m14 human melanoma cells  (ATCC)

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Identification of differentially expressed genes based on ImmuneScore and StromalScore. ( A-C ) Overall survival analysis of high and low Immune/Stromal/ESTIMATE Scores in patients with metastatic melanoma using the ESTIMATE algorithm. ( D , E ) Heatmap depicting the top 100 DEmRNAs with the most significant p-values between high and low Immune/Stromal Scores (|log Fold change|>1.9, p < 0.001). Blue represents downregulation, while red represents upregulation. ( F ) Venn diagram showing the upregulated and downregulated genes commonly intersecting between Stromal and Immune Scores, with green indicating immune-related DEmRNAs and purple indicating stromal-related DEmRNAs. ( G ) Construction of a protein-protein interaction network for overlapping genes using the STRING online tool (confidence score > 0.900) and Cytoscape software. ( H ) The top 15 genes ranked by nodes in the PPI network are shown in the graph. ( I ) Univariate Cox analysis was conducted on genes with a node count of 15 or more in the PPI network to identify differentially expressed genes significantly associated with prognosis

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Software

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: The expression of PTPN6 and its correlation analysis with survival. ( A ) Boxplot showing differential expression of PTPN6 in metastatic melanoma samples and normal skin tissue samples. ( B ) Correlation analysis of PTPN6 expression in metastatic melanoma samples with overall survival. ( C ) Correlation analysis of PTPN6 expression in metastatic melanoma samples with progression-free survival. ( D ) RT-qPCR assay analyzed the transcription level of PTPN6 in human keratinocyte cell line HaCaT and human metastatic melanoma cell lines A2058 and M14. The RT-qPCR results showed the relative mRNA expression of PTPN6 normalized to GAPDH. ( E ) Western blotting assay analyzed the expression of PTPN6 in human keratinocyte cell line HaCaT and human metastatic melanoma cell lines A2058 and M14. (** p < 0.01, *** p < 0.001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing, Quantitative Proteomics, Quantitative RT-PCR, Western Blot

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Overexpression of PTPN6 inhibited the proliferation and viability of metastatic melanoma cells in vitro. ( A–C ) RT-qPCR and Western blotting assays were performed to evaluate the mRNA and protein expression of PTPN6 in A2058 and M14 cells after transfection with PTPN6. The RT-qPCR results showed the relative mRNA expression of PTPN6 normalized to GAPDH. ( D , E ) CCK8 assay was used to determine the impact of PTPN6 overexpression on the proliferation of A2058 and M14 cells. ( F , G ) EdU assay was conducted to assess the impact of PTPN6 overexpression on the viability of A2058 and M14 cells. Representative images were randomly selected from three independent experiments. Data are presented as the mean ± standard deviation (SD). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Transfection, CCK-8 Assay, EdU Assay, Standard Deviation

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Overexpression of PTPN6 suppressed the migration and invasion of metastatic melanoma cells in vitro. ( A , B ) Wound healing assay was performed to determine the impact of PTPN6 overexpression on the migration of A2058 and M14 cells. ( C , D ) Transwell migration and Matrigel invasion assays were used to assess the impact of PTPN6 overexpression on the migration and invasion of A2058 and M14 cells. ( E , F ) Western blotting assay was conducted to verify the expression of migration and invasion-related genes, including MMP2, MMP9, E-cadherin, and Vimentin, in control, NC, and PTPN6 overexpression groups in A2058 and M14 cells. Representative images were randomly selected from three independent experiments. Data are presented as mean ± standard deviation (SD). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Over Expression, Migration, In Vitro, Wound Healing Assay, Western Blot, Expressing, Control, Standard Deviation

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Overexpression of PTPN6 inhibited the activity of the JAK2-STAT3 signaling pathway in metastatic melanoma cells. ( A-D ) Western blotting assay was used to assess the expression of p-JAK2, JAK2, p-STAT3, and STAT3 after transfection with the PTPN6 overexpression plasmid. (**** p < 0.0001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Over Expression, Activity Assay, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: PTPN6 may affect immune cell infiltration and immune therapy response in metastatic melanoma. ( A ) TIMER 2.0 online analysis tool was used to examine the relationship between PTPN6 and the levels of infiltration of 14 immune cells and tumor purity in metastatic melanoma. ( B ) The correlation between PTPN6 and immune checkpoint therapy (alone or in combination with PD-1 or CTLA-4) response was analyzed using the TCIA database. ( C , D ) Western blotting assay was used to detect PD-L1 expression after PTPN6 overexpression (* p < 0.05, ** p < 0.01)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Western Blot, Expressing, Over Expression

