imr90 human lung fibroblast  (ATCC)

Journal: Materials Today Bio

Article Title: Polystyrene nanoplastics-induced lung epithelial cells ferroptosis promotes pulmonary fibrosis via YY1/FTL axis

doi: 10.1016/j.mtbio.2025.102738

Figure Lengend Snippet: PS-NPs-Induced Ferroptosis in BEAS-2B Cells and Paracrine Fibroblast Activation: (A–B) Western blot of GPX4 and FTL in BEAS-2B cells after 24 h exposure to PS-NPs at 0, 50, 100, and 200 μg/ml (C–D) Time-course of GPX4 and FTL expression in cells treated with 200 μg/ml PS-NPs for 0, 3, 6, 12, and 24 h. (E) Transmission electron microscopy images of control and PS-NPs treated BEAS-2B cells showing characteristic mitochondrial shrinkage. (F–G) Restoration of GPX4 following co-treatment with Fer-1. (H) C11-BODIPY581/591 fluorescence showing Fer-1–mediated attenuation of lipid peroxidation. (I) FerroOrange staining demonstrating reduction of labile Fe 2+ upon Fer-1 co-incubation. (J–O) COL1 and α-SMA mRNA (qPCR) and protein (western blot) levels in MRC-5 fibroblasts after 24 h treatment with conditioned medium from BEAS-2B cells exposed to PS-NPs ± Fer-1. Statistical significance is denoted as *p < 0.05 and **p < 0.01.

Article Snippet: The human bronchial epithelial cell (BEAS-2B) and human embryonic lung fibroblast (MRC-5) were commercially purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Transmission Assay, Electron Microscopy, Control, Fluorescence, Staining, Incubation

Journal: Materials Today Bio

Article Title: Polystyrene nanoplastics-induced lung epithelial cells ferroptosis promotes pulmonary fibrosis via YY1/FTL axis

doi: 10.1016/j.mtbio.2025.102738

Figure Lengend Snippet: YY1 Silencing Reverses PS-NPs–Induced Ferroptosis and Paracrine Fibrogenesis: (A–C) Western blots showing restoration of GPX4 protein in BEAS-2B cells after YY1 knockdown and 24 h PS-NPs exposure. (D–E) C11-BODIPY581/591 fluorescence demonstrating reduced lipid peroxidation upon YY1 silencing. (F) FerroOrange staining indicating decreased labile Fe 2+ accumulation after YY1 knockdown. (G–H) Double immunofluorescence staining of YY1 with the epithelial marker SPC and the myofibroblast marker α-SMA in lung sections from PS-NPs-treated mice. (I–L) Western blot and qRT-PCR analyses of COL1 and α-SMA in MRC-5 fibroblasts treated for 24 h with conditioned medium from BEAS-2B cells exposed to PS-NPs ± YY1 silencing. Statistical significance is denoted as *p < 0.05 and **p < 0.01.

Article Snippet: The human bronchial epithelial cell (BEAS-2B) and human embryonic lung fibroblast (MRC-5) were commercially purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Knockdown, Fluorescence, Staining, Double Immunofluorescence Staining, Marker, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression

