependent activity in preventing cell proliferation. Experimental compounds NL221-75 and NL350-02 were as effective as FDA-approved controls in preventing cell proliferation. Data are plotted as percent inhibition of proliferation; and ( D ) hERG inhibition experiments performed on CHO-cells. Cobimetinib demonstrated low nanomolar inhibition of hERG. NL34-113, NL221-75, and NL350-02 did not inhibit hERG at the concentrations tested.
    Human A375 Metastatic Melanoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Application of Pharmacokinetic Prediction Platforms in the Design of Optimized Anti-Cancer Drugs"

    Article Title: Application of Pharmacokinetic Prediction Platforms in the Design of Optimized Anti-Cancer Drugs

    Journal: Molecules

    doi: 10.3390/molecules27123678

    In vitro screening of novel NL compounds: ( A ) hit compounds were screened at 10 micromolar concentrations for 24 h. All but one compound demonstrated significant activity and were selected for a dose response analysis; ( B ) compounds were screened at increasing concentrations to assay for activity. NL221-75 and NL350-02 were as potent as FDA-approved controls, demonstrating low nanomolar range activity; ( C ) MTT A375 cells were treated with increasing concentrations of test items and cell proliferation was determined at 24 h using the MTT method. All compounds demonstrated dose-dependent activity in preventing cell proliferation. Experimental compounds NL221-75 and NL350-02 were as effective as FDA-approved controls in preventing cell proliferation. Data are plotted as percent inhibition of proliferation; and ( D ) hERG inhibition experiments performed on CHO-cells. Cobimetinib demonstrated low nanomolar inhibition of hERG. NL34-113, NL221-75, and NL350-02 did not inhibit hERG at the concentrations tested.
    Figure Legend Snippet: In vitro screening of novel NL compounds: ( A ) hit compounds were screened at 10 micromolar concentrations for 24 h. All but one compound demonstrated significant activity and were selected for a dose response analysis; ( B ) compounds were screened at increasing concentrations to assay for activity. NL221-75 and NL350-02 were as potent as FDA-approved controls, demonstrating low nanomolar range activity; ( C ) MTT A375 cells were treated with increasing concentrations of test items and cell proliferation was determined at 24 h using the MTT method. All compounds demonstrated dose-dependent activity in preventing cell proliferation. Experimental compounds NL221-75 and NL350-02 were as effective as FDA-approved controls in preventing cell proliferation. Data are plotted as percent inhibition of proliferation; and ( D ) hERG inhibition experiments performed on CHO-cells. Cobimetinib demonstrated low nanomolar inhibition of hERG. NL34-113, NL221-75, and NL350-02 did not inhibit hERG at the concentrations tested.

    Techniques Used: In Vitro, Activity Assay, Inhibition

    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

    dopaminergic neuroblastoma cell line  (ATCC)


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    ATCC dopaminergic neuroblastoma cell line
    Dopaminergic Neuroblastoma Cell Line, 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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    86
    ATCC crl 1619ig 2 luc2
    In vitro screening of novel NL compounds: ( A ) hit compounds were screened at 10 micromolar concentrations for 24 h. All but one compound demonstrated significant activity and were selected for a dose response analysis; ( B ) compounds were screened at increasing concentrations to assay for activity. NL221-75 and NL350-02 were as potent as FDA-approved controls, demonstrating low nanomolar range activity; ( C ) MTT <t>A375</t> cells were treated with increasing concentrations of test items and cell proliferation was determined at 24 h using the MTT method. All compounds demonstrated dose-dependent activity in preventing cell proliferation. Experimental compounds NL221-75 and NL350-02 were as effective as FDA-approved controls in preventing cell proliferation. Data are plotted as percent inhibition of proliferation; and ( D ) hERG inhibition experiments performed on CHO-cells. Cobimetinib demonstrated low nanomolar inhibition of hERG. NL34-113, NL221-75, and NL350-02 did not inhibit hERG at the concentrations tested.
    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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    95
    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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    95
    ATCC dopaminergic neuroblastoma cell line
    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.
    Dopaminergic Neuroblastoma Cell Line, 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


