melanoma cells  (ATCC)

Journal: Human Mutation

Article Title: A Macrophage/Monocyte‐Related Four‐Gene Signature for Prognostic Assessment of Uveal Melanoma: BTBD6 , C2CD4B , CCL24 , and S100A4

doi: 10.1155/humu/4978880

Figure Lengend Snippet: Role of four prognostically relevant genes. (A) Quantitative reverse transcription PCR for the calculated levels of four prognostically relevant genes in human epidermal melanocytes. (B) Validation of the knockdown efficiency of C2CD4B. (C, D) Scratch and Transwell assays exploring the migration and invasion of A375 cells in vitro. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, p > 0.05.

Article Snippet: Melanoma cell line A375 (CRL‐1619) and human epidermal melanocytes (PCS‐200‐013) were purchased from American Type Culture Collection (ATCC, Manassas, Virginia) and cultured as follows.

Techniques: Reverse Transcription, Biomarker Discovery, Knockdown, Migration, In Vitro

Journal: bioRxiv

Article Title: PRMT1 arginine methylation of MCM4 restricts ssDNA gap formation during DNA replication

doi: 10.64898/2026.06.03.729374

Figure Lengend Snippet: (A) STRING network of the 82 proteins enriched at replisomes in iPOND-mass spectrometry. Edges represent known high-confidence protein-protein interactions and edge thickness is weighted based on the confidence of the interaction. Manually curated gene functions are indicated by node colour: blue, DNA replication; orange, chromatin-associated; grey, other. n =2 biological replicates. (B) Gene ontology (GO) biological process analysis for the 82 high-confidence proteins that localize to active replisomes. The fold-enrichment for each GO term is indicated, colours indicate corrected p -values, and the size of the circles corresponds to the number of genes annotated to each GO term. (C) UpSet plot showing the intersections of our study and iPOND-mass spectrometry datasets from Wessel et al . Filled circles connected by vertical lines denote the datasets represented by each intersection. Vertical bars indicate the number of proteins in each intersection, and horizontal bars indicate the total number of proteins in each dataset. Intersections are ordered by decreasing size and proteins unique to each dataset are indicated by the absence of vertical bars. (D) EdU incorporation following siRNA knockdown of the indicated genes. Following knockdown, cells were pulse-labelled with EdU. Nuclear EdU intensity was measured by fluorescence microscopy and is plotted in arbitrary units (AU). Circles represent the mean nuclear EdU intensity of each replicate, and the black bars indicate the average of the replicates. Knockdowns with statistically supported decreases in nuclear EdU intensity relative to the control siRNA ( p < 0.05; one-tailed Student’s t-test) are indicated *. siPCNA served as the positive control. n =3 biological replicates. (E) HEK293T cells were subjected to the iPOND workflow, and the input extracts and streptavidin pulldowns were immunoblotted for the presence of PRMT1, PCNA (DNA replication fork control), and histone H3 (chromatin control). The control without covalent attachment of streptavidin to EdU (no click), the EdU pulse, and the thymidine chase are shown. The positions of molecular weight standards, in kDa, are indicated to the left.

Article Snippet: HEK293T (ATCC), HEK293 (ATCC) and A375 (gift from Dr. Mikko Taipale) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Wisent) supplemented with 10% Fetal Bovine Serum (FBS, Wisent) and 1% penicillin/streptomycin (PS, Wisent).

Techniques: Mass Spectrometry, Protein-Protein interactions, Knockdown, Fluorescence, Microscopy, Control, One-tailed Test, Positive Control, Molecular Weight

Journal: bioRxiv

Article Title: PRMT1 arginine methylation of MCM4 restricts ssDNA gap formation during DNA replication

