Figure 5.

Up-regulation of Hif-1a expression by hypoxia is dependent on sialorphin. CSM cells were pretreated for 48 h with control-siRNA or vcsa1-siRNA, and cells were then exposed for 8 h to CoCl₂ (A–C) or low oxygen tension (D–E) to mimic hypoxia. A) Gene expression of Hif-1a and rpl24 was determined in the presence or absence of CoCl₂, in CSM cells pretreated with control-siRNA or vcsa1-siRNA. Three separate experiments were performed, with each gene being measured in triplicate. Data were normalized to the housekeeping gene, rpl24, and expressed relative to nonhypoxic CSM cells treated with control-siRNA. Data are expressed as means ± sd. Note that in the vcsa1-siRNA treated cells, there was no significant difference between cells maintained under hypoxic and nonhypoxic conditions. *P < 0.05 for increase vs. controls; ^vP < 0.05 for decrease vs. controls. B) Representative immunoblot for the expression of Hif-1a and GAPDH. C) Densitometric analysis of 3 separate experiments to determine expression of Hif-1a and GAPDH. Expression of Hif-1a was normalized to the housekeeping gene, GAPDH. Data represent mean ± sd fold change in expression of Hif-1a, compared between cells in hypoxic conditions with nonhypoxic cells for each treatment condition. *P < 0.05 for increase vs. controls; ^vP < 0.05 for decrease vs. controls. D) Gene expression of Hif-1a and rpl24 was determined in CSM cells grown in low oxygen tension (1% O₂, 5% CO₂, and 94% N₂) in a 95% humidified atmosphere or under normoxic conditions (20% O₂) pretreated with control-siRNA or vcsa1-siRNA. Three separate experiments were performed, with each gene being measured in triplicate. Data were normalized to the housekeeping gene, rpl24, and expressed relative to nonhypoxic CSM cells treated with control-siRNA. Data are expressed as means ± sd. Note that in the vcsa1-siRNA treated cells, there was no significant difference between cells maintained under hypoxic and nonhypoxic conditions. *P < 0.05 vs. control. E) Representative immunoblot for the expression of Hif-1a and GAPDH. F) Densitometric analysis of 3 separate experiments to determine expression of Hif-1a and GAPDH. Expression of Hif-1a was normalized to the housekeeping gene, GAPDH. Data represent mean ± sd fold change in expression of Hif-1a, compared between cells in hypoxic conditions with nonhypoxic cells for each treatment condition. *P < 0.05 vs. controls.