    CRISPR/Cas9 engineering of isogenic A375 models of drug-resistant melanoma. ( A ) Schematic diagram of the CRISPR/Cas9 editing strategy used for the introduction of point mutations associated with BRAF and MEK inhibitor resistance into A375 melanoma cells. Two guide RNAs in complex with Cas9 (sg1 and sg2, scissors) were used to create double-stranded breaks in the intronic regions (black lines) to either side of the target exon (black box). A donor plasmid containing a copy of the target exon with the desired point mutation (star) and flanking intronic sequences was used as a repair template. This strategy ensures that any indels resulting from imperfect sequence repair at the Cas9 cut sites (red lines) are spliced out during mRNA processing and do not affect the resulting cellular protein; ( B ) Sanger sequencing of genomic DNA from the resulting KRAS G13D heterozygous (left), NRAS Q61K heterozygous (middle), and MEK1 Q56P homozygous (right) A375 isogenic lines.

    Journal: Cancers

    Article Title: CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy

    doi: 10.3390/cancers14215449

    Figure Lengend Snippet: CRISPR/Cas9 engineering of isogenic A375 models of drug-resistant melanoma. ( A ) Schematic diagram of the CRISPR/Cas9 editing strategy used for the introduction of point mutations associated with BRAF and MEK inhibitor resistance into A375 melanoma cells. Two guide RNAs in complex with Cas9 (sg1 and sg2, scissors) were used to create double-stranded breaks in the intronic regions (black lines) to either side of the target exon (black box). A donor plasmid containing a copy of the target exon with the desired point mutation (star) and flanking intronic sequences was used as a repair template. This strategy ensures that any indels resulting from imperfect sequence repair at the Cas9 cut sites (red lines) are spliced out during mRNA processing and do not affect the resulting cellular protein; ( B ) Sanger sequencing of genomic DNA from the resulting KRAS G13D heterozygous (left), NRAS Q61K heterozygous (middle), and MEK1 Q56P homozygous (right) A375 isogenic lines.

    Article Snippet: The melanoma cell line A375 (ATCC, CRL-1619) and the derived isogenic lines A375 KRAS G13D (ATCC, CRL-1619IG-1), A375 NRAS Q61K (ATCC, CRL-1619IG-2) and A375 MEK1 Q56P (ATCC, CRL-1619IG-3) were grown and maintained in ATCC-formulated Dulbecco’s Modified Eagle’s Medium (ATCC, 30–2002) with 10% fetal bovine serum (ATCC, 30–2020).

    Techniques: CRISPR, Plasmid Preparation, Mutagenesis, Sequencing

    Isogenic melanoma models are resistant to BRAF inhibitors but not to BRAF non-specific chemotherapeutics in 2D tissue culture. ( A ) Dabrafenib resistance of A375 melanoma models in 2D tissue culture; ( B ) Vemurafenib resistance of A375 melanoma models in 2D tissue culture; ( C ) No resistance to the BRAF non-specific chemotherapeutic doxorubicin in 2D tissue culture; ( D ) Immunoblot demonstrating BRAF inhibitor resistance in EGFR pathway signaling in NRAS Q61K and KRAS G13D A375 melanoma models. Cells were treated with 1.0 μM of the indicated drug for 90 min prior to harvesting protein; ( E ) Dose-response curve for the MEK inhibitor trametinib in the MEK1 Q56P melanoma model in 2D tissue culture; ( F ) The same curve as in ( E ) for the MEK inhibitor binimetinib.

