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Study shows that treatment of cells with ketone bodies increased the proteolysis …


Biology Articles » Biochemistry » Ketone Bodies Stimulate Chaperone-mediated Autophagy » Figures

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- Ketone Bodies Stimulate Chaperone-mediated Autophagy

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FIG. 1. Effect of BOH and acetoacetate on the degradation of total cellular protein. Protein degradation is calculated by labeling cellular protein and measuring the release of [3H]leucine over time and comparing the acid-soluble radioactivity to the total radioactivity, as described (24). a, MR-90 cells in the presence and absence of serum treated or not treated with 4 mM BOH. b, MR-90 cells in the presence and absence of serum treated or not treated with 4 mM acetoacetate. In panel a, time points from BOH-treated cells show statistically significant increased proteolysis under all conditions (p ≤ 0.001). In panel b, acetoacetate treatment at 4 and 26 h in the presence of serum shows statistically increased degradation compared with control (p ≤ 0.001).

Figure 1

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FIG. 2. Stability of lysosomes isolated from untreated and BOH-treated IMR-90 cells. Lysosomes were isolated from IMR-90 cells maintained in media without serum, treated with or without 4 mM BOH, and subject to a latency test to determine stability (see "Materials and Methods"). Error bars represent S.D. of ±1; BOH-treated and control lysosomes were not significantly different.

Figure 2

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FIG. 3. The degradation of [14C]GAPDH and [14C]RNase A by isolated lysosomes. Lysosomes were isolated from cells maintained in media without serum and treated with (white bars) or without (black bars) 4 mM BOH. a, degradation of [14C]GAPDH was monitored by the conversion of acid-precipitable radioactivity into acid-soluble radioactivity (0 and 14 µg, not significant; *, p ≤ 0.001 for 28 and 50 µg). b, degradation of [14C]RNase A was monitored as for [14C]GAPDH (0 and 14 µg, not significant; *, p ≤ 0.001 for 28 and 50 µg).

figure 3

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FIG. 4. Degradation of [14C]GAPDH and [14C]RNase A by isolated lysosomes treated with varying concentrations of BOH. Lysosomes were isolated from IMR-90 cells maintained in media without serum and treated with increasing concentrations of BOH; n = 3 and 2 for each time point of GAPDH and RNase A, respectively. Error bars indicate S.D. of ±1 for GAPDH. The average and the range are shown for RNase A duplicates. All time points with intact lysosomes were statistically increased compared with broken lysosomes (p ≤ 0.001). Proteolysis is reported as percent of untreated lysosomes.

Figure 4

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FIG. 5. Protein levels of lyhsc70 and lamp2a in cells treated with or without BOH. Lysosomes (Ly) were isolated from IMR-90 cells maintained in media without serum, treated with (+) or without (-) BOH, and subjected to immunoblot. Protein (100 µg) was loaded per well. Bands were quantitated using scanning densitometry. There was no statistical difference between lamp2a or hsc70 in the treated or untreated groups. Mtx, matrix; Mem, membrane. These results are representative of three different experiments.

Figure 5

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FIG. 6. Analysis of GAPDH uptake into lysosomes of BOH-treated and untreated cells. Lysosomes were isolated from IMR-90 cells maintained in media without serum and treated (+) or not treated (-) with BOH. Shown is a Western blot analysis of lysosomes subjected to a GAPDH/proteinase K protection assay.

Figure 6

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FIG. 7. Preincubation of substrates, but not lysosomes, with BOH increases degradation. Substrate (sample set 3) or lysosomes (sample set 2) were preincubated with 4 mM BOH for 20 min prior to transport assay. Control lysosomes were untreated with BOH (sample set 4) or contained only substrate (sample set 1); n = 3 for all experiments. Error bars indicate S.D. of ±1. The increased proteolysis in sample set 3 is statistically significant (p ≤ 0.001) for both GAPDH and RNase A. Values for sample sets 2 and 4 were not significantly different for either GAPDH or RNase A.

Figure 7

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FIG. 8. OxyBlotTM analysis of GAPDH and RNase A. a, GAPDH was immunoprecipitated using anti-GAPDH antibody from cells maintained in media without serum and supplemented with (+) or without (-) 4 mM BOH for 24 h. b, RNase A was incubated with BOH for either 4 or 24 h and subjected to OxyBlotTM analysis followed by SDS-PAGE and immunoblotting

Figure 8

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FIG. 9. Effects of butanone, butanol, and glycerol on total cellular proteolysis. a, IMR-90 cells treated with compounds similar to BOH, 4 mM butanone and 4 mM butanol, showed no effect on proteolysis. b, similarly, 5 mM glycerol had no effect on total cellular proteolysis; n = 3 for all time points. Error bars indicate S.D. of ±1. Differences in time points are not statistically significant (p > 0.05).

Figure 9

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Source: J. Biol. Chem., Vol. 280, Issue 27, 25864-25870, July 8, 2005


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