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Knockdown of PTPN6 promoted the proliferation, migration, and invasion of metastatic melanoma cells in vitro. ( A , B ) Western blotting assay was performed to evaluate the protein expression of PTPN6 in A2058 and M14 cells after transfection with GV341-PTPN6-3FLAG-SV40-puromycin lentivirus. ( C , D ) Western blotting assay was performed to evaluate the protein expression of PTPN6 in A2058 and M14 cell lines stably overexpressing PTPN6 after transiently transfected siPTPN6 or siNC. ( E , F ) CCK8 assay was used to determine the impact of PTPN6 knockdown on the proliferation of cells. ( G , H ) Wound healing assay was performed to determine the impact of PTPN6 knockdown on the migration of cells. ( I , J ) Transwell migration and Matrigel invasion assays were used to assess the impact of PTPN6 knockdown on the migration and invasion of cells. Representative images were randomly selected from three independent experiments. Data are presented as mean ± standard deviation (SD). (** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Knockdown, Migration, In Vitro, Western Blot, Expressing, Transfection, Stable Transfection, CCK-8 Assay, Wound Healing Assay, Standard Deviation

Journal: Nanomaterials

Article Title: Parenteral Nanoemulsions Loaded with Combined Immuno- and Chemo-Therapy for Melanoma Treatment

doi: 10.3390/nano12234233

Figure Lengend Snippet: MTT assay performed after 72 h of incubation with the formulations under study. B16 cells: upper left panel; D4M cells: upper right panel; M14 cells: lower left panel; A2058 cells: lower right panel. Abbreviations: IL: Intralipid ® 10%; SOR: sorafenib; TMZ: temozolomide; MIX: drug combination (temozolomide dodecyl ester, sorafenib, ICOS-Fc). Concentrations employed: SOR A = 16 μM; B = 10 μM; C = 8 μM; D = 4.5 μM; E = 0.8 μM. TMZ A = 48 μM; B = 32 μM; C = 24 μM; D = 16 μM; E = 2.4 μM. Statistical analysis: ^ p < 0.05 SOR vs. IL SOR; * p < 0.05 TMZ vs. IL TMZ; § p < 0.05 MIX vs. IL MIX.

Article Snippet: A2058 and M14 human melanoma cells and B16-F10 murine melanoma cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: MTT Assay, Incubation

Journal: Nanomaterials

Article Title: Parenteral Nanoemulsions Loaded with Combined Immuno- and Chemo-Therapy for Melanoma Treatment

doi: 10.3390/nano12234233

Figure Lengend Snippet: Clonogenic assay with B16 (upper left panel), D4M (upper right panel), M14 (lower left panel), and A2058 (lower right panel) melanoma cells. Abbreviations: IL: Intralipid ® 10%; SOR: sorafenib; TMZ: temozolomide; MIX: drug combination (temozolomide dodecyl ester, sorafenib, ICOS-Fc). Cells were treated with the formulations under study for 3 h. Afterwards, the medium was changed, and cells were cultured in drug-free medium for an additional 7 days. Concentrations employed: SOR A = 16 μM; B = 10 μM; C = 8 μM; D = 4.5 μM; E = 0.8 μM. TMZ A = 48 μM; B = 32 μM; C = 24 μM; D = 16 μM; E = 2.4 μM. Statistical analysis: ^ p < 0.05 SOR vs. IL SOR; * p < 0.05 TMZ vs. IL TMZ; § p < 0.05 MIX vs. IL MIX.

Article Snippet: A2058 and M14 human melanoma cells and B16-F10 murine melanoma cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Clonogenic Assay, Cell Culture

Journal: Nanomaterials

Article Title: Parenteral Nanoemulsions Loaded with Combined Immuno- and Chemo-Therapy for Melanoma Treatment

doi: 10.3390/nano12234233

Figure Lengend Snippet: Hypothesized sorafenib (SOR) mechanism of action: ( a ) SOR release profile in culture medium, and ( b ) SOR internalization migration assay (B16, M14 melanoma cells): *** p < 0.005, * p < 0.05, # p < 0.01. ( c ) Hypothesized cellular pathways. Abbreviations: FCS: fetal calf serum; IL: Intralipid ® 10%; RAF: rapidly accelerated fibrosarcoma kinases; RTK: receptor tyrosine kinases.