    Journal: Cancers

    Article Title: CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy

    doi: 10.3390/cancers14215449

    Figure Lengend Snippet: Isogenic melanoma models are resistant to BRAF inhibitors but not to BRAF non-specific chemotherapeutics in 2D tissue culture. ( A ) Dabrafenib resistance of A375 melanoma models in 2D tissue culture; ( B ) Vemurafenib resistance of A375 melanoma models in 2D tissue culture; ( C ) No resistance to the BRAF non-specific chemotherapeutic doxorubicin in 2D tissue culture; ( D ) Immunoblot demonstrating BRAF inhibitor resistance in EGFR pathway signaling in NRAS Q61K and KRAS G13D A375 melanoma models. Cells were treated with 1.0 μM of the indicated drug for 90 min prior to harvesting protein; ( E ) Dose-response curve for the MEK inhibitor trametinib in the MEK1 Q56P melanoma model in 2D tissue culture; ( F ) The same curve as in ( E ) for the MEK inhibitor binimetinib.

    Article Snippet: The melanoma cell line A375 (ATCC, CRL-1619) and the derived isogenic lines A375 KRAS G13D (ATCC, CRL-1619IG-1), A375 NRAS Q61K (ATCC, CRL-1619IG-2) and A375 MEK1 Q56P (ATCC, CRL-1619IG-3) were grown and maintained in ATCC-formulated Dulbecco’s Modified Eagle’s Medium (ATCC, 30–2002) with 10% fetal bovine serum (ATCC, 30–2020).

    Techniques: Western Blot

    Effect of tissue culture format on EGFR pathway signaling in drug-resistant melanoma models. ( A ) A375 WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P cells growing in a 2D monolayer (top) and as 3D spheroids (bottom). For spheroid formation, 500 cells from each line were seeded in each well of a 96-well ultra-low attachment spheroid microplate and grown for five days before imaging; ( B ) Immunoblots tracking EGFR pathway signaling in engineered A375 melanoma model cells in 2D and 3D tissue culture. Protein was harvested from each cell line growing in 2D culture, and cellular protein from 3D spheroids was collected from spheroids seeded at 500 cells per 96-well and grown for seven days. Total protein (20 μg) was loaded in each lane and samples were blotted for total EGFR, total MEK1/2, phospho-MEK1/2 (Ser217/221), total ERK1/2 (p44/42 MAPK), phospho-ERK1/2 (Thr202/Tyr204), AKT, phospho-AKT (Ser473), and GAPDH.

    Journal: Cancers

    Article Title: CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy

    doi: 10.3390/cancers14215449

    Figure Lengend Snippet: Effect of tissue culture format on EGFR pathway signaling in drug-resistant melanoma models. ( A ) A375 WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P cells growing in a 2D monolayer (top) and as 3D spheroids (bottom). For spheroid formation, 500 cells from each line were seeded in each well of a 96-well ultra-low attachment spheroid microplate and grown for five days before imaging; ( B ) Immunoblots tracking EGFR pathway signaling in engineered A375 melanoma model cells in 2D and 3D tissue culture. Protein was harvested from each cell line growing in 2D culture, and cellular protein from 3D spheroids was collected from spheroids seeded at 500 cells per 96-well and grown for seven days. Total protein (20 μg) was loaded in each lane and samples were blotted for total EGFR, total MEK1/2, phospho-MEK1/2 (Ser217/221), total ERK1/2 (p44/42 MAPK), phospho-ERK1/2 (Thr202/Tyr204), AKT, phospho-AKT (Ser473), and GAPDH.

    Article Snippet: The melanoma cell line A375 (ATCC, CRL-1619) and the derived isogenic lines A375 KRAS G13D (ATCC, CRL-1619IG-1), A375 NRAS Q61K (ATCC, CRL-1619IG-2) and A375 MEK1 Q56P (ATCC, CRL-1619IG-3) were grown and maintained in ATCC-formulated Dulbecco’s Modified Eagle’s Medium (ATCC, 30–2002) with 10% fetal bovine serum (ATCC, 30–2020).

