Pkc#cod#x003B4; Activation is Involved in ROS-Mediated Mitochondrial Dysfunction and Apoptosis in Cardiomyocytes Exposed to Advanced Glycation End Products (Ages) Yang Yao-Chih 1 ;Tsai Cheng-Yen 2, 3 ;Chen Chien-Lin 4 ;Kuo Chia-Hua 5, 6 ;Hou Chien-Wen 5 ;Cheng Shi-Yann 7, 8, 9 ;Aneja Ritu 10 ;Huang Chih-Yang 11 ;Kuo Wei-Wen 1 ; 1 Department of Biological Science and Technology, College of Biopharmaceutical and Food Sciences, China Medical University, Taiwan. 2 Department of Pediatrics, China Medical University Beigang Hospital, Taiwan. 3 School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taiwan. 4 Department of Life Sciences, National Chung Hsing University, Taiwan. 5 Laboratory of Exercise Biochemistry, University of Taipei, Taipei, Taiwan. 6 Graduate Institute of Physical Therapy and Rehabilitation Science, China Medical University, Taiwan. 7 Department of Medical Education and Research and Department of Obstetrics and Gynecology, China Medical University Beigang Hospital, Taiwan. 8 Department of Obstetrics and Gynecology, China Medical University An Nan Hospital, Taiwan. 9 Obstetrics and Gynecology, School of Medicine, China Medical University, Taichung, Taiwan. 10 Department of Biology, Georgia State University, Atlanta, GA 30303, USA. 11 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan ; Graduate Institute of Chinese Medical Science, School of Chinese Medicine, China Medical University, Taichung, Taiwan ; Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan. ; Figure 6. AGE-BSA-induced mitochondria damage and decreased biological function is through PKC#cod#x003B4; activation and colocalization in cardiac cells. A, B Cells were treated with the PKC#cod#x003B4; activator, bryostatin 1 100 nM or inhibitor, rottlerin, 3 #cod#x003BC;M for 24 h following exposure to AGE-BSA 300 #cod#x003BC;gml. C AGE-BSA-exposed cells were transfected with PKC#cod#x003B4; siRNA 1 #cod#x003BC;g for 24 h. D Cells were transfected with GFP-PKC#cod#x003B4;-WT plus rottlerin or AGE-BSA-exposed cells were transfected with GFP-PKC#cod#x003B4;-KD. Mitochondrial damage was evaluated by mitochondrial mass, which was analyzed by flow cytometry. E AGE-BSA-treated cells were administered the inhibitor rottlerin 3 #cod#x003BC;M or siPKC#cod#x003B4; for 24 h. The mitochondrial fission and colocalization with PKC#cod#x003B4; were examined by immune-fluorescence and analyzed by confocal microscopy. F Super resolution images of mitochondria structure in cells were analyzed with similar experimental procedures. Blue, DAPI 4, 6-diamidino-2-phenylindole-stained nuclei; red, MitoTracker Red CMXRos-stained mitochondria. G The levels of proteins associated with mitochondrial fission and mitophagy in cells were analyzed with similar experimental procedure. These are cropped blots, full-length blots are presented in Suppl. Figure S6. Neonatal rat ventricular myocyte NRVM and H9c2 were then analyzed for H Cells were treated with AGE-BSA for 24 hr and then transfected with the PKC#cod#x003B4; siRNA 1 #cod#x003BC;g for 24 hr. Cell lysates were immunoprecipitated using antibodies against pPKC#cod#x003B4; T505507 . Protein expression was detected by immunoblotting. I The cytosolic and mitochondrial fractions were isolated and subjected to immunoprecipitation followed by western blot analysis. These are cropped blots; full-length blots are presented in Suppl. Figure S7. J Cellular oxygen consumption rate O.C.R and extracellular acidification E.C.A.R using an XF24 bioenergetics assay Seahorse Bioscience, Billerica, MA. Data are expressed as the mean #cod#x000B1; SEM, n=3. #cod#x0002A; P #cod#x0003C;0.05, #cod#x0002A;#cod#x0002A; P #cod#x0003C;0.01, #cod#x0002A;#cod#x0002A;#cod#x0002A; P #cod#x0003C;0.001 compared with the control or GFP group, # P #cod#x0003C;0.05, ## P #cod#x0003C;0.01, ### P #cod#x0003C;0.001 compared with the AGE-BSA 300 #cod#x003BC;gmg group, #cod#x02020; P #cod#x0003C;0.05 compared with the Bry1-treated group, #cod#x02020;#cod#x02020; P #cod#x0003C;0.01, #cod#x02020;#cod#x02020;#cod#x02020; P #cod#x0003C;0.001 compared with the GFP-PKC#cod#x003B4;-WT overexpression group. null,null,0(0),null-null. Doi:10.14336/AD.2017.0924