Figure 3.

eATP induces P2RX7-dependent autophagy in myoblasts. (A) Representative western blots showing increased P2RX7 expression levels in dystrophic (Dmd^mdx) vs. wild-type (Wt) myoblasts, with a time-course of MAPK1-MAPK3 phosphorylation and autophagy induction (LC3-I to LC3-II shift) following exposure to 3 mM eATP. No detectable changes in mitophagy were observed as shown by COX4 expression levels. ACTB represents protein loading control. (B) Western blots of triplicate samples showing the dependency of autophagy induction on P2RX7 expression in myoblasts; note the lack of 17 to 14 kDa LC3 shift in Dmd^mdx p2rx7^−/- double-mutant myoblasts following 30 min exposure to 1 mM BzATP. (C) Western blots of triplicate samples showing different effects of inhibiting P2RX7 activation and MAPK1-MAPK3 phosphorylation on autophagy induction in Wt (left panel) and Dmd^mdx (right panel) myoblasts. Cells were preincubated for 30 min with the P2RX7 antagonist Coomassie brilliant blue-G (BBG) or U0126 (MEK inhibitor) prior to 30 min 1 mM BzATP treatment. The classical P2RX7-MAPK1-MAPK3 activation cascade is involved in, but does not appear to be the sole pathway of autophagy induction in dystrophic myoblasts. Note: The phospho-MAPK1-MAPK3 is a multichannel (green/red) fluorescent blot, where yellow signal denotes increased MAPK1-MAPK3 phosphorylation. The left-hand lane in the Wt blot is a positive control. (D) Fold change in LC3-II levels in response to treatments shown in (C). Mean +/- SE, n =3, P < 0 .05* and 0.0001***.