a375 human malignant melanoma cell line  (ATCC)

Journal: Current Issues in Molecular Biology

Article Title: Resveratrol Targets Glycolytic Enzymes HK II and PKM2 to Promote Concurrent Apoptotic and Necrotic Cell Death in Malignant Melanoma

doi: 10.3390/cimb47121006

Figure Lengend Snippet: Effects of resveratrol on cell viability and morphology in malignant melanoma cells. ( A ) Chemical structure of resveratrol (RSV). ( B ) Cell viabilities of non-tumorigenic human epidermal melanocytes (HEMn-MP) and malignant melanoma cell lines (G361 and SK-MEL-24) after 48 h of RSV treatment, assessed by MTT assay. Results are presented as means ± Standard error (SE) of three independent experiments (* p < 0.05). ( C ) Representative phase-contrast images showing morphological alterations in HEMn-MP, G361, and SK-MEL-24 cells following RSV exposure for 48 h (scale bar = 50 μm; original magnification ×400). RSV: resveratrol.

Article Snippet: Normal human epidermal melanocytes (HEMn-MP) were obtained from Cascade Biologics (Portland, OR, USA), and the human malignant melanoma cell lines G361 and SK-MEL-24 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: MTT Assay

Journal: Current Issues in Molecular Biology

Article Title: Resveratrol Targets Glycolytic Enzymes HK II and PKM2 to Promote Concurrent Apoptotic and Necrotic Cell Death in Malignant Melanoma

doi: 10.3390/cimb47121006

Figure Lengend Snippet: Resveratrol induces apoptotic and necroptotic cell death in malignant melanoma cells. G361 and SK-MEL-24 cells were treated with RSV (20, 40, 60 μM) for 48 h. ( A ) Representative fluorescence images of DAPI-stained nuclei (scale bar = 25 μm; original magnification ×200). Condensed or fragmented nuclei, indicative of apoptosis, are indicated by white arrows. ( B ) Quantification of apoptotic cell populations by Annexin-V/7-AAD staining using a Muse™ Cell Analyzer. Annexin-V-positive/7-AAD-negative cells were classified as early apoptotic, whereas Annexin-V-positive/7-AAD-positive cells were classified as late apoptotic. Data are presented as mean ± SE ( n = 3). Statistical significance: * p < 0.05, ** p < 0.01, **** p < 0.0001. RSV: resveratrol.

Article Snippet: Normal human epidermal melanocytes (HEMn-MP) were obtained from Cascade Biologics (Portland, OR, USA), and the human malignant melanoma cell lines G361 and SK-MEL-24 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence, Staining

Journal: Current Issues in Molecular Biology

Article Title: Resveratrol Targets Glycolytic Enzymes HK II and PKM2 to Promote Concurrent Apoptotic and Necrotic Cell Death in Malignant Melanoma

doi: 10.3390/cimb47121006

Figure Lengend Snippet: Resveratrol induced G0/G1 cell cycle arrest in malignant melanoma cells. G361 and SK-MEL-24 cells were treated with RSV (0, 20, 40, 60 µM) for 48 h. ( A ) Representative histograms of cell cycle distribution following staining with Muse™ Cell Cycle Reagent and analysis on a Muse™ Cell Analyzer. ( B ) Quantitative analysis of the percentage of cells in G0/G1, S, and G2/M phases. Data represent mean ± SE ( n = 3). ( C ) Western blot analysis of cell cycle regulatory proteins (cyclin D1, cyclin E1, and CDK4) after 48 h of RSV treatment. β-Actin served as a loading control. Relative band intensities were quantified against the control. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (post hoc test). RSV: resveratrol.

