For negative control experiments, the pRetroX Tight Pur vector was trans duced

Because concerns have been raised about the effect of primary alcohols as an index of PLD involvement in cell responses, we assessed the effects of small molecule inhibitors of PLD. Treatment of myotubes by FIPI, an inhibitor of both PLD isoforms, resulted in a marked atrophy, thereby confirming the involvement of PLD inhibition in the AP24534 Bcr-Abl 阻害剤 above observations. We then used PLD isoform specific inhibitors, and observed that PLD1 inhibition affected myotube chatacteristics, whereas PLD2 inhibition had no significant effect. Finally, the respective role of PLD isoforms was further assessed by using PLD1 or PLD2 siRNA. This approach confirmed that PLD1 depletion was more efficient than PLD2 depletion to decrease myotube area and CK activity.<br><br> Conversely, we found adenovirus mediated overexpression of PLD1 to significantly increase myotube area and CK ac tivity as compared with control cells, whereas PLD2 over expression had no significant effect. These observations confirmed that PLD1 positively regulates muscle cells. To verify that enzymatic activity is required AT-406 concentration for PLD1 trophic effects, we treated PLD1 overexpressing myotubes with PLD inhibitors. As expected, the dual PLD inhibitor FIPI and the PLD1 specific inhibitor both suppressed the hypertrophy induced by PLD1 over expression, whereas the PLD2 specific inhibitor had no sigificant effect. Next, we assessed the in vivo relevance of these obser vations. We injected a PLD1 encoding adenovirus in the right gastrocnemius of mice, the left gastrocnemius be ing injected with an adenovirus encoding GFP as a control.<br><br> Muscles were dissected 10 days following injec tion, and PLD1 overexpression was verified. Measurement of myofibre cross sectional area demonstrated a significant increase in myofibre size in PLD1 injected muscles as compared with GFP injected ones, as shown by a shift of the CSA distribution curve to wards higher values. Taking advantage of the HA tag fused akt3 阻害剤 to our PLD1 expressing construct, we then compared the respective CSA of myofibres express ing or not the fusion protein, in sections of PLD1 injected muscles. Immunofluorescent labeling of recombinant HA tagged PLD1 followed by CSA measurement confirmed a significant increase in the size of PLD1 expressing fibres. PLD and PA counteract the atrophic response of myotubes induced by catabolic agents Muscle cell atrophy can be induced in vivo and in vitro by synthetic glucocorticoids such as dexamethasone.<br><br> We investigated the effects of PLD isoform overexpression in dexamethasone treated myotubes. As expected, dexamethasone induced a marked atrophy of myotubes, as evidenced by reduced myotube size and CK activity. Interestingly, this atrophic effect was sup pressed in PLD1 overexpressing cells, but not affected by PLD2 overexpression. Moreover, inhib ition of PLD activity by FIPI restored the atrophic effect of dexamethasone in PLD1 overexpressing myotubes. Next, we mimicked PLD activation by adding exogenous PA to dexamethasone treated cells. We found PA addition able to partially restore both myotube size and CK activity. We then used another agent able to induce atrophy of muscle cells, the pro inflammatory cytokine TNF.