Figure 6

BEX2 binds TUFM and suppresses in mitochondrial activity. (A) BEX2-binding partners were evaluated by LC/MS in both mouse kidney (upper panel) and HuCCT1 cell (lower panel) lysates, respectively. These results indicate that mitochondria-related proteins HSPD1, TUFM, IVD, and PECR (arrows) are direct binding partners of BEX2. (B) Oxygen consumption ratio (OCR, pmol/min) was determined in control and BEX2-knockdown HuCCT1 cells using a Flux analyzer. The data was normalized to relative cell numbers. *P < 0.05. (C) ATP level was determined by Cell Titer Glo in control and BEX2-knockdown HuCCT1 cells under glucose-free RPMI medium for 1 h. a.u., arbitrary units. n = 5, *P < 0.05. (D) Oxygen consumption ratio (OCR) was determined in control and knockdown lines of BEX2-binding partners in HuCCT1 cells using a Flux analyzer. The data was normalized to relative cell numbers. n = 9. *P < 0.05. (E) Immunoprecipitation analysis. 293 T-FlpIn-Flag-BEX2 cells were lysed and fractionated into mitochondria/nucleus and cytosol. (F) Oxygen consumption ratio (OCR) was determined under the treatment of siRNA of BEX2 and TUFM by Flux Analyzer. The data was normalized to relative cell numbers. (G) Tumorigenicity assay. Control or TUFM knock down HuCCT1 cells were injected into NOG mice subcutaneously (10² cells per site. Six sites total.). Tumor volumes were measured weekly. (H) The sensitivity against gemcitabine was determined by MTT assay. The results were normalized to no gemcitabine treatment. n = 3. *P < 0.05 between siControl and siTUFM#1 or siTUFM#2. All the bars indicated means standard deviation.