doi: 10.64898/2026.06.03.729374

Figure Lengend Snippet: (A) PRMT1 depletion in CRISPRi lines. CRISPRi A375 cells carrying doxycycline-inducible sgRNAs for PRMT1 were sampled at the indicated times after the addition of doxycycline and immunoblotted to detect PRMT1 and tubulin. The positions of molecular weight standards, in kDa, are indicated to the left. (B) EdU incorporation in PRMT1 CRISPRi cells. CRISPRi cells carrying doxycycline-inducible sgRNAs for PRMT1 were pulse-labelled with EdU at the indicated times after the addition of doxycycline. EdU intensity per cell was measured by flow cytometry and is plotted in arbitrary units as box plots, with horizontal bars indicating the medians. Boxes span the first through third quartiles, whiskers extend to the last data points within 1.5 times the interquartile range, and outliers are plotted as circles. A minimum of 11,000 cells were measured per sample. n =2 biological replicates (replicate 2, Figure S1B). (C) DNA combing analysis of PRMT1 CRISPRi cells. PRMT1 was depleted for 5 days by expressing the indicated sgRNAs (+dox), and cells were sequentially pulse-labelled with CldU and IdU, followed by DNA fibre isolation and molecular combing. The IdU tract lengths were measured and plotted as replication fork rates. Parallel cultures without PRMT1 depletion are shown for comparison (-dox). Medians are indicated by horizontal red bars and p -values were calculated with a two-sided Mann-Whitney U test. (D) DNA combing analysis of HEK293T cells following type I PRMT inhibition. Cells were treated with MS023 at the indicated concentrations for 48 hours, and sequentially pulse-labelled with CldU and IdU, followed by DNA fibre isolation and molecular combing. The IdU tract lengths were measured and plotted as replication fork rates. Parallel cultures without PRMT1 depletion are shown for comparison (-dox). Medians are indicated by horizontal red bars and p -values were calculated with a two-sided Mann-Whitney U test. (E) Rescue of EdU incorporation in PRMT1 CRISPRi cells. CRISPRi sgPRMT1-7 cells were treated with doxycycline for 5 days (closed circles) to deplete PRMT1, prior to transfection with the empty vector (EV), FLAG-PRMT1 (WT), or FLAG-PRMT1 E153Q (catalytic-dead; E153Q) prior to pulse-labelling with EdU, EdU intensity per cell was measured by flow cytometry and is plotted in arbitrary units as box plots, with horizontal bars indicating the medians. Boxes span the first through third quartiles, whiskers extend to the last data points within 1.5 times the interquartile range, and outliers are plotted as circles. A parallel culture without PRMT1 depletion is shown for comparison (open circle). n =2 biological replicates (replicate 2, Figure S1H). (F) Whole cell lysates from the samples in (E) were immunoblotted to detect asymmetric arginine dimethylation (α-ASYM26), PRMT1, and GAPDH. The positions of molecular weight standards, in kDa, are indicated to the left.

Article Snippet: HEK293T (ATCC), HEK293 (ATCC) and A375 (gift from Dr. Mikko Taipale) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Wisent) supplemented with 10% Fetal Bovine Serum (FBS, Wisent) and 1% penicillin/streptomycin (PS, Wisent).

Techniques: Molecular Weight, Flow Cytometry, Expressing, Isolation, Comparison, MANN-WHITNEY, Inhibition, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: PRMT1 arginine methylation of MCM4 restricts ssDNA gap formation during DNA replication