Article Snippet: A2058 and M14 human melanoma cells and B16-F10 murine melanoma cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 expression in melanoma cell lines and Rsf-1 knockdown efficiency. (A) Western blotting and RT-qPCR analysis revealed the endogenous expression levels of Rsf-1 in three melanoma cell lines (MV3, M14 and A375). (B) Western blotting and RT-qPCR analysis demonstrated that Rsf-1 siRNA transfection significantly decreased Rsf-1 expression levels in MV3 and A375 cells, while Rsf-1 plasmid transfection upregulated the protein and mRNA expression of Rsf-1 in M14 cells. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. Rsf-1, remodeling and spacing factor 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Standard Deviation, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates melanoma cell viability and invasion. (A) An MTT assay (96-well plate) revealed that Rsf-1 depletion decreased the viability of MV3 and A375 cells; conversely, Rsf-1 overexpression increased M14 cell viability. (B) A colony formation assay (culture dish diameter, 6 cm) demonstrated that the colony number was reduced in MV3 and A375 cells transfected with Rsf-1 siRNA, while Rsf-1 overexpression promoted colony formation ability in M14 cells. (C) A Transwell invasion assay (24-well plate) revealed that the number of invading cells decreased following Rsf-1 depletion in MV3 and A375, and increased following Rsf-1 overexpression in M14 cells. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. Magnification, ×200. Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: MTT Assay, Over Expression, Colony Assay, Transfection, Transwell Invasion Assay, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates cell cycle progression of melanoma and expression of MMP2, cyclin E and p-IκB. (A) Cell cycle analysis revealed that Rsf-1 depletion increased the percentage of G1 phase cells and decreased that of S phase cells in MV3 and A375 cell groups; Rsf-1 overexpression in M14 cells exhibited opposing effects. (B) Western blotting demonstrated that Rsf-1 depletion decreased the levels of MMP2, cyclin E and p-IκB in MV3 and A375 cell lines. Rsf-1 overexpression upregulated expression of MMP2, cyclin E and p-IκB in M14 cells. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. MMP2, matrix metalloproteinase-2; p, phosphorylated; Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: Expressing, Cell Cycle Assay, Over Expression, Western Blot, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates chemoresistance and the MMP of melanoma cells. (A) An MTT assay revealed that cell viability was decreased following Rsf-1 depletion in MV3 and A375 cells treated with cisplatin. Rsf-1 overexpression promoted cell viability in M14 cells treated with cisplatin. (B) Annexin V/propidium iodide analysis revealed that the percentage of apoptotic cells was significantly increased in Rsf-1-depleted MV3 and A375 cells compared with controls. Rsf-1 overexpression downregulated cisplatin-induced apoptosis in M14 cells. (C) Rsf-1 overexpression reduced MMP depolarization in M14 cells, while Rsf-1 depletion increased depolarization in MV3 and A375 cells treated with cisplatin. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. FITC, fluorescein isothiocyanate; MMP, mitochondrial membrane potential, Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: MTT Assay, Over Expression, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates Bcl-2 expression via NF-κB signaling. (A) Western blotting revealed that Bax expression levels increased, whereas cIAP1, cIAP2 and Bcl-2 expression decreased significantly following Rsf-1 depletion in MV3 and A375 cells. Rsf-1 overexpression in M14 cells exhibited opposing effects. (B) NF-κB inhibition significantly downregulated p-IκB and NF-κB p65 protein levels in M14 cells. NF-κB inhibition also eradicated the effects of Rsf-1 overexpression on Bcl-2 upregulation. Total IκB expression was markedly altered. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; cIAP1, cellular inhibitor of apoptosis protein 1; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells; p, phosphorylated; Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: Expressing, Western Blot, Over Expression, Inhibition, Standard Deviation, Small Interfering RNA

