Figure 2.

Jumpy blocks autophagic degradation. (A) Proteolysis of long-lived proteins in C2C12 myoblasts. C2C12 cells were transfected with control (scramble) or Jumpy siRNA (siJumpy), labelled overnight in media containing [³H] leucine, washed, incubated for 2 h in complete media (containing cold leucine) and incubated for 4 h in full or starvation media. Leucine release was calculated from radioactivity in the tricarboxylic acid-soluble form relative to total cell radioactivity. Results shown represent mean±s.e.m. for combined data from three independent experiments. (B) Quantitation of p62 protein levels. C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA and p62 levels analysed by immunoblotting, quantitated by densitometry and represented as a percentage of control. Data are mean±s.e.m. (n=4 independent experiments). (C) C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA, incubated for 4 h with or without 100 nM Bafilomycin A1 in full media, lysed, probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=3). (D) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2) or Beclin siRNA (lanes 3, 4) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3) or Jumpy siRNA (lanes 2, 4). Cells were lysed, probed for p62, Beclin and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=5). (E–M) C2C12 cells were transfected for 48 h with GFP, GFP-Jumpy (Jumpy), GFP-MTM1 (MTM1) or GFP-MTMR2 (MTMR2), fixed and immunostained with anti-p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of GFP (E), GFP-Jumpy (F), GFP-MTM1 (G), GFP-MTMR2 (H) transfected cells, immunostained for p62 (I), (J), (K) and (L), respectively. Scale bars, 5 μm. White lines represent outline of the cells. Quantitation of p62 puncta per cell (M). Bars, SEM (n=3 independent experiments, with an average of 30 cells per experiments). (N, O) C2C12 cells were transfected for 48 h with GFP, GFP-Jumpy (Jumpy), GFP-MTM1 (MTM1) or GFP-MTMR2 (MTMR2), lysed, analysed for p62, GFP and actin by immunoblotting (N) and percentage of p62 were quantitated (mean±s.e.m., n=3) (O). (P, Q) C2C12 cells were transfected for 48 h with GFP or GFP-Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and actin by immunoblotting (P) and percentage of p62 were quantitated (mean±s.e.m., n=4) (Q). ^*P<0.05, ^***P<0.001, †ns (t-test).