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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    human melanoma a375 cell line  (ATCC)


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    ATCC human melanoma a375 cell line
    A. Representative images of human melanoma <t>A375,</t> fibrosarcoma HT1080, or chondrosarcoma Sarc cells analysed by phase contrast microscopy. Original magnifications: 400x. Scale bar: 100 μm . B. Time-dependent proliferation of the considered human cell lines. Cells (2 × 10 3 cells/well) were seeded on E-plates and allowed to grow for 70 h in serum containing medium. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index. Data represent mean ± SD (standard deviation) from a quadruplicate experiment. C. Cell migration of the indicated human cell lines monitored by the xCELLigence system. Cells were seeded on CIM-plates and allowed to migrate towards serum free medium (basal cell migration, black line) or medium plus 10% FBS. Cell migration was monitored in real-time for 12 h and expressed as Cell Index. Data represent mean ± SD from a quadruplicate experiment.
    Human Melanoma A375 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A Macroscopic Mathematical Model for Cell Migration Assays Using a Real-Time Cell Analysis"

    Article Title: A Macroscopic Mathematical Model for Cell Migration Assays Using a Real-Time Cell Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0162553

    A. Representative images of human melanoma A375, fibrosarcoma HT1080, or chondrosarcoma Sarc cells analysed by phase contrast microscopy. Original magnifications: 400x. Scale bar: 100 μm . B. Time-dependent proliferation of the considered human cell lines. Cells (2 × 10 3 cells/well) were seeded on E-plates and allowed to grow for 70 h in serum containing medium. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index. Data represent mean ± SD (standard deviation) from a quadruplicate experiment. C. Cell migration of the indicated human cell lines monitored by the xCELLigence system. Cells were seeded on CIM-plates and allowed to migrate towards serum free medium (basal cell migration, black line) or medium plus 10% FBS. Cell migration was monitored in real-time for 12 h and expressed as Cell Index. Data represent mean ± SD from a quadruplicate experiment.
    Figure Legend Snippet: A. Representative images of human melanoma A375, fibrosarcoma HT1080, or chondrosarcoma Sarc cells analysed by phase contrast microscopy. Original magnifications: 400x. Scale bar: 100 μm . B. Time-dependent proliferation of the considered human cell lines. Cells (2 × 10 3 cells/well) were seeded on E-plates and allowed to grow for 70 h in serum containing medium. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index. Data represent mean ± SD (standard deviation) from a quadruplicate experiment. C. Cell migration of the indicated human cell lines monitored by the xCELLigence system. Cells were seeded on CIM-plates and allowed to migrate towards serum free medium (basal cell migration, black line) or medium plus 10% FBS. Cell migration was monitored in real-time for 12 h and expressed as Cell Index. Data represent mean ± SD from a quadruplicate experiment.

    Techniques Used: Microscopy, Standard Deviation, Migration

    Initial data and parameters of the mathematical model.
    Figure Legend Snippet: Initial data and parameters of the mathematical model.

    Techniques Used: Concentration Assay, Diffusion-based Assay, Transmission Assay

    human melanoma a375 cell line  (ATCC)


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    ATCC human melanoma a375 cell line
    Verification of the protein expression of the 6 FAMGs using the HPA database. (A) Immunohistochemical images show the protein expression of ACSL5, ALOX5AP, CD1D, CD74, IL4I1, and TBXAS1 in CM and skin tissue. (B) Immunofluorescence show that ACSL5, CD1D, CD74, IL4I1 , and TBXAS1 are mainly located in the cytosol and nucleoplasm. (C) qRT-PCR, showing the relative expression of ACSL5, CD1D, CD74, IL4I1 and TBXAS1 were significant differences between 2 normal human cell lines and 3 melanoma cell lines. *P≥0.05; **P≥0.01.
    Human Melanoma A375 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification of fatty acid metabolism–related molecular subtype biomarkers and their correlation with immune checkpoints in cutaneous melanoma"

    Article Title: Identification of fatty acid metabolism–related molecular subtype biomarkers and their correlation with immune checkpoints in cutaneous melanoma

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.967277

    Verification of the protein expression of the 6 FAMGs using the HPA database. (A) Immunohistochemical images show the protein expression of ACSL5, ALOX5AP, CD1D, CD74, IL4I1, and TBXAS1 in CM and skin tissue. (B) Immunofluorescence show that ACSL5, CD1D, CD74, IL4I1 , and TBXAS1 are mainly located in the cytosol and nucleoplasm. (C) qRT-PCR, showing the relative expression of ACSL5, CD1D, CD74, IL4I1 and TBXAS1 were significant differences between 2 normal human cell lines and 3 melanoma cell lines. *P≥0.05; **P≥0.01.
    Figure Legend Snippet: Verification of the protein expression of the 6 FAMGs using the HPA database. (A) Immunohistochemical images show the protein expression of ACSL5, ALOX5AP, CD1D, CD74, IL4I1, and TBXAS1 in CM and skin tissue. (B) Immunofluorescence show that ACSL5, CD1D, CD74, IL4I1 , and TBXAS1 are mainly located in the cytosol and nucleoplasm. (C) qRT-PCR, showing the relative expression of ACSL5, CD1D, CD74, IL4I1 and TBXAS1 were significant differences between 2 normal human cell lines and 3 melanoma cell lines. *P≥0.05; **P≥0.01.

