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ATCC media b16 cells

Figure 1. Melanin synthesis causes H2AX f oci f ormation DNA strand breaks and abasic sites formation. ( A ) Immunofluorescence of <t>B16</t> cells treated with PTU and tyrosine with phosphoryla ted H2AX antibod y. Nuclear DNA stained with DAPI (blue) and H2AX in (red). Experiment was performed with two biological replicates and a representati v e image is depicted. Scale bar 10 m. ( B ) Quantitation of mean fluorescence intensity per cell of H2AX from two biological replicates of pigmented day 7 PTU or tyrosine treated cells (shown in A). Data r epr esented as a box plot, horizontal line r epr esents mean and whiskers r epr esent SEM. Ordinary one-way ANOVA was perf ormed f or multiple comparisons. Adjusted P -v alue: * P -v alue < 0.05, *** P - value < 0.001, **** P -value < 0.0001. ( C ) (Top) Cell pellet of day 7 B16 mouse melanoma cells grown at low density (100 cells / cm 2 ). Cells were left untreated for control treated with tyrosinase inhibitor 200 M phenylthiourea (PTU) or 1mM tyrosinase substrate L-tyrosine (Tyr) for 7 days. Number of cells, mean ± SEM across three biological replicates is depicted below the image of the cell pellet. Numbers r epr esent mean ± SEM cell counts across biological triplicates. (Bottom) Number of abasic sites in the genomic DNA was estimated by an aldehyde specific conjugation of biotin and subsequent detection using streptavidin based detection. Using standards, abasic sites per 10 5 bp is estimated. Bars r epr esent mean ± SEM across duplicate biological experiments, each conducted in triplicates. Ordinary one-w ay ANOVA w as performed for multiple comparisons * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001. ( D ) Single cell electrophoresis followed by comet analysis of B16 cells undergoing varying le v els of pigmentation in the presence of PTU and tyrosine (alkaline comet assay). Experiment was carried out at mid phase (day 5) and late phase (day 7) of pigmentation. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Two-way ANOVA was performed. Adjusted P values; ns non-significant, * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001, **** P -value < 0.0001. ( E ) Neutral comet assay on B16 unpigmented (day 0) and pigmented (day 7) cells. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Student’s unpaired t-test was performed. P values ns non-significant. ( F ) Single cell electrophoresis followed by comet analysis of B16 cells untr eated, tr eated with DMSO for 24 h, melanin synthesis ( ex-cellulo L-tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM dihydroxyindole (DHI) for 24 h (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-way ANOVA was performed. Adjusted P values: * P -value < 0.05, **** P -value < 0.00001.

Media B16 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 543 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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media b16 cells - by Bioz Stars, 2026-10

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1) Product Images from "Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes."

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes.

Journal: Nucleic acids research

doi: 10.1093/nar/gkad704

Figure 1. Melanin synthesis causes H2AX f oci f ormation DNA strand breaks and abasic sites formation. ( A ) Immunofluorescence of B16 cells treated with PTU and tyrosine with phosphoryla ted H2AX antibod y. Nuclear DNA stained with DAPI (blue) and H2AX in (red). Experiment was performed with two biological replicates and a representati v e image is depicted. Scale bar 10 m. ( B ) Quantitation of mean fluorescence intensity per cell of H2AX from two biological replicates of pigmented day 7 PTU or tyrosine treated cells (shown in A). Data r epr esented as a box plot, horizontal line r epr esents mean and whiskers r epr esent SEM. Ordinary one-way ANOVA was perf ormed f or multiple comparisons. Adjusted P -v alue: * P -v alue < 0.05, *** P - value < 0.001, **** P -value < 0.0001. ( C ) (Top) Cell pellet of day 7 B16 mouse melanoma cells grown at low density (100 cells / cm 2 ). Cells were left untreated for control treated with tyrosinase inhibitor 200 M phenylthiourea (PTU) or 1mM tyrosinase substrate L-tyrosine (Tyr) for 7 days. Number of cells, mean ± SEM across three biological replicates is depicted below the image of the cell pellet. Numbers r epr esent mean ± SEM cell counts across biological triplicates. (Bottom) Number of abasic sites in the genomic DNA was estimated by an aldehyde specific conjugation of biotin and subsequent detection using streptavidin based detection. Using standards, abasic sites per 10 5 bp is estimated. Bars r epr esent mean ± SEM across duplicate biological experiments, each conducted in triplicates. Ordinary one-w ay ANOVA w as performed for multiple comparisons * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001. ( D ) Single cell electrophoresis followed by comet analysis of B16 cells undergoing varying le v els of pigmentation in the presence of PTU and tyrosine (alkaline comet assay). Experiment was carried out at mid phase (day 5) and late phase (day 7) of pigmentation. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Two-way ANOVA was performed. Adjusted P values; ns non-significant, * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001, **** P -value < 0.0001. ( E ) Neutral comet assay on B16 unpigmented (day 0) and pigmented (day 7) cells. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Student’s unpaired t-test was performed. P values ns non-significant. ( F ) Single cell electrophoresis followed by comet analysis of B16 cells untr eated, tr eated with DMSO for 24 h, melanin synthesis ( ex-cellulo L-tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM dihydroxyindole (DHI) for 24 h (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-way ANOVA was performed. Adjusted P values: * P -value < 0.05, **** P -value < 0.00001.

