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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Int J Impot Res. 2015 Apr 2;27(4):140–145. doi: 10.1038/ijir.2015.5

Figure 3. Up regulation of CD73 expression by hypoxia is dependent on sialorphin.

Figure 3

(A) In preliminary experiments CSM cells were treated for 48 hours with different concentrations of vcsa1-siRNA for 48 hours and expression of vcsa1 determined by quantitative-RT-PCR and expressed as fold change relative to the untreated cells. All concentrations gave a significant decrease in vcsa1 expression (*= P<0.05). (B) CSM cells were pretreated for 48 hours with 10 nM control-siRNA or 10 nM vcsa1-siRNA, and cells were then exposed for 8 hours to CoCl2 to mimic hypoxia. Gene expression of CD73 and rpl24 was determined in the presence or absence of CoCl2, in CSM cells pre-treated with control-siRNA or vcsa1-siRNA. Three separate experiments were performed with each gene being measured in triplicate. Data were normalized to the house keeping gene, rpl24, and expressed relative to the non-hypoxic CSM cells treated with control-siRNA. Data is expressed as mean ± Std. Dev. *=P<0.05 significantly increased, v= P<0.05 significantly decreased fold-change in gene expression compared to control (non-hypoxic CSM cells treated with control-siRNA). (C, lower panel) A representative immunoblot for the expression of CD73 and GAPDH. (C, upper panel) Densitometric analysis was performed from three separate experiments to determine expression of CD73 and GAPDH. Expression of CD73 was normalized to the housekeeping gene, GAPDH. The mean fold-change (± Std. Dev.) in expression of CD73 was compared between cells in hypoxic conditions with non-hypoxic cells for each of the treatment conditions *=P<0.05 significantly increased fold-change in protein expression compared to non-hypoxic controls.