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. 2015 May 11;6:7007. doi: 10.1038/ncomms8007

Figure 6. BafA1 blocks autophagosome–lysosome fusion through inhibition of dSERCA.

Figure 6

(a) Representative confocal images of control (GFP negative) and dSERCA-depleted (GFP-positive clones) fat body cells from well-fed and 4 h starved larvae. Depletion of dSERCA leads to accumulation of mCh-Atg8a punctae (red, and shown in greyscale at right) under fed conditions with no further accumulation in starved samples. (b) Quantification of size of Atg8-positive vesicles in control and dSERCA-depleted fat body cells from experiment in a. Error bars mark s.e.m. for each genotype (N≥5). **P<0.05 and ***P<0.001, Student's t-test. (c) Localization of LAMP1-GFP and mCh-Atg8a in control and dSERCA-depleted fat body from 4 h starved larvae. Insets illustrate the presence or absence of autolysosomes. Autolysosomes identified as mCh-Atg8a-positive structures surrounded by LAMP1-GFP were observed only in control. N≥9 for each genotype. (d) Quantification of co-localization between LAMP1-GFP and mCh-Atg8a. Pearson's correlation coefficient Rr was calculated using a minimum of nine representative images for each genotype. Error bars mark s.e.m. for each condition. ***P<0.001, Student's t-test. (e) Quantification of GCaMP3 fluorescent intensity relative to internal control DsRed. Fat body cells incubated ex vivo for 1 h with EGTA (10 mM) showed a significant reduction of GCaMP3/DsRed ratio, while cells treated with BafA1 (200 nM) or thapsigargin (200 nM) presented a strong increase compared with control. Error bars mark s.e.m. for each condition, N≥25. **P<0.005, ***P<0.001, Student's t-test. (f) Morphology of LAMP1-GFP and mCh-Atg8a structures in control and Seipin-overexpressing cells. Fat body were dissected from larvae either starved for 4 h in vivo or incubated for 6 h ex vivo in M3 media in the presence or absence of BafA1 (200 nM). In a, c and f, nuclei are labelled with DAPI; blue; scale bars, 10 μm. (g) Quantification of the presence of elongated mCh-Atg8a- and LAMP1-GFP-positive tubular structures in fat body cells as shown in f. Error bars mark s.e.m. for each condition, N≥16. **P<0.05 ***P<0.005, Student's t-test.