Journal: Cellular and Molecular Immunology

Article Title: Human fused NKG2D–IL-15 protein controls xenografted human gastric cancer through the recruitment and activation of NK cells

doi: 10.1038/cmi.2015.81

Figure Lengend Snippet: Binding activity of human dsNKG2D–IL-15 with MICA. (a) The human dsNKG2D–IL-15 protein was identified by the NKG2D antibody or IL-15 antibody through a western blot assay. (b) Plates coated with human dsNKG2D–IL-15, human NKG2D–Ig proteins, and IL-15 were bound by a human NKG2D antibody or an IL-15 antibody and were detected by ELISA. (c) Wells were coated with a soluble MICA protein (1 μg) or soluble RAE-1ε (1 μg). Human dsNKG2D–IL-15 was added and incubated at 37°C for 1 h. The NKG2D mAb, NKG2D pAb, or IL-15 pAb and HRP-conjugated secondary antibodies were added sequentially to the wells. The OD 450 value of each well was read after the addition of the substrate and stop solution. (d) All tumor cells were incubated with various concentrations of human dsNKG2D–IL-15, stained with a fluorescent IL-15 mAb and detected by flow cytometry. Gray area: isotype control; dotted line: IL-15 (5 μg mL−1) incubation and IL-15 mAb staining; and dashed line: human dsNKG2D–IL-15 incubation and IL-15 mAb staining. (e) B16-MICA cells were incubated with human or mouse dsNKG2D–IL-15 and detected with an IL-15 pAb, NKG2D mAb, or NKG2D pAb with or without MICA mAb neutralization. (f) B16BL6–MICA cells were co-incubated with serial concentrations of the human dsNKG2D–IL-15, mouse dsNKG2D–IL-15 or human NKG2D–Ig proteins (5 μg mL−1), stained with goat anti-human IgG antibody, and detected by flow cytometry. *P < 0.05, **P < 0.01.

Article Snippet: The K562, HeLa, and B16BL6 cell lines were all from ATCC.

Techniques: Binding Assay, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Staining, Flow Cytometry, Control, Neutralization

Journal: Cellular and Molecular Immunology

Article Title: Human fused NKG2D–IL-15 protein controls xenografted human gastric cancer through the recruitment and activation of NK cells

doi: 10.1038/cmi.2015.81

Figure Lengend Snippet: Anti-tumor effects of human dsNKG2D–IL-15depended mainly on NK cells. (a) B16BL6–MICA or B16 cells were injected into the C57BL/6 mice (n = 6, 2 × 106 cells). Human dsNKG2D–IL-15 (60 μg), mouse dsNKG2D–IL-15 (60 μg), or PBS was also intraperitoneally injected daily into these mice from days 5 to 21. Tumor growth was measured daily and was shown as the means ± SD. The upper asterisks represent the comparison between human dsNKG2D–IL-15 and PBS, and the lower asterisks represent the comparison between human dsNKG2D–IL-15 and mouse dsNKG2D–IL-15. All mice were killed on day 22, and their spleens were collected. The frequencies of splenic NK1.1+NKG2D+ cells (b), CD8+NKG2D+ T cells and CD8+CD44+ T cells (c) were detected by flow cytometry. *P < 0.05, **P < 0.01. (d) Growth curve of B16-MICA transplanted tumors in mice treated with human dsNKG2D–IL-15 from day 5 following either NK or CD8+ T cell depletion (n = 5). The upper asterisks represent the comparison between human dsNKG2D–IL-15 and NK cell depletion, and the lower asterisks represent the comparison between human dsNKG2D–IL-15 and CD8+ T-cell depletion. The experiments were performed twice.

Article Snippet: The K562, HeLa, and B16BL6 cell lines were all from ATCC.

