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. Author manuscript; available in PMC: 2018 Jun 11.
Published in final edited form as: Nat Cell Biol. 2017 Dec 11;20(2):135–143. doi: 10.1038/s41556-017-0007-x

Figure 4. A screen of ribosome stress agents identifies sodium arsenite and reversine as ribophagy inducers.

Figure 4

(a) HEK293 RPS3-Keima cells were exposed to (Tor1, 150nM, 24h; Reversine, 0.5μM, 48h; Sodium Arsenite (AS), 10μM, 24h; H2O2, 250μM, 24h; Cycloheximide, 10μM, 5h; Diazaborine, 200μM, 24h; Bortezomib, 250nM, 5h; Puromycin, 1μM, 5h; p97 inhibitor CB5083, 100nM, 24h; Tunicamycin, 0.6μM, 24h), and 561/488 ratio measured. (n=10,000 cells/condition) (b) HEK293 RPS3-Keima:WT, ATG5−/−, and BECN1−/− cells treated as in a.. Mean 561/488 ratios are plotted. (n=3 independent experiments, Supplemetary Table 2). (c) Frequency distributions of 561/488 ratios measured in HEK293 RPS3-Keima:ATG5+/+, ATG5−/− cells treated with AS. (n=4200 cells/condition) (d) The average 561/488 ratios from triplicate experiments as in panel c. (e) HEK293 RPS3-Keima:ATG5−/− cells transduced with a lentivirus expressing either ATG5 or conjugation defective ATG5K130R mutant were treated with AS (10μM, 24h). The average 561/488 ratios of biological triplicate experiments are shown. Mean of two independent experiments is shown for ATG5K130R cells. (f) Immunoblotting of indicated cells treated with AS (20μM, 20h). (g) The average 561/488 ratios of the indicated cell lines treated with Reversine (0.5μM, 48h) ± BafA (50nM, 1h). (n=3 independent experiments) (h) Frequency distributions of 561/488 ratios for HCT116 RPL28-Keima cells with or without Reversine treatment. (n=10,000 cells/condition) (i) Immunoblot of HCT116 RPL28-Keima cells treated with Reversine ± SAR405. (j) Imaging of HCT116 RPL28-Keima:ATG5+/+, ATG5−/− cells ± Reversine (0.5μM, 48h). (Scale bar=20μm) (k) Quantification of the images in panel j. Mean ± S.E.M. (Total number of cells are indicated as n) (l) The average 561/488 ratios of cells treated with Reversine (0.5μM, 48h). (n=3 independent experiments) (m) Immunoblots of indicated cell lines treated as in panel l. Mean ± S.E.M. is shown in panel b,d,e,g,l. (****p<0.0001, ***p<0.001, **p<0.01, panel b:One-way ANOVA, panel d,e,g,l:Two-way ANOVA) All experiments were repeated three times unless otherwise indicated. Statistical source data are in Supplementary Table 2. Unprocessed original scans are shown in Supplementary Fig. 6.