human metastatic melanoma cell lines a375  (ATCC)


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    ATCC human metastatic melanoma cell lines a375
    Regulation of NACHT, LRR, and PYD domains-containing protein 1 ( NLRP1 ) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ( a ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. ( b ) IL-1β secretion from <t>A375</t> cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI - 1040 for 24 h. ( c ) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). ( d – f ) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. ( d ) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. ( e ) NLRP1 mRNA expression. ( f ) NLRP3 mRNA expression. ( g – i ) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter ( g ). ( h ) ATF3 was used as a positive control of ATF4 genomic targets. ( i ) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.
    Human Metastatic Melanoma Cell Lines A375, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma"

    Article Title: NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma

    Journal: Pharmaceuticals

    doi: 10.3390/ph14010023

    Regulation of NACHT, LRR, and PYD domains-containing protein 1 ( NLRP1 ) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ( a ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. ( b ) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI - 1040 for 24 h. ( c ) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). ( d – f ) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. ( d ) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. ( e ) NLRP1 mRNA expression. ( f ) NLRP3 mRNA expression. ( g – i ) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter ( g ). ( h ) ATF3 was used as a positive control of ATF4 genomic targets. ( i ) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.
    Figure Legend Snippet: Regulation of NACHT, LRR, and PYD domains-containing protein 1 ( NLRP1 ) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ( a ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. ( b ) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI - 1040 for 24 h. ( c ) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). ( d – f ) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. ( d ) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. ( e ) NLRP1 mRNA expression. ( f ) NLRP3 mRNA expression. ( g – i ) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter ( g ). ( h ) ATF3 was used as a positive control of ATF4 genomic targets. ( i ) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Positive Control, Binding Assay, Negative Control

    a375 human metastatic melanoma cell line  (ATCC)


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    ATCC a375 human metastatic melanoma cell line
    Following transfection with control pre-miR or pre-miR-21, cells were plated on Radius Migration Assay ECM-coated plates that have uniform wounds. Photographs were taken of the wound immediately following migration initiation and 14 hours later. Photographs of representative experiments for each cell line on the collagen-coated wells are shown ( A ). Migration was measured as the percent of the original wound area on the collagen-coated wells (n = 4) ( B ). Boyden chamber assays were used to evaluate invasive activity of melanoma cells transfected with control pre‑miR or pre-miR-21. Photographs of representative experiments for each cell line are shown ( C ). Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel (n = 4) ( D ). Following transfection with control pre-miR or pre-miR-21 at 3.125, 6.25, or 12.5 nM, cells were plated on Boyden chamber assays to evaluate invasive activity of <t>A375</t> melanoma cells at lower doseage of oligonucleotide. Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel ( E ). Error bars represent standard error. * p < 0.05.
    A375 Human Metastatic Melanoma Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor"

    Article Title: MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115919

    Following transfection with control pre-miR or pre-miR-21, cells were plated on Radius Migration Assay ECM-coated plates that have uniform wounds. Photographs were taken of the wound immediately following migration initiation and 14 hours later. Photographs of representative experiments for each cell line on the collagen-coated wells are shown ( A ). Migration was measured as the percent of the original wound area on the collagen-coated wells (n = 4) ( B ). Boyden chamber assays were used to evaluate invasive activity of melanoma cells transfected with control pre‑miR or pre-miR-21. Photographs of representative experiments for each cell line are shown ( C ). Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel (n = 4) ( D ). Following transfection with control pre-miR or pre-miR-21 at 3.125, 6.25, or 12.5 nM, cells were plated on Boyden chamber assays to evaluate invasive activity of A375 melanoma cells at lower doseage of oligonucleotide. Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel ( E ). Error bars represent standard error. * p < 0.05.
    Figure Legend Snippet: Following transfection with control pre-miR or pre-miR-21, cells were plated on Radius Migration Assay ECM-coated plates that have uniform wounds. Photographs were taken of the wound immediately following migration initiation and 14 hours later. Photographs of representative experiments for each cell line on the collagen-coated wells are shown ( A ). Migration was measured as the percent of the original wound area on the collagen-coated wells (n = 4) ( B ). Boyden chamber assays were used to evaluate invasive activity of melanoma cells transfected with control pre‑miR or pre-miR-21. Photographs of representative experiments for each cell line are shown ( C ). Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel (n = 4) ( D ). Following transfection with control pre-miR or pre-miR-21 at 3.125, 6.25, or 12.5 nM, cells were plated on Boyden chamber assays to evaluate invasive activity of A375 melanoma cells at lower doseage of oligonucleotide. Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel ( E ). Error bars represent standard error. * p < 0.05.