Figure Legend Snippet: Figure 1. Melanin synthesis causes H2AX f oci f ormation DNA strand breaks and abasic sites formation. ( A ) Immunofluorescence of B16 cells treated with PTU and tyrosine with phosphoryla ted H2AX antibod y. Nuclear DNA stained with DAPI (blue) and H2AX in (red). Experiment was performed with two biological replicates and a representati v e image is depicted. Scale bar 10 m. ( B ) Quantitation of mean fluorescence intensity per cell of H2AX from two biological replicates of pigmented day 7 PTU or tyrosine treated cells (shown in A). Data r epr esented as a box plot, horizontal line r epr esents mean and whiskers r epr esent SEM. Ordinary one-way ANOVA was perf ormed f or multiple comparisons. Adjusted P -v alue: * P -v alue < 0.05, *** P - value < 0.001, **** P -value < 0.0001. ( C ) (Top) Cell pellet of day 7 B16 mouse melanoma cells grown at low density (100 cells / cm 2 ). Cells were left untreated for control treated with tyrosinase inhibitor 200 M phenylthiourea (PTU) or 1mM tyrosinase substrate L-tyrosine (Tyr) for 7 days. Number of cells, mean ± SEM across three biological replicates is depicted below the image of the cell pellet. Numbers r epr esent mean ± SEM cell counts across biological triplicates. (Bottom) Number of abasic sites in the genomic DNA was estimated by an aldehyde specific conjugation of biotin and subsequent detection using streptavidin based detection. Using standards, abasic sites per 10 5 bp is estimated. Bars r epr esent mean ± SEM across duplicate biological experiments, each conducted in triplicates. Ordinary one-w ay ANOVA w as performed for multiple comparisons * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001. ( D ) Single cell electrophoresis followed by comet analysis of B16 cells undergoing varying le v els of pigmentation in the presence of PTU and tyrosine (alkaline comet assay). Experiment was carried out at mid phase (day 5) and late phase (day 7) of pigmentation. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Two-way ANOVA was performed. Adjusted P values; ns non-significant, * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001, **** P -value < 0.0001. ( E ) Neutral comet assay on B16 unpigmented (day 0) and pigmented (day 7) cells. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Student’s unpaired t-test was performed. P values ns non-significant. ( F ) Single cell electrophoresis followed by comet analysis of B16 cells untr eated, tr eated with DMSO for 24 h, melanin synthesis ( ex-cellulo L-tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM dihydroxyindole (DHI) for 24 h (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-way ANOVA was performed. Adjusted P values: * P -value < 0.05, **** P -value < 0.00001.

Techniques Used: Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Control, Conjugation Assay, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Neutral Comet Assay