doi: 10.64898/2026.06.03.729374

Figure Lengend Snippet: (A) Venn diagram of the overlap between the Larsen et al methylproteomics study33 and replisome-enriched proteins from . Proteins whose monomethylation abundance changed upon PRMT1 depletion are in the blue circle, and proteins localized to DNA replication forks are in the grey circle. The 14 proteins present in both datasets are shown to the right, with known substrates of PRMT1 underlined and MCM subunits in bold. (B) Asymmetric arginine dimethylation of MCM subunits analyzed by immunoblotting. HEK293T cells were transfected to express the indicated V5-tagged MCM proteins or MRE11-V5, nuclear extracts were prepared (input), and immunoprecipitated with an anti-V5 antibody (α -V5 IP). Immunoblots were probed to detect the V5-tagged proteins and asymmetric arginine dimethylation (α-ASYM26). The positions of molecular weight standards, in kDa, are indicated to the left. (C) Asymmetric arginine dimethylation of the MCM complex analyzed following PRMT1 depletion or inhibition. Whole-cell (left) and nuclear (right) extracts were prepared from CRISPRi sgPRMT1-6 cells following a 5-day PRMT1 depletion (+dox) or 2 days of PRMT1 inhibition with 1 µM MS023. An α -MCM2 antibody was used to precipitate the MCM complex from the nuclear extracts (α -MCM2 IP), and the whole-cell extracts and IPs were immunoblotted. The immunoblots were probed to detect MCM2, MCM4, PRMT1, GAPDH, and asymmetric arginine demethylation GAPDH, and asymmetric arginine dimethylation (α-ASYM26). The positions of molecular weight standards, in kDa, are indicated to the left. (D) Methylation of recombinant MCM4 with purified GST-PRMT1. MCM4 was incubated with wild-type (WT) or catalytically inactive (E153Q) GST-PRMT1 for 3 hours at 30°C. The samples were immunoblotted to detect MCM4, GST-PRMT1, and asymmetric arginine dimethylation (α-ASYM26). The positions of molecular weight standards, in kDa, are indicated to the left. (E) Multiple sequence alignment of Homo sapiens, Mus musculus, Rattus norvegicus , and Saccharomyces cerevisiae N-terminal MCM4 protein sequences. The putative GAR motif is highlighted with a yellow box, and the red arrows mark arginine residues detected as dimethylated by mass spectrometry . (F) Asymmetric arginine dimethylation of MCM4 variants. HEK293T cells were transfected to express V5-tagged MCM4 (WT) or MCM4 R10K (R10K). Nuclear extracts were prepared (input), precipitated with anti-V5 antibody (α-V5 IP), and immunoblotted to detect the V5-MCM4 proteins and asymmetric arginine dimethylation (α-ASYM26). The positions of molecular weight standards, in kDa, are indicated to the left. (G) Assessment of MCM4 variant interactions with the MCM2-7 complex. FLAG-tagged wild-type MCM4 (WT), MCM4 R10K (R10K), MCM4 Kall (K all), or MCM4 Aall (A all), or the empty vector (EV) were integrated in HEK293 Flp-In T-Rex cells, and expression was induced for 16 hours. The MCM4-FLAG proteins were immunoprecipitated (α-FLAG IP) and analyzed by mass spectrometry. The number of spectral counts and relative abundance of each MCM2-7 complex member is plotted.

Article Snippet: HEK293T (ATCC), HEK293 (ATCC) and A375 (gift from Dr. Mikko Taipale) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Wisent) supplemented with 10% Fetal Bovine Serum (FBS, Wisent) and 1% penicillin/streptomycin (PS, Wisent).

Techniques: Western Blot, Transfection, Immunoprecipitation, Molecular Weight, Inhibition, Methylation, Recombinant, Purification, Incubation, Sequencing, Mass Spectrometry, Variant Assay, Plasmid Preparation, Expressing

Journal: Frontiers in Immunology

Article Title: Melanoma stem cells drive macrophage reprogramming to a hybrid phenotype, modulating melanoma stemness and compromising NK cell-mediated immunity

doi: 10.3389/fimmu.2026.1698412

Figure Lengend Snippet: Transcriptomic profiling of melanoma SCs identifies stemness-associated and cell line-specific molecular programs. (A) PCA of RNA-seq data from A375 and WM115 cells cultured under adherent conditions (2D) or as melanospheres (3D). Each dot represents one sample. (B) ssGSEA enrichment scores for the Melanoma stemness, WNT/β-catenin signaling, Hedgehog signaling, and Notch signaling gene sets in A375 and WM115 cells cultured in 2D or 3D conditions. Data are presented as the mean ± SEM. Each dot represents one sample. *p<0.05 by Welch’s t-test, followed by Benjamini-Hochberg correction for multiple testing across signatures. (C) Left, number of up-regulated and down-regulated DEGs identified in melanospheres compared to matched adherent cultures in A375 and WM115 cells (padj < 0.05). Right, filtered volcano plots showing DEGs (padj < 0.05, |log2FC| > 1, and baseMean > 50) in A375 and WM115 melanospheres relative to 2D cultures. The 10 most upregulated and 10 most downregulated genes are labelled. (D) Bubble plots of the top 10 most statistically significant (padj < 0.05) Reactome pathways enriched among genes up-regulated in A375 and WM115 melanospheres relative to matched 2D cultures determined by GSEA analysis. The X-axis represents the Gene Ratio and Y-axis indicates enriched pathway terms. Bubble area represents the gene count and bubble color indicates the adjusted p value. (E) VIPER analysis showing the top 15 inferred transcription factor activity in melanospheres relative to matched 2D cultures in A375 and WM115 cells. Red bars indicate transcription factors inferred to be activated in 3D cultures, whereas blue bars indicate transcription factors inferred to be activated in 2D cultures.

