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. Author manuscript; available in PMC: 2015 Mar 26.
Published in final edited form as: Biochem J. 2014 Mar 1;458(2):203–211. doi: 10.1042/BJ20131350

Figure 1. Expression of CBS in U87-MG cells under hypoxic conditions.

Figure 1

(A) Cells were exposed to 20% or 1% O2 for 24 h and total RNA was isolated. Reverse transcription and real-time quantitative PCR (RT-qPCR) were performed to determine levels of mRNA encoding cystathionine β-synthase (CBS), cystathionine γ-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (MPST). Levels of SLC2A1 mRNA, which is encoded by a known HIF target gene, were also determined as a positive control. Data shown are mean ± S.E.M., n = 3; *p < 0.05 vs cells exposed to 20% O2. (B) immunoblot assays were performed to analyze CBS and actin protein levels. U87-MG cells were exposed to 20% or 1% O2 for 72 h and whole cell lysates were prepared. Asterisks show nonspecific band and arrow indicates CBS. (C) Primary cultures of human aortic (left panel) and lung microvascular (right panel) endothelial cells (ECs) were exposed to 20% or 1% O2 for 24 h and qRT-PCR assays were performed to quantify levels of the indicated mRNAs, normalized to the levels at 20% O2 (mean ± S.E.M., n = 3). MPST mRNA was not detected in aortic ECs. (D) U87-MG cells were exposed to 20% or 1% O2 for 24 h in the presence of vehicle (0.1% DMSO), 5 μM acriflavine (ACF), or 400 nM digoxin. Total RNA was isolated and RT-qPCR assays were performed using primers specific for SLC2A1, CBS, and RPL13A mRNA. Data shown are mean ± S.E.M., n = 3; *p < 0.05 vs vehicle-treated cells exposed to 20% O2; #p < 0.05 vs vehicle-treated cells exposed to 1% O2.