Figure 4. CRISPR-based genetic ablation of tyrosinase prevents melanogenesis, reduces DNA breaks and curtails Pol induction. ( A ) Immunofluorescence images of B16 WT and B16 Tyrosinase mutant (Tyr fs118 ) cells that w ere allow ed to pigment for 7 days. Bright field images indicate the presence of melanin granules. Labelling using 5-ethynyl-2 ′ -deoxyuridine (EdU) (red) and H2AX antibody (green). Merged images show co-localization of H2AX with EdU (yellow). A zoomed in view of a single cell is shown as an inset. Scale bars r epr esent 10 m. ( B ) (Top) Cell pellets of B16 WT and Tyrosinase mutant (Tyr fs118 ) cells on days 5, 6 and 7 of pigmentation. (Bottom) Quantitation of average of the number of H2AX puncta that are single positi v e or double positi v e (colocalizing with EdU puncta) per cell across 50 cells in B16 WT and B16 Tyr fs118 cells is depicted as a violin plot across three biological replicates. The percent population of cells with indicated puncta is mentioned alongside. ( C ) Growth curve analysis of B16 WT and Tyr fs118 cells on days 0, 5, 6 and 7 of pigmentation. Each point r epr esents mean ± SEM across biological triplicates. Two-w ay ANOVA w as performed. Adjusted P values: * P -value < 0.05, *** P -value < 0.001. ( D ) B16 WT and Tyr fs118 cells on days 5 and 7 of pigmentation were subjected to single cell electrophoresis and alkaline comet assay was performed. Mean tail moment distribution across each population of duplicate biological experiments with at least 50 comets analyzed is depicted by a violin plot. Two-w ay ANOVA w as performed. Adjusted P values ns non-significant: **** P -value < 0.0001. ( E ) Western blot analysis of whole-cell lysates of B16 WT and B16 Tyr fs118 cells on day 5 and day 7 stages of pigmentation. Numbers below the blot correspond to day 5 GAPDH normalized expression of the indicated protein. Experiments were performed in biological duplicates with similar results.

Figure Legend Snippet: Figure 4. CRISPR-based genetic ablation of tyrosinase prevents melanogenesis, reduces DNA breaks and curtails Pol induction. ( A ) Immunofluorescence images of B16 WT and B16 Tyrosinase mutant (Tyr fs118 ) cells that w ere allow ed to pigment for 7 days. Bright field images indicate the presence of melanin granules. Labelling using 5-ethynyl-2 ′ -deoxyuridine (EdU) (red) and H2AX antibody (green). Merged images show co-localization of H2AX with EdU (yellow). A zoomed in view of a single cell is shown as an inset. Scale bars r epr esent 10 m. ( B ) (Top) Cell pellets of B16 WT and Tyrosinase mutant (Tyr fs118 ) cells on days 5, 6 and 7 of pigmentation. (Bottom) Quantitation of average of the number of H2AX puncta that are single positi v e or double positi v e (colocalizing with EdU puncta) per cell across 50 cells in B16 WT and B16 Tyr fs118 cells is depicted as a violin plot across three biological replicates. The percent population of cells with indicated puncta is mentioned alongside. ( C ) Growth curve analysis of B16 WT and Tyr fs118 cells on days 0, 5, 6 and 7 of pigmentation. Each point r epr esents mean ± SEM across biological triplicates. Two-w ay ANOVA w as performed. Adjusted P values: * P -value < 0.05, *** P -value < 0.001. ( D ) B16 WT and Tyr fs118 cells on days 5 and 7 of pigmentation were subjected to single cell electrophoresis and alkaline comet assay was performed. Mean tail moment distribution across each population of duplicate biological experiments with at least 50 comets analyzed is depicted by a violin plot. Two-w ay ANOVA w as performed. Adjusted P values ns non-significant: **** P -value < 0.0001. ( E ) Western blot analysis of whole-cell lysates of B16 WT and B16 Tyr fs118 cells on day 5 and day 7 stages of pigmentation. Numbers below the blot correspond to day 5 GAPDH normalized expression of the indicated protein. Experiments were performed in biological duplicates with similar results.

Techniques Used: CRISPR, Immunofluorescence, Mutagenesis, Quantitation Assay, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Western Blot, Expressing

