Fig. 5. Trehalose arrests fibroblasts in the G2/M-phase of the cell cycle and triggers a senescence-like state with activation of Erk1/2 and Akt.

a Representative photos of human dermal fibroblasts treated with trehalose (10 and 100 mg/ml) or vehicle (PBS) for 24 h. Phase contrast micrographs, bar = 50 μm. b Fibroblasts were characterized for a potential senescent phenotype via SPiDER-βGAL (green), γH2AX (red), and DAPI (blue) 24 h after treatment with trehalose (100 mg/ml), vehicle, or hydrogen peroxide (800 nM). Scale bar = 50 μm. c, d Flow cytometric analysis of DNA damage was performed with the DNA Damage Detection Kit to monitor H2AX with phosphorylated Ser-139 (γH2AX) as a DNA damage marker. Alexa Fluor 647-conjugated anti-γH2AX and propidium iodide (PI) staining 24 h after vehicle, hydrogen peroxide (800 nM), or trehalose (100 mg/ml) treatment (c), and the numerical analysis was conducted for each experiment (d). Data were expressed as means ± SD and are representative of three independent experiments. e Western blotting showing the expression of ERK1/2, p-ERK1/2, AKT, p-AKT, and β-actin in human dermal fibroblasts treated with trehalose (10 and 100 mg/ml) or vehicle for 24 h. f Twenty-four hours after the trehalose (100 mg/ml) or vehicle treatment, human dermal fibroblasts were stained with PI, and the percentage of G2/M cells was measured by flow cytometry. g, h Flow cytometric analysis of cell cycle distribution was performed with FITC-conjugated anti-BrdU and 7-AAD staining 24 h after vehicle or trehalose (100 mg/ml) treatment (g), and numerical analysis was conducted for each experiment (h). Data were expressed as means ± SD and are representative of three independent experiments. i Seven days after air exposure, human dermal fibroblasts in the living skin equivalent were stained with PI, and the percentage of G2/M cells was measured by flow cytometry. Representative FACS images. Data were representative of two independent experiments.