    Techniques: Imaging, Western Blot

    A375 Ras mutant melanoma models are resistant to BRAF inhibitors in 3D tissue culture and A375 MEK1 melanoma model is resistant to both MEK and BRAF inhibitors in 3D tissue culture. ( A ) BRAF inhibitor resistance in A375 RAS mutant melanoma models in 3D tissue culture. For each indicated cell type, 500 cells were seeded in each well of a ULA spheroid microplate and grown for three days in the absence of drug. After 72 h the indicated BRAF-specific inhibitor or the BRAF-nonspecific chemotherapeutic agent doxorubicin was added (Db = dabrafenib 25 nM, Vb = vemurafenib 50 nM, Dx = doxorubicin 100 nM) and the spheroids were grown for an additional three days. Spheroids were then stained with 2 μM Calcein AM green and NucBlue live-cell nuclear marker for 90 min and then imaged; ( B ) Average spheroid size of RAS mutant melanoma models following drug treatment relative to the un-drugged condition. Statistical analysis was performed using two-way ANOVA with multiple comparisons, each condition represents at least n = 3 spheroids; ( C ) MEK and BRAF inhibitor resistance in A375 MEKQ56P spheroid melanoma model. Drugged spheroids were handled as in ( A ) and treated with 25 nM dabrafenib, 50 nM vemurafenib, and 50 nM of the MEK inhibitors trametinib (Tb) and binimetinib (Bb), respectively, or 100 nM doxorubicin; ( D ) Average numbers of nuclei per spheroid were calculated relative to the un-drugged condition. Results from at least n = 3 spheroids for each condition were averaged and plotted, statistical analysis was performed using two-way ANOVA with multiple comparisons. ****, p < 0.0001.

    Journal: Cancers

    Article Title: CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy

    doi: 10.3390/cancers14215449

    Figure Lengend Snippet: A375 Ras mutant melanoma models are resistant to BRAF inhibitors in 3D tissue culture and A375 MEK1 melanoma model is resistant to both MEK and BRAF inhibitors in 3D tissue culture. ( A ) BRAF inhibitor resistance in A375 RAS mutant melanoma models in 3D tissue culture. For each indicated cell type, 500 cells were seeded in each well of a ULA spheroid microplate and grown for three days in the absence of drug. After 72 h the indicated BRAF-specific inhibitor or the BRAF-nonspecific chemotherapeutic agent doxorubicin was added (Db = dabrafenib 25 nM, Vb = vemurafenib 50 nM, Dx = doxorubicin 100 nM) and the spheroids were grown for an additional three days. Spheroids were then stained with 2 μM Calcein AM green and NucBlue live-cell nuclear marker for 90 min and then imaged; ( B ) Average spheroid size of RAS mutant melanoma models following drug treatment relative to the un-drugged condition. Statistical analysis was performed using two-way ANOVA with multiple comparisons, each condition represents at least n = 3 spheroids; ( C ) MEK and BRAF inhibitor resistance in A375 MEKQ56P spheroid melanoma model. Drugged spheroids were handled as in ( A ) and treated with 25 nM dabrafenib, 50 nM vemurafenib, and 50 nM of the MEK inhibitors trametinib (Tb) and binimetinib (Bb), respectively, or 100 nM doxorubicin; ( D ) Average numbers of nuclei per spheroid were calculated relative to the un-drugged condition. Results from at least n = 3 spheroids for each condition were averaged and plotted, statistical analysis was performed using two-way ANOVA with multiple comparisons. ****, p < 0.0001.

    Article Snippet: The melanoma cell line A375 (ATCC, CRL-1619) and the derived isogenic lines A375 KRAS G13D (ATCC, CRL-1619IG-1), A375 NRAS Q61K (ATCC, CRL-1619IG-2) and A375 MEK1 Q56P (ATCC, CRL-1619IG-3) were grown and maintained in ATCC-formulated Dulbecco’s Modified Eagle’s Medium (ATCC, 30–2002) with 10% fetal bovine serum (ATCC, 30–2020).

