(a) Frequency distributions of 561/488 nm excitation ratios measured
on HEK293 RPS3-Keima cells lacking ATG5 or BECN1 were compared after Torin1 or
Torin1/BafA treatment using flow cytometry. (n = 10,000
cells per condition) (b) Average 561/488 nm excitation ratios
calculated from the biological triplicate experiments from panel a. Mean
± S.E.M. (****p
< 0.0001, **p < 0.01, Two-way
ANOVA) (c) Confocal images of live HEK293 RPS3-Keima cells lacking
ATG5 after Torin1 (150 nM, 24h) or Torin1 (150 nM, 24h)/SAR405 (1 μM,
24h) co-treatment. (Scale bar = 20 μm) (d) Number
of red Keima puncta/cell was measured from the live-cell images of HEK293
RPS3-Keima WT, ATG5−/−, or
BECN1−/− cells taken after Torin1 (150 nM, 24h)
or Torin1 (150 nM, 24h)/SAR405 (1 μM, 24h) co-treatment. Mean ±
S.E.M. (Total number of cells from three biologically independent samples are
indicated in the graph) (e,f) HEK293 RPS3-Keima cells (with or
without ATG5) were incubated with HBSS in the presence of lysosomal hydrolase
inhibitors (L.H.I., E64d and Pepstatin, 30 μM each) for the indicated
times prior to live cell imaging. (Scale bar = 20 μm)
(g) Unbiased quantification of the live-cell images obtained as
shown in panels e and f. (h) Unbiased quantification of red Keima
puncta obtained from live HCT116 RPL28-Keima cells (with and without ATG5) as
shown in Supplemental Fig.
2g,h. In panels g and h, total number of cells from three
biologically independent samples are indicated in the graph, and Mean ±
S.E.M. is shown. (i) Electron microscopy images of HEK293
RPS3-Keima WT, ATG5−/− and
BECN1−/− cells 4.5 h after HBSS treatment in the
presence of BafA (50 nM). Red arrow: ribosomes in autophagosomes or
autophagolysosomes, yellow arrow: ribosomes bound to ER in cytosol (Scale bar
= 500 μm). The data shown represents two independent
experiments. Statistical source data for b can be found in Supplementary Table 2.
All experiments were repeated at least three times unless otherwise
indicated.