Journal: bioRxiv

Article Title: Progressive matrix stiffening of tyramine-modified silk fibroin hydrogels governs stage-specific pulmonary fibroblast activation

doi: 10.64898/2026.06.01.729382

Figure Lengend Snippet: A) Hydrogels were composed of either 0% or 50% mixture of tyramine-modified silk and were exposed to media without or with 5 ng/mL TGFβ. After hydrogels were cast, 25,000 NHLF cells were seeded on the surface. B) SEM imaging of the surface of the silk hydrogels incubated without cells for Day 0 and Day 14. C) Live/Dead imaging of Calcein AM for live cells (green) and Ethidium Homodimer-1 for dead cells (red) indicated cytocompatibility. D) Alamar Blue Assay for metabolic activity indicated that 0% gels showed an increase in activity over 14 days, while 50% gels plateaued from day 7 to 14. E) Mammalian β-Galactosidase Assay for senescence revealed no change for any timepoints tested over 14 days. N=3-4, Error bars = SEM.TCP = Tissue Culture Plate, T-= without TGFβ, T+ = 5 ng/mL of TGFβ. Two-way ANOVA, Tukey’s: ns = not significant p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: Normal human lung fibroblasts (NHLFs, PCS-201-013, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium, high glucose (DMEM, ThermoFisher) supplemented with 10% Gibco Fetal Bovine Serum, Premium (FBS, ThermoFisher), 1% Gibco non-essential amino acids (ThermoFisher), and 1% Gibco antibiotic–antimycotic (ThermoFisher).

Techniques: Modification, Imaging, Incubation, Alamar Blue Assay, Activity Assay

Journal: bioRxiv

Article Title: Progressive matrix stiffening of tyramine-modified silk fibroin hydrogels governs stage-specific pulmonary fibroblast activation

doi: 10.64898/2026.06.01.729382

Figure Lengend Snippet: Top row: NHLFs on 0% SF-TA hydrogels at Day 2, Day 7, and Day 14, exhibiting a proliferative, inflammatory phenotype consistent with early fibroblast activation, characterized by secretion of IL-6, IL-8, and MCP-1 (dots) and progressive collagen deposition (fibers). Bottom row: NHLFs on 50% SF-TA hydrogels at Day 2, Day 7, and Day 14, exhibiting metabolic plateau and lower inflammatory cytokine secretion, gradual collagen secretion, with TGFβ-dependent myofibroblast activation. Together, these two substrate conditions recapitulate distinct stages of the fibrotic disease continuum.

Article Snippet: Normal human lung fibroblasts (NHLFs, PCS-201-013, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium, high glucose (DMEM, ThermoFisher) supplemented with 10% Gibco Fetal Bovine Serum, Premium (FBS, ThermoFisher), 1% Gibco non-essential amino acids (ThermoFisher), and 1% Gibco antibiotic–antimycotic (ThermoFisher).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Host DNA repair factors empower a mechanism of antiviral nucleoside analog resistance

doi: 10.64898/2026.05.21.726832

Figure Lengend Snippet: Primary lung fibroblasts (MRC-5) were infected with WT or F412V at an MOI of 1. At 2 hpi, virus was washed out and media were added containing either 0.1% DMSO or 0.1% DMSO with 1.6 µM GCV. At 96 hpi, cells were harvested and total DNA was isolated for Illumina sequencing. After sequence alignment and removal of reads mapping to the human genome, viral reads were quantified and plotted for each condition. (A and B) Representative sequence coverage plots of total unique (dark blue) viral reads from WT (A) and F412V (B) infected cells treated with DMSO vehicle (top) or GCV in vehicle (bottom). The red dotted line represents the location of the oriLyt (location ∼140,000bp in the genome). Top (cyan) and bottom (purple) strand reads are depicted separately. Only unique reads that map to only one location in the reference genome were counted. (C) Total reads per million (top) and reads distant from oriLyt (bottom) were quantified. Non-parametric Wilcoxon testing was used to determine statistical significance. The resulting p-values were adjusted using the Benjamini-Hochberg method: **, p<0.01; ns, not significant) (D) Sequence coverage decay plots representing the normalized coverage at the oriLyt location up to 50kb moving away from the oriLyt in both directions. (E) Distribution of decay slopes and pairwise comparisons of adjusted p-values of WT and F412V treated with either DMSO or GCV. The slopes estimated per sample were compared across groups using pairwise permutation testing (with BH correction) to assess significant differences in replication bias patterns between conditions: *, p<0.05; ns, not significant.

