Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Synthesis, in silico modelling, and in vitro biological evaluation of substituted pyrazole derivatives as potential anti-skin cancer, anti-tyrosinase, and antioxidant agents

doi: 10.1080/14756366.2023.2205042

Figure Lengend Snippet: Cytotoxicity of pyrazole (celecoxib analogs), isoxazole, pyrazolone, and positive control compounds P1 – P25 (structures, Figure 3 ) against cells of human cutaneous melanoma and non-melanoma skin cancer lines relative to standard control noncancerous immortalised HaCaT cells.

Article Snippet: Human-derived GFP-expressing melanoma A375 and epidermoid carcinoma A431 cell lines were purchased from Angio-Proteomie (Boston, MA).

Techniques: Positive Control, Control

Journal: Molecular Oncology

Article Title: RIPK 4 function interferes with melanoma cell adhesion and metastasis

doi: 10.1002/1878-0261.70220

Figure Lengend Snippet: RIPK4 downregulation impairs the lung colonization capacity of melanoma cells. (A) Western blot analysis of RIPK4 protein levels in A375 RIPK4.KO (clones #1 and #2) and WM266.4 RIPK4.KO cells, together with their respective negative controls, including densitometric quantification. GAPDH served as a loading control. Data are presented as mean ± SD from three independent biological replicates ( n = 3). (B) Representative H&E, PRAME, and RIPK4 immunohistochemical staining of lung sections obtained from NOD/SCID mice injected intravenously with A375 neg ( n = 13), A375 RIPK4.KO (clone #1; n = 12), WM266.4 neg ( n = 9), and WM266.4 RIPK4.KO ( n = 10) cells. Scale bar = 500 μm. (C) Dot plot representing the number and total area of metastatic foci in lung sections. Animals were randomly assigned to experimental groups, and histological analysis was performed blinded. (D) Higher‐magnification immunohistochemical staining of RIPK4 in melanoma lesions within the lung, derived from xenografts of RIPK4.KO and corresponding negative‐control cells. Dotted lines separate images from two different melanoma lesions. Scale bar = 50 μm. Statistical analysis was performed using ANOVA or two‐tailed unpaired Student's t ‐test, **** P < 0.0001.

Article Snippet: Human melanoma cell line A375 (RRID: CVCL_0132) was obtained from ATCC (Manassas, VA, USA) in 2020, and WM266.4 (RRID: CVCL_2765) was kindly provided by the Department of Medical Biochemistry, Jagiellonian University Medical College (Kraków, Poland) in 2006.

Techniques: Western Blot, Clone Assay, Control, Immunohistochemical staining, Staining, Injection, Derivative Assay, Negative Control, Two Tailed Test

Journal: Molecular Oncology

Article Title: RIPK 4 function interferes with melanoma cell adhesion and metastasis

doi: 10.1002/1878-0261.70220

Figure Lengend Snippet: RIPK4 regulates melanoma cell motility via adhesion molecules. (A) GO enrichment analysis of differentially expressed genes (DEGs) identified by RNA‐seq following RIPK4 knockout in A375 cells. The bubble plot displays the top 30 significantly enriched biological processes. The size of each bubble represents the number of DEGs associated with each term, and the colour indicates statistical significance. (B) Western blot analysis of N‐cadherin, MCAM, EphA2 and JAM‐C expression levels in RIPK4.KO and control cells. GAPDH served as a loading control. A representative GAPDH band is shown. (C) Densitometric quantification of analysed protein levels, normalized to the corresponding GAPDH band obtained from the same membrane and the same sample. Data are presented as mean ± SD from three independent biological replicates ( n = 3). (D) Analysis of A375 and WM266.4 cells with RIPK4 knockout (RIPK4.KO) and their respective negative controls. Transmigration (top) and invasion (bottom) assays. Scale bar = 100 μm. Bar plots show quantification of cell migration and invasion, presented as mean ± SD from three independent experiments ( n = 3). Statistical analysis was performed using ANOVA or two‐tailed unpaired Student's t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human melanoma cell line A375 (RRID: CVCL_0132) was obtained from ATCC (Manassas, VA, USA) in 2020, and WM266.4 (RRID: CVCL_2765) was kindly provided by the Department of Medical Biochemistry, Jagiellonian University Medical College (Kraków, Poland) in 2006.

