Journal: Nature Communications

Article Title: PRC2-AgeIndex as a universal biomarker of aging and rejuvenation

doi: 10.1038/s41467-024-50098-2

Figure Lengend Snippet: a Average DNAm levels at CpGs across the whole genome (left panel), at LMRs ranked by the level of their PRC2 binding in hESCs (center panel) and within high-PRC2 regions (right panel) from 8 different tumor and normal samples. Data were obtained from TCGA WGBS datasets, (see Supplementary Data ). Error bars on the right show 95% confidence intervals for average DNAm across high-PRC2 LMRs for each sample. b Average DNAm levels at CpGs across the whole genome (left panel) and at LMRs ranked by the level of their PRC2 binding in hESCs (right panel) from young vs old human oligodendrocytes. (WGBS dataset, GSE107729, n = 6). c Average DNAm levels at CpGs across the whole genome (left panel) and at LMRs ranked by the level of their PRC2 binding in hESCs (middle panel), correlation between average DNAm at high-PRC2 LMRs and the number of passages (right panel, shaded area of line plot represents 95% CI) in in vitro cultured fibroblasts (GSE79798, n = 5). High-PRC2 LMRs are highlighted with dotted red boxes.

Article Snippet: Neonatal fibroblasts were purchased from Cell Applications (#106-05n) and old fibroblasts were isolated from skin tissue collected from deceased healthy individuals.

Techniques: Binding Assay, In Vitro, Cell Culture

Journal: Nature Communications

Article Title: PRC2-AgeIndex as a universal biomarker of aging and rejuvenation

doi: 10.1038/s41467-024-50098-2

Figure Lengend Snippet: a Average DNAm levels of neonatal and old passaged fibroblasts, and CD4 T-cells, at CpGs across the whole genome (left panel) and at the LMRs ranked by the level of EZH2-binding data of the same respective tissue (middle panels). Bottom panels show heatmaps of LMR rank number, ordered by EZH2 binding in their respective tissue (top heatmap) and in hESCs (bottom heatmap). Heatmaps are colored by EZH2 binding in the same respective tissue, i.e. purple/orange represents high and low-ranked neonatal/old LMRs ordered by neonatal/old fibroblasts PRC2 binding, respectively, and red/blue represent high and low-ranked CD4 T-cells LMRs ordered by CD4 T-cells PRC2 binding, respectively. Right panel shows the correlation of mean methylation of high-PRC2 LMRs against age or passage number. b Average methylation levels at LMRs ranked by PRC2 binding in hESCs for neonatal fibroblasts (left panels) and old fibroblasts (right panels). c Average DNAm levels of LMRs calculated from neonatal and old samples merged (all passages), ranked by PRC2 binding in hESCs (left panel) and correlation between methylation and age for high-PRC2 LMRs of both neonatal and old in vitro passaged fibroblasts (right panel). d Heatmap of normalized read density of the high-PRC2 neonatal/old LMRs in neonatal and old fibroblasts (passage 2). WGBS Neonatal and old fibroblasts were generated by our lab (GSE253987, n = 3 for neonatal samples (one donor) and n = 3 for old samples (one donor)), CD4 T-cell samples are the same as used in Fig. (6 samples 18–86 years old, GSE79798 and 2 samples 0 and 103 years old, GSE31263). ChIP data for fibroblast and CD4 T-cells were generated by our lab (GSE253987, two donors pooled at passage 2 for neonatal fibroblasts, three donors pooled at passage 2 for old fibroblasts, three donors pooled for CD4 T-cells). High-PRC2 LMRs are highlighted with dotted red boxes. Line plots with >3 samples plotted have a shaded area representing 95% CI.

Article Snippet: Neonatal fibroblasts were purchased from Cell Applications (#106-05n) and old fibroblasts were isolated from skin tissue collected from deceased healthy individuals.

Techniques: Binding Assay, Methylation, In Vitro, Generated

Journal: iScience

Article Title: Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics

doi: 10.1016/j.isci.2023.106381

Figure Lengend Snippet: SM induces human dendritic cell maturation which is further enhanced by cancer cell necroptosis (A) Schematic representation of the in vitro co-culture assay. moDCs alone (B) or in co-culture with spheroids (tumor cells and hd-fibroblasts) (D and E) were treated with the compound conditions depicted. 72 h post-treatment, cells were harvested and analyzed via flow cytometry. (B) Relative proportion of activated vs non-activated moDC treated alone. (C) TSNE plot depicting islands of phenotypically similar cells based on CD45, CD11c, HLA-DR, CD86, CD83, and PDL1 as markers. Included are the moDCs treated in monoculture, as well as the different co-cultures. Analyzed cells separate into three subpopulations: activated moDC – blue, non-activated moDC - light gray, tumor cells - black. (D) Relative proportion of tumor and moDCs in the different co-cultures. (E) Relative proportion of activated vs non-activated moDCs in co-culture. Error bars, mean ± SD of triplicate independent wells for each condition of at least 2 independent experiments using different donors. ∗∗p value <0.01, by two-tailed t-test. TNFα: 0.1 ng/mL, SM: 1 μM, zVad: 5 μM. (F) Heatmap depicting cytokines measured in the supernatant of spheroids composed of tumor cells with or without CAFs, 72 h post-treatment. Data are averages from 3 independent wells and black squares indicate levels measured below the detection limit. SM: 0.25-1 μM; TNFα: 0.1 ng/mL, zVad: 20 μM.

Article Snippet: The human dermal fibroblasts (hd-fibroblasts - ATCC® PCS-201-010TM) were cultured in Fibroblast Basal Medium (ATCC PCS201030) supplemented 2% heat-inactivated FBS and Fibroblast Growth Kit-Serum Low (ATCC, #PCS-201-041) without HLL Supplement and rh EGF/TGTb1.

Techniques: In Vitro, Co-culture Assay, Co-Culture Assay, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics

doi: 10.1016/j.isci.2023.106381

Figure Lengend Snippet: SM modulates fibroblasts by downregulating myofibroblast-like CAF markers and upregulating pro-inflammatory soluble mediators CAF 2D monoculture was treated with increasing concentrations of SM or TGFβ. (A) Fold change in FAP and αSMA expression, analyzed via flow cytometry, 72 h post-treatment. (B) CAF confluence monitored overtime using IncuCyte. Error bars, mean ± SD of 3 independent experiments. (C) Heatmap depicting fold change in cytokine and chemokine concentration in the supernatant of treated CAFs (normalized to control). Results from 2 independent experiments. Black squares indicate levels measured below the detection limit.

Article Snippet: The human dermal fibroblasts (hd-fibroblasts - ATCC® PCS-201-010TM) were cultured in Fibroblast Basal Medium (ATCC PCS201030) supplemented 2% heat-inactivated FBS and Fibroblast Growth Kit-Serum Low (ATCC, #PCS-201-041) without HLL Supplement and rh EGF/TGTb1.

Techniques: Expressing, Flow Cytometry, Concentration Assay, Control

Journal: iScience

Article Title: Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics

doi: 10.1016/j.isci.2023.106381

Figure Lengend Snippet: Key resources table

Article Snippet: The human dermal fibroblasts (hd-fibroblasts - ATCC® PCS-201-010TM) were cultured in Fibroblast Basal Medium (ATCC PCS201030) supplemented 2% heat-inactivated FBS and Fibroblast Growth Kit-Serum Low (ATCC, #PCS-201-041) without HLL Supplement and rh EGF/TGTb1.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software, Simple Western, High Content Screening

Journal: Pharmaceuticals

Article Title: Ethanolic Fenugreek Extract: Its Molecular Mechanisms against Skin Aging and the Enhanced Functions by Nanoencapsulation

doi: 10.3390/ph15020254

Figure Lengend Snippet: Anti-aging activities’ evaluation of fenugreek extract: ( a ) In vitro collagenase inhibition of fenugreek extract (denoted as Extract) and liponiosome encapsulating fenugreek extract (denoted as LNF). ( b ) Effect of fenugreek extract on collagen production. Human dermal fibroblast cells were treated with 125 µg/mL of fenugreek extract and rutin, and 50 µg/mL of vitamin C as a positive control and 0.005% DMSO as a control (vehicle), for 7 and 14 days. Data are reported as means ± SD ( n = 3). *, p < 0.05; ***, p < 0.001.

Article Snippet: Human dermal fibroblast cells were purchased from the American Type Culture Collection: ATCC (PCS-201-010) and immortalized human keratinocytes (HaCaT) were purchased from Cell Lines Service, Germany (Cat. No. 300493).

Techniques: In Vitro, Inhibition, Positive Control, Control

Journal: Pharmaceuticals

Article Title: Ethanolic Fenugreek Extract: Its Molecular Mechanisms against Skin Aging and the Enhanced Functions by Nanoencapsulation

doi: 10.3390/ph15020254

Figure Lengend Snippet: Evaluation of LNF activities: ( a ) Cytotoxicity induced by LNF: The effect of blank, LNF, and fenugreek extract on cell viability. Human dermal fibroblast cells were treated with different concentrations of blank (liponiosome without fenugreek extract denoted as Blank), LNF, and fenugreek extract for 24 h. Data are means ± SD ( n = 3). ( b ) Collagen production induced by LNF: The effect of LNF on collagen production. Human dermal fibroblast cells were treated with 0.005% DMSO as a control (vehicle), 7 µg/mL of extract, and 100 µg/mL of LNF (7 µg/mL of extract equivalence), together with blank particles, for 7 and 14 days. Data are represented as means ± SD ( n = 3). *, p < 0.05.

