Research Paper Volume 11, Issue 4 pp 1110—1128

The ubiquitin ligase KBTBD8 regulates PKM1 levels via Erk1/2 and Aurora A to ensure oocyte quality

Figure 2. KBTBD8 regulates PKM1 levels and is essential for oocyte quality. A. Representative images of PKM1 and tubulin immunofluorescence in control and KBTBD8-depleted oocytes. Both cytoplasmic and spindle pole PKM1 signals decreased after KBTBD8-depletion. B. PKM1 and PKM2 expression in control and KBTBD8-depleted (KBTBD8-DE) oocytes. GAPDH was used as a loading control. C. RT-PCR assessment of PKM1 and PKM2 mRNAs. GAPDH was used as control. D. DNA sequencing verified that the bands in (C) are PKM1 and PKM2. E. Mitochondrial staining (MitoTracker Red) in control and KBTBD8-depleted oocytes. F. Cytoplasmic ATP contents in control and KBTBD8-depleted oocytes. G. Mitochondrial membrane potential (JC1 staining) in control and KBTBD8-depleted oocytes. H. Quantification of JC1 aggregate/monomer ratios. I. Apoptosis detection (Annexin V staining) in control and KBTBD8-depleted oocytes. Apoptosis increased significantly after KBTBD8 depletion. J. Quantification of Annexin V signal. K. ROS generation in control and KBTBD8-depleted oocytes. L. ROS quantification. Scale bars, 20 µm. * p ˂ 0.05.