    Techniques Used: Expressing, Immunohistochemical staining, Immunofluorescence, Quantitative RT-PCR

    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

    a375 mek1 q56p  (ATCC)


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    ATCC a375 mek1 q56p
    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), NRAS Q61K heterozygous (middle), and <t>MEK1</t> <t>Q56P</t> homozygous (right) A375 isogenic lines.
    A375 Mek1 Q56p, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a375 mek1 q56p/product/ATCC
    Average 93 stars, based on 1 article reviews
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    a375 mek1 q56p - by Bioz Stars, 2024-05
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    Images

    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

    lines a375 kras g13d  (ATCC)


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    Structured Review

    ATCC lines a375 kras g13d
    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 <t>KRAS</t> <t>G13D</t> heterozygous (left), NRAS Q61K heterozygous (middle), and MEK1 Q56P homozygous (right) A375 isogenic lines.
    Lines A375 Kras G13d, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    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
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    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

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

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    crl 1619ig 2  (ATCC)


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    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

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

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    crl 1619ig  (ATCC)


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    ATCC human melanoma a375 cell line
    A. Representative images of human melanoma <t>A375,</t> fibrosarcoma HT1080, or chondrosarcoma Sarc cells analysed by phase contrast microscopy. Original magnifications: 400x. Scale bar: 100 μm . B. Time-dependent proliferation of the considered human cell lines. Cells (2 × 10 3 cells/well) were seeded on E-plates and allowed to grow for 70 h in serum containing medium. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index. Data represent mean ± SD (standard deviation) from a quadruplicate experiment. C. Cell migration of the indicated human cell lines monitored by the xCELLigence system. Cells were seeded on CIM-plates and allowed to migrate towards serum free medium (basal cell migration, black line) or medium plus 10% FBS. Cell migration was monitored in real-time for 12 h and expressed as Cell Index. Data represent mean ± SD from a quadruplicate experiment.
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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.
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    ATCC a375 mek1 q56p
    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), NRAS Q61K heterozygous (middle), and <t>MEK1</t> <t>Q56P</t> homozygous (right) A375 isogenic lines.
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    ATCC lines a375 kras g13d
    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 <t>KRAS</t> <t>G13D</t> heterozygous (left), NRAS Q61K heterozygous (middle), and MEK1 Q56P homozygous (right) A375 isogenic lines.
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    ATCC crl 1619ig 2
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    Image Search Results


    A. Representative images of human melanoma A375, fibrosarcoma HT1080, or chondrosarcoma Sarc cells analysed by phase contrast microscopy. Original magnifications: 400x. Scale bar: 100 μm . B. Time-dependent proliferation of the considered human cell lines. Cells (2 × 10 3 cells/well) were seeded on E-plates and allowed to grow for 70 h in serum containing medium. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index. Data represent mean ± SD (standard deviation) from a quadruplicate experiment. C. Cell migration of the indicated human cell lines monitored by the xCELLigence system. Cells were seeded on CIM-plates and allowed to migrate towards serum free medium (basal cell migration, black line) or medium plus 10% FBS. Cell migration was monitored in real-time for 12 h and expressed as Cell Index. Data represent mean ± SD from a quadruplicate experiment.

    Journal: PLoS ONE

    Article Title: A Macroscopic Mathematical Model for Cell Migration Assays Using a Real-Time Cell Analysis

    doi: 10.1371/journal.pone.0162553

    Figure Lengend Snippet: A. Representative images of human melanoma A375, fibrosarcoma HT1080, or chondrosarcoma Sarc cells analysed by phase contrast microscopy. Original magnifications: 400x. Scale bar: 100 μm . B. Time-dependent proliferation of the considered human cell lines. Cells (2 × 10 3 cells/well) were seeded on E-plates and allowed to grow for 70 h in serum containing medium. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index. Data represent mean ± SD (standard deviation) from a quadruplicate experiment. C. Cell migration of the indicated human cell lines monitored by the xCELLigence system. Cells were seeded on CIM-plates and allowed to migrate towards serum free medium (basal cell migration, black line) or medium plus 10% FBS. Cell migration was monitored in real-time for 12 h and expressed as Cell Index. Data represent mean ± SD from a quadruplicate experiment.

    Article Snippet: Human melanoma A375 cell line purchased from American Type Culture Collection (ATCC) was cultured in RPMI 1640 medium (Lonza, Milan, Italy), supplemented with 3 mM L-glutamine (Invitrogen-Gibco ® /Life Technologies, Monza, Italy), 2% penicillin/streptomycin and 10% fetal bovine serum (FBS).

    Techniques: Microscopy, Standard Deviation, Migration

    Initial data and parameters of the mathematical model.

    Journal: PLoS ONE

    Article Title: A Macroscopic Mathematical Model for Cell Migration Assays Using a Real-Time Cell Analysis

    doi: 10.1371/journal.pone.0162553

    Figure Lengend Snippet: Initial data and parameters of the mathematical model.

    Article Snippet: Human melanoma A375 cell line purchased from American Type Culture Collection (ATCC) was cultured in RPMI 1640 medium (Lonza, Milan, Italy), supplemented with 3 mM L-glutamine (Invitrogen-Gibco ® /Life Technologies, Monza, Italy), 2% penicillin/streptomycin and 10% fetal bovine serum (FBS).

    Techniques: Concentration Assay, Diffusion-based Assay, Transmission Assay

    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

    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

    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