    Techniques Used: Transfection, Migration, Activity Assay

    A375 cells were transfected in vitro with an LNA oligonucleotide targeting miR-21 or a control LNA oligonucleotide. Cells were collected 24 hours post‑transfection for total RNA isolation. The RNA was converted to cDNA, and Real‑Time PCR for miR-21 confirmed reduced miR-21 expression ( A ). Transfected cells were then injected subcutaneously into athymic nude mice, and tumor growth was followed. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( B ). At the completion of the study, tumors were harvested, fixed and embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). N = 4 for control LNA tumors and n = 6 for anti‑miR‑21 LNA tumors. Error bars represent standard error. * p < 0.05.
    Figure Legend Snippet: A375 cells were transfected in vitro with an LNA oligonucleotide targeting miR-21 or a control LNA oligonucleotide. Cells were collected 24 hours post‑transfection for total RNA isolation. The RNA was converted to cDNA, and Real‑Time PCR for miR-21 confirmed reduced miR-21 expression ( A ). Transfected cells were then injected subcutaneously into athymic nude mice, and tumor growth was followed. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( B ). At the completion of the study, tumors were harvested, fixed and embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). N = 4 for control LNA tumors and n = 6 for anti‑miR‑21 LNA tumors. Error bars represent standard error. * p < 0.05.

    Techniques Used: Transfection, In Vitro, Isolation, Real-time Polymerase Chain Reaction, Expressing, Injection, Immunohistochemistry

    A375 cells were subcutaneously injected into the right flank of athymic nude mice and tumor development was measured. Each tumor was treated with four 50 µL injections of PBS, control LNA, or anti-miR-21 LNA at 500 nM at days 0, 4, 7, 11 (arrows). Day 0 is the day of the first treatment injection. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( A ). Following the completion of the study, tumors were harvested. Total RNA was extracted from the tumors. The RNA was converted to cDNA, and Real-Time PCR for miR-21 confirmed reduced miR-21 expression ( B ). Tumors were also fixed, embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). Tumors were evaluated for mean areas of confluent necrosis ( D ) and mean mitotic indices ( E ). N = 5 for each group. Error bars represent standard error.
    Figure Legend Snippet: A375 cells were subcutaneously injected into the right flank of athymic nude mice and tumor development was measured. Each tumor was treated with four 50 µL injections of PBS, control LNA, or anti-miR-21 LNA at 500 nM at days 0, 4, 7, 11 (arrows). Day 0 is the day of the first treatment injection. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( A ). Following the completion of the study, tumors were harvested. Total RNA was extracted from the tumors. The RNA was converted to cDNA, and Real-Time PCR for miR-21 confirmed reduced miR-21 expression ( B ). Tumors were also fixed, embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). Tumors were evaluated for mean areas of confluent necrosis ( D ) and mean mitotic indices ( E ). N = 5 for each group. Error bars represent standard error.

    Techniques Used: Injection, Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry

    human a375 metastatic melanoma cells  (ATCC)


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

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

    Journal: Molecules

    doi: 10.3390/molecules27123678

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

    Techniques Used: In Vitro, Activity Assay, Inhibition

    human metastatic melanoma cell lines a375  (ATCC)


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    ATCC human metastatic melanoma cell lines a375
    Human Metastatic Melanoma Cell Lines A375, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human metastatic melanoma cell lines a375  (ATCC)


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    ATCC human metastatic melanoma cell lines a375
    Regulation of NACHT, LRR, and PYD domains-containing protein 1 ( NLRP1 ) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ( a ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. ( b ) IL-1β secretion from <t>A375</t> cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI - 1040 for 24 h. ( c ) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). ( d – f ) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. ( d ) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. ( e ) NLRP1 mRNA expression. ( f ) NLRP3 mRNA expression. ( g – i ) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter ( g ). ( h ) ATF3 was used as a positive control of ATF4 genomic targets. ( i ) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.
    Human Metastatic Melanoma Cell Lines A375, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1 article reviews
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    1) Product Images from "NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma"

    Article Title: NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma

    Journal: Pharmaceuticals

    doi: 10.3390/ph14010023

    Regulation of NACHT, LRR, and PYD domains-containing protein 1 ( NLRP1 ) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ( a ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. ( b ) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI - 1040 for 24 h. ( c ) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). ( d – f ) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. ( d ) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. ( e ) NLRP1 mRNA expression. ( f ) NLRP3 mRNA expression. ( g – i ) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter ( g ). ( h ) ATF3 was used as a positive control of ATF4 genomic targets. ( i ) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.
    Figure Legend Snippet: Regulation of NACHT, LRR, and PYD domains-containing protein 1 ( NLRP1 ) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ( a ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. ( b ) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI - 1040 for 24 h. ( c ) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). ( d – f ) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. ( d ) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. ( e ) NLRP1 mRNA expression. ( f ) NLRP3 mRNA expression. ( g – i ) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter ( g ). ( h ) ATF3 was used as a positive control of ATF4 genomic targets. ( i ) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Positive Control, Binding Assay, Negative Control

    human metastatic melanoma cell line a375  (ATCC)


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    ATCC human metastatic melanoma cell line a375
    Human Metastatic Melanoma Cell Line A375, 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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    a375 human metastatic melanoma cell line  (ATCC)