Article Snippet: Primary human lung MRC-5 fibroblasts (ATCC CCL-171) were cultured in DMEM supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Infection, Virus, Isolation, Illumina Sequencing, Sequencing

Journal: bioRxiv

Article Title: Host DNA repair factors empower a mechanism of antiviral nucleoside analog resistance

doi: 10.64898/2026.05.21.726832

Figure Lengend Snippet: Growth-arrested MRC-5 fibroblasts were transduced with lentiviruses to express shRNAs targeting Luc or three Y-family polymerases: polη, polι, or polκ in combination (A-C) or individually (D and E). (A) Following two days of selection by puromycin (2 µg/mL), cells were lysed and RNA was harvested for analysis by RT-qPCR. Knockdown efficiency was determined by ΔΔCt analysis between Luc and experimental conditions with cellular H6PD as a housekeeping control. Statistical significance was determined by two-tailed, paired t -test with Bonferroni correction. Asterisks (* P <0.05, ** P < 0.01) represent statistically significant differences relative to Luc. (B) Transduced fibroblasts were infected at an MOI of 1 with WT or F412V. At 2 hpi, virus was washed out and media was replaced containing various concentrations of GCV. At four days post infection, cells and supernatant were collected for analysis by TCID 50 . For each condition, virus yield at each GCV concentration was compared to the DMSO control and plotted as percent yield reduction for three biological replicate experiments. Curves were similarly fitted for WT and F412V in each individual experiment to obtain ED 50 values and CIs for each condition. (C) The ED 50 values from three biological replicate experiments were used for a ratio paired t test comparing viruses within each condition. ED 50 mean values are plotted with standard deviation error bars. (D-E) Transduced fibroblasts were infected with (D) WT or (E) F412V at an MOI of 1. At 2 hpi, virus was washed out and media was replaced containing either 0.1% DMSO or 0.1% DMSO plus 1.6 µM GCV, and at 4 days post infection, cells and supernatant were collected for analysis by TCID 50 . Percent virus yield in GCV-treated conditions compared to the DMSO control in each condition was plotted for five biological replicate experiments. Statistical significance was determined by a ratio paired t test: *, p<0.05; ns, not significant.

Article Snippet: Primary human lung MRC-5 fibroblasts (ATCC CCL-171) were cultured in DMEM supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Transduction, Selection, Quantitative RT-PCR, Knockdown, Control, Two Tailed Test, Infection, Virus, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Host DNA repair factors empower a mechanism of antiviral nucleoside analog resistance

doi: 10.64898/2026.05.21.726832

Figure Lengend Snippet: (A) MRC-5 fibroblasts were infected at an MOI of 1 with WT or F412V. At 2 hpi, virus was washed out and media was replaced containing various concentrations of CDV. At four days post infection, cells and supernatant were collected for analysis by TCID 50 . For each condition, virus yield at each CDV concentration was compared to the DMSO control and plotted as percent yield reduction for three biological replicate experiments. Curves were similarly fitted for WT and F412V in each individual experiment to obtain ED 50 values and CIs for each condition. (B-C) Growth-arrested MRC-5 fibroblasts were transduced with lentiviruses to express shRNAs targeting Luc or polκ. These fibroblasts were infected with (B) WT or (C) F412V at an MOI of 1. At 2 hpi, virus was washed out and media was replaced containing either 0.1% DMSO or 0.1% DMSO plus 2.4 µM GCV, and at 4 days post infection, cells and supernatant were collected for analysis by TCID 50 . Percent virus yield in CDV-treated conditions compared to the DMSO control in each condition was plotted for three biological replicate experiments. Statistical significance was determined by a ratio paired t test: *, p<0.05; ns, not significant.

Article Snippet: Primary human lung MRC-5 fibroblasts (ATCC CCL-171) were cultured in DMEM supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Infection, Virus, Concentration Assay, Control, Transduction

Journal: Cell Death Discovery

Article Title: Trifluoperazine causes mast cell apoptosis through a secretory granule-mediated pathway

doi: 10.1038/s41420-026-03122-x

Figure Lengend Snippet: A Human lung smooth muscle cells (HLSMCs), lung fibroblasts (HLFs), and small airway epithelial cells (HSAECs) were treated with TFP at the indicated concentrations for 24 h. Human peripheral blood eosinophils and neutrophils were treated with TFP for 2 h. Cell viability was assessed by staining the cells with Annexin V (AnnV) and DRAQ7. Viable cells, AnnV − DRAQ7 − ; apoptotic cells, AnnV + DRAQ7 − ; necrotic/late apoptotic cells, AnnV + DRAQ7 + . HSAECs, n = 6 from three independent experiments; HLSMCs, eosinophils, neutrophils, n = 4 from four independent experiments; HLFs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA for HLSMCs, HLFs; Kruskal-Wallis for HSAECs; Friedman test for eosinophils, neutrophils). B – C Bone marrow-derived MCs (BMMCs) and peritoneal cell-derived MCs (PCMCs) treated under the same conditions as in ( A ) for 24 h. BMMCs, n = 5 from two independent experiments; PCMCs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA). Untreated (control) cells were used for statistical comparisons to all other groups in all figures. The bar charts show mean values + SEM or median + interquartile range. * P < 0.05; ** P < 0 .01; **** P < 0.0001. D Effect of TFP on DNA degradation. MCs were preincubated with bafilomycin A1 (Baf A1) (20 nM) for 2 h followed by treatment with TFP (10 μΜ) for 2 h. DNA was extracted from MCs and fragmentation was assessed by agarose gel electrophoresis. St standard marker.

Article Snippet: Primary human lung fibroblasts (HLFs) (PCS-201-013) and primary human lung smooth muscle cells (HLSMCs)(PCS-130-010) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Control, Agarose Gel Electrophoresis, Marker