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A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications

Guo, Sujuan, Liang, Yanping, Murphy, Susan F, Huang, Angela, Shen, Haihong, Kelly, Deborah F, Sobrado, Pablo, Sheng, Zhi·2015·Autophagy
DOI 10.1080/15548627.2015.1017181PMC PMC4502761
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Figure 1. The Cyto-ID dye specifically labeled autophagic compartments with minimal staining of lysosomes and endosomes. Colocalization of the Cyto-ID fluorescence dye, Hoechst 33342, and mCherry-LC3B (A), LAMP1-mRFP (B), or mRFP-RAB5A (C) in HeLa cells treated without or with 10 μM PP242 for 4 h.
Figure 1. The Cyto-ID dye specifically labeled autophagic compartments with minimal staining of lysosomes and endosomes. Colocalization of the Cyto-ID fluorescence dye, Hoechst 33342, and mCherry-LC3B (A), LAMP1-mRFP (B), or mRFP-RAB5A (C) in HeLa cells treated without or with 10 μM PP242 for 4 h.CC BY-NC · PubMed Central
Figure 2. The Cyto-ID dye but not EGFP-LC3 was suitable for spectrophotometry. (A) Light sensitivity of the Cyto-ID dye or EGFP-LC3. HeLa cells transfected with or without EGFP-LC3 were treated with 10 μM PP242 for 4 h. HeLa cells with no EGFP-LC3 were further stained with the Cyto-ID. Images at the same field were taken at different time points (1 to 80 sec) after exposing cells to the light. (B) Quantification of fluorescent dots in images presented in A. The average intensities were obtained from 5 fluorescent dots and the error bars depicts means ± s.d. (C) Fluorescence intensities of the Cyto-ID or EGFP-LC3 recorded by a spectrophotometer at various time points.
Figure 2. The Cyto-ID dye but not EGFP-LC3 was suitable for spectrophotometry. (A) Light sensitivity of the Cyto-ID dye or EGFP-LC3. HeLa cells transfected with or without EGFP-LC3 were treated with 10 μM PP242 for 4 h. HeLa cells with no EGFP-LC3 were further stained with the Cyto-ID. Images at the same field were taken at different time points (1 to 80 sec) after exposing cells to the light. (B) Quantification of fluorescent dots in images presented in A. The average intensities were obtained from 5 fluorescent dots and the error bars depicts means ± s.d. (C) Fluorescence intensities of the Cyto-ID or EGFP-LC3 recorded by a spectrophotometer at various time points.CC BY-NC · PubMed Central
Figure 3. The Cyto-ID assay quantitatively measured the size of autophagic compartments. (A) A schematic outline of the Cyto-ID-based fluorescence spectrophotometric assay. (B) Imatinib-induced autophagy. K562 cells were treated with 1 or 2 μM imatinib for 16 h. (C) PP242-induced autophagy. HEK293 and OVCAR8 cells were treated with PP242 at 2 or 5 μM overnight, respectively. (D) Starvation-induced autophagy. K562 or HEK293 cells were incubated in Earle's Balanced Salt Solution for 30 min or 12 h, respectively. (E) Imatinib-induced autophagy affected by 3-MA. K562 cells were treated with 2 μM imatinib in the absence or presence of 10 mM 3-MA for 4 h. (F) PP242-induced autophagy affected by 3-MA. HEK293 or OVCAR8 cells were treated with 4 or 10 μM PP242, respectively, in the absence or presence of 10 mM 3-MA for 4 h. All experiments were repeated 3 times and error bars depict means ± s.d.; * P < 0.05.
Figure 3. The Cyto-ID assay quantitatively measured the size of autophagic compartments. (A) A schematic outline of the Cyto-ID-based fluorescence spectrophotometric assay. (B) Imatinib-induced autophagy. K562 cells were treated with 1 or 2 μM imatinib for 16 h. (C) PP242-induced autophagy. HEK293 and OVCAR8 cells were treated with PP242 at 2 or 5 μM overnight, respectively. (D) Starvation-induced autophagy. K562 or HEK293 cells were incubated in Earle's Balanced Salt Solution for 30 min or 12 h, respectively. (E) Imatinib-induced autophagy affected by 3-MA. K562 cells were treated with 2 μM imatinib in the absence or presence of 10 mM 3-MA for 4 h. (F) PP242-induced autophagy affected by 3-MA. HEK293 or OVCAR8 cells were treated with 4 or 10 μM PP242, respectively, in the absence or presence of 10 mM 3-MA for 4 h. All experiments were repeated 3 times and error bars depict means ± s.d.; * P < 0.05.CC BY-NC · PubMed Central
Figure 4. The Cyto-ID assay estimated autophagy flux. (A) Imatinib-induced autophagy flux at different time points measured by the Cyto-ID assay. (B) Imatinib-induced autophagy flux at different time points measured by the LC3B or SQSTM1 immunoblotting assay. (C) Chloroquine-blocked autophagy flux at steady state. The chloroquine treatments were 5 μM in HEK293 for 4 h, 20 μM in OVCAR8 for 4 h, or 20 μM in K562 for 4 h, respectively. (D) Chloroquine-blocked autophagy flux at different time points analyzed by the Cyto-ID assay. (E) Chloroquine-blocked autophagy flux at different time points analyzed by the LC3B or SQSTM1 immunoblotting assay. All experiments were repeated 3 times and error bars depict means ± s.d. *P < 0.05.
