Fig 1: 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
Fig 2: The expression of AFF1, AFF4, and ELL3 in the neonatal mouse cochlea. (A,B) The mRNA and protein expression of AFF1, AFF4, and ELL3 in P3 mouse cochleae were detected by RT-PCR (A) and western blotting (B), respectively. (C) The gray levels comparison of western blot. (D) The mRNA expression of AFF1, AFF4, and ELL3 in Lgr5+ cells. Brain samples of P3 mice were used as the positive control, and GAPDH was used as the internal reference. n.s., not significant.
Fig 3: 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
Fig 4: Proposed model of Ell3 activity in somatic stem cells
Fig 5: Suppression of Ell3 expression induces stem cell senescence. a Quantitative reverse transcription PCR (qRT-PCR) analysis was performed on ADSCs and BM-MSCs at the indicated culture passage. The numbers of b ADSCs and ES-MSCs as well as those of c MCF7 and MCF10A cells transfected with siNS or siEll3 were counted on the indicated days after transfection. Cell cycle analysis of d ADSCs and BM-MSCs as well as e MCF7 and MCF10A cells transfected with siNS or siEll3 was performed by FACS 48 h after siRNA transfection (left panel). Quantitation of the cell cycle analysis results is presented as a graph (right panel). f The mitochondrial membrane potentials of ADSCs, BM-MSCs, MCF7 cells, and MCF10A cells transfected with siNS or siEll3 were evaluated by JC-1 staining (left) and flow cytometry analysis (right). Scale bar 25 μm. g β-gal staining was performed with ADSCs and BM-MSCs transfected with siNS or siEll3. β-gal (+) cells were imaged under a light microscope (left) and quantified (right). Scale bar 20 μm. 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; BM-MSCs, bone marrow-derived mesenchymal stem cells
Supplier Page from Abcam for Anti-ELL3 antibody