Techniques: RNA Sequencing, Knock-Out, Western Blot, Expressing, Control, Membrane, Transmigration Assay, Migration, Two Tailed Test

Journal: Molecular Oncology

Article Title: RIPK 4 function interferes with melanoma cell adhesion and metastasis

doi: 10.1002/1878-0261.70220

Figure Lengend Snippet: RIPK4 interferes with the adhesive status of melanoma cells in 3D spheroid model. (A) Phase‐contrast images of spheroids formed by RIPK4.KO and negative‐control cells. Scale bar = 500 μm. (B) Quantification of spheroid area. Each dot represents an individual spheroid (20 spheroids per condition). Data are shown as mean ± SD from four independent biological experiments ( n = 4), each performed in quintuplicate. (C) Western blot analysis of EphA2, ITGAV, ITGA2 and THBS1 protein levels in RIPK4.KO and control spheroids. GAPDH was used as a loading control. Data are presented as mean ± SD from three independent biological experiments for integrin αV and integrin α2 ( n = 3‐4) and five independent biological experiments for EphA2 and THBS1 ( n = 4‐5), with 10 spheroids pooled per sample. (D) Relative mRNA expression levels of ECM‐related genes in spheroids assessed using the TaqMan Array Human Extracellular Matrix and Adhesion Molecules panel. Data are presented as fold change relative to the negative control from two independent biological experiments ( n = 2), with six spheroids pooled per sample. These data are also visualized using Venn diagrams showing genes uniquely upregulated, uniquely downregulated, or shared between the groups. Circles represent A375 RIPK4.KO clone #1 (blue), clone #2 (pink), and WM266.4 RIPK4.KO (green) cells. Statistical analysis was performed using ANOVA or two‐tailed unpaired Student's t ‐test * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human melanoma cell line A375 (RRID: CVCL_0132) was obtained from ATCC (Manassas, VA, USA) in 2020, and WM266.4 (RRID: CVCL_2765) was kindly provided by the Department of Medical Biochemistry, Jagiellonian University Medical College (Kraków, Poland) in 2006.

Techniques: Adhesive, Negative Control, Western Blot, Control, Expressing, Two Tailed Test

Journal: Molecular Oncology

Article Title: RIPK 4 function interferes with melanoma cell adhesion and metastasis

doi: 10.1002/1878-0261.70220

Figure Lengend Snippet: RIPK4 knockout promotes amoeboid phenotype of melanoma cells. (A) Morphology and cytoskeleton architecture of A375 RIPK4.KO (clone #1), WM266.4 RIPK4.KO and their respective negative controls. Vinculin (green) is visualized using TIRF microscopy; F‐actin cytoskeleton (red) and cell nuclei (blue) are visualized with epifluorescence microscopy. Corresponding differential interference contrast (DIC) images present cells' morphology. Bleb‐like structures are indicated with white arrows. Scale bars: 20 μm. (B) Motility of melanoma A375 RIPK4.KO (clone #1), WM266.4 RIPK4.KO and their respective negative control cells under 2D conditions. For each condition, data are presented as mean ± SD for n = 225 cells pooled from three independent experiments. Statistical analysis was performed using two‐tailed unpaired Student's t ‐test. (C) RNA‐seq data from A375 RIPK4.KO (clone #1) and control cells, presented as heatmaps showing DEGs associated with the amoeboid mode of cell migration (upper panel; fold change > 1.5, FDR‐adj. P < 0.05) and the ROCK‐myosin II pathway (lower panel; genes highlighted in green indicate FDR‐adj. P < 0.05). Red‐blue gradient indicates the expression of transcript – red: upregulation; blue: downregulation. (D) Protein levels of phospho‐MLC2 and MLC2 detected by western blot. GAPDH was used as a loading control. A representative GAPDH band is shown. Bars represent mean ± SD from at least four independent biological replicates ( n ≥ 4). Statistical analysis was performed using a two‐tailed unpaired Student's t ‐test, * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Human melanoma cell line A375 (RRID: CVCL_0132) was obtained from ATCC (Manassas, VA, USA) in 2020, and WM266.4 (RRID: CVCL_2765) was kindly provided by the Department of Medical Biochemistry, Jagiellonian University Medical College (Kraków, Poland) in 2006.

