Overexpressed Thrombospondin 2 Induced Osteogenic Differentiation of Valve Interstitial Cells via Inhibition of Akt/NF-κB Signaling Pathway to Promote Calcific Aortic Valve Disease Development

Figure 5

THBS2 negatively regulated Akt/NF-κB signaling pathway during OD procedure of VICs. (a–c) Representative images of protein bands during Western blot (a) and the levels of p-Akt/Akt (b) and p-p65/p65 (c) of VICs transfected with shTHBS2 and THBS2 overexpression plasmid were assessed by Western blot after the induction of OD. GAPDH was a loading control. (d) Relative expression of p65 in nucleus of VICs transfected with shTHBS2 and THBS2 overexpression plasmid was assessed by Western blot after the induction of OD. Lamin B1 was a loading control. (e) Relative mRNA expressions of THBS2, Akt, and p65 in VICs transfected with shTHBS2 and treated with LY294002 were tested by qRT-PCR after the induction of OD. GAPDH was a loading control. (f) Representative images of protein bands during Western blot. (g and h) The levels of p-Akt/Akt (g) and p-p65/p65 (h) of VICs transfected with shTHBS2 and treated with LY294002 were determined through Western blot after the induction of OD. GAPDH was a loading control. (i) Relative protein expression of THBS2 in VICs transfected with shTHBS2 and treated with LY294002 was determined through Western blot after the induction of OD. GAPDH was a loading control. (j) and (k) Relative mRNA (j) and protein (k) expressions of p65 in nucleus of VICs transfected with shTHBS2 and treated with LY294002 were detected by Western blot after the induction of OD. Lamin B1 was a loading control. , , vs. vector group; vs. shTHBS2 group; ; ; vs. LY294002 group. All experiments were repeated independently at least three times. Data were performed as the . Abbreviations: THBS2: thrombospondin 2; OD: osteogenic differentiation; VICs: valve interstitial cells; p-: phosphorylation-; shTHBS2: short hairpin THBS2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR: quantitative reverse transcription-polymerase chain reaction.

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