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    ATCC a375 human metastatic melanoma cell line
    (A) MTS proliferation assay measuring cell proliferation after 48 or 72 hours of <t>A375</t> cell line transduced with an empty vector control (A375 EV) or A375 transduced with vectors specific for each NRAS isoform (A375 iso 1–5). (B) Quantification of matrigel invasion assay, (C) Visualization of soft agar colony formation assay at day 4, 4x magnification of 96 well plate and quantification of cell growth by fluorescent staining, and (D) Immunoblot probing for p-ERK, p-AKT, total ERK and AKT and β-actin of same panel of A375 cell lines (left), and densitometry analysis for n=3 immunoblots (right). Bars represent three biological replicates, displayed as mean ± s.e.m. * p <0.05, ** p<0.01, *** p< 0.001.
    A375 Human Metastatic Melanoma Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "NRAS mRNA is differentially spliced to give five distinct isoforms: implications for melanoma therapy"

    Article Title: NRAS mRNA is differentially spliced to give five distinct isoforms: implications for melanoma therapy

    Journal: Melanoma research

    doi: 10.1097/CMR.0000000000000623

    (A) MTS proliferation assay measuring cell proliferation after 48 or 72 hours of A375 cell line transduced with an empty vector control (A375 EV) or A375 transduced with vectors specific for each NRAS isoform (A375 iso 1–5). (B) Quantification of matrigel invasion assay, (C) Visualization of soft agar colony formation assay at day 4, 4x magnification of 96 well plate and quantification of cell growth by fluorescent staining, and (D) Immunoblot probing for p-ERK, p-AKT, total ERK and AKT and β-actin of same panel of A375 cell lines (left), and densitometry analysis for n=3 immunoblots (right). Bars represent three biological replicates, displayed as mean ± s.e.m. * p <0.05, ** p<0.01, *** p< 0.001.
    Figure Legend Snippet: (A) MTS proliferation assay measuring cell proliferation after 48 or 72 hours of A375 cell line transduced with an empty vector control (A375 EV) or A375 transduced with vectors specific for each NRAS isoform (A375 iso 1–5). (B) Quantification of matrigel invasion assay, (C) Visualization of soft agar colony formation assay at day 4, 4x magnification of 96 well plate and quantification of cell growth by fluorescent staining, and (D) Immunoblot probing for p-ERK, p-AKT, total ERK and AKT and β-actin of same panel of A375 cell lines (left), and densitometry analysis for n=3 immunoblots (right). Bars represent three biological replicates, displayed as mean ± s.e.m. * p <0.05, ** p<0.01, *** p< 0.001.

    Techniques Used: Proliferation Assay, Transduction, Plasmid Preparation, Invasion Assay, Soft Agar Assay, Staining, Western Blot

    (A) A375 cells that over-express either NRAS isoforms 1–5 or an empty vector control (A375 EV) were injected s.c. into athymic nude mice (1×106 cells). and tumor growth was followed. On day 27, mice were sacrificed and tumors were removed and measured for volume (B) and weight (C). Bars depict mean ± s.e.m.
    Figure Legend Snippet: (A) A375 cells that over-express either NRAS isoforms 1–5 or an empty vector control (A375 EV) were injected s.c. into athymic nude mice (1×106 cells). and tumor growth was followed. On day 27, mice were sacrificed and tumors were removed and measured for volume (B) and weight (C). Bars depict mean ± s.e.m.

    Techniques Used: Plasmid Preparation, Injection

    a375 human metastatic melanoma cell line  (ATCC)


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    ATCC a375 human metastatic melanoma cell line
    A375 Human Metastatic Melanoma Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a375 human metastatic melanoma cell line  (ATCC)


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    ATCC a375 human metastatic melanoma cell line
    (A) MTS proliferation assay measuring cell proliferation after 48 or 72 hours of <t>A375</t> cell line transduced with an empty vector control (A375 EV) or A375 transduced with vectors specific for each NRAS isoform (A375 iso 1–5). (B) Quantification of matrigel invasion assay, (C) Visualization of soft agar colony formation assay at day 4, 4x magnification of 96 well plate and quantification of cell growth by fluorescent staining, and (D) Immunoblot probing for p-ERK, p-AKT, total ERK and AKT and β-actin of same panel of A375 cell lines (left), and densitometry analysis for n=3 immunoblots (right). Bars represent three biological replicates, displayed as mean ± s.e.m. * p <0.05, ** p<0.01, *** p< 0.001.
    A375 Human Metastatic Melanoma Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a375 human metastatic melanoma cell line/product/ATCC
    Average 86 stars, based on 1 article reviews
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    a375 human metastatic melanoma cell line - by Bioz Stars, 2024-05
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    1) Product Images from "NRAS mRNA is differentially spliced to give five distinct isoforms: implications for melanoma therapy"