Article Snippet: A375 human melanoma cells (CRL-1619TM), THP-1 human acute monocytic leukemia cells (TIB-202TM), NK-92 human natural killer cell line (CRL-2407TM), and K562 human chronic myelogenous leukemia cells (CCL-243TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: RNA Sequencing, Cell Culture, Activity Assay

Journal: Frontiers in Immunology

Article Title: Melanoma stem cells drive macrophage reprogramming to a hybrid phenotype, modulating melanoma stemness and compromising NK cell-mediated immunity

doi: 10.3389/fimmu.2026.1698412

Figure Lengend Snippet: Distinct secretory profiles and monocyte chemoattractant capacity of A375 and WM115 SCs. (A) PCA of multiplex ELISA assay data obtained from CM of A375 and WM115 melanospheres. Each dot represents one sample. (B) Multiplex ELISA assay quantification of soluble factors in CM from A375 and WM115 SCs. Data are presented as the mean ± SEM. Each dot represents one sample. (C) Transwell migration assay of THP-1 cells exposed to control medium, A375 SC-CM, or WM115 SC-CM, in the absence or presence of the CCR2 antagonist RS504393. Data are presented as mean ± SEM. Each dot represents one sample. Statistical analysis was performed by two-way ANOVA followed by Bonferroni-adjusted post hoc comparisons. Asterisks indicate comparisons between inhibitor-treated and untreated conditions within each medium. **p < 0.01, ***p < 0.001.

Article Snippet: A375 human melanoma cells (CRL-1619TM), THP-1 human acute monocytic leukemia cells (TIB-202TM), NK-92 human natural killer cell line (CRL-2407TM), and K562 human chronic myelogenous leukemia cells (CCL-243TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Transwell Migration Assay, Control

Journal: Frontiers in Immunology

Article Title: Melanoma stem cells drive macrophage reprogramming to a hybrid phenotype, modulating melanoma stemness and compromising NK cell-mediated immunity

doi: 10.3389/fimmu.2026.1698412

Figure Lengend Snippet: Transcriptomic characterization of THP-1-derived macrophages exposed to melanoma SC-CM. (A) PCA of RNA-seq data from M0 macrophages and macrophages exposed to CM from A375 or WM115 melanospheres. (B) Number of upregulated and downregulated DEGs (padj<0.05) in A375 SC-CM-educated and WM115 SC-CM-educated macrophages compared with M0 macrophages. (C) Venn diagrams showing the overlap of upregulated and downregulated DEGs between A375 SC-CM-educated and WM115 SC-CM-educated macrophages, each compared with M0 macrophages. (D) Filtered volcano plots showing DEGs (padj < 0.05, |log2FC| > 1, and baseMean > 50) in A375 SC-CM-educated versus M0 macrophages and WM115 SC-CM-educated versus M0 macrophages. The 10 most upregulated and 10 most downregulated genes are labelled. (E) Bubble plots of the top 10 most statistically significant (padj < 0.05) Reactome pathways enriched among genes upregulated in A375 SC-CM-educated macrophages and WM115 SC-CM-educated macrophages determined by GSEA analysis. The X-axis represents the Gene Ratio and Y-axis indicates enriched pathway terms. Bubble area represents the gene count, and bubble color indicates the adjusted p value. (F) VIPER analysis showing the top 15 inferred transcription factor activity in A375 SC-educated and WM115 SC-educated macrophages compared to M0 macrophages. Red bars indicate transcription factors inferred to be activated in melanoma SC-educated macrophages, whereas blue bars indicate transcription factors inferred to be activated in M0 macrophages.

Article Snippet: A375 human melanoma cells (CRL-1619TM), THP-1 human acute monocytic leukemia cells (TIB-202TM), NK-92 human natural killer cell line (CRL-2407TM), and K562 human chronic myelogenous leukemia cells (CCL-243TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Derivative Assay, RNA Sequencing, Activity Assay