    Techniques: Mutagenesis, Staining, Marker

    MEK1 Q56P melanoma model is sensitive to combination BRAF/MEK inhibitor treatment in both 2D and 3D tissue culture. ( A ) Dose-response curves for A375 WT cells in 2D tissue culture with dabrafenib (grey line), trametinib (black line), or a combination of dabrafenib and trametinib (red line, molarity indicates total drug concentration); ( B ) Dose-response curves for NRAS Q61K cells in 2D tissue culture with dabrafenib, trametinib, or a combination of dabrafenib and trametinib; ( C ) Dose-response curves for MEK1 Q56P cells in 2D tissue culture with dabrafenib, trametinib, or combination; ( D ) Dose-response curves for MEK1 Q56P cells in 2D tissue culture with dabrafenib, trametinib, or combination. Lower survival with combination indicates synergistic drug killing in this line; ( E ) Immunoblot demonstrating synergistic inhibition of the MEK/ERK signaling pathway in MEK1 Q56P melanoma model cells in 2D tissue culture. Cells were treated with either 1.0 μM of the indicated inhibitor compound or with 0.5 μM each of each indicated drug for 90 min prior to harvesting protein (Db = dabrafenib 1.0 μM, Vb = vemurafenib 1.0 μM, Tb = trametinib 1.0 μM, Bb = binimetinib 1.0 μM, Db/Tb = dabrafenib 0.5 μM + trametinib 0.5 μM); ( F ) Model of synergistic inhibition of the RAS/RAF/MEK/ERK pathway by combination MEK and BRAF inhibitor treatment. Orange star indicates the primary BRAF V600E mutation which drives cell proliferation in the absence of BRAF inhibitor. Treatment with BRAF inhibitor results in secondary MEK1 Q65P mutation (black outlined orange star). In the presence of these two mutations, upstream pathway inhibition with BRAF inhibitor in combination with downstream pathway inhibition with MEK inhibitor (red arrows) leads to less cell survival and proliferation than is observed when each drug is used alone; ( G ) Susceptibility of MEK1 Q56P cells grown in 3D tissue culture to dabrafenib, trametinib, and combination drug treatment. For each indicated cell type, 500 cells were seeded in each well of a ULA spheroid microplate and grown for three days in the absence of drug. The spheroids were then treated with 12 nM dabrafenib, 4 nM trametinib, a combination of 6 nM dabrafenib and 2 nM trametinib, or vehicle control for an additional three days. Spheroids were then stained with 2 μM Calcein AM green and NucBlue live-cell nuclear marker for 90 min and then imaged; ( H ) Spheroid sizes were calculated relative to the un-drugged condition. Results from at least n = 3 spheroids for each condition were averaged and plotted, statistical analysis was performed using two-way ANOVA with multiple comparisons. ***, p < 0.001.

