Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: The Induction of Apoptosis in A375 Malignant Melanoma Cells by Sutherlandia frutescens

doi: 10.1155/2016/4921067

Figure Lengend Snippet: The effect of S. frutescens extract on the viability of (a) A375 melanoma, (b) Colo-800 melanoma, (c) HDF α fibroblast, and (d) Hek 293 cells after 24, 48, and 72 h of treatment. The viability was assessed using the alamarBlue assay and expressed as percentage relative to the control-treated cells (0 mg/mL). Error bars represent the SEM ( n ≥ 3) and ∗ indicates a significant difference from 0 mg/mL ( P < 0.05).

Article Snippet: The human primary dermal fibroblast cell line (HDF α ) was purchased from ScienCell, CA, USA.

Techniques: Alamar Blue Assay, Control

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of ADAMTS17 shows location and targeting of exon 3 by CRISPR/Cas9 gRNA to induce nonhomologous end joining. The nucleotide and amino acid sequence of the ADAMTS17 WT allele (green) and after AT insertion (red) are indicated. The dinucleotide insertion induced a frameshift, which resulted in a premature stop codon after 12 amino acids. (B) Sanger sequencing traces of a PCR product generated with primers flanking exon 3 showing the AT insertion (underlined) in the Adamts17 KO. (C) Quantitative real-time PCR using cDNA prepared from WT and Adamts17 KO lung tissue as a template shows significant reduction of ADAMTS17 mRNA in the KO (n = 3). (D) Micrographs of ADAMTS17 immunostaining of sections through WT and Adamts17 KO skin (left), DKO growth plates (middle), and of primary DKO mouse skin fibroblasts (right). The signal in the dermis around hair follicles, in growth plate chondrocytes, and in fibroblasts and their ECM originating from the monoclonal ADAMTS17 antibody was strongly reduced in KO and DKO tissues and cells, indicating lack of ADAMTS17 protein in Adamts17 KO mice. (E) Pie chart showing Mendelian distribution of genotypes recovered from Adamts17 Het intercrosses at the time of genotyping (P7–P10) (n = 94 mice). (F) Breeding scheme to generate WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. (G) Pie chart showing distribution of genotypes recovered from Adamts10 Het; Adamts17 Het intercrosses at P7–P10 (n = 180 mice). Statistical analysis was performed using Chi square calculation. (H) Kaplan–Meier survival analysis of DKO mice. The numbers of observed dead/total mice for the individual genotypes are indicated in brackets. Statistical significance was determined using a log-rank test. (I) Whole mount images of WT, 10KO, 10KO;17Het mice at 4 wk of age show progressive reduction in body size. (J) Bar graphs showing body weights of 4-wk-old mice of the indicated genotypes. The number of mice is indicated below the genotypes. (I, K) Bar graphs showing body weight normalized to average femur length for the genotypes that were significantly different in (I). Bars in (C) indicate mean values and whiskers the SD. In (J, K) floating bars indicate the 25th–75th percentile range, lines the mean value, and whiskers the SD. (C, J, K) Statistical differences in (C) were determined using a two-sided t test and (J, K) were using a one-way ANOVA with post hoc Tukey test. a, P < 0.05 compared with WT.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: CRISPR, Sequencing, Generated, Real-time Polymerase Chain Reaction, Immunostaining

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Micrographs of Masson’s trichrome–stained cross sections through dorsal skin from 4-wk-old WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. ED, epidermis; D, dermis; HD, hypodermis; PC, panniculus carnosus. (B, C, D) Bar graphs showing quantification of overall skin thickness (B) and the thicknesses of the epidermis, dermis, hypodermis (C), and panniculus carnosus (p. carnosus, (D)). Individual data points represent multiple measurements along the different skin layers from n = 3 mice/genotype. (E) Stacked bar graphs showing the relative proportions of individual skin layers. The percentage values are indicated. (F) Bar graphs show the quantification of hair follicle numbers in the skin for each genotype. (G, H) Bar graphs showing normalized gene expression in fragments per kilobase of transcript per million mapped reads (FPKM) for Adamts10 and Adamts17 in individual skin cell types at E14.5 (G) and P5 (H). Data were extracted from the Hair-GEL database ( ; ). (I, J, K) Micrographs showing the localization of ADAMTS17 mRNA (red/dark purple) in WT skin cross sections at E13.5 (I), E16.5 (J), and P0 (K) detected by RNAscope in situ hybridization with a probe specific for ADAMTS17 mRNA. Sections were counterstained with hematoxylin. (L) Micrograph of ADAMTS17 immunostaining (green) of cross sections through WT skin. Nuclei were stained with DAPI (blue). (M) Micrographs of primary mouse skin fibroblasts after immunostaining for fibrillin-1 (red) and fibronectin (green). Nuclei were counterstained with DAPI (blue). (M, N) Quantification of mean fluorescence intensity from (M) (n = 4 biological replicates). In (B, C, D, F), floating bars indicate 25th–75th percentile range, lines the mean value and whiskers the SD. In (N), the bars represent the mean value and the whiskers the SD. Statistical differences in (B, C, D, F, N) were determined using a one-way ANOVA with post hoc Tukey test. a, P < 0.05 compared with WT; b, P < 0.05 compared with Adamts10 KO; P < 0.05 compared with Adamts17 KO.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Staining, Gene Expression, RNAscope, In Situ Hybridization, Immunostaining, Fluorescence