Journal: Frontiers in Immunology

Article Title: Melanoma stem cells drive macrophage reprogramming to a hybrid phenotype, modulating melanoma stemness and compromising NK cell-mediated immunity

doi: 10.3389/fimmu.2026.1698412

Figure Lengend Snippet: ssGSEA-based inference of macrophage phenotypes induced by melanoma SC-CM. (A) Bar plots showing normalized ssGSEA scores for literature-derived TAM signatures and curated macrophage-state signatures in M0 macrophages and in macrophages exposed to A375 SC-CM or WM115 SC-CM. Curated signatures were derived from GSE115978 . Data are presented as the mean ± SEM. Each dot represents one sample. **p<0.01, ***p<0.001 by one-way ANOVA with Benjamini-Hochberg correction across signatures followed by Tukey’s multiple-comparison test. (B) Integrated matrix showing pairwise relationships among the signatures significantly modulated in vitro . The lower triangle reports Spearman correlation coefficients calculated from ssGSEA scores across samples, with asterisks indicating Benjamini-Hochberg-adjusted significance levels (*p<0.05, **p<0.01, ***p<0.001). The upper triangle reports overlap coefficients based on gene-set composition. Abbreviations: IFN, interferon-primed TAMs; Inflamm, inflammatory cytokine-enriched TAMs; Angio, pro-angiogenic TAMs; Reg, immune regulatory TAMs. (C) Bar plots representing MPI (macrophage polarization index) and AMDI (activation-induced macrophage differentiation index) values from MacSpectrum analysis of M0 macrophages and macrophages exposed to CM from A375 or WM115 melanospheres. Data are presented as the mean ± SEM. Each dot represents one sample. *p<0.05, **p<0.01 by Kruskal-Wallis test with Holm correction across the two endpoints, followed by Dunn’s post hoc test with Holm correction.

Article Snippet: A375 human melanoma cells (CRL-1619TM), THP-1 human acute monocytic leukemia cells (TIB-202TM), NK-92 human natural killer cell line (CRL-2407TM), and K562 human chronic myelogenous leukemia cells (CCL-243TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Derivative Assay, Comparison, In Vitro, Activation Assay

Journal: Frontiers in Immunology

Article Title: Melanoma stem cells drive macrophage reprogramming to a hybrid phenotype, modulating melanoma stemness and compromising NK cell-mediated immunity

doi: 10.3389/fimmu.2026.1698412

Figure Lengend Snippet: Functional characterization of THP-1-derived macrophages exposed to melanoma SC-CM. (A) PCA performed on multiplex ELISA assay of soluble mediator profiles from M0 macrophages and macrophages exposed to CM from A375 or WM115 melanospheres. Each dot represents one sample. (B) Multiplex ELISA assay quantification of soluble mediators secreted by M0 macrophages and by macrophages previously exposed to CM from A375 or WM115 melanospheres. Data are presented as the mean ± SEM. Each dot represents one sample. *p<0.05 by Kruskal-Wallis test with Benjamini-Hochberg correction across analytes, followed by Dunn’s post hoc test with Holm correction; pairwise p values were additionally Benjamini-Hochberg-corrected across analytes within each contrast. (C) Percentage of lysis of NK-92 cells against K562 target cells after pre-exposure to CM from M0 macrophages or from macrophages educated with A375 or WM115 SC-CM, assessed by Calcein-AM release cytotoxicity assay at the indicated E:T ratios. Data are presented as the mean ± SEM. Each dot represents one sample. The results are representative of one of at least three independent experiments showing similar results. **p<0.01, ***p<0.001 by Tukey-adjusted comparisons among experimental groups within each E:T ratio following two-way ANOVA. (D) Percentage of lysis of NK-92 cells against K562 target cells after pre-exposure to untreated or heat-treated CM from M0 macrophages or from macrophages educated with A375 or WM115 melanoma SC-CM assessed by Calcein-AM release cytotoxicity assay. Data are presented as the mean ± SEM. Each dot represents one sample. The results are representative of one of at least three independent experiments showing similar results. ***p<0.001 by Tukey-adjusted comparisons between treatment conditions within each experimental group following two-way ANOVA. (E) RT-qPCR analysis of KLF4, SOX10, POU5F1, SOX9, SOX2 in A375 and WM115 SC-enriched cultures after exposure to CM from M0 macrophages or from macrophages previously educated with melanoma SC-CM. Bar plots show mean ± SEM of 2 −ΔCt RT-qPCR values. The results are representative of one of at least three independent experiments showing similar results. *p<0.05, **p<0.01, ***p<0.001 by Welch’s t-test, followed by Holm correction for multiple testing across genes within each cell line.

Article Snippet: A375 human melanoma cells (CRL-1619TM), THP-1 human acute monocytic leukemia cells (TIB-202TM), NK-92 human natural killer cell line (CRL-2407TM), and K562 human chronic myelogenous leukemia cells (CCL-243TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Functional Assay, Derivative Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Lysis, Cytotoxicity Assay, Quantitative RT-PCR