Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation

doi: 10.3390/ijms21031022

Figure Lengend Snippet: EV secretion was increased in replicative senescent dermal fibroblasts. ( A ) Senescence-associated (SA)-β-gal assay in young HDFs (PDL < 10) and senescent HDFs (PDL > 50). Scale bar = 50 μm. Data are means ± SD of three independent experiments on two independent senescent cell lines (*** p < 0.001). ( B ) Western blot analysis of p21 and p16 in young versus senescent HDFs. GAPDH was the loading control. ( C ) Levels of EVs derived from equal numbers of young and senescent HDFs. Protein concentrations in isolated EVs were determined by BCA assay. Data are means ± SD of four independent experiments using two independent senescent cell lines (*** p < 0.001). ( D ) Dynamic light scattering analysis of EVs derived from young and senescent HDFs. ( E ) Western blot analyses of EV markers. Five micrograms of EV protein were subjected to immunoblot analysis with anti-CD81, anti-CD9, anti-Alix, and anti-HSP90. GAPDH was the loading control.

Article Snippet: Human dermal fibroblasts derived from the skin of a 33-year old adult (HDFs) or neonatal foreskin (HDFs-Neo) were purchased (Lonza, Basel, Switzerland) and cultured in DMEM supplemented with 10% FBS (Lonza, Basel, Switzerland).

Techniques: Western Blot, Derivative Assay, Isolation, BIA-KA

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation

doi: 10.3390/ijms21031022

Figure Lengend Snippet: EV biogenesis increased in senescent dermal fibroblasts. ( A ) Schematic image of sandwich ELISA using anti-CD81 antibody to detect EVs. ( B ) Conditioned media used to culture equal numbers of young and senescent HDFs were harvested at the indicated time points. EV levels were quantitated by sandwich ELISA for CD81. Data are means ± SD of three independent experiments using a senescent cell line (*** p < 0.001). ( C ) HDFs were labeled with a fluorescent lipid molecule N-Rh-PE (red) for the indicated time periods and co-stained with anti-CD63 antibody (green). Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI; blue). Fluorescence images were taken under a confocal microscope. Representative images are shown. Scale bar = 20 μm.

Article Snippet: Human dermal fibroblasts derived from the skin of a 33-year old adult (HDFs) or neonatal foreskin (HDFs-Neo) were purchased (Lonza, Basel, Switzerland) and cultured in DMEM supplemented with 10% FBS (Lonza, Basel, Switzerland).

Techniques: Sandwich ELISA, Labeling, Staining, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation

doi: 10.3390/ijms21031022

Figure Lengend Snippet: Senescent HDF-derived EVs attenuate the dermal effect on keratinocyte differentiation but evoke proinflammatory cytokine IL-6. Human epidermal keratinocytes (HEKs) in culture were treated with EVs derived from 5 × 10 5 young and senescent HDFs for 4 days. HEKs cultured without EV treatment for 4 d served as the control (-). Cells were analyzed for mRNA ( A ) and protein ( B ) expression of keratinocyte differentiation-related (KRT1, LOR) or barrier function-related (BLMH) markers by qRT-PCR and western blot analyses, respectively. The mRNA levels were normalized to those of RPL13A. GAPDH was the loading control for western blot analysis. KRT1, keratin 1; LOR, loricrin; BLMH, bleomycin hydrolase. ( C , D ) HEKs were treated with EVs derived from 5 × 10 5 young and senescent HDFs. Conditioned media at 24 h and 4 d post-treatment with EVs were harvested for the determination of secreted IL-8 ( C ) and IL-6 ( D ) levels, respectively, with specific ELISA kits. Data (in A , C , D ) are means ± SD of three independent experiments using two senescent cell lines (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant).

Article Snippet: Human dermal fibroblasts derived from the skin of a 33-year old adult (HDFs) or neonatal foreskin (HDFs-Neo) were purchased (Lonza, Basel, Switzerland) and cultured in DMEM supplemented with 10% FBS (Lonza, Basel, Switzerland).

Techniques: Derivative Assay, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay