human lung fibroblast cell line ccd8lu  (ATCC)

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Research strategy. (a) Fibroblast activation is understood to be a result of many microenvironmental cues, such as cell–cell communication, fibrillar structure, biochemical cues, and matrix stiffness, which can either drive healthy wound healing or persistence and fibrosis. There is a need for innovative tools for understanding why certain signals drive homeostasis vs disease. (b) Simplified gene cassettes depict the two key genes for fluorescent reporting: a red fluorescent protein expressed under the constitutive PGK promoter and a green fluorescent protein conditionally expressed with the activation of the ACTA2 promoter associated with alpha smooth muscle actin expression. (c) Fibroblasts are transduced at a high titer to create reporter cell populations, where some cells receive more gene cassette copies than others, but always at a one-to-one ratio of red and green fluorescence. (d) Transduced cells then express constitutive DsRed-Express2 and conditionally express ZsGreen, where the one-to-one ratio of red and green genes delivered to each cell allows for normalization by red fluorescence intensity. (e) To expand the reporter system to track temporal fibroblast behavior, three versions of the gene cassette were utilized, where Intermediate and Fast are modified to degrade on different timescales.

Article Snippet: Human lung fibroblast lines were purchased from ATCC (CCL151 male healthy, CCL134 female IPF, CCL210 female healthy).

Techniques: Activation Assay, Expressing, Fluorescence, Modification

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Comparison of αSMA expression with immunostaining assessment. (a) Stable reporter lung fibroblasts (CCL151, male healthy; CCL210, female healthy; and CCL134, female IPF) were cultured on glass for 48 h, fixed, and stained for alpha smooth muscle actin. (b) Quantification of immunostained images demonstrates that samples from all reporter lines (Stable, Intermediate, and Fast) of all cell types had 100% of the cells positive for diffuse staining for alpha smooth muscle actin (n = 3 wells per condition, >50 cells per well). (c) To further differentiate proto-myofibroblasts from mature myofibroblasts, cells with organized αSMA fibers (CCL151-stable reporter) were visually assessed and (d) quantified across all reporter cell lines (n = 3 well per condition, >50 cells per well). (e)–(g) Reporter intensity normalized on a per cell basis was compared for cells with and without organized αSMA fibers for CCL151 (e), CCL210 (f), and CCL134 (g) cell lines. (per cell line, data from all wells were pooled for distributions). Scale bars are 200 μ m. Statistical comparisons in (d) were assessed by one-way ANOVA and comparisons in (e)–(g) were assessed by Welch's t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Human lung fibroblast lines were purchased from ATCC (CCL151 male healthy, CCL134 female IPF, CCL210 female healthy).

Techniques: Comparison, Expressing, Immunostaining, Cell Culture, Staining

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Comparison of reporters between cells of different tissue origin. ZsGreen(+) population fractions for CCL151 (male healthy lung fibroblasts), CCL210 (female healthy lung fibroblasts), and CCL134 (female IPF lung fibroblasts) Stable, Intermediate, and Fast reporters after 48 h culture were assessed by flow cytometry (initial seeding density 5000 cell/cm 2 ). Statistical differences were determined within reporters by one-way ANOVA. Error bars represent standard deviation, n ≥ 3 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Human lung fibroblast lines were purchased from ATCC (CCL151 male healthy, CCL134 female IPF, CCL210 female healthy).

Techniques: Comparison, Flow Cytometry, Standard Deviation

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Impact of culture density on the reporting of fibroblast activation. CCL151, CL210, and CCL134 lines at different initial seeding densities and culture timepoints were investigated by flow cytometry with (a) Stable, (b) Intermediate, and (c) Fast reporters. Data for 5000 cells/cm 2 48 h conditions are repeated here from for ease of comparison. Statistical differences were assessed by one-way ANOVA within each cell line and reporter. Error bars depict standard deviation with n ≥ 3 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Human lung fibroblast lines were purchased from ATCC (CCL151 male healthy, CCL134 female IPF, CCL210 female healthy).

Techniques: Activation Assay, Flow Cytometry, Comparison, Standard Deviation

Journal: APL Bioengineering

Article Title: Dynamic reporters for probing real-time activation of human fibroblasts from single cells to populations

doi: 10.1063/5.0166152

Figure Lengend Snippet: Mechanical properties of the culture substrate impact extent of fibroblast activation. (a) Workflow for assessing the impact of culture surface mechanical properties on αSMA reporting behavior; CCL151 fibroblasts of all reporter types (Stable, Intermediate, and Fast) were cultured on surfaces with different stiffness and assessed using flow cytometry. Error bars represent standard deviation of n ≥ 3 replicates. (b) Stable, (c) Intermediate, and (d) Fast reporter ZsGreen(+) population fractions after culture on soft (E∼2 kPa) and stiff (E∼50 kPa) substrates over two passages. (e) Stable, (f) Intermediate, and (g) Fast box-cox transformed (λ = 0.075) distributions of normalized reporter intensities for individual cell events, where arrows point to shifts in population from P1 to P2. Statistical analysis in panels (b)–(d) were assessed by two-way ANOVA, with passage and culture substrate as factors. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Human lung fibroblast lines were purchased from ATCC (CCL151 male healthy, CCL134 female IPF, CCL210 female healthy).

Techniques: Activation Assay, Cell Culture, Flow Cytometry, Standard Deviation, Transformation Assay

Journal: International journal of molecular sciences

Article Title: miR-145 as a Potential Biomarker and Therapeutic Target in Patients with Non-Small Cell Lung Cancer.

doi: 10.3390/ijms241210022

Figure Lengend Snippet: Figure 2. Upregulated miR-145 inhibits cell proliferation, migration, and invasion. (A) The relative expression of miR-145 was significantly lower in NSCLC cell lines (A549 and NCI-H358) when compared to a normal human lung fibroblast cell line (CCD-11Lu) measured using RT-qPCR. (B) miR- 145 mimic transfection significantly inhibited cell proliferation in NCI-H358 cells at the last three time points (p = 0.026, 0.004, and 0.004 vs. pre-transfection, respectively). (C) miR-145 inhibits NSCLC migration in A549 cells 24 h after transfection with miR-145 mimic. miR-145 also inhibits NSCLC invasion in A549 cells 48 h after transfection with miR-145 mimic. (D) Flow cytometry analysis was used to examine the relative cell cycle composition of NCI-H358 cells transfected with miR- 145 mimic or miR-NC, our results showed no significant difference between miR-145 and controls. Each experiment was performed in triplicate and data are shown as mean ± standard error of the mean. NC, negative control; NSCLC, non-small cell lung cancer; and RT-qPCR, reverse transcription quantitative real-time polymerase chain reaction.

Article Snippet: The human NSCLC cell lines (A549 and NCI-H358) and normal human lung fibroblast cell line (CCD-11Lu) were purchased from American Type Culture Collection (Manassas, Int.

Techniques: Migration, Expressing, Quantitative RT-PCR, Transfection, Flow Cytometry, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction