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1.
Figure 3

Figure 3. From: Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease.

Effect of ERT on glycogen clearance in skeletal muscle of GAA−/− and MLCcre:Atg7F/F:GAA−/− mice. (A) Quantitation of glycogen levels in gastrocnemius (fast) muscle from GAA−/−, MLCcre:Atg7F/F:GAA−/− and HS Acre:Atg5F/F:GAA−/− mice before and after ERT (values are mean ± sd.; n = 18−33 for the untreated mice and n = 9−14 for the treated mice). (B) PAS-stained sections (shown in black/white) of gastrocnemius muscle from 4 month-old mice. (C) Immunostaining of single psoas (fast) fibers stained for LAMP-1 (green) and LC3 (red) from 4 month-old ERT-treated GAA−/− and MLCcre:Atg7F/F:GAA−/− mice. Comparison of the lower part with that of Figure 1D shows the shrinking of lysosomes after ERT. Bar: 20 microns.

Nina Raben, et al. Autophagy. 2010 November 16;6(8):1078-1089.
2.
Figure 5

Figure 5. From: Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease.

Increase in the levels of Beclin 1 and changes in the phosphorylation status of GSK-3β and glycogen synthase (GS) in muscle from GAA−/− mice. Western blot of protein lysates from gastrocnemius (fast) muscles derived from WT and GAA−/− mice with the indicated antibodies. (A) Upregulation of Beclin 1 indicates that basal autophagy in GAA−/− is induced. (B and C) A markedly decreased phosphorylation (activation) of GSK-3β (on Ser9) is observed in muscle from GAA−/− mice without changes in the amount of total GSK-3β. (D) Increased phosphorylation (inactivation) of GS is shown with anti-phospho-GS antibody (top part) and by an upward shift on the gel with anti-GS antibody. Images shown are representative blots from at least four samples.

Nina Raben, et al. Autophagy. 2010 November 16;6(8):1078-1089.
3.
Figure 1

Figure 1. From: Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease.

Autophagy is suppressed in fast muscles from MLCcre:Atg7F/F:GAA−/− mice. (A) Western blot of protein lysates from gastrocnemius (fast) muscles derived from WT, GAA−/− and MLCcre:Atg7F/F:GAA−/− mice with LC3 (top part) or with Atg7 antibody (middle part) (n = 10 for each age group). (B) Western blot of lysates from soleus (slow) muscle derived from 6-month-old GAA−/− (lane 1) and MLCcre:Atg7F/F:GAA−/− (lane 2) mice with LC3. LC3-II is consistently present in slow muscle from MLCcre:Atg7F/F:GAA−/− mice although the levels are lower than in GAA−/− mice. The data are representative of at least five independent experiments. GAPDH serves as a loading control. (C) Western blot of protein lysates from gastrocnemius (fast) muscles derived from WT, GAA−/− and MLCcre:Atg7F/F:GAA−/− mice with p62 antibody (n = 10 for each age group). Muscle samples were taken from 3–4.5 month-old mice. (D) Immunostaining of single psoas (fast) fibers stained for LAMP-1 (green) and LC3 (red). Muscle samples were taken from 4-month-old mice. Bar: 20 microns.

Nina Raben, et al. Autophagy. 2010 November 16;6(8):1078-1089.
4.
Figure 2

Figure 2. From: Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease.

Electron microscopy confirms the disappearance of autophagic buildup in fast muscles of MLCcre:Atg7F/F:GAA−/− mice and provides evidence for the presence of residual autophagy-related vesicles. (A and B) Two non-overlapping images of a single continuous area of autophagic accumulation in the psoas muscle of a 10 month-old GAA−/− mouse illustrate the impressive size of such areas, which were not encountered in muscle from age-matched MLCcre:Atg7F/F:GAA−/− mice (C–E). However, limited accumulations of nonlysosomal vesicular material are seen in the psoas muscle of MLCcre:Atg7F/F:GAA−/− mice (arrowheads in C and E). Vesicles are often found adjacent to glycogen-laden lysosomes (C, white asterisk) and to accumulations of Ub-proteins (C, black asterisk). (D) shows a detailed view of multi-membrane vesicles, some of which (D, small arrowheads) may contain glycogen. (F) shows a higher magnification image of the area in (E); one of the vesicles (small arrowheads) resembles a pre-autophagosome membrane cup. Bars: (A and B): 1 micron; (C and E): 500 nanometers; (D and F): 100 nanometers.

Nina Raben, et al. Autophagy. 2010 November 16;6(8):1078-1089.
5.
Figure 6

Figure 6. From: Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease.

Expression of constitutively active GSK-3β (caGSK-3β) stimulates autophagy in C2C12 cells. (A) Immunostaining of fixed C2C12 myoblasts with LC3 shows an increase in the number of autophagosomes in the cells expressing caGSK-3β (pHan-caGSK-3β) compared to the cells with the vector alone (pHan). The cells were starved in Krebs-Ringer solution for 4 h. (B) Western blot of lysates from myotubes (three-day cultures) expressing caGSK-3β or vector alone with LC3. Myotubes were starved or treated with bafilomycin for 4 h. An increase in the amount of LC3-II is observed under all three conditions in the caGSK-3β cells. The numbers correspond to the densitometry values for LC3-II and α-tubulin (loading control). The expression of hemagglutinin (HA) confirms the expression of caGSK-3β. Images shown are representative blots from at least four experiments. (C) The effect of GSK-3β on autophagy in C2C12 myotubes was assessed as a fold increase of LC3-II /α-tubulin ratio in the cells with or without caGSK-3β. (D) Western blot of lysates from myotubes expressing caGSK-3β or vector alone with antibody against phosphorylated 4E-BP1.

Nina Raben, et al. Autophagy. 2010 November 16;6(8):1078-1089.
6.
Figure 4

Figure 4. From: Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease.

Decrease in the amount of potentially toxic Ub-proteins in muscle from autophagy-deficient GAA−/− strains upon therapy. (A) Western blot of protein lysates from gastrocnemius (fast) muscle with anti-ubiquitin (FK2) antibody. No difference in the levels of Ub-proteins is observed between untreated (lane 1) and ERT-treated (lane 2) GAA−/− mice. (B) Western blot of lysates from gastrocnemius muscle, prepared as detergent (Triton X-100)-soluble (s) and -nonsoluble fractions (ns), with FK2. Lane 1-WT; Lane 2-HS Acre:Atg5F/F:WT; Lane 3-HS Acre:Atg5F/F:GAA−/− untreated; Lane 4-HS Acre:Atg5F/F:GAA−/− treated. Note that there is a modest increase in the amount of Ub-proteins in muscle-specific autophagy-deficient WT mice (lane 2). (C) Single fibers stained for Ub (red) and LAMP-1 (green) show a dramatic decrease in the amount of Ub-proteins upon therapy. Muscle samples were taken from 4-month-old mice. Bars: 20 microns (top two parts); 10 microns (two lower parts). Occasional fibers from autophagy-deficient GAA−/− strains still contain enlarged lysosomes. The distribution of Ub-proteins in untreated fast muscle of MLCcre:Atg7F/F:GAA−/− mice (shown in Suppl. Fig. 2) is similar to that in the HS Acre:Atg5F/F:GAA−/− mice.

Nina Raben, et al. Autophagy. 2010 November 16;6(8):1078-1089.

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