Journal: Cell reports

Article Title: SILAC Analysis Reveals Increased Secretion of Hemostasis-Related Factors by Senescent Cells

doi: 10.1016/j.celrep.2019.08.049

Figure Lengend Snippet: (A) A schematic outlining the strategy for SILAC-based identification of SASP proteins. See for time lines of how cells were made quiescent or senescent and how quiescent and senescent conditioned media (CM) were produced. (B) Reactome pathway analysis of proteins secreted at >2-fold levels by senescent compared with quiescent HCA2 cells. Pathways related to hemostasis are identified by boxes. (C) Heatmap showing individual levels of proteins, identified by SILACs, that participate in hemostasis and are secreted by quiescent (QUI) and senescent (SEN [IR]) HCA2 normal human fibroblasts. (D) CM were collected from quiescent (QUI) and senescent (SEN [IR]) cells, concentrated, and analyzed using western blotting for the indicated SASP factors. Cells remaining on the plate at the time of CM collection were counted for normalization. Mindin protein levels and Ponceau S staining were used as loading controls. Fold changes are mean ± SEM. *p < 0.05 and **p < 0.01. See also and .

Article Snippet: WI-38 and IMR-90 are normal primary human embryonic lung fibroblasts (obtained from the American Type Culture Collection).

Techniques: Multiplex sample analysis, Produced, Western Blot, Staining

Journal: Cell reports

Article Title: SILAC Analysis Reveals Increased Secretion of Hemostasis-Related Factors by Senescent Cells

doi: 10.1016/j.celrep.2019.08.049

Figure Lengend Snippet: (A) mRNA was extracted from HCA2 cells made quiescent (QUI) or induced to senesce by doxorubicin (SEN [DOXO]), and analyzed using qPCR for the indicated genes, using actin mRNA for normalization. The data are shown in a.u., and the bars show mean ± SEM. *p < 0.05 and **p < 0.01, two-tailed, unpaired t test. (B) CM were collected from cells treated as in (A), concentrated and analyzed using western blotting for the indicated SASP factors. Cells were counted for normalization. Mindin protein levels and Ponceau S staining were used as loading controls. (C) IMR-90 fibroblasts were depleted of mtDNA by culture in ethidium bromide until senescent (21 days) (labeled as MiDAS). RNA was extracted and analyzed using qPCR for the indicated factors. Heatmap represents individual values for three experiments. *p < 0.05, one-way ANOVA. See also .

Article Snippet: WI-38 and IMR-90 are normal primary human embryonic lung fibroblasts (obtained from the American Type Culture Collection).

Techniques: Two Tailed Test, Western Blot, Staining, Labeling

Journal: Cell reports

Article Title: SILAC Analysis Reveals Increased Secretion of Hemostasis-Related Factors by Senescent Cells

doi: 10.1016/j.celrep.2019.08.049

Figure Lengend Snippet:

Article Snippet: WI-38 and IMR-90 are normal primary human embryonic lung fibroblasts (obtained from the American Type Culture Collection).

Techniques: Recombinant, Modification, Sequencing, Multiplex sample analysis, Labeling, Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Reverse Transcription, Software

Journal: Biotechnology and bioengineering

Article Title: Engineering dense tumor constructs via cellular contraction of extracellular matrix hydrogels.

doi: 10.1002/bit.28561

Figure Lengend Snippet: FIGURE 1 Harnessing fibroblast contractility to engineer dense carcinoma constructs. (a) Schematic illustration of fibroblast‐driven contraction of collagen fibers around cancer spheroids. Blue shapes represent fibroblasts, black lines represent collagen fibers, and green clusters represent spheroids. (b) Side by side comparison of H&E‐stained sections of dense breast carcinoma constructs (left) and biopsied mammary ductal carcinoma tissue (right). Scale bars = 80 μm. (c) Time‐course imaging of gross contraction and compaction of dense carcinoma constructs. Scale bars = 200 μm. (d) Box plot of the relative area change over time for gels seeded at 2.5 × 105 HLF/mL (n = 6) and 5 × 105/mL (n = 7). **p < 0.01. ***p < 0.001. See Section 2 for details of statistical analyses. (e) Box plot of the corresponding increase in estimated collagen density over time for gels seeded for acellular gels and 5 × 105 HLF/mL (n = 5 per group).

Article Snippet: Normal human lung fibroblasts (HLF) (American Type Culture Collection [ATCC] and Lonza) were cultured in fibroblast basal medium with low serum kit (ATCC) and 2% fetal bovine serum (FBS, ATCC).

