FIGURE 1: p38β MAPK activation is necessary and sufficient for autophagy activation by tumor in skeletal muscle cells. (A) Inhibition of p38 MAPK attenuates LLC-conditioned medium (LCM)-induced autophagy activation in C2C12 myotubes. C2C12 myotubes were pretreated with SB202190 (SB, 10 mM) or vehicle (0.1% DMSO) for 30 min prior to treatment with LCM or control medium (NL20) for 8 h. Autophagy activation was evaluated by Western blotting analysis of LC3 in cell lysate. (B) Inhibition of p38 MAPK attenuates autophagy activation in skeletal muscle of LLC tumor-bearing mice. In 7 days of LLC implant to mice, SB202190 was i.p. injected (5 mg/kg) daily with DMSO (50%) as vehicle control for 14 days. Lysate of TA collected on 21 days of LLC implant was analyzed by Western blotting for LC3 (samples from 3 mice per group were loaded on each gel). (C) LCM-induced autophagy activation in myotubes is dependent on p38β MAPK. C2C12 myoblasts were transfected with control, p38α MAPK or p38β MAPK-specific siRNA. After differentiation, myotubes were treated with LCM or control medium for 8 h. Autophagy activation was evaluated by Western blotting analysis of LC3. (D) Expression of constitutively active p38β MAPK stimulates autophagy flux in C2C12 myotubes. Plasmids encoding a constitutively active mutant of p38α MAPK or p38β MAPK with the HA tag were transfected into C2C12 myoblasts, with empty vector as control. After differentiation, myotubes were treated with chloroquine (CQ, 20 mM) for 8 h. LC3, p62 and expression of the p38 MAPK mutants were monitored by Western blotting. (E) Constitutively active p38β MAPK activates autophagy in mouse muscle. Plasmids encoding the constitutively active mutant of p38α MAPK or p38β MAPK fused with the HA tag were transfected into the TA of mice. Empty vector was transfected into the contralateral TA. On day 14 expression of the p38 MAPK mutants and autophagy activation was analyzed by Western blotting (data from two mice per group are shown). Data were analyzed by one way ANOVA. * denotes a difference between bracketed groups or from controls (p < 0.05). (F) Constitutively active p38β MAPK stimulates autophagosome formation in mouse muscle. A plasmid encoding GFP-LC3 was transfected into mouse TA muscle, and co-transfected with the plasmid encoding the constitutively active mutant of p38α MAPK or p38β MAPK in the contralateral TA. In 7 days, TA muscle was collected. Frozen sections were prepared and stained with DAPI. Autophagosome formation was evaluated by confocal microscopy. Bars represent 50 mm.