Activation of Notch1 signaling alleviates dysfunction of bone marrow-derived mesenchymal stem cells induced by cigarette smoke extract

Figures & data

Table 1 Primer sequences used for real-time polymerase chain reaction

Gene	Primer sequence	Product length
Hes-1	Forward: 5′TCGGTGGTTACTTTGTTGCTT3′	122 bp
	Reverse: 5′AGGCGCAATCCAATATGAAC3′
Hey1	Forward: 5′GCGCGGACGAGAATGGAAA3′	231 bp
	Reverse: 5′TCAGGTGATCCACAGTCATCTG3′
HeyL	Forward: 5′CAGCCCTT CGCAGATGCAA3′	101 bp
	Reverse: 5′CCAATCGTCGCAATTCAGAAAG3′
SDF-1α	Forward: 5′ATGCCCCTGCCGATTCTTTG3′	116 bp
	Reverse: 5′TGTTGTTGCTTTTCAGCCTTGC3′
CXCR4	Forward: 5′GAAGTGGGGTCTGGAGACTAT3′	125 bp
	Reverse: 5′TTGCCGACTATGCCAGTCA AG3′
G-CSF	Forward: 5′ACTTTCTGCTCAAGGACGCA3′	85 bp
	Reverse: 5′AATGGCCTCTTGTCCTGACC3′
CSF3R	Forward: 5′GGACAAGAAACCGACCCACT3′	191 bp
	Reverse: 5′CCACTACTTCTGGGCTGGTG3′

Abbreviations: CSF3R, colony stimulating factor 3 receptor; G-CSF, granulocyte-colony stimulating factor; SDF-1, stromal cell-derived factor-1.

Figure 1 MSCs were phenotypically characterized by means of flow cytometry.

Note: The results demonstrated that MSCs were uniformly negative for CD34, CD45, CD80 and CD86, and positive for CD29, CD73, CD90, and CD105 expression.
Abbreviation: MSCs, mesenchymal stem cells.

Figure 2 Effects of CSE and N1ICD overexpression on MSC viability, Notch signaling pathway activity, and the PI3K/Akt pathway.

Notes: MSCs were incubated with 10% CSE over an extended period. Their viability was assessed with CCK-8. Notch signaling pathway activity was detected by real-time polymerase chain reaction, and activation of the PI3K/Akt pathway was evaluated by Western blotting. (A) CSE exposure for <24 h had little influence on cell viability and N1ICD overexpression can improve MSCs’ resistance to CSE cytotoxic effects. (B) The Notch signaling pathway was inhibited by incubation with CSE; N1ICD overexpression increased the Hey1 gene expression level. (C and D) CSE treatment downregulated the p-Akt expression level, while N1ICD overexpression increased the level of p-Akt in MSCs. *P<0.05 vs control group, ^#P<0.05 vs CSE group.
Abbreviations: CCK-8, cell counting kit-8; CSE, cigarette smoke extract; LY, LY294002; MSCs, mesenchymal stem cells; N1ICD, intracellular domain of Notch1; OD, optical density.

Figure 3 PI3K/Akt is required for N1ICD-mediated proliferation.

Notes: (A and B) Ki67 staining showed that N1ICD overexpression rescued the inhibition of proliferation induced by CSE, while blockade of the PI3K/Akt pathway decreased N1ICD-mediated MSC proliferation. (C and D) After treatment with CSE for 24 h, cyclin D1, p-Rb, and E2F-1 protein expression decreased, while that of p21 increased in the CSE group. N1ICD overexpression reversed this trend, but N1ICD effects were inhibited by blockade of PI3K/Akt. *P<0.05 vs control group; ^#P<0.05 vs CSE group.
Abbreviations: CSE, cigarette smoke extract; DAPI, 4′,6-diamidino-2-phenylindole; LY, LY294002; MSC, mesenchymal stem cells; N1ICD, intracellular domain of Notch1.

Figure 4 N1ICD improved CXCR4 expression and the subsequent migration capacity of MSCs via PI3K/Akt.

Notes: (A and B) CXCR protein expression was increased in the N1ICD group, but was inhibited by blockade of the PI3K/Akt pathway. (C and D) MSC migration capacity was analyzed with a transwell assay. (E) The expression of genes related to the SDF-1α/CXCR4 and G-CSF/CSF3R pathways was detected by real-time polymerase chain reaction. *P<0.05 vs control group; ^#P<0.05 vs CSE group.
Abbreviations: CSF, colony stimulating factor; CSE, cigarette smoke extract; LY, LY294002; MSC, mesenchymal stem cells; N1ICD, intracellular domain of Notch1.

Figure 5 The PI3K/Akt pathway is involved in the anti-apoptotic effects of N1ICD in MSCs after CSE treatment.

Notes: Apoptosis was confirmed by TUNEL assay (A, B). Cleaved caspase-3 immunocytochemistry (C, D). The expression of proteins associated with the apoptotic pathway was also detected. CSE led to an increase in the pro-apoptotic Bax/anti-apoptotic Bcl-2 ratio and cleaved caspase-3 levels. N1ICD overexpression decreased the Bax/Bcl-2 ratio and reduced cleaved caspase-3 expression (E, F). *P<0.05 vs control group; ^#P<0.05 vs CSE group.
Abbreviations: CSE, cigarette smoke extract; Cl. caspase-3, cleaved caspase-3; DAPI, 4′,6-diamidino-2-phenylindole; LY, LY294002; MSC, mesenchymal stem cells; N1ICD, intracellular domain of Notch1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling.

Figure S1 Isolated MSCs presented a fibroblast-like shape (A) and exhibited adipocyte (B), osteoblast (C), and chondrocyte (D) differentiation capacity.

Abbreviation: MSC, mesenchymal stem cells.