Techniques: Knock-Out, Microscopy, Epifluorescence Microscopy, Negative Control, Two Tailed Test, RNA Sequencing, Control, Migration, Expressing, Western Blot

Journal: Molecular Oncology

Article Title: RIPK 4 function interferes with melanoma cell adhesion and metastasis

doi: 10.1002/1878-0261.70220

Figure Lengend Snippet: RIPK4 knockout promotes an incomplete amoeboid phenotype in collagen matrices. (A) Microphotographs of melanoma cells in 3D, presented as maximum projections of Z‐stacks captured at increasing magnifications and resolutions, showing the F‐actin cytoskeleton (red) and cell nuclei (blue). Top rows depict wider fields of view, while the bottom row shows higher magnifications of selected regions. Scale bar: 100 μm (top row and middle row) or 50 μm (bottom row). (B) Motile activity of melanoma A375 RIPK4.KO (clone #1 and #2) and WM266.4 RIPK4.KO cells and their negative controls in 3D during 3 h of time‐lapse recording. For each condition, motility was analysed in 75 individual cells, and data are presented as mean ± SD. Statistical analysis was performed using one‐way ANOVA or a two‐tailed unpaired Student's t ‐test, *P < 0.05, **** P < 0.0001.

Article Snippet: Human melanoma cell line A375 (RRID: CVCL_0132) was obtained from ATCC (Manassas, VA, USA) in 2020, and WM266.4 (RRID: CVCL_2765) was kindly provided by the Department of Medical Biochemistry, Jagiellonian University Medical College (Kraków, Poland) in 2006.

Techniques: Knock-Out, Activity Assay, Two Tailed Test

Journal: Molecular Oncology

Article Title: RIPK 4 function interferes with melanoma cell adhesion and metastasis

doi: 10.1002/1878-0261.70220

Figure Lengend Snippet: RIPK4 rescue restores a mesenchymal phenotype of A375 RIPK4.KO cells in collagen matrices. (A) A375 RIPK4.KO (clone #1) cells were transfected with a full‐length RIPK4 construct fused to a GFP tag, or with an empty GFP‐expressing vector as a control. (B) Representative image of GFP following transfection. Scale bar = 250 μm. (C) Western blot analysis of RIPK4, phospho‐MLC2 and total MLC2 levels. GAPDH was used as a loading control. Densitometric quantification is presented as bar plots showing the mean ± SD from three independent biological replicates ( n = 3). (D) Quantification of migration of individual GFP‐positive cells in 3D collagen gel over 3 h of time‐lapse imaging. Data are presented as mean ± SD, each dot represents a single cell from n = 50 cells (empty vector/EGFP) or n = 45 cells (RIPK4 rescue). (E) Microphotographs of melanoma cells in 3D, presented as maximum projections of Z‐stacks (upper row) or as magnified single‐plane images (bottom row), showing the F‐actin cytoskeleton (red), GFP (green) and cell nuclei (blue). Scale bar: 25 μm (upper row) and 10 μm (bottom row). Statistical analysis was performed using two‐tailed unpaired Student's t ‐test, ** P < 0.01, *** P < 0.001.

Article Snippet: Human melanoma cell line A375 (RRID: CVCL_0132) was obtained from ATCC (Manassas, VA, USA) in 2020, and WM266.4 (RRID: CVCL_2765) was kindly provided by the Department of Medical Biochemistry, Jagiellonian University Medical College (Kraków, Poland) in 2006.

