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Fig. 3 | Stem Cell Research & Therapy

Fig. 3

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

Fig. 3

Ectopic Ell3 expression induces ADSC apoptosis. a A control or Ell3-expressing plasmid was transfected into ADSCs by lipofectamine, and cell morphology was observed under a light microscope 48 h after transfection. Scale bar 50 μm. b The numbers of ADSCs transfected with the control or Ell3-expressing plasmid were counted on the indicated days after transfection. c The effect of ectopic Ell3 expression on the expression of the indicated genes was evaluated by qRT-PCR analysis. The expression of each gene in ADSCs transfected with the control plasmid was used as the control. d Immunoblot analysis was performed to analyze the effect of ectopic Ell3 expression on the protein accumulation of each gene. e The apoptosis of ADSCs transfected with the control or Ell3-expressing plasmid was analyzed by Annexin V staining and flow cytometric analysis. f Relative expression of total Ell3 and exogenic Ell3 was evaluated by qRT-PCR. The expression of exogenic Ell3 was analyzed by qRT-PCR with primers targeting the 5′ UTR of the Ell3 expression plasmid (forward primer: 5′-ATC CAC TAG TCC AGT GTG GT-3′, reverse primer: 5′-TGA AGG AGA GGC AGG AC-3′). g Live and dead staining was performed with ADSCs cultured in conditioned media prepared from culture media from ADSCs transfected with the control or Ell3-expressing plasmid. Recipient cells were either siNS- or siEll3-transfected ADSCs. Live (green) and dead [6] cells were imaged under a light microscope (left). The relative ratio of live and dead cells was evaluated by counting stained cells and presented in a graph (right). Scale bar 20 μm. h The relative expression level of Ell3 in the ADSCs from the experiments in g was analyzed by qRT-PCR. The experiments were repeated three times independently, and the results presented as bars represent the mean ± s.d. Abbreviations: C, control plasmid; Ell3 OE, Ell3-expressing plasmid; C-CM, conditioned media prepared from the culture media of ADSCs transfected with the control plasmid; OE-CM, conditioned media prepared from the culture media of ADSCs transfected with the Ell3-expressing plasmid; siNS, nonspecific siRNA; siEll3, siRNA targeting Ell3; ADSCs, adipose stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells

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