Article Snippet: Normal human epidermal melanocytes (HEMn-MP) were obtained from Cascade Biologics (Portland, OR, USA), and the human malignant melanoma cell lines G361 and SK-MEL-24 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Western Blot, Control

Journal: Current Issues in Molecular Biology

Article Title: Resveratrol Targets Glycolytic Enzymes HK II and PKM2 to Promote Concurrent Apoptotic and Necrotic Cell Death in Malignant Melanoma

doi: 10.3390/cimb47121006

Figure Lengend Snippet: Resveratrol modulates glycolytic enzymes and induces apoptosis and necroptosis in malignant melanoma cells. G361 and SK-MEL-24 cells were treated with RSV (0, 20, 40, 60 µM) for 48 h. ( A ) Western blot analysis of HK II, PKM2, and apoptosis-related proteins (cleaved caspase-3, caspase-3, Bax, and Bcl-2). β-Actin served as loading control. ( B ) Western blot analysis of necroptosis-associated proteins, including phosphorylated MLKL (p-MLKL) and phosphorylated RIP (p-RIP). Total MLKL and total RIP were used as loading controls. ( C ) Intracellular ATP levels. ( D ) Hexokinase enzymatic activity. ( E ) Caspase-3/7 activity. Data are expressed as mean ± SE ( n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). HK II: Hexokinase II; PKM2: pyruvate kinase M2; RSV: resveratrol.

Article Snippet: Normal human epidermal melanocytes (HEMn-MP) were obtained from Cascade Biologics (Portland, OR, USA), and the human malignant melanoma cell lines G361 and SK-MEL-24 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Control, Activity Assay

Journal: Current Issues in Molecular Biology

Article Title: Resveratrol Targets Glycolytic Enzymes HK II and PKM2 to Promote Concurrent Apoptotic and Necrotic Cell Death in Malignant Melanoma

doi: 10.3390/cimb47121006

Figure Lengend Snippet: Resveratrol suppresses the migratory ability of malignant melanoma cells. A wound healing assay was performed in G361 ( A ) and SK-MEL-24 ( B ) cells treated with RSV (0, 20, 40, 60 μM) for 48 h. Assays were conducted under both 5% FBS-supplemented and serum-free conditions to minimize confounding effects of proliferation. Wound areas were imaged at 0 h and 48 h to assess cell migration (scale bar = 50 μm; original magnification ×400). Data are presented as mean ± SE ( n = 3). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (post hoc test). RSV: resveratrol.

Article Snippet: Normal human epidermal melanocytes (HEMn-MP) were obtained from Cascade Biologics (Portland, OR, USA), and the human malignant melanoma cell lines G361 and SK-MEL-24 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Wound Healing Assay, Migration

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircANKRD52 Augments the Growth and Invasion of Melanoma Cells by Sponging miR ‐141‐3p and Upregulating PRKACB

doi: 10.1111/jcmm.70909

Figure Lengend Snippet: Identification of a circANKRD52 in melanoma cells. (A) The genomic loci of circANKRD52. (B) RT‐qPCR analysis of the expression levels of circANKRD52 and ANKRD52 after treatment with RNase R in A375 and SK‐MEL‐28 cells. (C) RT‐qPCR analysis of the transcriptional stability of circANKRD52 and ANKRD52 after treatment with Actinomycin D in A375 cells. (D) RT‐qPCR analysis of the location of circANKRD52 in A375 and SK‐MEL‐28 cells. (E) FISH analysis of the location of circANKRD52 in A375 and SK‐MEL‐28 cells. Data shown are the mean ± SEM of three experiments. **** p < 0.0001.

Article Snippet: Human malignant melanoma cell lines A375, Hs294T, SK‐MEL‐28 were procured from American Type Culture Collection (ATCC, Manassas, VA, USA) and melanocytes were used as the control cells.

Techniques: Quantitative RT-PCR, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircANKRD52 Augments the Growth and Invasion of Melanoma Cells by Sponging miR ‐141‐3p and Upregulating PRKACB

doi: 10.1111/jcmm.70909

Figure Lengend Snippet: CircANKRD52 augments proliferation and invasion of melanoma cells. (A) EdU analysis of the cell proliferation activity after transfection with oe‐circANKRD52 into A375 cells or sh‐circANKRD52 into SK‐MEL‐28 cells. (B) Transwell analysis of the cell invasion capabilities after transfection with oe‐circANKRD52 into A375 cells or sh‐circANKRD52 into SK‐MEL‐28 cells. (C) Flow cytometry analysis of the cell apoptosis after transfection with oe‐circANKRD52 into A375 cells or sh‐circANKRD52 into SK‐MEL‐28 cells. (D) Tube formation assay analysis of the angiogenesis ability after transfection with oe‐circANKRD52 into A375 cells or sh‐circANKRD52 into SK‐MEL‐28 cells. (E) Western blot analysis of the protein levels of VEGF after transfection with oe‐circANKRD52 into A375 cells or sh‐circANKRD52 into SK‐MEL‐28 cells. Data shown are the mean ± SEM of three experiments. * p < 0.05.

Article Snippet: Human malignant melanoma cell lines A375, Hs294T, SK‐MEL‐28 were procured from American Type Culture Collection (ATCC, Manassas, VA, USA) and melanocytes were used as the control cells.

Techniques: Activity Assay, Transfection, Flow Cytometry, Tube Formation Assay, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircANKRD52 Augments the Growth and Invasion of Melanoma Cells by Sponging miR ‐141‐3p and Upregulating PRKACB

doi: 10.1111/jcmm.70909

Figure Lengend Snippet: The circANKRD52 acts as a sponge of miR‐141‐3p in melanoma cells. (A) The RNA pull‐down analysis of the binding between circANKRD52 probe and miRNA in A375 cells. (B) RT‐qPCR analysis of the expression levels of miR‐141‐3p in melanoma and the adjacent normal tissues. (C) Pearson's correlation analysis of the correlation between miR‐141‐3p and circANKRD52 in melanoma tissues. (D) RT‐qPCR analysis of the expression of miR‐141‐3p in melanoma cell lines (A375, Hs294T, and SK‐MEL‐28). (E) The dual luciferase reporter assay indicated the binding between circANKRD52 3′UTR and miR‐141‐3p. (F) RT‐qPCR analysis of the expression of miR‐141‐3p after transfection with oe‐circANKRD52 into A375 cells or sh‐circANKRD52 into SK‐MEL‐28 cells. (G) RIP analysis of the binding ability between circANKRD52 and Ago2 after transfection with oe‐circANKRD52 into A375 cells or sh‐circANKRD52 into SK‐MEL‐28 cells. (H) RNA pull‐down assay analysis of the binding between Bio‐circANKRD52 WT and miR‐141‐3p. Data shown are the mean ± SEM of three experiments. * p < 0.05.

Article Snippet: Human malignant melanoma cell lines A375, Hs294T, SK‐MEL‐28 were procured from American Type Culture Collection (ATCC, Manassas, VA, USA) and melanocytes were used as the control cells.

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Luciferase, Reporter Assay, Transfection, Pull Down Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircANKRD52 Augments the Growth and Invasion of Melanoma Cells by Sponging miR ‐141‐3p and Upregulating PRKACB

doi: 10.1111/jcmm.70909

Figure Lengend Snippet: The miR‐141‐3p reverses circANKRD52‐induced melanoma cell proliferation and invasion. (A) EdU analysis of the cell proliferation activity after co‐transfection with oe‐circANKRD52 and miR‐141‐3p mimic into A375 cells or sh‐circANKRD52 and miR‐141‐3p inhibitor into SK‐MEL‐28 cells. (B) Transwell analysis of the cell invasion capabilities after co‐transfection with oe‐circANKRD52 and miR‐141‐3p mimic into A375 cells or sh‐circANKRD52 and miR‐141‐3p inhibitor into SK‐MEL‐28 cells. (C) Flow cytometry analysis of the cell apoptosis after co‐transfection with oe‐circANKRD52 and miR‐141‐3p mimic into A375 cells or sh‐circANKRD52 and miR‐141‐3p inhibitor into SK‐MEL‐28 cells. (D) Tube formation assay analysis of the angiogenesis ability after co‐transfection with oe‐circANKRD52 and miR‐141‐3p mimic into A375 cells or sh‐circANKRD52 and miR‐141‐3p inhibitor into SK‐MEL‐28 cells. (E) Western blot analysis of the protein levels of VEGF after co‐transfection with oe‐circANKRD52 and miR‐141‐3p mimic into A375 cells or sh‐circANKRD52 and miR‐141‐3p inhibitor into SK‐MEL‐28 cells. Data shown are the mean ± SEM of three experiments. * p < 0.05.