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of ADAMTS10 and ADAMTS17, which is identical. The degenerate (ADAMTS10) and canonical (ADAMTS17) furin-processing sites and the localization of the catalytic residue Glu-390 in ADAMTS17 (17) that was mutated into Ala to generate proteolytically inactive ADAMTS17-EA (17-EA) are indicated. The domain organization of the catalytic (17-PCD) and ancillary (17-AD) domain constructs is indicated. (B) Schematic representation of experimental design for coculture of human dermal fibroblasts (HDF) with HEK293 cells stably expressing 17- or 17-EA (left) or co-transfection of 17- or 17-EA–encoding plasmids with FN1 or COL6A2-encoding plasmids in HEK293 cells (right). (C) Volcano plot showing N-terminally labeled peptides identified by N-terminomics method TAILS in conditioned medium from ADAMTS17-expressing HEK293 cells cocultured with HDFs. Peptides present only in samples from WT ADAMTS17 (red) or enriched in conditioned medium from WT ADAMTS17 cocultures compared with the cocultures with proteolytically inactive ADAMTS17-EA suggest ADAMTS17 substrates. (D) Venn diagram showing overlap of ADAMTS17-cleaved proteins (TAILS) from coculture systems (left) and binding partners for the ADAMTS17 ancillary domain (17-AD) identified by yeast-2-hybrid screening with a human placenta–derived cDNA library (right). Note that fibronectin (FN1) and COL6 were independently identified in both screens. (E) Domain organization of fibronectin (FN1, NP_997647 ) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence) and the localization of the peptide identified by TAILS (red bar, red amino acid sequence). (F) MS2 spectrum of the N-terminally labeled FN1 peptide (GNSVNEGLNQPTDDSCFDPYTVSHYAVGDEWER) showing b- and y ions. (G) Western blot detection of endogenous fibronectin in conditioned medium (Med) and cell lysates (Lys) collected after coculture of 17- or 17-EA–expressing HEKs with HDFs. A monoclonal (green) and four different polyclonal (red) anti-fibronectin antibodies were used. (H) Western blot detection of recombinant fibronectin (rFN) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA with rFN in HEK293 cells. A polyclonal anti-fibronectin antibody (red) and a monoclonal anti V5-tag antibody (green) were used to detect rFN.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Residue, Construct, Stable Transfection, Expressing, Cotransfection, Labeling, Binding Assay, Derivative Assay, cDNA Library Assay, Sequencing, Western Blot, Recombinant