    Article Title: NRAS mRNA is differentially spliced to give five distinct isoforms: implications for melanoma therapy

    Journal: Melanoma research

    doi: 10.1097/CMR.0000000000000623

    (A) MTS proliferation assay measuring cell proliferation after 48 or 72 hours of A375 cell line transduced with an empty vector control (A375 EV) or A375 transduced with vectors specific for each NRAS isoform (A375 iso 1–5). (B) Quantification of matrigel invasion assay, (C) Visualization of soft agar colony formation assay at day 4, 4x magnification of 96 well plate and quantification of cell growth by fluorescent staining, and (D) Immunoblot probing for p-ERK, p-AKT, total ERK and AKT and β-actin of same panel of A375 cell lines (left), and densitometry analysis for n=3 immunoblots (right). Bars represent three biological replicates, displayed as mean ± s.e.m. * p <0.05, ** p<0.01, *** p< 0.001.
    Figure Legend Snippet: (A) MTS proliferation assay measuring cell proliferation after 48 or 72 hours of A375 cell line transduced with an empty vector control (A375 EV) or A375 transduced with vectors specific for each NRAS isoform (A375 iso 1–5). (B) Quantification of matrigel invasion assay, (C) Visualization of soft agar colony formation assay at day 4, 4x magnification of 96 well plate and quantification of cell growth by fluorescent staining, and (D) Immunoblot probing for p-ERK, p-AKT, total ERK and AKT and β-actin of same panel of A375 cell lines (left), and densitometry analysis for n=3 immunoblots (right). Bars represent three biological replicates, displayed as mean ± s.e.m. * p <0.05, ** p<0.01, *** p< 0.001.

    Techniques Used: Proliferation Assay, Transduction, Plasmid Preparation, Invasion Assay, Soft Agar Assay, Staining, Western Blot

    (A) A375 cells that over-express either NRAS isoforms 1–5 or an empty vector control (A375 EV) were injected s.c. into athymic nude mice (1×106 cells). and tumor growth was followed. On day 27, mice were sacrificed and tumors were removed and measured for volume (B) and weight (C). Bars depict mean ± s.e.m.
    Figure Legend Snippet: (A) A375 cells that over-express either NRAS isoforms 1–5 or an empty vector control (A375 EV) were injected s.c. into athymic nude mice (1×106 cells). and tumor growth was followed. On day 27, mice were sacrificed and tumors were removed and measured for volume (B) and weight (C). Bars depict mean ± s.e.m.

    Techniques Used: Plasmid Preparation, Injection

    human metastatic melanoma cell line a375  (ATCC)


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    ATCC human metastatic melanoma cell line a375
    Human Metastatic Melanoma Cell Line A375, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human metastatic melanoma cell line a375  (ATCC)


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    ATCC human metastatic melanoma cell line a375
    IC50 of dehydrocorydaline treatment for 48 h in the inhibition of cell proliferation in ( A ) <t>A375</t> melanoma cells; ( B ) MV3 melanoma cells; and ( C ) PIG1 normal melanocytes determined by MTT assay.
    Human Metastatic Melanoma Cell Line A375, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human metastatic melanoma cell line a375 - by Bioz Stars, 2024-05
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    1) Product Images from "Dehydrocorydaline inhibits cell proliferation, migration and invasion via suppressing MEK1/2-ERK1/2 cascade in melanoma"

    Article Title: Dehydrocorydaline inhibits cell proliferation, migration and invasion via suppressing MEK1/2-ERK1/2 cascade in melanoma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S183558

    IC50 of dehydrocorydaline treatment for 48 h in the inhibition of cell proliferation in ( A ) A375 melanoma cells; ( B ) MV3 melanoma cells; and ( C ) PIG1 normal melanocytes determined by MTT assay.
    Figure Legend Snippet: IC50 of dehydrocorydaline treatment for 48 h in the inhibition of cell proliferation in ( A ) A375 melanoma cells; ( B ) MV3 melanoma cells; and ( C ) PIG1 normal melanocytes determined by MTT assay.

    Techniques Used: Inhibition, MTT Assay

    Dehydrocorydaline inhibits cell growth and proliferation in human melanoma cells. ( A ) Cell morphology of A375 and MV3 melanoma cells after treating with DMSO or the indicated concentrations of DHC for 48 h. Scale bar, 100 μm. ( B and C ) The effect of DHC on the proliferation rates of A375 and MV3 cells determined by cell counting in the microscope. (D) The effect of different concentrations of DHC treatment for 48 h on the proliferation rate of PIG1 cells determined by MTT assay. ( E and F ) The effect of DHC on the viability of A375 and MV3 cells. ( G ) Images and quantifications of A375 and MV3 cells positive for BrdU staining after treating with DMSO or 40 μM DHC for 24 h. Scale bar, 100 μm. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. * p <0.05, ** p <0.01, *** p <0.001, **** p< 0.0001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.
    Figure Legend Snippet: Dehydrocorydaline inhibits cell growth and proliferation in human melanoma cells. ( A ) Cell morphology of A375 and MV3 melanoma cells after treating with DMSO or the indicated concentrations of DHC for 48 h. Scale bar, 100 μm. ( B and C ) The effect of DHC on the proliferation rates of A375 and MV3 cells determined by cell counting in the microscope. (D) The effect of different concentrations of DHC treatment for 48 h on the proliferation rate of PIG1 cells determined by MTT assay. ( E and F ) The effect of DHC on the viability of A375 and MV3 cells. ( G ) Images and quantifications of A375 and MV3 cells positive for BrdU staining after treating with DMSO or 40 μM DHC for 24 h. Scale bar, 100 μm. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. * p <0.05, ** p <0.01, *** p <0.001, **** p< 0.0001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