Figure 5. Normal human epidermal melanocytes (NHEM) respond to pigmentation induced DNA breaks by elevating Pol . ( A ) NHEM cells wer e tr eated with 200 M PTU or 1mM tyrosine for 7 days for differential pigmentation. (Top) Cell pellet, (bottom) western blot analysis of cell lysates with POLK, HSC70, phosphorylated H2AX, total H2AX and beta actin antibodies. Numbers below the blot correspond to control normalized expression of the indi- cated protein. Experiments were performed in biological duplicates. ( B ) Immunofluorescence of NHEM treated with PTU or tyrosine with phosphorylated H2AX antibody. Nuclear DNA stained with DAPI (blue) and H2AX in (red). Experiments were performed with two biological replicates. Scale bar 10 m. ( C ) Quantitation of mean fluorescence intensity per cell of H2AX from two biological replicates of NHEM treated with PTU or tyrosine (shown in B). Ordinary one-way ANOVA was performed for multiple comparisons. Adjusted P values: * P -value < 0.05, **** P -value < 0.0001. ( D ) PTU and tyrosine treated NHEM cells were subjected to single cell electrophoresis and comet analysis (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-w ay ANOVA w as per- formed. Adjusted P values: ** P -value < 0.001, **** P -value < 0.00001. ( E ) Heat map of expression (fold change) in mRNA le v els of top two translesion polymerases (that were enriched in B16 microarray) (top), and a panel of known DNA replication stress response genes by qRT-PCR analysis in NHEM (Contr ol, PTU or tyr osine trea ted). Da ta r epr esented as mean of triplicate biological e xperiments. ( F ) Western b lot analysis of NHEM treated with DMSO or 50 nM AZ20, a selecti v e inhibitor of ATR kinase, for 24 h. Numbers below the blot correspond to control normalized expression of the indicated protein wrt beta-actin. Experiments were performed in biolo gical duplicates. ( G ) mRN A le v els of Polk in unpigmented B16 cells mock transfected, or with either control DNA, melanin modified DNA (plasmid DNA was incubated with L-DOPA and tyrosinase and column purified after 24 h) (Mel + DNA), DNA mixed with pre-synthesized melanin and coulmn purified [DNA+(Mel)], in-vitro melanin synthesis ( ex-cellulo L -tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM DHI (DHI) for 24 h. Bars r epr esent per cent mRNA le v els compared to contr ol acr oss biological triplicates. Ordinary one-w ay ANOVA w as performed. Adjusted P v alues: * P -v alue < 0.05. (Inset) Western blot analysis of B16 cells transfected with onl y DN A (Con DNA) or melanin-modified DNA (Mel + DNA) with Pol antibody normalized to HSC70. Experiments were performed in biological triplicates.

Figure Legend Snippet: Figure 5. Normal human epidermal melanocytes (NHEM) respond to pigmentation induced DNA breaks by elevating Pol . ( A ) NHEM cells wer e tr eated with 200 M PTU or 1mM tyrosine for 7 days for differential pigmentation. (Top) Cell pellet, (bottom) western blot analysis of cell lysates with POLK, HSC70, phosphorylated H2AX, total H2AX and beta actin antibodies. Numbers below the blot correspond to control normalized expression of the indi- cated protein. Experiments were performed in biological duplicates. ( B ) Immunofluorescence of NHEM treated with PTU or tyrosine with phosphorylated H2AX antibody. Nuclear DNA stained with DAPI (blue) and H2AX in (red). Experiments were performed with two biological replicates. Scale bar 10 m. ( C ) Quantitation of mean fluorescence intensity per cell of H2AX from two biological replicates of NHEM treated with PTU or tyrosine (shown in B). Ordinary one-way ANOVA was performed for multiple comparisons. Adjusted P values: * P -value < 0.05, **** P -value < 0.0001. ( D ) PTU and tyrosine treated NHEM cells were subjected to single cell electrophoresis and comet analysis (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-w ay ANOVA w as per- formed. Adjusted P values: ** P -value < 0.001, **** P -value < 0.00001. ( E ) Heat map of expression (fold change) in mRNA le v els of top two translesion polymerases (that were enriched in B16 microarray) (top), and a panel of known DNA replication stress response genes by qRT-PCR analysis in NHEM (Contr ol, PTU or tyr osine trea ted). Da ta r epr esented as mean of triplicate biological e xperiments. ( F ) Western b lot analysis of NHEM treated with DMSO or 50 nM AZ20, a selecti v e inhibitor of ATR kinase, for 24 h. Numbers below the blot correspond to control normalized expression of the indicated protein wrt beta-actin. Experiments were performed in biolo gical duplicates. ( G ) mRN A le v els of Polk in unpigmented B16 cells mock transfected, or with either control DNA, melanin modified DNA (plasmid DNA was incubated with L-DOPA and tyrosinase and column purified after 24 h) (Mel + DNA), DNA mixed with pre-synthesized melanin and coulmn purified [DNA+(Mel)], in-vitro melanin synthesis ( ex-cellulo L -tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM DHI (DHI) for 24 h. Bars r epr esent per cent mRNA le v els compared to contr ol acr oss biological triplicates. Ordinary one-w ay ANOVA w as performed. Adjusted P v alues: * P -v alue < 0.05. (Inset) Western blot analysis of B16 cells transfected with onl y DN A (Con DNA) or melanin-modified DNA (Mel + DNA) with Pol antibody normalized to HSC70. Experiments were performed in biological triplicates.

