FIGURE 4.

Exacerbated autophagosome accumulation in cultured myotubes from Tpcn2^−/− mice under nutrient deprivation and inhibition of vacuolar H⁺-ATPase. A, differentiated myotubes derived from the wild type or Tpcn2^−/− neonates were subjected to autophagy flux measurement (see “Experimental Procedures”). Baf A1 at concentration of 200 nm was used to inhibit the vacuolar H⁺-ATPase in the lysosome. HBSS was used to induce starvation of the cells. Cell lysates (40 μg of proteins/lane) were used for immunoblotting with anti-LC3 or anti-α-tubulin. B, relative levels of LC3-II/tubulin. Data are expressed as -fold induction relative to that of the wild type at basal condition (medium) (n = 4 experiments). Data are means ± S.E. (*, p < 0.01). C, Tpcn2^−/− myoblasts were transfected with either gfp-tpcn2 plasmid (odd-numbered lanes) or pcms-eGFP plasmid (even-numbered lanes) for 48 h and then subjected to autophagy flux analysis. Cell lysates (30 μg of proteins/lane) were collected for immunoblots with anti-LC3 or anti-α-tubulin. D, relative levels of LC3-II/tubulin. Data (means ± S.E.) are expressed as LC3-II/tubulin normalized to that obtained from pcms-eGFP-transfected Tpcn2^−/− cells cultured under basal conditions (medium; lane 2) (n = 3 experiments). Error bars represent S.E.