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. Author manuscript; available in PMC: 2016 Mar 20.
Published in final edited form as: Radiat Res. 2015 May 14;183(6):594–609. doi: 10.1667/RR14016.1

Figure 9. Knockdown of HIF-1α sensitizes P7NP cells to radiation and leads to a radiation-induced mitochondrial biogenesis defect.

Figure 9

Primary cells from P7NP tumors were isolated as described in Figure 3. All experiments were performed with in vitro cultures under 21% oxygen. A) Efficiency of Hif-1α knockdown by shRNA was measured by measuring HIF-1α protein level in nuclear extracts of parental P7NP sarcoma cells and P7NP cells stably expressing Hif-1α shRNA cultured under 0.5% oxygen for 24 hours. This showed that the efficiency of Hif-1α knockdown in P7NP sarcoma cells obtained from 3 different tumors was beyond the level of detection by Western Blot. B) Knockdown of Hif-1α by shRNA resulted in significantly increased radiation sensitivity compared to the parental controls after exposure to 6Gy under 21% oxygen as measured by clonogenic survival, by a two-tailed student t-test. Each symbol shape represents a parental control P7NP cells and its corresponding knockdown daughter sarcoma cells. C) The mitochondrial DNA (mtDNA) content was measured via real-time PCR using DNA extracted from parental P7NP and P7NP cells with Hif-1α knockdown at 72 hours after 6Gy irradiation, and normalized to 18S. P7NP cells had more mtDNA content at 72 hours following irradiation as compared to P7NP cells with Hif-1α shRNA, by a two-tailed student t-test. Each symbol shape represents a parental control P7NP and its corresponding knockdown daughter sarcoma cells. * p<0.05. ** p<0.01.