Article Snippet: Human dermal fibroblast cells were purchased from the American Type Culture Collection: ATCC (PCS-201-010) and immortalized human keratinocytes (HaCaT) were purchased from Cell Lines Service, Germany (Cat. No. 300493).

Techniques: Control

Journal: Pharmaceuticals

Article Title: Ethanolic Fenugreek Extract: Its Molecular Mechanisms against Skin Aging and the Enhanced Functions by Nanoencapsulation

doi: 10.3390/ph15020254

Figure Lengend Snippet: Inhibition of UV-induced MMPs and interleukin secretion on co-cultured skin cells by fenugreek extract and LNF: ( a ) Effect of UV-induced cytotoxicity after the pretreatments of resveratrol, rutin, fenugreek extract, blank nanoparticles (liponiosome without fenugreek extract), LNF nanoparticles, and 0.005% DMSO as a control (vehicle). HaCAT and human dermal fibroblast cells were co-cultured and pretreated with 7 µg/mL of extract and 100 µg/mL of LNF (7 µg/mL of extract equivalence), together with 100 µg/mL of blank, 10 µg/mL of resveratrol, 7 µg/mL of rutin as a positive, and 0.005% of DMSO as a control (vehicle), for 24 h before UV exposure. Data are means ± SD ( n = 3). *, p < 0.05. ( b ) The levels of UV-induced MMP1 and MMP9 secretions after fenugreek extract and LNF treatments. Data are reported as means ± SD ( n = 3). *, p < 0.05. ( c ) The levels of UV-induced IL-6 and IL-8 secretions after fenugreek extract and LNF treatments. Data are reported as means ± SD ( n = 3). *, p < 0.05.

Article Snippet: Human dermal fibroblast cells were purchased from the American Type Culture Collection: ATCC (PCS-201-010) and immortalized human keratinocytes (HaCaT) were purchased from Cell Lines Service, Germany (Cat. No. 300493).

Techniques: Inhibition, Cell Culture, Control

Journal: Cells

Article Title: Sevoflurane Dampens Acute Pulmonary Inflammation via the Adenosine Receptor A2B and Heme Oxygenase-1

doi: 10.3390/cells11071094

Figure Lengend Snippet: Impact of sevoflurane on the migratory behavior of human polymorphonuclear neutrophils (PMNs) in vitro. ( A ) Model of adhesion and transmigration assay to evaluate the neutrophilic diapedesis and to determine the impact of sevoflurane on PMN migration. ( B ) Human PMNs from healthy volunteers were isolated and treated by sevoflurane, respectively, sevoflurane and a specific adenosine A2B receptor (Adora2b) antagonist (PSB1115). The migration through a monolayer of human endothelial (HMEC) and ( C ) epithelial monolayer (H441) was initiated by the potent chemoattractant macrophage inflammatory protein-2 (MIP-2). ( D ) The impact of sevoflurane on reactive oxygen species activity of human PMNs was assessed by the quantification of dihydroethidium (DHE) activity (mean fluorescence intensity; MFI) ( n = 6–8). ( E ) The gene expression of the human adhesion-related molecules, intercellular adhesion molecule 1 (ICAM1), ( F ) vascular cell adhesion molecule 1 (VCAM1), and ( G ) Adora2b expression were detected by RT-PCR ( n = 6–10). Statistical analyses were one-way ANOVA + Bonferroni correction or Kruskal-Wallis test. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001, and **** p < 0.0001.

Article Snippet: The transmigration of human PMNs through a human endothelial (HMEC1; ATCC ® CRL-3243TM; Manassas, VA, USA) and human epithelial (NCI-H441; ATCC ® HTB-174; USA) monolayer were performed as described previously [ ].

