a549 cells  (ATCC)

Journal: bioRxiv

Article Title: Loss of the Y chromosome drives epigenetic and transcriptomic plasticity in lung adenocarcinoma

doi: 10.64898/2026.06.02.729627

Figure Lengend Snippet: (A) Experimental overview illustrating the generation of isogenic single-cell LOY and ROY clones from parental A549 cells and the multi-omic analysis of clones and patients. (B) Representative WGS profiles of ROY (top, orange) and LOY (bottom, blue) clones. Left: zoomed-in view of the Y chromosome. Right: genome-wide Circos plots (right). (C) Volcano plot of differentially expressed genes between LOY (n=4) and ROY (n=4) clones (Y-linked genes excluded). X-axis: log2 fold change; y-axis: -log 10 (p-value). Blue: significantly upregulated genes in LOY; orange: downregulated in LOY. (D) Dot plot showing the top 6 Hallmark gene sets with FDR<0.25 from preranked GSEA of RNA-seq data. X-axis: normalized enrichment score (NES); y-axis: gene sets. Dot size indicates gene count; color represents -log 10 (FDR). (E) Heatmap of lead genes contributing to the EMT gene set enrichment. TPM values are z-scored across clones. (F) Dot plot of Hallmark gene sets from GSEA of full proteome data at 70% confluency (left) and 100% confluency (right). X-axis: normalized enrichment score (NES); y-axis: gene sets. Dot size indicates protein count; color represents -log 10 (FDR). ( G ) Confocal microscopy images of clones stained for N-cadherin (green). Nuclei are counterstained with DAPI (4′,6-diamidino-2-phenylindole, blue). Scale bar 20 µm (H) . Flow cytometry histograms of CD90 expression for LOY (blue) and ROY (orange) clones, with isotype controls shown. (I) Dot plot of top 10 Hallmark gene sets from preranked GSEA of TCGA LUAD samples (LOY vs. ROY). (J) Dot plot the top 8 positively enriched Hallmark gene sets from GSEA of the LuCA single-cell dataset (LOY vs. ROY tumor cells).

Article Snippet: A549 cells were purchased from Cytion GmbH.

Techniques: Single Cell, Clone Assay, Genome Wide, RNA Sequencing, Confocal Microscopy, Staining, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Loss of the Y chromosome drives epigenetic and transcriptomic plasticity in lung adenocarcinoma

doi: 10.64898/2026.06.02.729627

Figure Lengend Snippet: (A) WGS profiles of four ROY (top) and four LOY clones (Circos plots) compared to bulk A549 parental cells. (B) Boxplot showing expression of six Y-linked genes and X-linked homologs across LOY (n=4, blue) and ROY (n=4, orange) clones (log 2 (TPM+1)). Box represents median and quartiles; whiskers show min/max values. (D) Protein detection metrics. Top: proteins detected per sample across confluency conditions. Bottom: mean log2 intensity distribution per group and condition. ( D ) Heatmap of proteotypic peptide intensities for three Y-linked proteins and their X-linked homologs across conditions. ( E ) Volcano plot of differentially abundant proteins at 70% (top) and 100% confluency (bottom). Blue: upregulated in LOY; orange: downregulated in LOY. X-axis: log2 fold change; y-axis: -log 10 (p-value). ( F ) Confocal microscopy images for all eight clones stained for E-cadherin (green) and DAPI (blue). Scale bar: 20 µm.

Article Snippet: A549 cells were purchased from Cytion GmbH.

Techniques: Clone Assay, Expressing, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Loss of the Y chromosome drives epigenetic and transcriptomic plasticity in lung adenocarcinoma

doi: 10.64898/2026.06.02.729627

Figure Lengend Snippet: (A) Experimental overview of epigenetic characterization using DiMeLo-seq in isogenic A549 ROY and LOY clones. (B) Unsupervised principal component analysis (PCA) of the 50,000 most variable autosomal CpGs. Each dot represents a clone (blue: LOY; orange: ROY). (C) Boxplot of differentially methylated CpG islands in LOY (blue) and ROY (orange) cells. (D) Heatmap of CpG island methylation levels for autosomal CpG islands within 10 kb of a transcription start site (TSS), filtered to one representative CpG island per gene with the largest absolute methylation difference. Columns represent individual clones, and rows represent associated genes with representative CpG islands. (E) GSEA dot plot of promoter-associated DMRs (±5 kb from TSS, Hallmark gene set). Positive normalized enrichment scores (NES) indicate enrichment of genes with hypomethylated promoters in LOY clones. ( F ) DNA methylation tracks at THY1 (top) and LOX (bottom) loci. (G) Preranked GSEA of promoter H3K4me3 signal (±2 kb from TSS, MSigDB Hallmark collection). Positive NES indicates increased H3K4me3 in LOY cells. ( H ) Locus-specific profiles of H3K4me3 signal (6mA) averaged for LOY and ROY clones at THY1 (top) and LOX (bottom).

Article Snippet: A549 cells were purchased from Cytion GmbH.