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside inhibits Srx expression, leading to the hyperoxidation of Prxs caused by reactive oxygen species (ROS) accumulation. ( A , B ) Melanoma M14 and A375 cells were treated with frugoside in ( A ) time-dependent (0.5 μg/mL) and ( B ) dose-dependent manners for 24 h, and the cells were subjected to a western blot analysis with antibodies against Srx, cytosolic Prx (Prx2), mitochondrial Prx (Prx3), and the loading control, tubulin. ( C , D ) M14 ( C ) and A375 ( D ) cells were treated with 200 μM H 2 O 2 for 10 min with 0.5 μg/mL frugoside. After 10 min, H 2 O 2 was washed out, and cells were incubated for the indicated times in a fresh medium. As a control of H 2 O 2 -stimulated samples, sample C was harvested without H 2 O 2 stimulation. The lysates were assessed by a western blot analysis using antibodies against the indicated proteins. ( E , F ) M14 cells were treated with different doses of frugoside for 12 h ( E ) or 0.5 μg/mL of frugoside for different lengths of time ( F ). Dichlorofluorescein (DCF) fluorescence was analyzed with a fluorescence microscope after staining with 5 μM CM-H2DCFDA at 37 °C for 30 min. Representative fluorescent microscope images are shown, with quantified data given as a graph in the right panel. Data represent mean ± SD. p values were derived to assess statistical significance and are indicated as follows: ** p < 0.01; and *** p < 0.001. Original magfication, 100×.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Western Blot, Control, Incubation, Fluorescence, Microscopy, Staining, Derivative Assay

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside induces mitochondria-mediated cell death in melanoma cells. ( A , B ) M14 ( A ) and A375 ( B ) cells were treated with frugoside at the indicated time and dose. The cytotoxicity level was measured by the CCK-8 assay. ( C – F ) M14 and A375 cells were treated with frugoside at the indicated dose for 24 h. The viability of frugoside-treated M14 ( C ) and A375 ( D ) cells was determined by a fluorescence-activated cell sorting (FACS) analysis after propidium iodide (PI)/Annexin V-fluorescein isothiocyanate (V-FITC) staining. The apoptotic cell population (Sub-G1) was measured by PI staining and a flow cytometry analysis in frugoside-treated M14 ( E ) and A375 ( F ) cells. ( G , H ) M14 and A375 cells were treated with various doses of frugoside for 24 h ( G ) or for the indicated periods ( H ; 0.5 μg/mL). The lysates were assessed by a western blot analysis using antibodies against the indicated proteins. ( I , J ) M14 ( I ) and A375 ( J ) cells were treated with frugoside in a dose-dependent manner for 24 h. Cytosolic and mitochondrial fractions were prepared from the frugoside treated cells. The identity of the cytosolic and mitochondrial fractions was verified by the specific marker proteins, tubulin and Prx3. ( K ) M14 and A375 cells were treated with frugoside at the indicated doses for 24 h. The cells were analyzed by a western blot analysis with antibodies against Bcl2 and tubulin. p values were derived to assess statistical significance and are indicated as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: CCK-8 Assay, Fluorescence, FACS, Staining, Flow Cytometry, Western Blot, Marker, Derivative Assay

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside results in mitochondrial dysfunction via ROS overproduction. ( A–F ) Melanoma cells were treated with frugoside in dose- (24 h) and time- (0.5 μg/mL dosage) dependent manners. Stimulated cells were analyzed using flow cytometry for mitochondrial conditions, including the membrane potential, mitochondrial ROS, and calcium overload. The mitochondrial membrane potential was measured using a FACSCanto II apparatus after tetraethylrhodamine ethyl ester (TMRE) staining in M14 and A375 cells treated with various doses of frugoside ( A ) over time ( B ). Mitochondrial ROS was measured in frugoside-treated melanoma cells in dose- ( C ) and time- ( D ) dependent manners via MitoSOX staining. The mitochondrial calcium level was measured using Rhod2-AM fluorescent dye and quantified (dose: E , time: F ). Values are presented as mean ± SD. ( G–I ) Melanoma cells were treated with 0.5 μg/mL frugoside for 12 h and then stained with TMRE, MitoSOX, and Rhod2-AM dye. The membrane potential ( G ), mitochondrial ROS ( H ), and mitochondrial calcium level ( I ) were also analyzed using fluorescence microscopy. p values were derived to assess statistical significance and are indicated as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001. Original magfication, 100×.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Flow Cytometry, Membrane, Staining, Fluorescence, Microscopy, Derivative Assay