Figure 4. The Cyto-ID assay estimated autophagy flux. (A) Imatinib-induced autophagy flux at different time points measured by the Cyto-ID assay. (B) Imatinib-induced autophagy flux at different time points measured by the LC3B or SQSTM1 immunoblotting assay. (C) Chloroquine-blocked autophagy flux at steady state. The chloroquine treatments were 5 μM in HEK293 for 4 h, 20 μM in OVCAR8 for 4 h, or 20 μM in K562 for 4 h, respectively. (D) Chloroquine-blocked autophagy flux at different time points analyzed by the Cyto-ID assay. (E) Chloroquine-blocked autophagy flux at different time points analyzed by the LC3B or SQSTM1 immunoblotting assay. All experiments were repeated 3 times and error bars depict means ± s.d. *P < 0.05.CC BY-NC · PubMed Central
Figure 5. The Cyto-ID assay determined the status of autophagy flux. (A) Autophagy flux affected by imatinib and chloroquine. K562 cells were treated with 1 μM imatinib and/or 2.5 μM chloroquine for 6 h. (B) Autophagy flux assessed by the LC3B immunoblotting assay. (C) Autophagy flux affected by PP242 and chloroquine. HEK293 cells were treated with 1 μM PP242 together with 0.5 μM chloroquine for 1 h. OVCAR8 cells were treated with 2 μM PP242 and 0.25 μM chloroquine for 1 h. (D) Autophagy flux assessed by the LC3B immunoblotting assay. All experiments were repeated 3 times and error bars depict means ± s.d.; * P < 0.05.
Figure 5. The Cyto-ID assay determined the status of autophagy flux. (A) Autophagy flux affected by imatinib and chloroquine. K562 cells were treated with 1 μM imatinib and/or 2.5 μM chloroquine for 6 h. (B) Autophagy flux assessed by the LC3B immunoblotting assay. (C) Autophagy flux affected by PP242 and chloroquine. HEK293 cells were treated with 1 μM PP242 together with 0.5 μM chloroquine for 1 h. OVCAR8 cells were treated with 2 μM PP242 and 0.25 μM chloroquine for 1 h. (D) Autophagy flux assessed by the LC3B immunoblotting assay. All experiments were repeated 3 times and error bars depict means ± s.d.; * P < 0.05.CC BY-NC · PubMed Central
Figure 6. The Cyto-ID was more sensitive than MDC in quantifying autophagic compartments. (A) Background levels of the Cyto-ID or MDC fluorescence in nonautophagic K562 cells. (B) Imatinib-induced autophagy. K562 cells were treated with 1 μM imatinib for 16 h and autophagy was assayed by the Cyto-ID- or MDC-based spectrophotometric assay. (c) Chloroquine-blocked autophagy flux in K562 cells.
Figure 6. The Cyto-ID was more sensitive than MDC in quantifying autophagic compartments. (A) Background levels of the Cyto-ID or MDC fluorescence in nonautophagic K562 cells. (B) Imatinib-induced autophagy. K562 cells were treated with 1 μM imatinib for 16 h and autophagy was assayed by the Cyto-ID- or MDC-based spectrophotometric assay. (c) Chloroquine-blocked autophagy flux in K562 cells.CC BY-NC · PubMed Central
Figure 7. The Cyto-ID assay yielded a more reliable quantification of autophagic compartments. (A) PP242-induced autophagy at different time points assessed by the Cyto-ID assay. HEK293 or OVCAR8 cells were treated with PP242 (4 or 10 μM, respectively) at the indicated time points. (B) PP242-induced autophagy at different time points analyzed by the LC3B immunoblotting assay. All experiments were repeated 3 times and error bars depict means ± s.d.
