Conclusions Myotoxicity induced by ATR and SIM is associated with the reduced GGOH-dependent prenylation of RAP1 protein. Lower myotoxicity is reflected from the respective increase in AKT 1 (S463) and GSK-3(S9) phosphorylation. Geranylgeranyltransferases (GGTs) control myocyte viability through GGOH, which in excess is likely myotoxic. Cytoprotective autophagy is usually elevated in myocytes during myogenesis. Lite Version 5.2.5, LI-COR BiotechnologyGmbH, Bad Homburg, Germany) and the open-source image processing bundle Fiji (ImageJ). Variations in the phosphorylation state of specific proteins were identified probing the Western blot membranes with main antibodies to the respective phosphorylated forms AKT1 (P-AKT1 (Ser473)) and GSK-3(P-GSK-3(Ser9)) in comparison to the total protein expression levels of the relevant proteins (AKT1 (T-AKT1) and GSK-3(T-GSK-3 0.001). As anticipated, a different pattern of response was observed between differentiating and already differentiated myotubes. While both MEV (100? 0.05), none of them were able to save ATR-mediated toxicity in differentiated myotubes. Neither FOH (10? 0.05), the compound rescues the statin effect in differentiated myotubes ( 0.05). Open in a separate window Number 1 Effect of nonsterol isoprenoids and soluble cholesterol treatments on C2C12 muscle mass cell viability. Nonsterol isoprenoids and soluble cholesterol differentially save C2C12 myoblasts from statin- or M 0.0001 for ATR; 0.0001 for SIM; 0.0002 for M 0.0001 (ATR, ATR?+?MEV, ATR?+?GGOH, ATR?+?FOH, and ATR?+?Chol-PEG); 0.0001 (SIM, SIM?+?MEV, SIM?+?GGOH, SIM?+?FOH, and SIM?+?Chol-PEG); 0.0001 (M 0.0001 for ATR; 0.0001 for SIM; 0.0001 for M 0.05, ?? 0.01, and ??? 0.001 for comparison with nontreated control cells. Results are means??SEM of three indie experiments. A different pattern was observed in the case of SIM-induced cytotoxicity (Number 1(b)). GGOH was capable of rescuing toxicity only in proliferating myoblasts and MEV was inefficient individually of the differentiation state. Decursin DOH (1? 0.001), while only UBOH improved SIM-reduced cell viability in differentiating myotubes while FOH in differentiated myotubes. FOH was able to save SIM-induced toxicity only in differentiated myotubes ( 0.001). To gain insight into the cellular pathways translating into the reduced cell viability depicted in Numbers 1(a) and 1(b), the apoptotic index (AI) was determined based on the analysis of nuclei morphology depicted in the micrographs illustrated in Supplementary data 2. As can be observed from your bar charts, ATR did not modify the value of AI with regard to nontreated control cells (Number 2(a)). GGOH and FOH at day time 1, FOH at day time 3, while Chol-PEG at day time 5 significantly raised AI versus the nontreated settings (Number 2(a)). SIM could hardly impact AI, but at day time 1, FOH and Chol-PEG significantly elevated a portion of apoptotic cells (Number 2(b)). Open in a separate window Number 2 Effect of nonsterol isoprenoids and soluble cholesterol treatments Decursin on apoptotic index (AI) in C2C12 myoblasts affected by statins or M 0.0001 for ATR; 0.0001 for SIM; 0.0001 for M 0.0001), SIM, SIM?+?MEV, SIM?+?GGOH, SIM?+?FOH, and SIM?+?Chol-PEG (= 0.0002), M 0.0001). Connection: 0.0001 for ATR; 0.0001 for SIM; 0.0001 for M 0.05, ?? 0.01, ??? 0.001 for comparison between the means. Results are means of three self-employed experiments. 3.2. Decursin Effect of M 0.001). The highest AI values were found after 3- and 5-day time treatment with M 0.001). Neither MEV, GGOH, FOH, nor Chol-PEG significantly reduced the percentage of apoptotic cells, albeit Chol-PEG seemed the most efficient. 3.3. Statin- and MSignaling Pathway IC50 concentrations of statins and Mphosphorylation at serine 9 (P-GSK-3cascade takes on a fundamental part in muscle mass cell viability [37] in which P-GSK-3protein expression levels (Number 3). Total Rabbit polyclonal to ZNF791 protein was extracted from differentiating C2C12 myoblasts revealed for 24, 72, or 120?h to statins or M(P-GSK-3(P-GSK-3(P-GSK-3 0.0001 for ATR; = 0.0006 for SIM; = 0.0521 for M= 0.9520); SIM, SIM?+?MEV, SIM?+?GGOH, SIM?+?FOH, and SIM?+?Chol-PEG (= 0.9423); M= 0.7228). Connection: 0.0001 for ATR; = 0.0006 for SIM; = 0.42 for Moptical denseness ratio followed by Bonferroni’s multiple comparisons was employed to analyze the data. The results of [time (proliferating myoblasts, differentiating myotubes, differentiated myotubes)] amounted to = 0.0059 Decursin for ATR; 0.0001 for SIM; Decursin and 0.0001 for M 0.0001); SIM, SIM?+?MEV, SIM?+?GGOH, SIM?+?FOH, and SIM?+?Chol-PEG (= 0.7074); M= 0.9568). Connection: = 0.0033 for ATR; =.