normal human lung fibroblast cell lines imr 90  (ATCC)

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: Cartilage

Article Title: In Vitro Model Exploring the Mechanisms of Dextrose Prolotherapy: Fibroblasts Exposed to Clinical Concentrations of Dextrose Exhibit Significant Rebound Effects 48 Hours After Exposure

doi: 10.1177/19476035251408601

Figure Lengend Snippet: Direct effect of dextrose exposure on fibroblast metabolic activity immediately following treatment. XTT assay, displaying optical density (OD) readings of fibroblasts treated with 5%, 10%, 15%, 20%, and 25% dextrose compared to media control for 15, 30, 60, and 120 minutes of exposure, run in duplicate, n = 5, error bars represent SEM, * P <0.05.

Article Snippet: The MRC-5 cells, a human lung fibroblast cell line (ATCC; cat# CCL-171), were cultured in complete medium made from Eagle’s Minimum Essential Medium (ATCC; cat# 3002003) supplemented with 10% fetal bovine serum (Atlanta Biologicals; cat# S11150 ), 1% penicillin/streptomycin (ATCC; cat# 30-2300), and 2 mM L-glutamine (ATCC; cat# 30-2214) according to the protocol for subculture recommended by ATCC.

Techniques: Activity Assay, XTT Assay, Control

Journal: Cartilage

Article Title: In Vitro Model Exploring the Mechanisms of Dextrose Prolotherapy: Fibroblasts Exposed to Clinical Concentrations of Dextrose Exhibit Significant Rebound Effects 48 Hours After Exposure

doi: 10.1177/19476035251408601

Figure Lengend Snippet: Direct effect of dextrose exposure on fibroblast metabolic activity 48 hours after treatment. After fibroblasts were treated with 5%, 10%, 15%, 20%, and 25% dextrose compared to media control for 15, 30, 60, and 120 minutes of exposure, the supernatant fluid was replaced with fresh media and the cells were maintained at 37°C, 5% CO 2 for 48 hours prior to running the XTT assay, displaying optical density (OD) readings, run in triplicate, n = 5, error bars represent SEM, * P < 0.05.

Article Snippet: The MRC-5 cells, a human lung fibroblast cell line (ATCC; cat# CCL-171), were cultured in complete medium made from Eagle’s Minimum Essential Medium (ATCC; cat# 3002003) supplemented with 10% fetal bovine serum (Atlanta Biologicals; cat# S11150 ), 1% penicillin/streptomycin (ATCC; cat# 30-2300), and 2 mM L-glutamine (ATCC; cat# 30-2214) according to the protocol for subculture recommended by ATCC.

Techniques: Activity Assay, Control, XTT Assay

Journal: Cartilage

Article Title: In Vitro Model Exploring the Mechanisms of Dextrose Prolotherapy: Fibroblasts Exposed to Clinical Concentrations of Dextrose Exhibit Significant Rebound Effects 48 Hours After Exposure

doi: 10.1177/19476035251408601

Figure Lengend Snippet: Percent change in fibroblast metabolic activity comparing immediately following dextrose treatment to 48 hours following treatment. MRC-5 fibroblasts were exposed to concentrations of 5%, 10%, 15%, 20%, or 25% dextrose for 15, 30, 60, or 120 min. Absorbance was measured by XTT assay immediately following these treatment conditions and then also 48 hours following each of these treatment conditions. Average absorbance values using the XTT assay were calculated for each type of treatment: (1) immediately following direct treatment with dextrose and (2) 48 hours following direct treatment. The percent change was calculated as a ratio of the 48-hour average absorbance value minus the immediate average absorbance value divided by the average absorbance value immediately following treatment for each matched dextrose concentration and exposure time. These values were normalized to the corresponding media control for each duration of dextrose treatment (15, 30, 60, or 120 min). Averaged values represent 5 independent experiments.

Article Snippet: The MRC-5 cells, a human lung fibroblast cell line (ATCC; cat# CCL-171), were cultured in complete medium made from Eagle’s Minimum Essential Medium (ATCC; cat# 3002003) supplemented with 10% fetal bovine serum (Atlanta Biologicals; cat# S11150 ), 1% penicillin/streptomycin (ATCC; cat# 30-2300), and 2 mM L-glutamine (ATCC; cat# 30-2214) according to the protocol for subculture recommended by ATCC.

Techniques: Activity Assay, XTT Assay, Concentration Assay, Control

Journal: Cartilage

Article Title: In Vitro Model Exploring the Mechanisms of Dextrose Prolotherapy: Fibroblasts Exposed to Clinical Concentrations of Dextrose Exhibit Significant Rebound Effects 48 Hours After Exposure

doi: 10.1177/19476035251408601

Figure Lengend Snippet: Indirect effect of dextrose exposure on the metabolic activity of nascent fibroblasts 48 hours after treatment. After fibroblasts were treated with 5%, 10%, 15%, 20%, and 25% dextrose compared to media control for 15, 30, 60, and 120 minutes of exposure, the supernatant fluid was replaced with fresh media and the cells were maintained at 37°C, 5% CO 2 for 8 hours prior to collecting the supernatant fluid. Nascent fibroblasts (not exposed to dextrose) were then incubated with this supernatant fluid from dextrose-treated cells for 48 hours. XTT assay, displaying optical density (OD) readings of nascent fibroblasts 48 hours after being exposed to supernatant fluid taken from fibroblasts directly treated with dextrose, run in triplicate, n = 5, error bars represent SEM. * P < 0.05.

Article Snippet: The MRC-5 cells, a human lung fibroblast cell line (ATCC; cat# CCL-171), were cultured in complete medium made from Eagle’s Minimum Essential Medium (ATCC; cat# 3002003) supplemented with 10% fetal bovine serum (Atlanta Biologicals; cat# S11150 ), 1% penicillin/streptomycin (ATCC; cat# 30-2300), and 2 mM L-glutamine (ATCC; cat# 30-2214) according to the protocol for subculture recommended by ATCC.

Techniques: Activity Assay, Control, Incubation, XTT Assay