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Fig. 2 | Stem Cell Research & Therapy

Fig. 2

From: Ell3 functions as a critical decision maker at the crossroad between stem cell senescence and apoptosis

Fig. 2

Ell3 suppression shifts the differentiation potential of ADSCs into adipogenesis by inducing Bcl-2 accumulation. a The adipogenic, osteogenic, and chondrogenic lineage differentiation efficiency of ADSCs transfected with siNS or siEll3 was evaluated by oil red O staining, Alcian Blue staining, and alizarin red S staining, respectively, under 2D culture and 3D pellet cultures. The cells were then cultured for 2 weeks (adipogenic) and 3 weeks (osteogenic, chondrogenic), and the medium was changed every 2 days. Scale bar 20 μm. b The expression of each lineage marker gene in ADSCs transfected with siNS or siEll3 was analyzed by qRT-PCR and cultured in 2D differentiation condition for 48 h. c The expression of Ell3, p53, and Bcl-2 in ADSCs transfected with siNS or siEll3 for 48 h was analyzed by qRT-PCR (left) and immunocytochemical staining (right). d The protein levels of Ell3, p53, p21, p16, and phospho-Rb in ADSCs transfected with siNS or siEll3 for 48 h were analyzed by immunoblot (right). β-actin was used as the loading control. Scale bar 25 μm. e The effect of ABT-737 treatment on the adipogenic and osteogenic lineage differentiation efficiencies of ADSCs transfected with siNS or siEll3 was evaluated by oil red O staining and alizarin red S staining under 2D culture (left). Adipogenesis was quantified by the extracting oil red O stain from cells according to the protocol, and osteogenesis was quantified using the ImageJ program (right). The cells were cultured for 2 weeks (adipogenic differentiation) and 3 weeks (osteogenic differentiation), and the medium containing 0.25 μM ABT-737 was changed every 2 days. Scale bar 25 μm. f ADSCs transfected with siNS or siEll3 for 48 h were treated with 0.25 μM ABT-747 for 24 h, and the expression of PPAR-γ and Runx2 was detected by qRT-PCR. The experiments were repeated three times independently, and the results presented as bars represent the mean ± s.d. Abbreviations: siNS, nonspecific siRNA; siEll3, siRNA targeting Ell3; ADSCs, adipose stem cells; ES-MSCs, embryonic stem cell-derived mesenchymal-like stem cells

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