    Techniques Used: Cell Counting, Microscopy, MTT Assay, BrdU Staining, Two Tailed Test

    Dehydrocorydaline induces cell cycle arrest at G1 phase in human melanoma cells. ( A and B ) The cell cycle of A375 and MV3 cells was analyzed by flow cytometry after treating with DMSO or the indicated concentrations of DHC for 48 h. ( C and D ) Western blot assay was performed to assess the cell cycle-related protein levels in A375 and MV3 cells, respectively. Protein levels were calculated based on the grayscale value of protein bands and normalized with the grayscale value of GAPDH bands. Cells were treated with the indicated concentrations (0, 20, 40, 80 μM) of DHC for 48 h or with 40 μM DHC treatment for indicated times (0, 24, 48, 72 h) of DHC; GAPDH was used as a control. ( E ) The cell cycle of PIG1 cells was analyzed by flow cytometry after treating with DMSO or 40 μM DHC for 48 h. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. ** p <0.01, *** p <0.001. Abbreviations: n.s.=not significant; DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
    Figure Legend Snippet: Dehydrocorydaline induces cell cycle arrest at G1 phase in human melanoma cells. ( A and B ) The cell cycle of A375 and MV3 cells was analyzed by flow cytometry after treating with DMSO or the indicated concentrations of DHC for 48 h. ( C and D ) Western blot assay was performed to assess the cell cycle-related protein levels in A375 and MV3 cells, respectively. Protein levels were calculated based on the grayscale value of protein bands and normalized with the grayscale value of GAPDH bands. Cells were treated with the indicated concentrations (0, 20, 40, 80 μM) of DHC for 48 h or with 40 μM DHC treatment for indicated times (0, 24, 48, 72 h) of DHC; GAPDH was used as a control. ( E ) The cell cycle of PIG1 cells was analyzed by flow cytometry after treating with DMSO or 40 μM DHC for 48 h. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. ** p <0.01, *** p <0.001. Abbreviations: n.s.=not significant; DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Techniques Used: Flow Cytometry, Western Blot, Two Tailed Test

    Dehydrocorydaline inhibits cell migration and invasion in melanoma cells. ( A ) Cell migration rate detected by wound-healing assay of A375 and MV3 cells after treating with DMSO or 40 μM DHC for the indicated time. Scale bar, 100 μm. ( B ) The effect of 40 μM DHC on the wound closure in A375 and MV3 cells. ( C ) The effect of transwell migration assays in A375 and MV3 cells after treating with DMSO or 40 μM DHC for 24 h. Scale bar, 100 μm. Migration rates were normalized by proliferation. ( D ) The effect of transwell invasion assays in A375 and MV3 cells after treating with DMSO or 40 μM DHC for 72 h. Scale bar, 100 μm. Invasion rates were normalized by proliferation. ( E and F ) Western blot analysis of the metastasis-related protein levels in A375 and MV3 cells, respectively. Protein levels were calculated based on the grayscale value of protein bands and normalized with the grayscale value of GAPDH bands. Cells were treated with the indicated concentrations (0, 20, 40, 80 μM) of DHC for 48 h or with 40 μM DHC treatment for indicated times (0, 24, 48, 72 h) of DHC; GAPDH was used as a control. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
    Figure Legend Snippet: Dehydrocorydaline inhibits cell migration and invasion in melanoma cells. ( A ) Cell migration rate detected by wound-healing assay of A375 and MV3 cells after treating with DMSO or 40 μM DHC for the indicated time. Scale bar, 100 μm. ( B ) The effect of 40 μM DHC on the wound closure in A375 and MV3 cells. ( C ) The effect of transwell migration assays in A375 and MV3 cells after treating with DMSO or 40 μM DHC for 24 h. Scale bar, 100 μm. Migration rates were normalized by proliferation. ( D ) The effect of transwell invasion assays in A375 and MV3 cells after treating with DMSO or 40 μM DHC for 72 h. Scale bar, 100 μm. Invasion rates were normalized by proliferation. ( E and F ) Western blot analysis of the metastasis-related protein levels in A375 and MV3 cells, respectively. Protein levels were calculated based on the grayscale value of protein bands and normalized with the grayscale value of GAPDH bands. Cells were treated with the indicated concentrations (0, 20, 40, 80 μM) of DHC for 48 h or with 40 μM DHC treatment for indicated times (0, 24, 48, 72 h) of DHC; GAPDH was used as a control. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Techniques Used: Migration, Wound Healing Assay, Western Blot, Two Tailed Test

    Dehydrocorydaline suppresses tumor growth in xenograft model of melanoma cells. ( A and B ) The colony formation was examined by soft agar assay (1,000 cells/well) in A375 or MV3 cells after treating with DMSO or 40 μM DHC for 14 days. Scale bar, 1 mm. ( C and D ) A375 and MV3 cells were injected into the flank of BALB/c nude mice. When tumors were palpable, mice were orally administrated with 100 mg/kg DHC or DMSO every day 12 times after the tumor plumped, and mice body weight was measured. ( E and F ) The tumor volume of xenograft tumors formed by the A375 and MV3 cells which were treated with DHC (100 mg/kg) or DMSO was measured. ( G and H ) The weight of the tumor was measured after DHC or DMSO treatment. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. * p <0.05, *** p <0.001, **** p <0.0001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide.
    Figure Legend Snippet: Dehydrocorydaline suppresses tumor growth in xenograft model of melanoma cells. ( A and B ) The colony formation was examined by soft agar assay (1,000 cells/well) in A375 or MV3 cells after treating with DMSO or 40 μM DHC for 14 days. Scale bar, 1 mm. ( C and D ) A375 and MV3 cells were injected into the flank of BALB/c nude mice. When tumors were palpable, mice were orally administrated with 100 mg/kg DHC or DMSO every day 12 times after the tumor plumped, and mice body weight was measured. ( E and F ) The tumor volume of xenograft tumors formed by the A375 and MV3 cells which were treated with DHC (100 mg/kg) or DMSO was measured. ( G and H ) The weight of the tumor was measured after DHC or DMSO treatment. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. * p <0.05, *** p <0.001, **** p <0.0001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide.

    Techniques Used: Soft Agar Assay, Injection, Two Tailed Test

    Dehydrocorydaline downregulates MEK1/2-ERK1/2 cascade in melanoma cells. ( A and B ) Western blot analysis of the expression of MAPK cascade in A375 and MV3 cells, respectively. Protein phosphorylation levels were calculated based on the grayscale value of phosphorylated protein bands and normalized with the grayscale value of total protein bands. Cells were treated with the indicated concentrations (0, 20, 40, 80 μM) of DHC for 48 h or with 40 μM DHC treatment for indicated times (0, 24, 48, 72 h) of DHC; GAPDH was used as a control. ( C ) The effect of 40 μM DHC and 50 μM ERK1/2 activator t-butylhydroquinone (tBHQ) on the viability of A375 and MV3 cells, respectively. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. * p <0.05, ** p<0.01, *** p <0.001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase; tBHQ, t-butylhydroquinone.
    Figure Legend Snippet: Dehydrocorydaline downregulates MEK1/2-ERK1/2 cascade in melanoma cells. ( A and B ) Western blot analysis of the expression of MAPK cascade in A375 and MV3 cells, respectively. Protein phosphorylation levels were calculated based on the grayscale value of phosphorylated protein bands and normalized with the grayscale value of total protein bands. Cells were treated with the indicated concentrations (0, 20, 40, 80 μM) of DHC for 48 h or with 40 μM DHC treatment for indicated times (0, 24, 48, 72 h) of DHC; GAPDH was used as a control. ( C ) The effect of 40 μM DHC and 50 μM ERK1/2 activator t-butylhydroquinone (tBHQ) on the viability of A375 and MV3 cells, respectively. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t -test was carried out. * p <0.05, ** p<0.01, *** p <0.001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase; tBHQ, t-butylhydroquinone.

    Techniques Used: Western Blot, Expressing, Two Tailed Test

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    ATCC human metastatic melanoma cell lines a375
    Regulation of NACHT, LRR, and PYD domains-containing protein 1 ( NLRP1 ) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ( a ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. ( b ) IL-1β secretion from <t>A375</t> cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI - 1040 for 24 h. ( c ) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). ( d – f ) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. ( d ) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. ( e ) NLRP1 mRNA expression. ( f ) NLRP3 mRNA expression. ( g – i ) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter ( g ). ( h ) ATF3 was used as a positive control of ATF4 genomic targets. ( i ) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.
    Human Metastatic Melanoma Cell Lines A375, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC a375 human metastatic melanoma cell line
    Following transfection with control pre-miR or pre-miR-21, cells were plated on Radius Migration Assay ECM-coated plates that have uniform wounds. Photographs were taken of the wound immediately following migration initiation and 14 hours later. Photographs of representative experiments for each cell line on the collagen-coated wells are shown ( A ). Migration was measured as the percent of the original wound area on the collagen-coated wells (n = 4) ( B ). Boyden chamber assays were used to evaluate invasive activity of melanoma cells transfected with control pre‑miR or pre-miR-21. Photographs of representative experiments for each cell line are shown ( C ). Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel (n = 4) ( D ). Following transfection with control pre-miR or pre-miR-21 at 3.125, 6.25, or 12.5 nM, cells were plated on Boyden chamber assays to evaluate invasive activity of <t>A375</t> melanoma cells at lower doseage of oligonucleotide. Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel ( E ). Error bars represent standard error. * p < 0.05.
    A375 Human Metastatic Melanoma Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human a375 metastatic melanoma cells
    In vitro screening of novel NL compounds: ( A ) hit compounds were screened at 10 micromolar concentrations for 24 h. All but one compound demonstrated significant activity and were selected for a dose response analysis; ( B ) compounds were screened at increasing concentrations to assay for activity. NL221-75 and NL350-02 were as potent as FDA-approved controls, demonstrating low nanomolar range activity; ( C ) MTT <t>A375</t> cells were treated with increasing concentrations of test items and cell proliferation was determined at 24 h using the MTT method. All compounds demonstrated dose-dependent activity in preventing cell proliferation. Experimental compounds NL221-75 and NL350-02 were as effective as FDA-approved controls in preventing cell proliferation. Data are plotted as percent inhibition of proliferation; and ( D ) hERG inhibition experiments performed on CHO-cells. Cobimetinib demonstrated low nanomolar inhibition of hERG. NL34-113, NL221-75, and NL350-02 did not inhibit hERG at the concentrations tested.
    Human A375 Metastatic Melanoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human metastatic melanoma cell line a375
    In vitro screening of novel NL compounds: ( A ) hit compounds were screened at 10 micromolar concentrations for 24 h. All but one compound demonstrated significant activity and were selected for a dose response analysis; ( B ) compounds were screened at increasing concentrations to assay for activity. NL221-75 and NL350-02 were as potent as FDA-approved controls, demonstrating low nanomolar range activity; ( C ) MTT <t>A375</t> cells were treated with increasing concentrations of test items and cell proliferation was determined at 24 h using the MTT method. All compounds demonstrated dose-dependent activity in preventing cell proliferation. Experimental compounds NL221-75 and NL350-02 were as effective as FDA-approved controls in preventing cell proliferation. Data are plotted as percent inhibition of proliferation; and ( D ) hERG inhibition experiments performed on CHO-cells. Cobimetinib demonstrated low nanomolar inhibition of hERG. NL34-113, NL221-75, and NL350-02 did not inhibit hERG at the concentrations tested.
    Human Metastatic Melanoma Cell Line A375, 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/human metastatic melanoma cell line a375/product/ATCC
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    Regulation of NACHT, LRR, and PYD domains-containing protein 1 ( NLRP1 ) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ( a ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. ( b ) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI - 1040 for 24 h. ( c ) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). ( d – f ) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. ( d ) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. ( e ) NLRP1 mRNA expression. ( f ) NLRP3 mRNA expression. ( g – i ) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter ( g ). ( h ) ATF3 was used as a positive control of ATF4 genomic targets. ( i ) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.

    Journal: Pharmaceuticals

    Article Title: NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma

    doi: 10.3390/ph14010023

    Figure Lengend Snippet: Regulation of NACHT, LRR, and PYD domains-containing protein 1 ( NLRP1 ) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ( a ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. ( b ) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI - 1040 for 24 h. ( c ) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). ( d – f ) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. ( d ) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. ( e ) NLRP1 mRNA expression. ( f ) NLRP3 mRNA expression. ( g – i ) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter ( g ). ( h ) ATF3 was used as a positive control of ATF4 genomic targets. ( i ) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.

    Article Snippet: Human metastatic melanoma cell lines A375 (CRL-1619) and SK-MEL-28 (HTB-72) were obtained from the American Type Culture Collection (Manassas, VA, USA) and 1205Lu cell line (1205Lu-01-0001) was purchased from Rockland (Limerick, PA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Positive Control, Binding Assay, Negative Control

    Following transfection with control pre-miR or pre-miR-21, cells were plated on Radius Migration Assay ECM-coated plates that have uniform wounds. Photographs were taken of the wound immediately following migration initiation and 14 hours later. Photographs of representative experiments for each cell line on the collagen-coated wells are shown ( A ). Migration was measured as the percent of the original wound area on the collagen-coated wells (n = 4) ( B ). Boyden chamber assays were used to evaluate invasive activity of melanoma cells transfected with control pre‑miR or pre-miR-21. Photographs of representative experiments for each cell line are shown ( C ). Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel (n = 4) ( D ). Following transfection with control pre-miR or pre-miR-21 at 3.125, 6.25, or 12.5 nM, cells were plated on Boyden chamber assays to evaluate invasive activity of A375 melanoma cells at lower doseage of oligonucleotide. Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel ( E ). Error bars represent standard error. * p < 0.05.