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside-mediated ROS accumulation induces sustained p38 mitogen-activated protein kinase (MAPK) activation, and this activation contributes to apoptotic cell death. ( A ) M14 cells were treated with frugoside in time- and dose-dependent manners as described in . The cell lysates were subjected to western blotting with antibodies specific to phosphorylated MAPKs as indicated. The same membrane was re-probed with antibodies to the following MAPKs: c-Jun N-terminal kinases (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK). ( B ) M14 cells were treated with 0.5 μg/mL frugoside in the presence or absence of the p38 MAPK inhibitor, SB202190, or MAP/ERK kinase (MEK) 1/2 inhibitor, U0126, as indicated. The cells were analyzed for cell death using FACSCanto II after PI/Annexin V-FITC staining. ( C ) M14 cells were treated with 0.5 µg/mL frugoside for 24 h after a 1 h of SB202190 pretreatment. The cell lysates were subjected to western blotting with antibodies against poly (ADP-ribose) polymerase (PARP), caspase-3, Bcl2, and tubulin. ( D ) M14 cells were pretreated with 2 mM N-acetyl cysteine (NAC) or 10 μM diphenyleneiodonium (DPI) for 1 h, followed by 1 μg/mL frugoside for 24 h. The cells were subjected to a FACS analysis after PI/Annexin V-FITC staining. ( E ) M14 cells were treated with frugoside at the indicated doses for 24 h in the presence of 2 mM NAC or 10 μM DPI following a 1 h pretreatment, and the cell lysates were subjected to western blotting with the indicated antibodies. ( F ) M14 cells were pretreated with 2 mM NAC or 10 μM DPI for 1 h, followed by 1 μg/mL frugoside for 24 h, and the cells were analyzed with a FACSCanto II after TMRE staining. ( G ) As described in ( D ), frugoside- and antioxidant-treated M14 cells were subjected to western blotting with antibodies against phosphorylated and non-phosphorylated p38 MAPK. p values were derived to assess statistical significance and are indicated as follows: ** p < 0.01; and *** p < 0.001.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activation Assay, Western Blot, Membrane, Staining, Derivative Assay

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Srx is essential for ROS-mediated cell death by frugoside. ( A ) HA-Srx and Mock plasmids were transfected into M14 and A375 cells. After transfection for 24 h, the cells were treated with or without frugoside (0.5 μg/mL) for 12 h, followed by H 2 O 2 treatment (200 μM) for 10 min. H 2 O 2 was removed by replacement with a fresh medium, then the cells were incubated for the indicated times. The lysates were assessed by a western blot analysis using antibodies against the indicated proteins. ( B , C ) M14 ( B ) and A375 ( C ) cells were transfected with HA-Srx-overexpressing or Mock plasmids, and the cells were treated with various doses of frugoside. Cell death was measured by a FACS analysis after PI/Annexin V-FITC staining. ( D ) M14 cells were transfected as indicated in ( B ), and the cells were treated with frugoside in dose- and time-dependent manners. The cell lysates were subjected to western blotting with specific antibodies for caspase-3, HA, phospho-p38, p38, and tubulin. ( E ) Srx-overexpressed M14 cells were treated with frugoside and H 2 O 2 , as described in ( A ), and the cells were separated into cytosolic and mitochondrial fractions. The fractionated lysates were subjected to western blotting with antibodies specific to Prx-SO 2 , Prx3, and tubulin.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Transfection, Incubation, Western Blot, Staining

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside inhibits tumorigenic ability in vitro and in vivo. ( A ) A total of 6 × 10 4 M14 or A375 cells were seeded in a six-well plate. After three days, the cells were treated with frugoside at the indicated dose for one week. The cells were stained with crystal violet solution. Original magfication, 100×. ( B ) M14 and A375 cells were subjected to soft agar assay after 72 h in the medium containing frugoside at the indicated dose, which was changed every five days. The number of colonies generated per 10,000 cells was counted three weeks later. Original magfication, 100×. ( C , D ) Frugoside reduced the tumor volume in M14 and A375-inoculated nude mice. To generate the xenograft model, six-week-old BALB/c nude mice were inoculated with 1 × 10 6 M14 ( C ) and A375 ( D ) cells. After two weeks, frugoside (100 µg/kg in 10% DMSO and 90% PBS) or vehicle were injected once every two days for 17 days. The M14 and A375-inoculated nude mice were sacrificed, and tumor tissue was collected. The photograph shows tumor masses extracted from each group of mice after 17 days (upper panel). ( E ) Schematic illustration: The anti-cancer mechanism of frugoside.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, In Vivo, Staining, Soft Agar Assay, Generated, Injection