Journal: International Journal of Molecular Sciences

Article Title: HPV-Mediated Resistance to TNF and TRAIL Is Characterized by Global Alterations in Apoptosis Regulatory Factors, Dysregulation of Death Receptors, and Induction of ROS/RNS

doi: 10.3390/ijms20010198

Figure Lengend Snippet: Pro-inflammatory cytokines TNF and TRAIL increase the production and release of reactive oxygen and nitrogen species (ROS/RNS) in cells expressing HPV oncogenes. The levels of ROS and RNS in the absence or presence of pro-inflammatory cytokines alone (2 nM TNF or 50 ng/mL TRAIL) or in combination (2 nM TNF and 50 ng/mL TRAIL) were determined in cultures of PHKs ( A ), PHKs transduced with HPV16 oncogenes ( B ), and in cervical cancer-derived cell lines SiHa (positive for HPV16) ( C ) and HeLa (positive for HPV18) ( D ) using the OxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs, Inc.), according to the manufacturer’s instructions. The results presented are representative of three independent experiments. Solid lines represent statistical differences between intracellular samples and dashed lines between supernatant samples as determined by Student’s t -test ( p -value ≤ 0.05).

Article Snippet: Cervical cancer-derived cell lines SiHa (HPV16, ATCC #HTB-35), HeLa (HPV18, ATCC #CCL-2) and C33 (HPV-negative, ATCC #HTB-31) were cultured in MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% BCS (Cultilab, Campinas, SP, Brazil) and maintained at 37 °C and 5% CO 2 .

Techniques: Expressing, Transduction, Derivative Assay, In Vitro

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: 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: 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

Journal: Allergologia et immunopathologia

Article Title: PTPRO activates TLR4/NF-κB signaling to intensify lipopolysaccharide-induced pneumonia cell injury.

doi: 10.15586/aei.v50i3.589

Figure Lengend Snippet: Figure 2 Cytotoxicity of PTPRO in HFL1. (A) Transfection with pcDNA-PTPRO or shPTPRO increased or decreased PTPRO expression, respectively. (B) Overexpression of PTPRO reduced cell viability of HFL1, while silencing of PTPRO did not affect cell viability. (C) Overexpression of PTPRO promoted cell apoptosis of HFL1, while silencing of PTPRO did not affect cell apoptosis. *p < 0.05, **p < 0.01.

Article Snippet: Human lung fibroblasts (HFL1) (ATCC, Manassas, VA, USA) were cultured in DMEM medium containing 10% fetal bovine Table 1 Primers.

Techniques: Transfection, Expressing, Over Expression

Journal: Allergologia et immunopathologia

Article Title: PTPRO activates TLR4/NF-κB signaling to intensify lipopolysaccharide-induced pneumonia cell injury.

doi: 10.15586/aei.v50i3.589

Figure Lengend Snippet: Figure 1 Lipopolysaccharide induced the elevation of PTPRO in HFL1. (A) Incubation with lipopolysaccharide reduced cell viability of HFL1 in a dose-dependent way. (B) Incubation with lipopolysaccharide enhanced protein expression of PTPRO in HFL1 in a dose-dependent way. *p < 0.05, ***p < 0.001.

Article Snippet: Human lung fibroblasts (HFL1) (ATCC, Manassas, VA, USA) were cultured in DMEM medium containing 10% fetal bovine Table 1 Primers.

Techniques: Incubation, Expressing

Journal: Allergologia et immunopathologia

Article Title: PTPRO activates TLR4/NF-κB signaling to intensify lipopolysaccharide-induced pneumonia cell injury.

doi: 10.15586/aei.v50i3.589

Figure Lengend Snippet: Figure 3 PTPRO contributed to lipopolysaccharide-induced apoptosis in HFL1. (A) Transfection with pcDNA-PTPRO reduced the cell viability of lipopolysaccharide-induced HFL1, while transfection with shPTPRO enhanced cell viability. (B) Overexpression of PTPRO increased cell apoptosis of lipopolysaccharide-induced HFL1, while transfection with shPTPRO inhibited cell apoptosis. (C) The apoptotic ratio of lipopolysaccharide-induced HFL1 that was transfected with pcDNA-PTPRO or shPTPRO. #p < 0.05; ##,^^p < 0.01; ***,###p < 0.001.

Article Snippet: Human lung fibroblasts (HFL1) (ATCC, Manassas, VA, USA) were cultured in DMEM medium containing 10% fetal bovine Table 1 Primers.

Techniques: Transfection, Over Expression

Journal: Allergologia et immunopathologia

Article Title: PTPRO activates TLR4/NF-κB signaling to intensify lipopolysaccharide-induced pneumonia cell injury.

doi: 10.15586/aei.v50i3.589

Figure Lengend Snippet: Figure 4 PTPRO contributed to lipopolysaccharide-induced inflammation in HFL1. (A) mRNA expressions of TNF-α, IL-1β, and IL-18 in lipopolysaccharide-induced HFL1 were enhanced by PTPRO overexpression, while they were reduced by PTPRO silence. (B) Protein expressions of TNF-α, IL-1β, and IL-18 in lipopolysaccharide-induced HFL1 were enhanced by PTPRO overexpression, while they were reduced by PTPRO silence.

Article Snippet: Human lung fibroblasts (HFL1) (ATCC, Manassas, VA, USA) were cultured in DMEM medium containing 10% fetal bovine Table 1 Primers.

Techniques: Over Expression

Journal: Allergologia et immunopathologia

Article Title: PTPRO activates TLR4/NF-κB signaling to intensify lipopolysaccharide-induced pneumonia cell injury.

doi: 10.15586/aei.v50i3.589

Figure Lengend Snippet: Figure 5 PTPRO contributed to lipopolysaccharide-induced activation of TLR4/NF-κB in HFL1. (A) Protein expressions of TLR4, p-p65, and p-IκBα in lipopolysaccharide-induced HFL1 were increased by PTPRO overexpression, while they were decreased by PTPRO silence. #,^p < 0.05; ^^p < 0.01, ***,###p < 0.001.

Article Snippet: Human lung fibroblasts (HFL1) (ATCC, Manassas, VA, USA) were cultured in DMEM medium containing 10% fetal bovine Table 1 Primers.

Techniques: Activation Assay, Over Expression