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of COL6A2 (NP_0018403) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence). (B) Domain organization of COL6A3 ( NP_004360 ) showing the localization of the peptide identified by MS (red bar, red amino acid sequence). (C) MS2 spectrum of the N-terminally labeled ADAMTS17-digested COL6A3 peptide (SDDEVDDPAVELkQFGVAPF) showing b- and y ions. (D) Western blot of endogenous (end.) COL6 (red) in conditioned medium (Med) and cell lysate (Lys) collected from cocultures of 17- or 17-EA–expressing HEK293 cells with HDFs. (E) Micrographs of endogenous COL6A1 deposition (red) in the ECM of HDFs cocultured with 17- or 17-EA–expressing HEK293 cells. Nuclei were stained with DAPI (blue). (F) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates). (G) Micrographs of endogenous COL6A1 deposition (red) in the ECM of HDF after culture in the presence of conditioned medium from 17- or 17-EA–expressing HEK293 cells. Nuclei were stained with DAPI (blue). (H) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates). (I) Western blot of recombinant COL66A2 (rCOL6) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA and rCOL6A2 in HEK293 cells using a monoclonal anti FLAG-tag antibody (green). (J) Micrographs of HDFs cultured in the presence of 50 μg/ml of purified recombinant 17-PCD and 17-AD protein (see for domain organization) costained for endogenous COL6A1 (red) and the Myc-tag of the recombinant ADAMTS17 protein fragments (green). Nuclei were stained with DAPI (blue). (K) Micrographs of adult HDFs and Weill–Marchesani syndrome (WMS) patient–derived dermal fibroblasts (WMS-DF) for endogenous COL6A1 (red). Nuclei were counterstained with DAPI (blue). (L) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates, 2–3 fields of view). (M) Western blot of endogenous COL6A1 (red) and GAPDH (green) in conditioned medium (Med) and cell lysate (Lys) collected from HDF and WMS-DF cultures. (N) Quantification of COL6A1 band mean fluorescence intensities normalized to GAPDH. In (E, G, M), bars represent the mean value and whiskers the SD. In (K), the floating bars indicate the 25th–75th percentile range, the lines the mean value, and whiskers the SD. Statistical differences in (E, G, K, M) were determined using a two-sided t test.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Sequencing, Labeling, Western Blot, Expressing, Staining, Fluorescence, Recombinant, FLAG-tag, Cell Culture, Purification, Derivative Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures

doi: 10.3389/fncel.2023.1003188

Figure Lengend Snippet: Phase contrast images of (A) HDFs and (B) resulting hiDFP colonies from 20/30-, 50- and 70-year-old subjects. Scale = 300 μm. HDF, human dermal fibroblast; hiDFP, human induced dorsal forebrain precursor.

Article Snippet: Nine adult human dermal fibroblast cell lines (HDFs) were obtained from Cell Applications Inc or The NINDS Human Cell and Data Repository.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures

doi: 10.3389/fncel.2023.1003188

Figure Lengend Snippet: Reprogrammed hiDFPs maintain aging-associated features. (A) SA-βgal activity assay of HDFs and hiDFPs. No statistical significance was determined by two-way mixed ANOVA. (B) Mitochondrial superoxide levels in HDFs and hiDFPs. Data represent mean ± SEM with n = 3. Simple main effect of reprogramming on mitochondrial superoxide levels determined by two-way mixed ANOVA with * for p < 0.05.

Article Snippet: Nine adult human dermal fibroblast cell lines (HDFs) were obtained from Cell Applications Inc or The NINDS Human Cell and Data Repository.

Techniques: Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures

doi: 10.3389/fncel.2023.1003188

Figure Lengend Snippet: Reprogrammed hiDFPs exhibit increased amounts of DNA methylation as measured by the production of 5-methylcytosine (5-mC). (A) Amount of methylated DNA in hiPSCs and hiDFPs relative to HDF. Significance was determined by one-way ANOVA with Tukey post-hoc test. (B) Amount of methylated DNA in HDFs and hiDFPs. A simple main effect of reprogramming irrespective of age group was determined by two-way mixed ANOVA. Data represent mean ± SEM with n = 3. * for p < 0.05, ** for p ≤ 0.01.

Article Snippet: Nine adult human dermal fibroblast cell lines (HDFs) were obtained from Cell Applications Inc or The NINDS Human Cell and Data Repository.

Techniques: DNA Methylation Assay, Methylation

Journal: Biology

Article Title: Chemical Composition, Antioxidant Capacity, and Anticancerous Effects against Human Lung Cancer Cells of a Terpenoid-Rich Fraction of Inula viscosa

doi: 10.3390/biology13090687

Figure Lengend Snippet: IVL DCM decreased the viability of A549 cells. ( A ) MTT assay was used to measure the viability of A549 and HDFn cells treated with the indicated concentrations of IVL DCM for 24, 48, or 72 h. Data represents the mean ± SEM of the percent viability of treated cells compared to vehicle-treated cells. Data are the mean of three independent experiments ( n = 3). *** p < 0.001, **** p < 0.0001. ( B ) A549 cells treated with the indicated concentrations of IVL DCM for 24 h were stained with DAPI nuclear stain. DAPI fluorescence was imaged at 20X magnification using a BioTek Cytation 5 reader. ( C ) A549 cells were treated with the indicated concentrations of IVL DCM for 24 h and then stained with crystal violet. The micrographs represent stained cells imaged at 20X magnification by light microscopy using the Invitrogen EVOS ® FL Cell Imaging System.