    Journal: PLoS ONE

    Article Title: MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor

    doi: 10.1371/journal.pone.0115919

    Figure Lengend Snippet: Following transfection with control pre-miR or pre-miR-21, cells were plated on Radius Migration Assay ECM-coated plates that have uniform wounds. Photographs were taken of the wound immediately following migration initiation and 14 hours later. Photographs of representative experiments for each cell line on the collagen-coated wells are shown ( A ). Migration was measured as the percent of the original wound area on the collagen-coated wells (n = 4) ( B ). Boyden chamber assays were used to evaluate invasive activity of melanoma cells transfected with control pre‑miR or pre-miR-21. Photographs of representative experiments for each cell line are shown ( C ). Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel (n = 4) ( D ). Following transfection with control pre-miR or pre-miR-21 at 3.125, 6.25, or 12.5 nM, cells were plated on Boyden chamber assays to evaluate invasive activity of A375 melanoma cells at lower doseage of oligonucleotide. Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel ( E ). Error bars represent standard error. * p < 0.05.

    Article Snippet: The A375 human metastatic melanoma cell line was obtained from American Type Cell Culture Collection (ATCC, Manassas, VA).

    Techniques: Transfection, Migration, Activity Assay

    A375 cells were transfected in vitro with an LNA oligonucleotide targeting miR-21 or a control LNA oligonucleotide. Cells were collected 24 hours post‑transfection for total RNA isolation. The RNA was converted to cDNA, and Real‑Time PCR for miR-21 confirmed reduced miR-21 expression ( A ). Transfected cells were then injected subcutaneously into athymic nude mice, and tumor growth was followed. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( B ). At the completion of the study, tumors were harvested, fixed and embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). N = 4 for control LNA tumors and n = 6 for anti‑miR‑21 LNA tumors. Error bars represent standard error. * p < 0.05.

    Journal: PLoS ONE

    Article Title: MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor

    doi: 10.1371/journal.pone.0115919

    Figure Lengend Snippet: A375 cells were transfected in vitro with an LNA oligonucleotide targeting miR-21 or a control LNA oligonucleotide. Cells were collected 24 hours post‑transfection for total RNA isolation. The RNA was converted to cDNA, and Real‑Time PCR for miR-21 confirmed reduced miR-21 expression ( A ). Transfected cells were then injected subcutaneously into athymic nude mice, and tumor growth was followed. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( B ). At the completion of the study, tumors were harvested, fixed and embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). N = 4 for control LNA tumors and n = 6 for anti‑miR‑21 LNA tumors. Error bars represent standard error. * p < 0.05.

    Article Snippet: The A375 human metastatic melanoma cell line was obtained from American Type Cell Culture Collection (ATCC, Manassas, VA).

    Techniques: Transfection, In Vitro, Isolation, Real-time Polymerase Chain Reaction, Expressing, Injection, Immunohistochemistry

    A375 cells were subcutaneously injected into the right flank of athymic nude mice and tumor development was measured. Each tumor was treated with four 50 µL injections of PBS, control LNA, or anti-miR-21 LNA at 500 nM at days 0, 4, 7, 11 (arrows). Day 0 is the day of the first treatment injection. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( A ). Following the completion of the study, tumors were harvested. Total RNA was extracted from the tumors. The RNA was converted to cDNA, and Real-Time PCR for miR-21 confirmed reduced miR-21 expression ( B ). Tumors were also fixed, embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). Tumors were evaluated for mean areas of confluent necrosis ( D ) and mean mitotic indices ( E ). N = 5 for each group. Error bars represent standard error.

    Journal: PLoS ONE

    Article Title: MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor

    doi: 10.1371/journal.pone.0115919

    Figure Lengend Snippet: A375 cells were subcutaneously injected into the right flank of athymic nude mice and tumor development was measured. Each tumor was treated with four 50 µL injections of PBS, control LNA, or anti-miR-21 LNA at 500 nM at days 0, 4, 7, 11 (arrows). Day 0 is the day of the first treatment injection. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( A ). Following the completion of the study, tumors were harvested. Total RNA was extracted from the tumors. The RNA was converted to cDNA, and Real-Time PCR for miR-21 confirmed reduced miR-21 expression ( B ). Tumors were also fixed, embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). Tumors were evaluated for mean areas of confluent necrosis ( D ) and mean mitotic indices ( E ). N = 5 for each group. Error bars represent standard error.

    Article Snippet: The A375 human metastatic melanoma cell line was obtained from American Type Cell Culture Collection (ATCC, Manassas, VA).

    Techniques: Injection, Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry

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

    Journal: Molecules

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

    doi: 10.3390/molecules27123678

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

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

    Techniques: In Vitro, Activity Assay, Inhibition