Techniques Used: Western Blot, Control, Expressing, Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Microarray, Quantitative RT-PCR, Transfection, Modification, Plasmid Preparation, Incubation, Purification, Synthesized, In Vitro

Figure 6. Silencing of Pol during pigmentation pre v ents replication stress response despite elevated DNA damage. ( A ) Cell pellets of control non-targeting (shNT) and Pol silenced (shPol ) B16 cells on day 0 (left) and day 7 (right) of pigmentation. ( B ) Immunofluorescence analysis of day 7 pigmented shNT and shPol cells with phosphorylated H2AX antibody (puncta labelled in green) and the nucleus is counterstained with DAPI (blue). Scale bars represent 10 m. ( C ) Quantitation of mean fluorescence intensity of H2AX (shown in B) from two biological replicates of shNT and shPol cells across day 0, 5 and 7 of pigmentation induction. Two-way ANOVA was performed for multiple comparisons. Adjusted P values * P -value < 0.05 *** P -value < 0.0005 **** P -value < 0.00001 ns non-significant. ( D ) shNT and shPol expressing pigmented B16 cells were subjected to single cell electrophoresis and comet analysis (alkaline comet) on days 0, 5 and 7 of pigmentation induction. Mean tail moment distribution across each population of duplicate biological experiments with at least 50 comets analyzed is depicted by a violin plot. Two-w ay ANOVA w as performed for multiple comparisons. Adjusted P values, ns non-significant, * P -value < 0.05. ( E ) Growth curve analysis of shNT and shPol expressing B16 cells on days 0, 5, 6 and 7 of pigmentation. Each point r epr esents mean ± SEM across biological triplicates. Two-way ANOVA was performed. Adjusted P values: * P -value < 0.05, *** P -value < 0.001. ( F ) Western blot analysis of DNA repair and cell cycle related proteins in shNT and shPol cells. Numbers below represent tubulin normalized fold changes wrt shNT. Experiments were performed in biological duplicates. ( G ) shNT and shPol expressing B16 cells were injected inside the flank of C57 / BL6 mice and allowed to grow as tumors. The volume of the tumor was non-invasi v ely monitored and plotted over time of biological triplicates mean ± SEM. Two-w ay ANOVA w as perf ormed f or multiple comparisons. Adjusted P v alues: * P -v alue < 0.05. ( H ) Heat ma p of expression (fold change) in mRN A le v els of a panel of known DNA replication stress response genes by qRT-PCR analysis in shNT (day 0-unpigmented and day 7-pigmented) and shPol (day 0-unpigmented and day 7-pigmented) B16 cells. ( I ) Western blot images and analysis of p-RPA2 and total RPA2 in shNT and shPol B16 cells (day 0 unpigmented and day 7 pigmented). Numbers below r epr esent beta-actin normalized fold changes wrt shNT at day 0. Experiments were performed in biolo gical triplicates. ( J ) Anal ysis of melanoma samples from TCGA data for mRNA expression of POLK (high, low or not detected) segregated into bar plots and proportion of mutations were plotted on y-axis. ( K ) Survival plot of melanoma patients with low or high expression of POLK from TCGA data. Analysis from Human Protein Atlas database. P air ed t -test P value 0.017.

