Journal: Science signaling

Article Title: Thrombospondin-1 promotes matrix homeostasis by interacting with collagen and lysyl oxidase precursors and collagen cross-linking sites.

doi: 10.1126/scisignal.aar2566

Figure Lengend Snippet: Fig. 5. Localization of TSP1, fibrillar collagen, and LOX in human der- mal fibroblast cultures. (A) Dual in- direct immunofluorescence staining of the indicated pairs of proteins in permeabilized or nonpermeabilized HDFs cultured for 72 hours. The re- gions shown at higher magnification in the insets are boxed by dotted lines in the main panels. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Images are representa- tive of four independent experiments. (B) Quantification by Pearson correla- tion (per cell) of colocalization of the indicated pairs of proteins in permea- bilized (P) HDF cultures at the indicated times. C, collagen I; T, TSP1; L, LOX. n = 4 independent experiments with at least 36 cells analyzed per condition per experiment. Data were analyzed by Kruskal-Wallis test and Dunn’s com parison. (C) Quantification by Pearson correlation (per field) of colocalization of the indicated pairs of proteins in nonpermeabilized (NP) HDF cultures at the indicated times. n = 4 indepen- dent experiments with at least six fields analyzed per condition per ex- periment. (D) Dual indirect immuno- fluorescence staining of the indicated pairs of proteins in permeabilized and nonpermeabilized HDFs after 10 days of culture. Images are representative of four independent experiments. The regions shown at higher magnifica- tion in the insets are boxed by dotted lines in the main panels. (E) Quantifi- cation by Pearson correlation (per field) of colocalization of the indicated pairs of proteins in HDFs after 10 days of culture. n = 4 independent experi- ments with at least six fields analyzed per condition per experiment. (F) In situ proximity ligation assay for the indicated pairs of proteins in HDFs cultured for 72 hours. Images are rep- resentative of four independent ex- periments. (G to L) Quantification of in situ proximity ligation signals for the indicated pairs of proteins over time in permeabilized (P) or nonper- meabilized (NP) HDFs. 2Ab, second- ary antibodies only. Each data point represents the mean from one exper- iment, and the bars indicate the mean. n = 4 (G to J) and 3 (K to L) indepen- dent experiments. At least 36 cells were analyzed per condition for each experiment. (A), (D), and (F) show re- sults for HDFs at passage 7 and are representative of four experiments on HDFs between passages 4 and 8. Scale bars, 50 m (main images) and 10 m (insets).

Article Snippet: Normal HDFs from foreskins of healthy juveniles (C-12300, PromoCell) were cultured in fibroblast growth medium (C-23010, PromoCell) with l-ascorbic acid (50 g/ml) and used for experiments between passages 3 and 8.

Techniques: Immunofluorescence, Staining, Cell Culture, Fluorescence, In Situ, Proximity Ligation Assay, Ligation

Journal: Science signaling

Article Title: Thrombospondin-1 promotes matrix homeostasis by interacting with collagen and lysyl oxidase precursors and collagen cross-linking sites.

doi: 10.1126/scisignal.aar2566

Figure Lengend Snippet: Fig. 7. KGHR-containing, TSP1-binding collagen triple-helical peptides increase myofibroblast differentiation through a TGF-R1–dependent mechanism. (A) Merged immunofluorescence images of HDFs cultured for 96 hours in the presence of HAc, TGF-1, collagen peptide II-8, or colla- gen peptide II-52 and stained for SMA (green) and nuclei (DAPI; blue). Images are representative of three independent experiments. (B) Quantification of the percentage of SMA-positive cells under the indicated conditions from three independent experiments. (C) Effects of the various treatments on cell numbers over time; n = 3 independent experiments. (D) Merged immunofluorescence images showing SMA (green) and DNA (blue) in HDFs cultured for 96 hours in the presence of HAc, TGF-1, or the indicated peptides. Images are representative of three independent experiments. (E) Quantification of SMA-positive cells per field after treatment with the indicated peptides. n = 3 independent experiments. (F) Effects of the indicated peptides on cell numbers at 96 hours. n = 3 independent experiments. (G) Merged immunofluorescence images showing SMA (green) and DNA (blue) in HDFs cultured for 96 hours in the presence of the inhibitor of TGF-R1 kinase activity SB-431542 (SB) or the -catenin signaling inhibitor PNU-74654 (PNU). Images are representative of three independent experiments. (H) Quantification of SMA-positive cells after 96 hours in the presence of the indicated inhibitors. n = 3 independent experiments. (I) Quantification of bioactive and total (determined after acid activation) active TGF-1 in media from HDFs cultured under the indicated conditions. UT, untreated. n = 3 independent experiments with duplicate samples per condition per experiment. In all graphs, each data point indicates the mean, and error bars indicate the SEM. In (B), (C), (E), (F), and (H), at least 30 cells were scored per condition per experiment. Data were analyzed by Dunnett’s multiple comparison test (one-way ANOVA) against HAc (B) or by Kruskal-Wallis test and pairwise tests against HAc (E and F) or against dimethyl sulfoxide (DMSO) (H). Scale bars, 50 m.