Techniques: Injection, Comparison, Flow Cytometry

Journal: Biomolecules

Article Title: A Cell-Based Evaluation of the Tyrosinase-Mediated Metabolic Activation of Leukoderma-Inducing Phenols, II: The Depletion of Nrf2 Augments the Cytotoxic Effect Evoked by Tyrosinase in Melanogenic Cells

doi: 10.3390/biom15010114

Figure Lengend Snippet: Nrf2 knockdown enhances the reduction in viability of melanoma cells following their exposure to leukoderma-inducing phenolic compounds in a tyrosinase-dependent manner. B16BL6 melanoma cells were transfected with a negative control siRNA ( Ctrl ) or a siRNA directed against Nrf2 (#1, MSS275988) or Tyr (#1, MSS212191) for 24 h. ( A , D ) Levels of Nrf2 and Tyr mRNA following 24 h incubation in control medium. ( B , C , E , F ) The viability of cells following exposure to the indicated concentration of compounds for 24 h or 48 h. Data are presented as the means ± SD of independent experiments (n = 3 for ( A ) or as the number indicated for ( C , F )) or the means ± SD (n = 3 wells) with similar results in more than two independent experiments (for ( B , D , E )). *, p < 0.05, versus control siRNA-transfected cells (for ( A , D )) or between respective values. B16BL6 melanoma cells (for G – J ) were transfected with a siRNA directed against Nrf2 (#2, MSS207018), Tyr (#2, MSS212190), or a negative control siRNA ( Ctrl ) for 24 h. B16-4A5 melanoma cells (for ( K – M )) were transfected with Nrf2 (#1) and Tyr (#1). ( G , K , M ). Levels of Nrf2 or Tyr mRNA following 24 h incubation in control medium. ( H , I , J , L ) Viability of the cells following exposure to the indicated concentration of compounds for 24 h or 48 h. Data represent means ± SD (n = 3 wells). Similar results were obtained in two independent experiments (for ( H , I , J , L )). For M, data represent the means ± SD of independent experiments (as the number indicated). *, p < 0.05, between respective values or versus control siRNA-transfected cells (for ( G , K )).

Article Snippet: Mouse melanoma cell lines B16BL6 and B16 melanoma 4A5 (B16-4A5) were purchased from the RIKEN BioResource Research Center (Tsukuba, Japan) and maintained in RPMI1640 medium and Dulbecco’s modified Eagle’s medium (DMEM), respectively, containing 10% FBS.

Techniques: Knockdown, Transfection, Negative Control, Incubation, Control, Concentration Assay

Journal: Biomolecules

Article Title: A Cell-Based Evaluation of the Tyrosinase-Mediated Metabolic Activation of Leukoderma-Inducing Phenols, II: The Depletion of Nrf2 Augments the Cytotoxic Effect Evoked by Tyrosinase in Melanogenic Cells

doi: 10.3390/biom15010114

Figure Lengend Snippet: Nrf2 knockdown reduces GSH , Slc7a11, and Nqo1 mRNA levels and effectively prevents their induction by phenolic compounds. B16BL6 melanoma cells were transfected with a negative control siRNA ( Ctrl ) or a siRNA directed against Nrf2 (#1) for 24 h. The cells were further incubated in the medium containing a vehicle or compounds (0.3 mM) for 24 h. ( A , B ) Cellular GSH levels. Data represent means ± SD (n = 3 wells). Similar results were obtained in two independent experiments. ( C , D ) Levels of Slc7a11 , Nqo1 , and Nrf2 mRNA. Data represent means ± SD of independent experiments (n = 3 for ( C ), or as the number indicated for ( D )). *, p < 0.05, versus control siRNA-transfected cells; #, p < 0.05, versus vehicle-treated cells.

Article Snippet: Mouse melanoma cell lines B16BL6 and B16 melanoma 4A5 (B16-4A5) were purchased from the RIKEN BioResource Research Center (Tsukuba, Japan) and maintained in RPMI1640 medium and Dulbecco’s modified Eagle’s medium (DMEM), respectively, containing 10% FBS.

Techniques: Knockdown, Transfection, Negative Control, Incubation, Control

Journal: Biomolecules

Article Title: A Cell-Based Evaluation of the Tyrosinase-Mediated Metabolic Activation of Leukoderma-Inducing Phenols, II: The Depletion of Nrf2 Augments the Cytotoxic Effect Evoked by Tyrosinase in Melanogenic Cells