Techniques: Clone Assay, Methylation, DNA Methylation Assay

Journal: bioRxiv

Article Title: Loss of the Y chromosome drives epigenetic and transcriptomic plasticity in lung adenocarcinoma

doi: 10.64898/2026.06.02.729627

Figure Lengend Snippet: (A) Experimental workflow for single-cell profiling of isogenic A549 ROY and LOY clones using Epi-CyTOF and scMultiome, alongside clinical data reanalysis . (B) Heatmap of Epi-CyTOF histone modification measurements. Values are z-scored across clones; blue: lower-than-average; red: higher-than-average levels. Hierarchical clustering was applied to histone marks and samples. (C) Boxplot showing the epigenetic heterogeneity metric derived from multidimensional Epi-CyTOF data. (D) Boxplot of EMT module scores from snRNA-seq, based on lead genes from . (E) Heatmaps of lead EMT gene expression (snRNA-seq, left) and corresponding chromatin accessibility at gene promoters (scATACseq, right). (F) Boxplot of transcriptional heterogeneity (QuoTHiC) in premalignant and tumor cells compared to normal AT2 cells in a cohort from the LuCA dataset , . (G) Boxplot comparing QuoTHiC scores across cell types and ROY/LOY status from the cohort shown in (F).

Article Snippet: A549 cells were purchased from Cytion GmbH.

Techniques: Single Cell, Clone Assay, Modification, Derivative Assay, Gene Expression

Journal: bioRxiv

Article Title: Loss of the Y chromosome drives epigenetic and transcriptomic plasticity in lung adenocarcinoma

doi: 10.64898/2026.06.02.729627

Figure Lengend Snippet: (A) Schematic of the in vitro phenotypic characterization of isogenic A549 ROY and LOY clones. (B) Dot plot showing the top 10 Hallmark gene sets with FDR<0.25 from preranked GSEA of RNA-seq data comparing A549 LOY and ROY under glucose deprivation. X-axis: normalized enrichment score (NES); y-axis: gene sets. Dot size indicates gene count; color represents -log 10 (FDR). ( C) Dot plot showing significant Hallmark gene sets from preranked GSEA of global proteome data comparing LOY vs. ROY clones under glucose (left) and glutamine (right) deprivation. Dot size indicates protein count; color represents -log 10 (FDR). (D) Colony formation capacity under glutamine deprivation. Left: Boxplot of mean colony number (n = 3 technical replicates/clone). Right: Representative crystal violet-stained images. (E ) Normalized dose-response curves of four LOY (blue) and four ROY (orange) clones assessed 24 hours post-irradiation (0-40 Gy). Viability was measured in triplicate using CellTiter-Blue and fitted using non-linear regression. (F) Clonogenic survival following 10 Gy. Left: boxplots showing mean colony counts across two independent experiments (n=3 replicates/clone/experiment). Right: representative images from experiment 1. Note: For all boxplots, the center line represents the median, box limits represent upper and lower quartiles, and whiskers represent minimum and maximum values. Statistical significance was assessed using an unpaired t-test (α=0.05).

Article Snippet: A549 cells were purchased from Cytion GmbH.

Techniques: In Vitro, Clone Assay, RNA Sequencing, Staining, Irradiation

Journal: bioRxiv

Article Title: Loss of the Y chromosome drives epigenetic and transcriptomic plasticity in lung adenocarcinoma

doi: 10.64898/2026.06.02.729627

Figure Lengend Snippet: (A) Boxplots displaying cell proliferation (BrdU ELISA) of four LOY (blue) and four ROY (orange) clones under normal growth conditions (glucose+, glutamine+), glucose deprivation (glucose-, glutamine+), or glutamine deprivation (glucose+, glutamine-). (B) Volcano plot of differentially expressed genes between LOY (n=4) and ROY (n=4) clones under glucose deprivation (top) and glutamine deprivation (bottom) (Y-linked genes excluded). X-axis: log2 fold change; y-axis: -log 10 (p-value). Blue: significantly upregulated genes in LOY; orange: downregulated in LOY. (C) Dot plot showing the top 2 Hallmark gene sets with FDR<0.25 from preranked GSEA of RNA-seq data comparing A549 LOY and ROY under glutamine deprivation. X-axis: normalized enrichment score (NES); y-axis: gene sets. Dot size indicates gene count; color represents -log 10 (FDR). (D) Volcano plots of differentially abundant proteins under glucose (left) and glutamine (right) deprivation. Blue: significantly upregulated in LOY; orange: downregulated in LOY. X-axis: log2 fold change; y-axis: -log 10 (p-value). (E) 10-day colony formation assay of four LOY (blue) and four ROY (orange) clones, standard growth conditions. Left: boxplot of mean colony number (n=3 technical replicates/clone). Right: representative images. (F) Normalized growth curves of four LOY (blue) and four ROY (orange) clones over 5 days following 10 Gy irradiation, fitted using non-linear regression. (G) Boxplot quantifying the area occupied by colonies after 10 Gy irradiation (experiment 1). (H) Boxplot quantifying the area occupied by non-irradiated controls (left) and representative images (right) from experiment 1. Box plots as in .

Article Snippet: A549 cells were purchased from Cytion GmbH.

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, RNA Sequencing, Colony Assay, Irradiation