Journal: Frontiers in Pharmacology

Article Title: Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin

doi: 10.3389/fphar.2020.00748

Figure Lengend Snippet: Shikonin inhibited melanoma tumor growth in zebrafish xenograft model. Fluorescently labeled (A) A375 or (B) A2058 cells were microinjected into the yolk sac of 2 dpf zebrafish embryos. These zebrafish were then transferred randomly to 24-well plates, 10 embryos per well with 0.5 ml of embryo medium containing indicated concentrations of shikonin or sorafenib for a treatment period of 48 h. Representative images of embryos were shown as merged bright-field and fluorescent images at 0, 24, and 48 h. Relative fluorescence intensity were analyzed by Image J software. Data were shown as mean ± SD from three independent experiments, ** P < 0.01. Blue arrows indicated pericardia. (C) H&E staining of A375 cell transplanted zebrafish. Tumor cells in yolk were shown (yellow squares).

Article Snippet: Human melanoma A375 and normal human liver-derived cells (MIHA) were purchased from American Type Culture Collection (ATCC, USA), and human melanoma A2058 cells were obtained from Cell Lines Service GmbH (CLS, Germany).

Techniques: Labeling, Fluorescence, Software, Staining

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin

doi: 10.3389/fphar.2020.00748

Figure Lengend Snippet: Shikonin reduced viability and induced apoptosis in human melanoma cells. A375 and A2058 cells were treated with indicated concentrations of shikonin. (A) Cell viability was measured by MTT assay after 24 or 48 h treatment. (B) Represented photos showed the effect of shikonin on clonogenic survival of A375 and A2058 cells. (C) Cell cycle phases of the treated cells were evaluated by flow cytometry and analyzed by ModFit software. The DNA content of cells at different cell phase was presented as the mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01. (D) Apoptosis was analyzed by flow cytometry after 24 h treatment. The percentage of apoptotic cells was presented as the mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01. (E) Cleavages of PARP and caspase-3 were detected by Western blot analysis after 24 h treatment (left panel) and relative expression levels (right panel) were analyzed by Image J software. Data were shown as mean ± SD from three independent experiments, * P < 0.05, ** P < 0.01.

Article Snippet: Human melanoma A375 and normal human liver-derived cells (MIHA) were purchased from American Type Culture Collection (ATCC, USA), and human melanoma A2058 cells were obtained from Cell Lines Service GmbH (CLS, Germany).

Techniques: MTT Assay, Flow Cytometry, Software, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin

doi: 10.3389/fphar.2020.00748

Figure Lengend Snippet: Shikonin inhibited melanoma cell migration and invasion. (A) A single scratch was created in the confluent monolayer of A375 or A2058 cells. The scratch was photographed at 0 and 24 h after shikonin treatment (left) and relative migrated areas (right) were analyzed by Image J software. (B) Cells were allowed to migrate through membranes without or with Matrigel basement in the presence of indicated concentration of shikonin. Representative photographs of migrated/invasive cells (left) and quantification of these cells (right) were shown. (C) Cells were treated with shikonin for 24 h and changes in Tubulin and F-actin were visualized using confocal microscope. Data were shown as mean ± SD from three independent experiments, * P < 0.05 and ** P < 0.01.

Article Snippet: Human melanoma A375 and normal human liver-derived cells (MIHA) were purchased from American Type Culture Collection (ATCC, USA), and human melanoma A2058 cells were obtained from Cell Lines Service GmbH (CLS, Germany).

Techniques: Migration, Software, Concentration Assay, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin

doi: 10.3389/fphar.2020.00748

Figure Lengend Snippet: Shikonin down-regulated protein levels and inhibited enzymatic activities of STAT3-targeted molecules in melanoma cells. A375 and A2058 cells were treated with indicated concentrations of shikonin for 24 h, and then total cell lysates were collected, expression levels of (A) Mcl-1, Bcl-2, (B) MMP-2, (D) Twist, Vimentin, and N-cadherin were evaluated by Western blot analysis (left) and relative expression levels were analyzed by Image J software (right). Data were shown as mean ± SD from three independent experiments, * P < 0.05 and ** P < 0.01. (C) The enzymatic activities of MMP-2 and MMP-9 were determined by gelatinase zymography.

