Journal: bioRxiv

Article Title: Uncovering senescent fibroblast heterogeneity connects DNA damage response to idiopathic pulmonary fibrosis

doi: 10.1101/2025.11.26.690792

Figure Lengend Snippet: (A) Schematic of 7 healthy and 9 idiopathic pulmonary fibrosis (IPF) primary, human, lung fibroblasts, between ages 29-75, induced to senesce by γ-irradiation (IR). RNA and DNA were collected for bulk RNA sequencing (RNA-seq) and ATAC-seq, respectively, 10-days post IR. (B) Volcano plot of differentially expressed genes (DEGs) in healthy IR vs CTL lung fibroblasts (left) and IPF IR vs CTL lung fibroblasts (right). DEG cutoffs were defined by log2FC < -0.4 or > 0.4, p-adjusted value < 0.05, and minimum expression of 4 transcripts per million. (C) Principal component analysis (PCA) of healthy and IPF lung fibroblasts with and without IR. (D) UpSet plots displaying the number of unique and overlapping genes between healthy and IPF lung fibroblasts with and without IR. (E) Gene set enrichment analysis performed using Wikipathway gene sets of upregulated (top) and downregulated (bottom) DEGs in healthy and IPF lung fibroblasts after IR.

Article Snippet: Primary lung fibroblasts were cultured in T25 flasks or 6-well or 12-well plates (Genesee Scientific, 25-100) for sequencing experiments or 96-well microplates for microscopy imaging, with media changes every 2–3 days.

Techniques: Irradiation, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Uncovering senescent fibroblast heterogeneity connects DNA damage response to idiopathic pulmonary fibrosis

doi: 10.1101/2025.11.26.690792

Figure Lengend Snippet: (A) Schematic of RNA collection time points at 0, 8, 24, 48, and 240-hours after IR. 6 healthy (donors: AJAZ, HLF250, AJDL, HLF872, HLF763, AJIQ) and 6 IPF (donors: 83A, 110A, IPF809, 86A, 127A, 155B) lung fibroblasts were used for this time course analysis. (B) Schematic for (E) detailing transcript counts at 5 time points prior to and after IR in healthy (turquoise line) and IPF (red line) lung fibroblasts. (C) Gene set enrichment analysis performed using The Molecular Signatures Database (MSigDB) hallmark gene set collection with DEGs from healthy and IPF lung fibroblasts at timepoints 0h prior to and 8h after IR. (D) E2F gene target differential expression heatmap with DEGs from healthy and IPF lung fibroblasts at timepoints 0h prior to and 8h after IR. (E) Relative transcript abundance of genes associated with DNA repair over time after IR between healthy and IPF lung fibroblasts.

Article Snippet: Primary lung fibroblasts were cultured in T25 flasks or 6-well or 12-well plates (Genesee Scientific, 25-100) for sequencing experiments or 96-well microplates for microscopy imaging, with media changes every 2–3 days.

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Uncovering senescent fibroblast heterogeneity connects DNA damage response to idiopathic pulmonary fibrosis

doi: 10.1101/2025.11.26.690792

Figure Lengend Snippet: (A) Schematic of immunocytochemistry (ICC) performed after IR in healthy and IPF lung fibroblasts. 3 healthy (donors: AJAZ, HLF250, AJDL) and 3 IPF (donors: 83A, 110A, 112A) lung fibroblasts were used for the time course analysis. (B) Harmony spot software analysis example of γH2AX foci in two nuclei at 63X magnification. Staining is in the left panel is a red γH2AX stain and the “Spots” middle panel is the software identifying foci in the nucleus from the γH2AX staining. Grayscale image of the red stain provided in the right panel showing algorithmic nuclear segmentation. (C-D) Healthy and IPF lung fibroblasts were fixed and stained by ICC for γH2AX (C), Rad51 (D), pATM, and PCNA at timepoints 15 min, 2h, and 24h after IR (pATM and PCNA images shown in Supp. Fig. 9). Images taken on the Harmony Operetta CLS system at 63X. (E-F) Foci and nuclear intensity quantification of images performed by the Harmony analysis system and Harmony spot finder analysis, respectively. Statistics were calculated using an unpaired t-test with Welch correction on each row and Holm-Šídák’s multiple comparisons test with alpha = 0.05.

Article Snippet: Primary lung fibroblasts were cultured in T25 flasks or 6-well or 12-well plates (Genesee Scientific, 25-100) for sequencing experiments or 96-well microplates for microscopy imaging, with media changes every 2–3 days.

Techniques: Immunocytochemistry, Software, Staining

Journal: bioRxiv

Article Title: Uncovering senescent fibroblast heterogeneity connects DNA damage response to idiopathic pulmonary fibrosis

doi: 10.1101/2025.11.26.690792

Figure Lengend Snippet: (A) Diagram of z-score comparisons between RNA-seq and scRNA-seq data. In brief, RNA-seq data has gene expression values converted to z-scores which is then summed against the z-scores from each cell type gene expression in the scRNA-seq data. The cell type from the scRNA-seq data with the highest summed z-score is the most similar, in terms of gene expression, to the in culture data. (B) Volcano plot of healthy and IPF RNA-seq data after senescence induction. Red bubbles are genes are listed in (C) used to identify cell populations in the scRNA-seq data comprising of the top 50 differentially expressed genes in our senescent healthy or IPF lung fibroblast data sets. (C) Hierarchical clustering of scRNA-seq data from Habermann et. al. 2020. The first row of columns indicates diagnosis (cHP: Chronic hypersensitivity pneumonitis, Unclassifiable ILD: interstitial lung disease, IPF: Idiopathic Pulmonary Fibrosis, NSIP: Nonspecific Interstitial Pneumonia). The data is then clustered based on cell type (low-res) from the scRNA-seq data in the second row of columns. The third row of columns indicates further cell type (high-res) detail based on gene expression markers. The darker the red color indicates more similarity to the scRNA-seq cluster. Outlined column (number 14) indicates a potential column of interest due to high similarity scores. (D) Wikipathway enrichment of senescence-associated secretory phenotype (SASP) related genes for all healthy and IPF DEGs. (E) Venn diagram showing the overlap between the SenMayo, Wikipathay SASP, and the healthy and IPF IR (IR_sig) gene sets. The IR gene signature is comprised of 163 total genes which is a subset of the entire healthy IR and IPF IR DEG list that showed the most consensus signature to the scRNA-seq ILD gene expression signature.

Article Snippet: Primary lung fibroblasts were cultured in T25 flasks or 6-well or 12-well plates (Genesee Scientific, 25-100) for sequencing experiments or 96-well microplates for microscopy imaging, with media changes every 2–3 days.

Techniques: RNA Sequencing, Gene Expression, Biomarker Discovery