Techniques: In Vitro, Transmigration Assay, Migration, Isolation, Activity Assay, Fluorescence, Gene Expression, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: BioMed Research International

Article Title: Analysis of In Vitro Cyto- and Genotoxicity of Barbatimão Extract on Human Keratinocytes and Fibroblasts

doi: 10.1155/2018/1942451

Figure Lengend Snippet: General experiment design. (A) Initially was obtained a hydroalcoholic extract using barbatimão bark samples. (B) Main bioactive compounds were quantified by HPLC-DA. (C) An exploratory noncelular GEMO assay was conducted to determine if barbatimão at 10 different concentrations could present some genotoxic or genoprotective capacity of the extract. (D) The in vitro protocols were performed in two commercial human cell lines of keratinocytes (HaCaT) and dermal fibroblasts (HFF-1). Additional protocols were conducted to evaluate barbatimão effects on DNA oxidation by quantification of DNA-8-OhdG levels and by intrinsic or extrinsic apoptosis induction by quantification and comparison with negative control group of gene BAX/Bcl-2 ratio and CASP 3 and 8 protein levels. Details of experimental design and assays used in this study are presented in Methods section.

Article Snippet: The in vitro investigation used two commercial cell lines: immortalized human keratinocytes (HaCaT) and neonatal foreskin human dermal fibroblasts (HFF-1) obtained from American Type Culture Collection (ATCC).

Techniques: In Vitro, Comparison, Negative Control

Journal: BioMed Research International

Article Title: Analysis of In Vitro Cyto- and Genotoxicity of Barbatimão Extract on Human Keratinocytes and Fibroblasts

doi: 10.1155/2018/1942451

Figure Lengend Snippet: Barbatimão preliminary assays: (a) genoprotective capacity determined by GEMO noncellular assay that uses DNA PicoGreen® fluorescent dye (Cadoná et al., 2014). This dye presents specific affinity to binding with double-strand (ds) DNA levels. In GEMO assay a solution containing isolated calf dsDNA and H 2 O 2 (1M) is produced. The H 2 O 2 trigger extensive DNA fragmentation that causes decreasing in the fluorescence and here is considered the control group represented by 0 value. Cell culture supplementation with barbatimão hydroalcoholic extract at different concentrations showed significant increase in the fluorescence indicating genoprotective effect of this extract; barbatimão's viability effect on (b) keratinocytes and (c) fibroblast 24 h cell cultures measured by MTT-assay. Data were analyzed by ANOVA one-way analysis of variance followed by Tukey post hoc test and all statistical tests with p ≤ 0.05 were considered significant. Statistical differences among treatments were identified by different alphabetical letters, whereas same letters indicated no differences between each treatment compared to the others.

Article Snippet: The in vitro investigation used two commercial cell lines: immortalized human keratinocytes (HaCaT) and neonatal foreskin human dermal fibroblasts (HFF-1) obtained from American Type Culture Collection (ATCC).

Techniques: Binding Assay, Isolation, Produced, Fluorescence, Control, Cell Culture, MTT Assay

Journal: BioMed Research International

Article Title: Analysis of In Vitro Cyto- and Genotoxicity of Barbatimão Extract on Human Keratinocytes and Fibroblasts

doi: 10.1155/2018/1942451

Figure Lengend Snippet: Comparison among genotoxic and apoptotic markers of keratinocytes and fibroblasts exposed to barbatimão hydroalcoholic extraction. (a) 8-OHdG levels; (b) BAX / Bcl-2 gene expression ratio quantified by qRT-PCR, that indicates modulation of intrinsic apoptotic events; (c) caspase 3 protein levels; (d) caspase 8 protein levels. Data were compared by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test. In each marker tested here, statistical differences at p ≤ 0.05 among 24 h cell cultures treatments were identified by different letters (A, B, C).

Article Snippet: The in vitro investigation used two commercial cell lines: immortalized human keratinocytes (HaCaT) and neonatal foreskin human dermal fibroblasts (HFF-1) obtained from American Type Culture Collection (ATCC).

Techniques: Comparison, Extraction, Gene Expression, Quantitative RT-PCR, Marker

Journal: BioMed Research International

Article Title: Analysis of In Vitro Cyto- and Genotoxicity of Barbatimão Extract on Human Keratinocytes and Fibroblasts

doi: 10.1155/2018/1942451

Figure Lengend Snippet: Modulation of oxidative markers on three different cell culture days of keratinocytes and fibroblasts exposed to two different hydroalcoholic barbatimão extracts to evaluate potential chronic genotoxicity of this extract on cells. (a) Reactive oxygen species (ROS) levels; (b) 8-OHdG levels. Data were compared by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test. In each marker tested here, statistical differences at p ≤ 0.05 among 24 h cell cultures treatments were identified by different letters (A, B, C).

Article Snippet: The in vitro investigation used two commercial cell lines: immortalized human keratinocytes (HaCaT) and neonatal foreskin human dermal fibroblasts (HFF-1) obtained from American Type Culture Collection (ATCC).

Techniques: Cell Culture, Marker