    Journal: Cancers

    Article Title: CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy

    doi: 10.3390/cancers14215449

    Figure Lengend Snippet: MEK1 Q56P melanoma model is sensitive to combination BRAF/MEK inhibitor treatment in both 2D and 3D tissue culture. ( A ) Dose-response curves for A375 WT cells in 2D tissue culture with dabrafenib (grey line), trametinib (black line), or a combination of dabrafenib and trametinib (red line, molarity indicates total drug concentration); ( B ) Dose-response curves for NRAS Q61K cells in 2D tissue culture with dabrafenib, trametinib, or a combination of dabrafenib and trametinib; ( C ) Dose-response curves for MEK1 Q56P cells in 2D tissue culture with dabrafenib, trametinib, or combination; ( D ) Dose-response curves for MEK1 Q56P cells in 2D tissue culture with dabrafenib, trametinib, or combination. Lower survival with combination indicates synergistic drug killing in this line; ( E ) Immunoblot demonstrating synergistic inhibition of the MEK/ERK signaling pathway in MEK1 Q56P melanoma model cells in 2D tissue culture. Cells were treated with either 1.0 μM of the indicated inhibitor compound or with 0.5 μM each of each indicated drug for 90 min prior to harvesting protein (Db = dabrafenib 1.0 μM, Vb = vemurafenib 1.0 μM, Tb = trametinib 1.0 μM, Bb = binimetinib 1.0 μM, Db/Tb = dabrafenib 0.5 μM + trametinib 0.5 μM); ( F ) Model of synergistic inhibition of the RAS/RAF/MEK/ERK pathway by combination MEK and BRAF inhibitor treatment. Orange star indicates the primary BRAF V600E mutation which drives cell proliferation in the absence of BRAF inhibitor. Treatment with BRAF inhibitor results in secondary MEK1 Q65P mutation (black outlined orange star). In the presence of these two mutations, upstream pathway inhibition with BRAF inhibitor in combination with downstream pathway inhibition with MEK inhibitor (red arrows) leads to less cell survival and proliferation than is observed when each drug is used alone; ( G ) Susceptibility of MEK1 Q56P cells grown in 3D tissue culture to dabrafenib, trametinib, and combination drug treatment. For each indicated cell type, 500 cells were seeded in each well of a ULA spheroid microplate and grown for three days in the absence of drug. The spheroids were then treated with 12 nM dabrafenib, 4 nM trametinib, a combination of 6 nM dabrafenib and 2 nM trametinib, or vehicle control for an additional three days. Spheroids were then stained with 2 μM Calcein AM green and NucBlue live-cell nuclear marker for 90 min and then imaged; ( H ) Spheroid sizes were calculated relative to the un-drugged condition. Results from at least n = 3 spheroids for each condition were averaged and plotted, statistical analysis was performed using two-way ANOVA with multiple comparisons. ***, p < 0.001.

    Article Snippet: The melanoma cell line A375 (ATCC, CRL-1619) and the derived isogenic lines A375 KRAS G13D (ATCC, CRL-1619IG-1), A375 NRAS Q61K (ATCC, CRL-1619IG-2) and A375 MEK1 Q56P (ATCC, CRL-1619IG-3) were grown and maintained in ATCC-formulated Dulbecco’s Modified Eagle’s Medium (ATCC, 30–2002) with 10% fetal bovine serum (ATCC, 30–2020).

    Techniques: Concentration Assay, Western Blot, Inhibition, Mutagenesis, Staining, Marker

    PD-L1 is constitutively expressed in KRAS G13D, but not in A375 WT, NRAS Q61K, or MEK1 Q56P melanoma models. ( A ) Flow cytometry analysis of cell surface PD-L1 expression in A375 WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P melanoma models grown in 2D tissue culture. Cells were treated overnight with 200 ng/µL interferon gamma (red), or mock treated (blue). The following day the cells were stained with either anti-PD-L1 or isotype control (grey); ( B ) PD-L1 immunoblot of total cellular protein from A375 WT, NRAS Q61K, and KRAS G13D cells grown in either 2D or 3D tissue culture; ( C ) Indirect immunofluorescence staining of PD-L1 in A375 WT, NRAS Q61K and KRAS G13D melanoma model lines.

    Journal: Cancers

    Article Title: CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy

    doi: 10.3390/cancers14215449

    Figure Lengend Snippet: PD-L1 is constitutively expressed in KRAS G13D, but not in A375 WT, NRAS Q61K, or MEK1 Q56P melanoma models. ( A ) Flow cytometry analysis of cell surface PD-L1 expression in A375 WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P melanoma models grown in 2D tissue culture. Cells were treated overnight with 200 ng/µL interferon gamma (red), or mock treated (blue). The following day the cells were stained with either anti-PD-L1 or isotype control (grey); ( B ) PD-L1 immunoblot of total cellular protein from A375 WT, NRAS Q61K, and KRAS G13D cells grown in either 2D or 3D tissue culture; ( C ) Indirect immunofluorescence staining of PD-L1 in A375 WT, NRAS Q61K and KRAS G13D melanoma model lines.