Figure Legend Snippet: Figure 6. Silencing of Pol during pigmentation pre v ents replication stress response despite elevated DNA damage. ( A ) Cell pellets of control non-targeting (shNT) and Pol silenced (shPol ) B16 cells on day 0 (left) and day 7 (right) of pigmentation. ( B ) Immunofluorescence analysis of day 7 pigmented shNT and shPol cells with phosphorylated H2AX antibody (puncta labelled in green) and the nucleus is counterstained with DAPI (blue). Scale bars represent 10 m. ( C ) Quantitation of mean fluorescence intensity of H2AX (shown in B) from two biological replicates of shNT and shPol cells across day 0, 5 and 7 of pigmentation induction. Two-way ANOVA was performed for multiple comparisons. Adjusted P values * P -value < 0.05 *** P -value < 0.0005 **** P -value < 0.00001 ns non-significant. ( D ) shNT and shPol expressing pigmented B16 cells were subjected to single cell electrophoresis and comet analysis (alkaline comet) on days 0, 5 and 7 of pigmentation induction. Mean tail moment distribution across each population of duplicate biological experiments with at least 50 comets analyzed is depicted by a violin plot. Two-w ay ANOVA w as performed for multiple comparisons. Adjusted P values, ns non-significant, * P -value < 0.05. ( E ) Growth curve analysis of shNT and shPol expressing B16 cells on days 0, 5, 6 and 7 of pigmentation. Each point r epr esents mean ± SEM across biological triplicates. Two-way ANOVA was performed. Adjusted P values: * P -value < 0.05, *** P -value < 0.001. ( F ) Western blot analysis of DNA repair and cell cycle related proteins in shNT and shPol cells. Numbers below represent tubulin normalized fold changes wrt shNT. Experiments were performed in biological duplicates. ( G ) shNT and shPol expressing B16 cells were injected inside the flank of C57 / BL6 mice and allowed to grow as tumors. The volume of the tumor was non-invasi v ely monitored and plotted over time of biological triplicates mean ± SEM. Two-w ay ANOVA w as perf ormed f or multiple comparisons. Adjusted P v alues: * P -v alue < 0.05. ( H ) Heat ma p of expression (fold change) in mRN A le v els of a panel of known DNA replication stress response genes by qRT-PCR analysis in shNT (day 0-unpigmented and day 7-pigmented) and shPol (day 0-unpigmented and day 7-pigmented) B16 cells. ( I ) Western blot images and analysis of p-RPA2 and total RPA2 in shNT and shPol B16 cells (day 0 unpigmented and day 7 pigmented). Numbers below r epr esent beta-actin normalized fold changes wrt shNT at day 0. Experiments were performed in biolo gical triplicates. ( J ) Anal ysis of melanoma samples from TCGA data for mRNA expression of POLK (high, low or not detected) segregated into bar plots and proportion of mutations were plotted on y-axis. ( K ) Survival plot of melanoma patients with low or high expression of POLK from TCGA data. Analysis from Human Protein Atlas database. P air ed t -test P value 0.017.

Techniques Used: Control, Immunofluorescence, Quantitation Assay, Fluorescence, Expressing, Electrophoresis, Western Blot, Injection, Quantitative RT-PCR

Chromatin Immunoprecipitation:

Article Title: Collective synthesis of 1,2,4-trisubstituted, meta- and ortho-substituted arene bioisosteres from bicyclobutanes.
Article Snippet: Replacing benzene rings with C(sp)-rich bioisosteres to yield compounds with improved drug-like properties is an attractive strategy in medicinal chemistry.. While many caged hydrocarbons have been validated as bioisosteres of orthoand meta-disubstituted benzenes, 3D analogues of 1,2,4-trisubstituted benzenes—the second most prevalent benzenoid pattern in drugs—remain elusive because vector fidelity and enantioselective access are still formidable challenges.. Here we report a practical route to (enantiomerically pure) 2-thiabicyclo[3.1.1]heptanes (thia-BCHeps) by cycloadditions of bicyclo[1.1.0]butanes with 1,4-dithiane2,5-diol.

Flow Cytometry:

Magnetic Resonance Imaging:

Multiple Displacement Amplification:

Mutagenesis:

Article Title: ApoM-Fc fusion proteins and uses thereof
Article Snippet: .. Tumor Metastasis Assay In order to evaluate the efficacy of ApoM-Fc mutant “Sponge” entities, a tumor metastasis assay will be performed using B16 melanoma cells obtained from the ATCC and tested for mycoplasma. ..

Cell Culture:

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity
Article Snippet: Cells were stimulated with anti-CD3/CD28 antibodies (2 and 1 μg/well, respectively, #BE0001/BE0015, Bioxcell) and IL-2 (50U, #202-IL-050, R&D) in a 24-well plate in complete RPMI-1640 medium containing 10% FBS, 1% penicillin/streptomycin, 1% glutamine, 1% Sodium Pyruvate, 1% non-essential amino acids, and 0.1% β-mercaptoethanol for 3-5days. .. Murine tumor cell lines B16 melanoma (RRID: CVCL_0159), MC38 colon cancer (RRID: CVCL_B288), and 4T1 (RRID: CRL_2539) were purchased from the ATCC and cultured in RPMI 1640 medium (Gibco) or DMEM medium (Gibco) with 10% FBS as well as 1% penicillin/streptomycin. .. Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.