doi: 10.1093/nar/gkaf1521

Figure Lengend Snippet: NSMF mitigates replication stress and prevents oncogene-induced senescence. ( A ) SA-β-galactosidase (SA-β-Gal) staining in stable NSMF knockdown (shNSMF #1 and #2) or control (shCtrl) HCT116 cells. Representative images (upper) and quantification of SA-β-gal positive cells (lower). Scale bar, 50 μm. Data are presented as mean ± SEM from 150 cells across seven images obtained from three independent experiments. ** P < .01, *** P < .001, one-way ANOVA followed by Dunnett’s multiple comparisons test. ( B ) GSEA plot showing enrichment of cellular senescence-related genes in RNA-seq data from Nsmf +/+ ; Apc Min/+ and Nsmf −/− ; Apc Min/+ intestinal tumor (upper). Heatmap visualization of differentially expressed senescence- and SASP-related genes between genotypes (lower). ( C ) qRT-PCR analysis of senescence-associated genes in intestinal tumors from Nsmf +/+ ; Apc Min/+ ( n = 4) and Nsmf −/− ; Apc Min/+ ( n = 3) mice. Data represent the mean ± SEM. * P < .05, ** P < .01, *** P < .001, unpaired two-tailed t -test with Holm–Sidak correction for multiple comparisons. ( D ) Western blot analysis of p16INK4A and p21CIP1 in intestinal tumor tissues from Nsmf +/+ ; Apc Min/+ and Nsmf −/− ; Apc Min/+ mice. GAPDH served as a loading control. ( E ) Schematic representation of the experimental design for the oncogene-induced senescence model. IMR-90 cells were transduced with lentiviruses encoding either GFP-vector or GFP-NSMF. Following selection, senescence was induced by expression of oncogenic Ras G12V . ( F ) Western blot analysis of the indicated proteins on day 4 after induction of oncogenic Ras G12V expression. α-Tubulin was used as a loading control. ( G ) SA-β-Gal staining in IMR-90 cells 8 days post-transduction. Representative images (left) and quantification of SA-β-Gal positive cells (right). Scale bar, 20um. Data are presented as mean ± SEM ( n = 4–6 independent images per sample). *** P < .001, n.s., not significant, one-way ANOVA followed by Tukey’s HSD test. ( H ) Immunofluorescence analysis of γH2AX in GFP-vector or GFP-NSMF expressing IMR-90 cells with or without Ras G12V . Quantification of γH2AX foci per GFP-positive cell was performed in at least 42 cells per group. Scale bar, 20 μm. Data are presented as median. ** P < .01, **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. All experiments were independently performed at least three times, and representative results are shown. ( I ) Correlation of NSMF expression with genomic instability in pan-cancer analysis. Genomic instability was assessed across 4315 pan-cancer samples from TCGA using multiple genomic instability features, including frequency of LOH, HRD-related LOH frequency, telomeric allelic imbalance, large-scale transitions, mutation burden per sample, and weighted genome integrity index. Tumors were categorized based on genomic instability scores as low (<25%, below first quartile), medium (25%–75%, between first and third quartile), or high (>75%, above third quartile). Statistical comparisons of NSMF expression levels across groups were performed using Wilcoxon rank-sum test. ( J ) Hypothetical model illustrating the role of NSMF in regulating replication stress, highlighting its critical function in alleviating excessive replication stress and preventing cytotoxic DNA damage. This regulatory activity supports a controlled level of genomic instability, thereby promoting CRC progression. Figure was created using BioRender.com.

Article Snippet: The normal colon-derived cell line CCD-18Co, human CRC cell lines HCT116, and the human lung fibroblast cell line IMR-90 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Staining, Knockdown, Control, RNA Sequencing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Transduction, Plasmid Preparation, Selection, Expressing, Immunofluorescence, Mutagenesis, Activity Assay

Journal: Nucleic Acids Research

Article Title: Human cytomegalovirus regulates host DNA repair machinery for viral genome integrity

doi: 10.1093/nar/gkaf1445

Figure Lengend Snippet: USP1 restricts CMV replication. MRC-5 fibroblasts were reverse-transfected with a siRNA pool targeting UAF1 or USP1 or an NTC. At two days post-transfection, cells were infected with CMV TB40/E-GFP wild-type (WT) or TB40/E-∆ UL138 STOP at an MOI of 1 and cells were harvested at 96 hpi. ( A ) Viral genome copy number was determined by qPCR using a TB40/E BAC standard curve and primer set designed for the region of the viral genome encoding the β2.7 transcript. The graph represents the fold change in viral genome copy number relative to siNTC. ( B ) Virus yields were measured by TCID 50 and normalized relative to the WT siNTC condition. For panels (A) and (B), statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparisons test. Asterisks (** P < .01, *** P < .001, **** P < .0001) represent statistically significant differences determined for four independent experiments. ns, nonsignificant. ( C ) Whole cell lysates were collected at two days post-transfection to determine efficiency of USP1 knockdown by immunoblotting using monoclonal antibodies for USP1 and tubulin as a loading control and secondary antibodies conjugated to DyLight™ 680 (mouse) or 800 (rabbit). Densitometry analysis of three biological replicates reveals an average 68% knockdown compared to siNTC. ( D ) At two days post-transfection, RNA was collected for RT-qPCR to determine the efficiency of UAF1 knockdown relative to siNTC. H6PD was used as a housekeeping control for cell number. Over all experiments, an average knockdown efficiency of 81% was achieved. A paired t- test was used to determine statistical significance, **** P < .0001, over multiple independent experiments.

Article Snippet: Primary human lung MRC-5 fibroblasts (ATCC CCL-171) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 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: Transfection, Infection, Virus, Knockdown, Western Blot, Bioprocessing, Control, Quantitative RT-PCR