Article Snippet: Normal HDFs from foreskins of healthy juveniles (C-12300, PromoCell) were cultured in fibroblast growth medium (C-23010, PromoCell) with l-ascorbic acid (50 g/ml) and used for experiments between passages 3 and 8.

Techniques: Binding Assay, Immunofluorescence, Cell Culture, Staining, Activity Assay, Activation Assay, Comparison

Journal: Oncotarget

Article Title: Evaluation of an Actinomycin D/VX-680 aurora kinase inhibitor combination in p53-based cyclotherapy

doi:

Figure Lengend Snippet: (A) Pretreatment with low dose ActD decreases the proportion of 8N cells caused by VX-680 in NHDF cells. ActD was added 24 hours post-seeding. VX-680 was added 48 hours post-seeding. Cells were harvested 96 hours after seeding. (B) NHDFs were treated with 4 nM ActD for 72 hours, fixed and stained with Giemsa (top panel). In the bottom panel, ActD was removed from the medium and cells were allowed to proliferate for 6 days before Giemsa staining.

Article Snippet: NHDF cells were bought from Promocell.

Techniques: Staining

Journal: Oncotarget

Article Title: Evaluation of an Actinomycin D/VX-680 aurora kinase inhibitor combination in p53-based cyclotherapy

doi:

Figure Lengend Snippet: (A and B) NHDF cells were treated for 48 hours with VX-680 alone (labelled as no preinc.), or treated for 48 hours with VX-680 after preincubating with the indicated amounts of ActD for 24 hours. Thereafter cells were fixed, stained Giemsa (A) and counted (B). Arrows in (A) indicate cells with abnormal nuclei. (C and D) NHDF cells treated as in A and B were allowed to recover for 6 days in drug-free medium. The growth of the cultures in drug-free medium is quantified in panel (C) by counting the total number of cells per field before and after the recovery period. The average number of cells per field with aberrant nuclei before and after the recovery period is shown in panel (D). Error bars correspond to standard deviations.

Article Snippet: NHDF cells were bought from Promocell.

Techniques: Staining

Journal: Oncotarget

Article Title: Evaluation of an Actinomycin D/VX-680 aurora kinase inhibitor combination in p53-based cyclotherapy

doi:

Figure Lengend Snippet: (A) NHDF cells were treated with the indicated amounts of ActD for 4 or 12 hours. p53 and p21 were analysed by Western blotting. α-tubulin was detected as a loading control. (B) Example of nuclear abnormalities in NHDF cells expressing a dominant negative form of p53. The percentage of cells with this sort of aberrant nuclei was 2.5 fold higher than in the corresponding cells with intact p53. Severely damaged cells, such as the one in the center of this picture, only appeared in the fibroblasts with dominant negative p53.

Article Snippet: NHDF cells were bought from Promocell.

Techniques: Western Blot, Control, Expressing, Dominant Negative Mutation

Journal: Scientific Reports

Article Title: Increasing the concentration of plasma molecules improves the biological activity of platelet-rich plasma for tissue regeneration

doi: 10.1038/s41598-025-88918-0

Figure Lengend Snippet: Cellular viability in NHDF cells. The viability levels of NHDF cells incubated with sPRP and BPCP are expressed as relative light units (RLUs), and each line represents a different donor ( n = 8). Serum-free (SF) condition served as negative control. Statistical analysis was calculated using one-way ANOVA ( p < 0.0001).

Article Snippet: NHDF cells (Lonza, Basel, Switzerland) were kept in an incubator at 37 °C in a 5% CO 2 atmosphere.

Techniques: Incubation, Negative Control