Techniques: Construct, Comparison, Staining, Imaging

Journal: Biotechnology and bioengineering

Article Title: Engineering dense tumor constructs via cellular contraction of extracellular matrix hydrogels.

doi: 10.1002/bit.28561

Figure Lengend Snippet: FIGURE 2 Effect of fibroblast cell type and seeding density on tissue contraction. (a–c) Time‐course imaging of gross contraction and compaction of dense carcinoma constructs formed using several types of fibroblasts. Constructs were seeded at 5 × 105/mL (n = 3 per group). Scale bars = 3 mm. Similar kinetics and magnitude of contraction using human dermal fibroblasts (HDF, a), human lung cancer associated fibroblasts (CAF, b), and human lung fibroblasts (HLF, c). (d and e) Time‐course imaging of gross contraction and compaction for up to 3 weeks in culture. Scale bars = 3 mm. Constructs were seeded at 0.5 × 105 HLF/mL (n = 6, panel d) and 5 × 105/mL (n = 7, e). (f) Relative area change over time as a measure of contraction for each type of fibroblast during 7 days of culture as shown in (a–c). No significant differences were measured. HDF constructs were slightly less contracted at 7 days, matching the qualitatively larger size in time course images. (g) Relative area change over time as a measure of contraction. Similar final volumes were reached with each seeding density. Contraction was more gradual with a seeding density of 0.5 × 105 HLF/mL.

Article Snippet: Normal human lung fibroblasts (HLF) (American Type Culture Collection [ATCC] and Lonza) were cultured in fibroblast basal medium with low serum kit (ATCC) and 2% fetal bovine serum (FBS, ATCC).

Techniques: Imaging, Construct

Journal: Biotechnology and bioengineering

Article Title: Engineering dense tumor constructs via cellular contraction of extracellular matrix hydrogels.

doi: 10.1002/bit.28561

Figure Lengend Snippet: FIGURE 5 Local anisotropy is similar in constructs formed with various types of contractile cells. (a) H&E‐staining of sectioned dense carcinoma constructs formed with cancer associated fibroblasts (CAF), human dermal fibroblasts (HDF), and human lung fibroblasts (HLF). Seven days, scale bars = 30 μm. (b) Collagen type I staining of sectioned dense carcinoma constructs formed with CAF, HDF, and HLF. Seven days, scale bars = 20 μm. (c) Box plot of the relative fluorescent intensity in binned regions of interest at the specified distances from spheroid for dense carcinoma constructs formed with CAF (white), HDF (light gray), and HLF (dark gray) (n = 9 per group). *p < 0.05, **p < 0.01. One way ANOVA. (d) Box plot of fiber orientation relative to spheroid tangent line and distance from spheroid for dense carcinoma constructs. Same shading as (d) to indicate fibroblast type; CAF (white), HDF (light gray), and HLF (dark gray) (n = 9 per group). One way ANOVA. ns, not significant.

Article Snippet: Normal human lung fibroblasts (HLF) (American Type Culture Collection [ATCC] and Lonza) were cultured in fibroblast basal medium with low serum kit (ATCC) and 2% fetal bovine serum (FBS, ATCC).

Techniques: Construct, Staining

Journal: Journal of Biological Chemistry

Article Title: Evidence That Factor VIII Forms a Bivalent Complex with the Low Density Lipoprotein (LDL) Receptor-related Protein 1 (LRP1)

doi: 10.1074/jbc.m116.754622

Figure Lengend Snippet: FIGURE 7. Effect of clusters II and IV on LRP1-mediated internalization of BDD-fVIII (A) and tPAPAI-1 (B) by WI-38 fibroblasts. WI-38 fibroblasts were seeded in 12-well tissue culture dishes (1 105 cells/well) and incubated with 20 nM 125I-BDD-fVIII (A) or 10 nM 125I-tPAPAI-1 (B) for 4 h at 37 °C in the presence orabsenceofLRP1clusterIIorIV(500nM).Followingincubation,theamountofradiolabeledligandinternalizedwasdetermined,andthedatawerenormalized to the amount of fVIII internalized in the absence of competitors. The results represent the mean of three independent experiments. Error bars represent S.E. (*, p 0.002, one-way analysis of variance with Tukey’s multiple comparison post-test).

Article Snippet: WI38 cells, human lung fibroblast cells, were obtained from American Type Culture Collection (ATCC) and maintained in DMEM (Corning) supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Incubation, Comparison