Article Snippet: Human melanoma A375 and normal human liver-derived cells (MIHA) were purchased from American Type Culture Collection (ATCC, USA), and human melanoma A2058 cells were obtained from Cell Lines Service GmbH (CLS, Germany).

Techniques: Expressing, Western Blot, Software, Zymography

Journal: Cell reports

Article Title: The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma.

doi: 10.1016/j.celrep.2019.05.069

Figure Lengend Snippet: Figure 6. MITF IRES-Mediated Translation Dictates Melanoma Phenotype and Metastasis Formation In Vivo (A) CRISPR/Cas9-mediated mutagenesis strategy employed to delete the SL3B region (50 UTR DSL3B) within the MITF 50 UTR in melanoma cells. (B and C) SL3B deletion abrogates MITF translational control without affecting its mRNA. (B) Representative protein analysis shows the levels of MITF and its direct target MLANA in 50 UTR wild-type (WT) or 50 UTR DSL3B HT144 and A2058 melanoma cells. (C) Graphs show relative MITF mRNA levels ± SD measured by qPCR in three independent experiments. No statistical significance was observed in HT144 or A2058 cells (p = 0.244 and p = 0.051, respectively; ANOVA). (D) MITF mRNA is significantly enriched in DDX3X immunoprecipitates from WT cells compared to DSL3B A2058 melanoma cells. Graph shows mean MITF fold change levels to control IgG normalized to input total RNA ± SD measured by qPCR in at least three independent RIP experiments. ****p < 0.0001 (t test). (E) Reduced proliferation of MITF 50 UTR DSL3B melanoma cells compared to controls. Graph shows relative proliferation rates ± SD in 50 UTR WT and DSL3B HT144 and A2058 cells at different time points. ****p < 0.0001; ANOVA with multiple comparison. (F) Increased melanoma cell migratory potential in MITF 50 UTR DSL3B melanoma cells. Representative images of the transwell assay indicate increased migratory potential of DSL3B HT144 and A2058 cells compared to isogenic controls. Graph shows quantification of mean cell migration in 50 UTR DSL3B to WT cells ± SD in two independent experiments. **p < 0.01 (t test). (G) Schematic of xenotransplantation experiments used to study the in vivo tumorigenic potential of MITF 50 UTR WT and DSL3B A2058 cells.

Article Snippet: Protein extracts were denatured in sample buffer containing (10%glycerol, 2% SDS, 62.5 mM Tris-HCL, pH 6.8) with 10% b-mercaptoethanol for 5 min at 99 C. For active RAC1 and RHO-A pulldown assays (Cell Biolabs), HT144 cells (140 000 cells) or A2058 cells (60 000 cells) were plated in 6-well plates (Nunc) in triplicates and reversed transfected with either control 10 ml of siRNA-a (Santa Cruz Biotechnology) or siRNA against DDX3X (Santa Cruz Biotechnology) using Lipofectamine 2000 (ThermoScientific, 6 ml/ sample) in antibiotics free media.

Techniques: In Vivo, CRISPR, Mutagenesis, Control, Comparison, Transwell Assay, Migration

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: Kinetics of intracellular Ca 2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB‐KO) were seeded on 96‐well plates, loaded with Fluo‐4 dye and stimulated with 100 nM EDN or PBS. Fluo‐4 fluorescence was monitored over 10 min ( n = 6). Representative example of three independent experiments. The error bars indicate ± standard deviation. Time course of AKT and CREB phosphorylation. Melanoma cell lines were stimulated with 100 nM EDN for 2–90 min. AKT and CREB phosphorylation and expression were evaluated by Western blot. Representative example of three independent experiments. Experiment design for the UACC257 phosphoproteomic study, showing time points, cell line derivatives, replicate structure and GIST composition. The GIST was generated by pooling all heavy SILAC plates (blue) and spiked into all UACC257 (red) and UACC257 EDNRB‐KO (green) samples. The A2058 cell line was also analysed in biological triplicates and a GIST but without EDNRB‐KO cells. High reproducibility in the UACC257 and A2058 data sets. CV distributions for triplicates were calculated and are provided in Fig D. All CV values were grouped according to cell line to represent data set‐specific CV distributions. Numbers and lines indicate the median.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Fluorescence, Standard Deviation, Phospho-proteomics, Expressing, Western Blot, Generated, Multiplex sample analysis