    Article Snippet: The melanoma cell line A375 (ATCC, CRL-1619) and the derived isogenic lines A375 KRAS G13D (ATCC, CRL-1619IG-1), A375 NRAS Q61K (ATCC, CRL-1619IG-2) and A375 MEK1 Q56P (ATCC, CRL-1619IG-3) were grown and maintained in ATCC-formulated Dulbecco’s Modified Eagle’s Medium (ATCC, 30–2002) with 10% fetal bovine serum (ATCC, 30–2020).

    Techniques: Flow Cytometry, Expressing, Staining, Western Blot, Immunofluorescence

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: AP-1 transcription factor network explains diverse patterns of cellular plasticity in melanoma cells

    doi: 10.1016/j.celrep.2022.111147

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Human: A375 NRAS(Q61K), Melanoma Cell Line , ATCC , Cat# CRL-1619IG-2, RRID: CVCL_JF22.

    Techniques: Recombinant, Blocking Assay, Expressing, Activity Assay, Software

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: AP-1 transcription factor network explains diverse patterns of cellular plasticity in melanoma cells

    doi: 10.1016/j.celrep.2022.111147

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Human: A375 NRAS(Q61K), Melanoma Cell Line , ATCC , Cat# CRL-1619IG-2, RRID: CVCL_JF22.

    Techniques: Recombinant, Blocking Assay, Expressing, Activity Assay, Software

    In vitro screening of novel NL compounds: ( A ) hit compounds were screened at 10 micromolar concentrations for 24 h. All but one compound demonstrated significant activity and were selected for a dose response analysis; ( B ) compounds were screened at increasing concentrations to assay for activity. NL221-75 and NL350-02 were as potent as FDA-approved controls, demonstrating low nanomolar range activity; ( C ) MTT A375 cells were treated with increasing concentrations of test items and cell proliferation was determined at 24 h using the MTT method. All compounds demonstrated dose-dependent activity in preventing cell proliferation. Experimental compounds NL221-75 and NL350-02 were as effective as FDA-approved controls in preventing cell proliferation. Data are plotted as percent inhibition of proliferation; and ( D ) hERG inhibition experiments performed on CHO-cells. Cobimetinib demonstrated low nanomolar inhibition of hERG. NL34-113, NL221-75, and NL350-02 did not inhibit hERG at the concentrations tested.

    Journal: Molecules

    Article Title: Application of Pharmacokinetic Prediction Platforms in the Design of Optimized Anti-Cancer Drugs

    doi: 10.3390/molecules27123678

    Figure Lengend Snippet: In vitro screening of novel NL compounds: ( A ) hit compounds were screened at 10 micromolar concentrations for 24 h. All but one compound demonstrated significant activity and were selected for a dose response analysis; ( B ) compounds were screened at increasing concentrations to assay for activity. NL221-75 and NL350-02 were as potent as FDA-approved controls, demonstrating low nanomolar range activity; ( C ) MTT A375 cells were treated with increasing concentrations of test items and cell proliferation was determined at 24 h using the MTT method. All compounds demonstrated dose-dependent activity in preventing cell proliferation. Experimental compounds NL221-75 and NL350-02 were as effective as FDA-approved controls in preventing cell proliferation. Data are plotted as percent inhibition of proliferation; and ( D ) hERG inhibition experiments performed on CHO-cells. Cobimetinib demonstrated low nanomolar inhibition of hERG. NL34-113, NL221-75, and NL350-02 did not inhibit hERG at the concentrations tested.

    Article Snippet: Human A375 metastatic melanoma cells (ATCC (Manassas, VA, USA); No. CRL-1619IG-2) were cultured in Gibco Dulbecco’s Modified Eagle Medium (DMEM) (ATCC; No. 30-2002) using 10% fetal bovine serum and 1% Penicillin-Streptomycin.

    Techniques: In Vitro, Activity Assay, Inhibition

    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