Article Snippet: Human malignant melanoma cell lines A375, Hs294T, SK‐MEL‐28 were procured from American Type Culture Collection (ATCC, Manassas, VA, USA) and melanocytes were used as the control cells.

Techniques: Activity Assay, Cotransfection, Flow Cytometry, Tube Formation Assay, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircANKRD52 Augments the Growth and Invasion of Melanoma Cells by Sponging miR ‐141‐3p and Upregulating PRKACB

doi: 10.1111/jcmm.70909

Figure Lengend Snippet: PRKACB is identified as a direct target of miR‐141‐3p in melanoma cells. (A) RT‐qPCR analysis of the PRKACB expression in melanoma and the adjacent normal tissues. (B) Pearson's correlation analysis of the correlations of PRKACB with miR‐141‐3p or circANKRD52 expression in melanoma tissues. (C, D) RT‐qPCR and Western blot analysis of the expression of PRKACB in A375, SK‐MEL‐28, and melanocytes. (E) The dual luciferase reporter assay indicated the binding between miR‐141‐3p and PRKACB 3'UTR. (F) RT‐qPCR analysis of the expression of PRKACB after transfection with oe‐circANKRD52 into A375 cells or sh‐circANKRD52 into SK‐MEL‐28 cells. (G) RT‐qPCR analysis of the expression of PRKACB after transfection with miR‐141‐3p mimic into A375 cells or miR‐141‐3p inhibitor into SK‐MEL‐28 cells. Data shown are the mean ± SEM of three experiments. * p < 0.05. # p < 0.05.

Article Snippet: Human malignant melanoma cell lines A375, Hs294T, SK‐MEL‐28 were procured from American Type Culture Collection (ATCC, Manassas, VA, USA) and melanocytes were used as the control cells.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Reporter Assay, Binding Assay, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircANKRD52 Augments the Growth and Invasion of Melanoma Cells by Sponging miR ‐141‐3p and Upregulating PRKACB

doi: 10.1111/jcmm.70909

Figure Lengend Snippet: The circANKRD52/PRKACB axis mediates melanoma cell proliferation and invasion. (A) EdU analysis of the cell proliferation activity after co‐transfection with oe‐circANKRD52 and sh‐PRKACB into A375 cells or sh‐circANKRD52 and oe‐PRKACB into SK‐MEL‐28 cells. (B) Transwell analysis of the cell invasion capabilities after co‐transfection with oe‐circANKRD52 and sh‐PRKACB into A375 cells or sh‐circANKRD52 and oe‐PRKACB into SK‐MEL‐28 cells. (C) Flow cytometry analysis of the cell apoptosis after co‐transfection with oe‐circANKRD52 and sh‐PRKACB into A375 cells or sh‐circANKRD52 and oe‐PRKACB into SK‐MEL‐28 cells. (D) Tube formation assay analysis of the angiogenesis ability after co‐transfection with oe‐circANKRD52 and sh‐PRKACB into A375 cells or sh‐circANKRD52 and oe‐PRKACB into SK‐MEL‐28 cells. (E) Western blot analysis of the protein levels of VEGF after co‐transfection with oe‐circANKRD52 and sh‐PRKACB into A375 cells or sh‐circANKRD52 and oe‐PRKACB into SK‐MEL‐28 cells. Data shown are the mean ± SEM of three experiments. * p < 0.05.

Article Snippet: Human malignant melanoma cell lines A375, Hs294T, SK‐MEL‐28 were procured from American Type Culture Collection (ATCC, Manassas, VA, USA) and melanocytes were used as the control cells.

Techniques: Activity Assay, Cotransfection, Flow Cytometry, Tube Formation Assay, Western Blot