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Control, Western Blot, Migration, Imaging, Single Cell, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effect of DOX on cell viability of A375 and MNT-1 cells. Cells were exposed to different concentrations of DOX for 24, 48, and 72 h, and cell viability was determined using MTT assay. Data shown are mean values ± standard deviation of three independent experiments with four technical replicates each. *—indicates statistical significance in comparison to the respective control ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: MTT Assay, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Inhibitory concentrations (ICs) obtained for 24, 48, and 72 h DOX exposure. Values are expressed in μM.

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effect of hyperthermia plus DOX on cell viability of A375 and MNT-1 cells. Cells were exposed to 43 °C for 30, 60, or 120 min, plus 0.012 μM or 0.043 μM and 0.68 μM or 1.38 μM during 24 h; 0.0056 μM or 0.0125 μM and 0.0066 μM or 0.0179 μM during 48 h; and 0.0012 μM or 0.0026 μM and 0.0042 μM or 0.0098 μM during 72 h; in cases of A375 or MNT-1, respectively. DMSO concentrations correspond to the equivalent percentage present in IC 20 of each cell line and time exposure. DOX concentrations correspond to the calculated IC 10 and IC 20 for each time exposure and for each cell line. Cell viability was determined using MTT assay. Data are shown as mean ± standard deviation of two independent experiments with four technical replicates each. *—indicates statistical significance in comparison to the control 37 °C; α indicates statistical significance in comparison to the respective control of each condition at 37 °C; and β indicates statistical significance of the combined treatment in comparison to hyperthermia alone ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: MTT Assay, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effect of hyperthermia plus DOX on morphology of A375 and MNT-1 cells. Cells were exposed to 43 °C for 30 min and 0.0125 μM or 0.0179 μM of DOX, in case of A375 or MNT-1 cells, respectively. ( A )—A375 cells; ( B )—MNT-1 cells.

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effects of hyperthermia combined with DOX on cell cycle distribution. Cells were exposed to 43 °C for 30 min and 0.0125 μM or 0.0179 μM of DOX, in case of A375 or MNT-1 cells, respectively. ( A ) Cell cycle distribution (%) in A375 and MNT-1 cells; ( B ) histograms representative of cell distribution of A375 and MNT-1 cells. Data shown are mean values ± standard deviation of two independent experiments with two technical replicates each and each replicate with at least 5000 events. *—indicates statistical significance in comparison to the control 37 °C; α indicates statistical significance in comparison to the respective control of each condition at 37 °C; and β indicates statistical significance of the combined treatment in comparison to hyperthermia alone ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effects of hyperthermia combined with DOX on production of intracellular ROS. Cells were exposed to 43 °C for 30 min and 0.0125 μM or 0.0179 μM of DOX for 48 h, in case of A375 or MNT-1 cells, respectively. ( A ) Relative abundance of intracellular ROS of A375 and MNT-1 cells; ( B ) histograms representative of abundance of intracellular ROS of A375 and MNT-1 cells. Data shown are mean values ± standard deviation of two independent experiments with two technical replicates each and each replicate with at least 5000 events. *—indicates statistical significance in comparison to the control 37 °C; α indicates statistical significance in comparison to the respective control of each condition at 37 °C; and β indicates statistical significance of the combined treatment in comparison to hyperthermia alone ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effects of hyperthermia in combination with DOX on apoptotic profile. Both cell lines were exposed to 43 °C for 30 min and A375 cells were treated with 0.0125 μM and MNT-1 cells with 0.0179 μM of DOX for 48 h. ( A ) Percentage of apoptotic cells after treatment in populations corresponding to viable and non-apoptotic, early and late apoptotic A375 and MNT-1 cells; ( B ) histograms representative of Annexin V-FITC. Data shown are mean values ± standard deviation of two independent experiments with two technical replicates each and each replicate with at least 5000 events. *— indicates statistical significance in comparison to the control 37 °C; α indicates statistical significance in comparison to the respective control of each condition at 37 °C; and β indicates statistical significance of the combined treatment in comparison to hyperthermia alone ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: Standard Deviation, Comparison, Control