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. 2017 Sep 1;31(17):1738–1753. doi: 10.1101/gad.302349.117

Figure 2.

Figure 2.

p73KD induces mitochondrial defects and activates AMPK signaling. (A) DAOY cells were transfected with scramble, sip73 (sip73*1, 2671; sip73*2, 115666), or siGLS-2 (siGLS-2*1, s25941; siGLS-2*2, s223735). OCR in response to a mitochondrial stress test was recorded to construct functional bioenergetic profiles. A minimum of five different samples was analyzed for each group. (**) P < 0.001; (***) P < 0.0001, unpaired two-sided t-test. Data from one representative experiment out of three are shown. (B) The reduced and oxidized glutathione ratio was measured in DAOY cells after transient transfection with scramble, sip73*1, sip73*2, siGLS-2*1, and siGLS-2*2 for 24 h. DAOY cells were infected with an empty vector or with shRNA (p73sKD*3 and p73sKD*5) alone or transfected with Flag-GLS-2-expressing vector for 48 h. n = 3. Data were mean ± SD. (*) P < 0.01; (**) P < 0.001; (***) P < 0.0001. (C) DAOY cells were transfected with scramble or sip73 (sip73*1, 2671; sip73*2, 115666). Extracellular acidification rate (ECAR) was measured in response to a mitochondrial stress test. A minimum of five different samples was analyzed for each group. (***) P < 0.0001, unpaired two-sided t-test. Data from one representative experiment out of three are shown. (D,E) DAOY cells were transfected with scramble or sip73*2. After 8 h of transfection, the medium was changed, and samples were collected after 6, 20, 30, and 40 h. The results show glucose consumption (D) or lactate production (E). Columns indicate mean ± SEM. n = 4. (**) P < 0.001; (***) P < 0.0001. (F) DAOY cells were transiently transfected with scramble, sip73*1, or sip73*2. After 48 h, cells were collected, and ATP levels were determined. Columns indicate mean ± SEM. n = 4. (***) P < 0.0001. (G) DAOY cells were transfected with scramble, sip73*1, or sip73*2. After 48 h a Western blot was performed for ACC, P-ACC, S6K, P-S6K, eEF-2, and P-eEF-2. Intensity analysis is shown below each lane; scramble was considered 1. (H) DAOY cells were transfected with scramble, sip73*1, or sip73*2. After 48 h, cells were treated for 1 h with 2.5 mg/mL puromycin. Next, cells were collected, and a Western blot of lysed cells was probed with a primary anti-puromycin antibody. Tubulin was used as a loading control. (2-DG) 2-deoxy-D-glucose.