Article Snippet: A549 human lung adenocarcinoma cells, the most common subtype of NSCLC, which accounts for approximately 85% of lung cancer cases [ ], human normal neonatal fibroblasts (HDFn), SK-OV-3 (human ovarian cancer), MCF-7 (human breast cancer), MDA-MB-231 (human breast cancer), HepG2 (human liver cancer), and HCT116 (human colorectal cancer) cells were obtained from ATCC and cultured at 37 °C and 5% CO 2 in a humidified incubator.

Techniques: MTT Assay, Staining, Fluorescence, Light Microscopy, Imaging

Journal: Antioxidants

Article Title: Effect of Pholiota nameko Polysaccharides Inhibiting Methylglyoxal-Induced Glycation Damage In Vitro

doi: 10.3390/antiox10101589

Figure Lengend Snippet: ( a ) PNPs protect Hs68 cells against MG treatment. # Followed by a significant difference with the controls ( p < 0.05); A: significant difference with the same concentration in different samples ( p < 0.05); a and b: significant difference with the same sample at different concentrations ( p < 0.05). ( b ) Effect of PNPs on cell growth rate. The cell growth rate was determined from generations 1 to 10. Experiments were conducted in triplicate (n = 3), and the data are expressed as the mean ± SD. The data for the linear regression equation plotted by each group are as follows: y = − 1.4 x + 417.5 (controls), y = − 3.2 x + 362.8 (MG-induced), y = − 1.9 x + 377 (PNP-40), y = − 1.6 x + 382.1 (PNP-60), and y = − 2 x + 384.7 (PNP-80). # Followed by a significant difference with the controls in the 10th generation ( p < 0.05); * followed by significant difference with the MG-treated group in the 10th generation ( p < 0.05).

Article Snippet: Human dermal fibroblast cell line Hs68 (Obtained from ATCC CRL-1635, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco ® , Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco ® , Grand Island, NY, USA) and antibiotics (100 U penicillin and 100 U/mL streptomycin, Gibco ® , Grand Island, NY, USA) under 5% CO 2 at 37 °C.

Techniques: Concentration Assay

Journal: Antioxidants

Article Title: Effect of Pholiota nameko Polysaccharides Inhibiting Methylglyoxal-Induced Glycation Damage In Vitro

doi: 10.3390/antiox10101589

Figure Lengend Snippet: ( a ) Effect of MG-induced ROS generation in Hs68 cells. Scale bar: 100 μm. ( b ) Quantitative analysis of MG-induced Hs68 cell ROS production with or without PNPs pretreatment. ROS scavenging effect of PNPs (1.5 mg/mL) on Hs68 cells against MG-induced ROS generation obtained from the Image J analysis. a–e: Significant difference with different groups ( p < 0.05).

Article Snippet: Human dermal fibroblast cell line Hs68 (Obtained from ATCC CRL-1635, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco ® , Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco ® , Grand Island, NY, USA) and antibiotics (100 U penicillin and 100 U/mL streptomycin, Gibco ® , Grand Island, NY, USA) under 5% CO 2 at 37 °C.

Techniques:

Journal: Molecules

Article Title: Volatile Natural Deep Eutectic Solvents (VNADESs) for Extraction of Shikonin Derivatives from Echium vulgare Roots and Evaluation of Biological Activity

doi: 10.3390/molecules31091434

Figure Lengend Snippet: Assessment of resazurin reduction in fibroblasts (HDFs) for ( a ) hexane (bars in yellow) and ( b ) TBa (bars in red) extracts. Control cells (green bars) without the addition of test samples, for which viability was assumed to be 100%. Data are presented as mean ± SD ( n = 3). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. Differences were considered statistically significant at p < 0.05 (*), p < 0.01(**), p < 0.001(***), relative to the control.

Article Snippet: Human dermal fibroblasts (HDFs) were obtained from CLS Cell Lines Service (Eppelheim, Germany) and were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Biological Industries, Cromwell, CO, USA) supplemented with sodium pyruvate, L-glutamine, glucose (4.5 g/L), and 10% fetal bovine serum (FBS; Genos, Łódź, Poland).

Techniques: Control