doi: 10.3390/biom15010114

Figure Lengend Snippet: Slc7a11 knockdown enhances the cytotoxicity induced by RD, RK, and pCRE, whereas Nqo1 knockdown or inhibition is ineffective for most compounds. B16BL6 melanoma cells were transfected with a negative control siRNA ( Ctrl ) or a siRNA directed against Slc7a11 or Nqo1 for 24 h. ( A ) Slc7a11 and Nqo1 mRNA levels following 24 h incubation in a control medium. ( B – D ) The viability of cells following exposure to the indicated concentration of compounds for 24 h or 48 h. ( E , F ) The viability of B16BL6 cells after exposure to the indicated concentration of phenolic compounds in the presence or absence of ES936 (500 nM) for 24 h. ( F ) Cells were pretreated with a vehicle or ES936 for 24 h before their exposure to the compounds with/without ES936. ( G ) Western blots and quantitation of tyrosinase and the loading control of β-actin in cells at 48 h after Nqo1 -siRNA transfection or 24 h after ES936 treatment. Data represent means ± SD (n = 3 wells) with similar results in two independent experiments (for ( A – F )) or means ± SD of independent experiments (n = 4 for RD in panel C, D or n = 3 for 4SCAP in panel D). *, p < 0.05, versus Ctrl -siRNA-transfected cells (for ( A )) or between respective values (for ( B – F )). B16-4A5 melanoma cells were transfected with a negative control siRNA ( Ctrl ) or a siRNA directed against Slc7a11 or Nqo1 for 24 h. ( H ) Levels of Slc7a11 and Nqo1 mRNA following 24 h incubation in a control medium. ( I ) The viability of cells following exposure to the indicated concentration of compounds for 24 h or 48 h. Data represent means ± SD (n = 3 wells). Similar results were obtained in two independent experiments (for ( I )). *, p < 0.05, versus Ctrl -siRNA-transfected cells.

Article Snippet: Mouse melanoma cell lines B16BL6 and B16 melanoma 4A5 (B16-4A5) were purchased from the RIKEN BioResource Research Center (Tsukuba, Japan) and maintained in RPMI1640 medium and Dulbecco’s modified Eagle’s medium (DMEM), respectively, containing 10% FBS.

Techniques: Knockdown, Inhibition, Transfection, Negative Control, Incubation, Control, Concentration Assay, Western Blot, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pterostilbene, a Dimethyl Derivative of Resveratrol, Exerts Cytotoxic Effects on Melanin-Producing Cells through Metabolic Activation by Tyrosinase

doi: 10.3390/ijms25189990

Figure Lengend Snippet: ( a ) Level of Tyr mRNA in siRNA-transfected cells. ( b ) 4SCAP, ( c ) RES ( 1 ), and ( d ) PTS ( 2 ) induce tyrosinase-dependent reductions in the viability of B16BL6 melanoma cells. B16BL6 melanoma cells were transfected with a negative control siRNA or with a siRNA directed against Tyr for 24 h. The cells were then treated with the indicated concentrations of compounds for 24 h, and their viability and mRNA levels (in vehicle-treated cells) were measured. Data represent means ± SD ( n = 3 wells). The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and representative. * p < 0.05 between siRNA control and siRNA Tyr at each treatment concentration (in µM). #1: p = 0.017 between RES ( 1 ) and PTS ( 2 ) in siRNA Ctrl. #2: p = 0.020 between RES ( 1 ) and PTS ( 2 ) in siRNA Tyr at 100 µM, respectively.

Article Snippet: B16BL6 mouse melanoma cells were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan) and were maintained in RPMI1640 medium containing 10% FBS.

Techniques: Transfection, Negative Control, Control, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Pterostilbene, a Dimethyl Derivative of Resveratrol, Exerts Cytotoxic Effects on Melanin-Producing Cells through Metabolic Activation by Tyrosinase

doi: 10.3390/ijms25189990

Figure Lengend Snippet: ( a , b ): Eumelanin and pheomelanin contents in B16BL6 melanoma cells. Data represent means ± SD ( n = 3 wells). p value between control (0 μM) and each treatment concentration (µM) at eumelanin and pheomelanin in B16BL6 melanoma cells. Statistically significant differences: * p < 0.01, ** p < 0.001. ( c , d ): Eumelanin and pheomelanin contents in the medium. p value between control (0 μM) and each treatment concentration (µM) at eumelanin and pheomelanin in the medium. * p < 0.01, ** p < 0.001. #1: p = 0.026 between RES ( 1 ) and PTS ( 2 ) in eumelanin values at 3 µM. #2: p = 0.002 between RES ( 1 ) and PTS ( 2 ) in pheomelanin values at 3 µM.

Article Snippet: B16BL6 mouse melanoma cells were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan) and were maintained in RPMI1640 medium containing 10% FBS.

Techniques: Control, Concentration Assay