Journal: Scientific reports

Article Title: Polyethylene glycol-phospholipid functionalized single-walled carbon nanotubes for enhanced siRNA systemic delivery.

doi: 10.1038/s41598-024-80646-1

Figure Lengend Snippet: Fig. 2. Significant gene silencing efficiency using SWNT-siRNA conjugates. (A) Graphical representation of SWNT-PL-PEG-S-S-siRNA. (B) Graphical representation of SWNT-PL-PEG-siRNA. (C, D and E) Evaluation of gene silencing effect by SWNT-PL-PEG-S-S-siGFP and SWNT-PL-PEG-siGFP conjugates in MCF-7, HEK-293T, H1299 and HeLa cells. Cells expressing stable green fluorescent protein (GFP) were treated with SWNT-PL-PEG-S-S-siGFP or SWNT-PL-PEG-siGFP conjugates at a concentration of 100 nM siGFP. The cell nuclei were stained with DAPI (blue). Microscopic observation was captured under 100x magnification. Bars represent mean ± SD of at least 3 independent experiments. * represents statistical significance as compared to vehicle controls (Student’s t-test, p < 0.01).

Article Snippet: Cell lines and cell culture The human breast cancer cell line MCF-7, human non-small cell lung carcinoma line H1299, and human cervical cancer cells HeLa were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Concentration Assay, Staining

Journal: Scientific reports

Article Title: Polyethylene glycol-phospholipid functionalized single-walled carbon nanotubes for enhanced siRNA systemic delivery.

doi: 10.1038/s41598-024-80646-1

Figure Lengend Snippet: Fig. 3. Dual-silencing efficacy of siRNA-conjugated SWNT delivery system. Fluorescence microscopic images of co-expressed GFP and RFP in H1299 cells incubated with 100 nM of (A) SWNT-PL-PEG-S-S-siRNA or (B) SWNT-PL-PEG-siRNA conjugates targeting GFP (siGFP) or RFP (siRFP) or LUC (siLUC; negative control) alone, or both (dual targeting; siGFP + siRFP) for 72 h. The cell nuclei were stained with DAPI. The furthest right panel was the combined images. Microscopic observation was captured under 100x magnification.

Article Snippet: Cell lines and cell culture The human breast cancer cell line MCF-7, human non-small cell lung carcinoma line H1299, and human cervical cancer cells HeLa were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Fluorescence, Incubation, Negative Control, Staining

Journal: Scientific reports

Article Title: Polyethylene glycol-phospholipid functionalized single-walled carbon nanotubes for enhanced siRNA systemic delivery.

doi: 10.1038/s41598-024-80646-1

Figure Lengend Snippet: Fig. 4. Energy dependent cellular internalization mechanisms of SWNT-siRNA conjugates independent of ABCB1. Graph shows the mean relative luminescent units (RLU) of (A and B) luciferase expressing H1299 cells co-treated with SWNT only (without siLUC), SWNT-PL-PEG-S-S-siLUC or SWNT-PL-PEG- siLUC with or without sub-lethal concentrations of various endocytosis inhibitors. NaN3, chlorpromazine, genistein and m-βCD are inhibitors for energy-dependent cellular uptake, clathrin-mediated endocytosis, caveolae-dependent endocytosis and macro-pinocytosis, respectively. Bars represent mean ± SD of at least 3 independent experiments. * represents statistical significance as compared to SWNT-siLUC (Student’s t-test, p < 0.01). (C) Western blot of ABCB1 and loading control GAPDH, cropped blots. Ectopic expression of ABCB1 (multiple drug resistance) protein in H1299-LUC cells was confirmed by the immunoblotting with increased expression of ABCB1 at 141 kDA, while GAPDH at 36 kDA served as the loading control. Full blots available in Supplementary Figure S2-S3. (D) Expression of ABCB1 did not affect the delivery of siRNA by the SWNT. Bars represent mean ± SD of at least 3 independent experiments. * represents statistical significance as compared to controls (Student’s t-test, p < 0.01).

Article Snippet: Cell lines and cell culture The human breast cancer cell line MCF-7, human non-small cell lung carcinoma line H1299, and human cervical cancer cells HeLa were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Luciferase, Expressing, Western Blot, Control

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: Genome Research

Article Title: Reorganization of the host epigenome by a viral oncogene

doi: 10.1101/gr.132308.111

Figure Lengend Snippet: Small e1a causes global deacetylation and redistribution of H3K18ac. ( A ) Venn diagram showing the overlap between significant peaks of H3K18ac in mock- (dark blue) and dl 1500-infected cells (light blue). ( B ) Venn diagram showing the overlap between significant peaks of H3K9ac in mock- (dark orange) and dl 1500-infected cells (yellow). ( C ) Patterns of H3K18ac and H3K9ac in the intergenic region between COPS8 and COL6A3 genes in mock-, dl 1500-infected, and asynchronous IMR90 cells. ( D ) Quantitative PCR (% of input) for EP300 and CREBBP in mock- and dl 1500-infected cells for the COL6A3 intergenic region, and CCNE2 and POLD3 promoters are shown as bar plots. Patterns of H3K18ac in mock- and dl 1500-infected cells at CCNE2 and POLD3 loci are also shown. For each histone modification, the y -axis indicates the number of input-normalized ChIP-seq reads across the locus ( x -axis). (Light blue arrows) New peaks of acetylation in e1a-expressing cells. (Dark arrows) Direction of transcription. ( E , F ) Overview of distribution of H3K18ac and H3K9ac peaks in mock- and dl 1500-infected cells in relation to gene structure are shown as pie charts. ( G ) Distribution of significant peaks of H3K18ac with respect to TSS in mock- (dark blue) and dl 1500-infected (light blue) cells. ( H ) Distribution of significant peaks of H3K9ac with respect to TSS in mock- (dark orange) and dl 1500-infected (yellow) cells.

Article Snippet: IMR90 human primary lung embryo fibroblasts (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 .

Techniques: Infection, Real-time Polymerase Chain Reaction, Modification, ChIP-sequencing, Expressing

Journal: Genome Research

Article Title: Reorganization of the host epigenome by a viral oncogene

doi: 10.1101/gr.132308.111

Figure Lengend Snippet: Analyses of RB-family protein genome-wide binding in contact-inhibited IMR90 fibroblasts. ( A ) Overview of RB1, RBL2, and RBL1 peak distributions in mock-infected cells in relation to gene structure are shown as pie charts. ( B ) Average binding profiles (significant counts) of the indicated RB-family members across the TSS regions of their respective target genes. ( C ) A 600-bp region around the peaks of RB1, RBL2, and RBL1 in mock-infected cells were analyzed for TF binding motifs using sitepro (Galaxy). Top three significant motifs for each family member are shown. All P -values are less than 1 × 10 −10 . ( D ) The distributions of the RB-family proteins across ±5 kb of TSS for genes with at least one RB-family member bound are shown as heat maps. The seven clusters are based on combinatorial binding patterns of the three proteins. ( E ) Relative gene expression changes of each of the seven clusters after e1a expression at 24 h p.i. are shown as box plots.

Article Snippet: IMR90 human primary lung embryo fibroblasts (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 .

Techniques: Genome Wide, Binding Assay, Infection, Gene Expression, Expressing

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