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: Validation of EDNRB ablation in UACC257 and A2058. EDNRB expression in WT and EDNRB‐KO cell lines was analysed by Western blot. Ten and 50 μg total protein were loaded for UACC257 and A2058, respectively. Specificity of TiO 2 enrichment, defined as percentage of phosphopeptide identifications over all identifications. The whiskers show 1.5*IQR (Tukey). LuciPHOr performance. Number of peptides with localised phosphates is compared to all phosphopeptides in the data set if phosphate positions were delocalised for different global FLR cut‐offs. CV of phosphopeptide quantification was benchmarked in six process replicates ( n = 6) of the full workflow. Peptides were quantified from the same result files using LFQ (Progenesis QI for proteomics) or the Skyline SILAC GIST workflow. CV violin plots for benchmarking samples and each triplicate in the UACC257 and A2058 data sets are shown. Lines and numbers indicate the medians for each group. Proportions of singly, doubly and triply phosphorylated peptides for each cell line. Proportions of phosphorylated amino acid residues for each data set.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Biomarker Discovery, Expressing, Western Blot, Phospho-proteomics, Targeted Proteomics, Multiplex sample analysis

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA. Colours indicate experimental groups, and shading is mapped to time. Overlap between UACC257 and A2058 data sets. From top to bottom: Identified proteins, identified phosphopeptides, EDN‐regulated phosphopeptides at q < 0.1, and q < 0.1 & FC > 1.5 up or down. Comparison of the kinetics of the 242 shared target phosphopeptides. Log 2 FC of EDN/Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Phospho-proteomics, Comparison

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: Hierarchical clustering of phosphopeptide MS1 intensity for UACC257 separates samples according to the presence and activation of the EDNRB receptor. Clustering was performed on the UACC257 data set in R following log 2 transformation of normalised MS1 intensities, imputation of missing values with the imputeLCMD R package and z ‐score transformation. PCA of the A2058 data set. Phosphopeptide MS1 intensities in A2058 samples were analysed by PCA using R. Colours indicate experimental groups, and shading is mapped to time. Examples kinetics of known (ERK1 T202/Y204) and novel phosphosites (MEK2 S23 & ROCK2 S1134/S1137) on relevant kinases in UACC257 (top) and A2058 (bottom) cells from phosphoproteomic data sets. Data are represented as mean ± SD. * q < 0.1. Two sided unpaired t ‐tests followed by Benjamini‐Hochberg multiple testing correction ( n = 3).

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Phospho-proteomics, Activation Assay, Transformation Assay

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: A, B EDN phosphoproteomic data were analysed in IPA. Significance of term enrichment is indicated through shading as −log ( P ‐value). Regulated pathways belonging to the classes “Canonical Pathways” (A) and “Disease and Biofunction” (B) with significant enrichment ( P < 10 −3 ) are shown for UACC257 and A2058. Regulated terms were grouped according to highest order ontology terms, which are shown on the left.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques:

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: A, B EDN target phosphopeptides for (A) UACC257 and (B) A2058 were segregated into six clusters each, using c‐means clustering (MFuzz). Each line represents one phosphopeptide, and cluster membership is indicated by colour. Most likely kinases for each phosphosite were predicted using NetworKIN, and enrichment of kinase substrates was calculated for each cluster. Enriched kinases are indicated above each cluster. Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster. Detailed information on the kinase enrichment is provided in Appendix Table S1 . C Average MS1 intensity z ‐score kinetics for 217 predicted CaMKII‐α sites that are shared between both cell lines. For comparison, average ( n = 6) calcium abundance kinetics from Fig A are shown.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Phospho-proteomics, Comparison