Figure 7. The Cyto-ID assay yielded a more reliable quantification of autophagic compartments. (A) PP242-induced autophagy at different time points assessed by the Cyto-ID assay. HEK293 or OVCAR8 cells were treated with PP242 (4 or 10 μM, respectively) at the indicated time points. (B) PP242-induced autophagy at different time points analyzed by the LC3B immunoblotting assay. All experiments were repeated 3 times and error bars depict means ± s.d.CC BY-NC · PubMed Central
Figure 8. An RNAi screen identified MAP3K6 and WNK2 as autophagy-modulating genes. (A) An RNAi screen. K562 cells were transduced with the viruses of nonsilencing (NS) shRNA or 88 individual shRNAs of human kinases. The line represents the cut-off (2-fold increase). (B) Validation using the LC3B immunoblotting assay. K562 cells were transduced with the viruses harboring nonsilencing (NS) or candidate shRNAs. (C) Knockdown of each candidate gene. The mRNA levels of candidate genes were measured using qRT-PCR. (D) Autophagy flux affected by candidate shRNAs and chloroquine. K562 cells with NS or candidate shRNAs were treated with or without 2.5 μM chloroquine for 4 h. (E) MTOR pathway affected by shRNAs against MAP3K6 or WNK2. All experiments were repeated 3 times and error bars depict means ± s.d.; *P < 0.05.
Figure 8. An RNAi screen identified MAP3K6 and WNK2 as autophagy-modulating genes. (A) An RNAi screen. K562 cells were transduced with the viruses of nonsilencing (NS) shRNA or 88 individual shRNAs of human kinases. The line represents the cut-off (2-fold increase). (B) Validation using the LC3B immunoblotting assay. K562 cells were transduced with the viruses harboring nonsilencing (NS) or candidate shRNAs. (C) Knockdown of each candidate gene. The mRNA levels of candidate genes were measured using qRT-PCR. (D) Autophagy flux affected by candidate shRNAs and chloroquine. K562 cells with NS or candidate shRNAs were treated with or without 2.5 μM chloroquine for 4 h. (E) MTOR pathway affected by shRNAs against MAP3K6 or WNK2. All experiments were repeated 3 times and error bars depict means ± s.d.; *P < 0.05.CC BY-NC · PubMed Central
Figure 9. A small molecule screen identified actinomycin D and sanguinarine as autophagy modulators. (A) PP242-induced autophagy in K562 cells. K562 cells were treated with 10 μM PP242 for 4 h. (B) A small molecule screen. K562 cells were added to a 96-well plate preloaded with either vehicle (DMSO) or 79 chemical compounds and then incubated for 4 h. The line represents the cut-off (2-fold increase). (C) Actinomycin D-induced autophagy in K562 cells. (D) Sanguinarine-induced autophagy in K562 cells. (E) Actinomycin D-affected autophagy flux in K562 cells. K562 cells were treated with a combination of 1 μM actinomycin D and 2.5 μM chloroquine for 4 h. (F) Sanguinarine-affected autophagy flux. K562 cells were treated with a combination of 2 μM sanguinarine and 2.5 μM chloroquine for 4 h. All experiments were repeated 3 times and error bars depict means ± s.d.; *P < 0.05.
Figure 9. A small molecule screen identified actinomycin D and sanguinarine as autophagy modulators. (A) PP242-induced autophagy in K562 cells. K562 cells were treated with 10 μM PP242 for 4 h. (B) A small molecule screen. K562 cells were added to a 96-well plate preloaded with either vehicle (DMSO) or 79 chemical compounds and then incubated for 4 h. The line represents the cut-off (2-fold increase). (C) Actinomycin D-induced autophagy in K562 cells. (D) Sanguinarine-induced autophagy in K562 cells. (E) Actinomycin D-affected autophagy flux in K562 cells. K562 cells were treated with a combination of 1 μM actinomycin D and 2.5 μM chloroquine for 4 h. (F) Sanguinarine-affected autophagy flux. K562 cells were treated with a combination of 2 μM sanguinarine and 2.5 μM chloroquine for 4 h. All experiments were repeated 3 times and error bars depict means ± s.d.; *P < 0.05.CC BY-NC · PubMed Central
Figure 10. Detection of autophagy in mice. (A) PP242 or chloroquine-mediated autophagy in mouse primary peripheral blood cells. Mice were treated with PP242 (60 mg/kg/d, IP) or chloroquine (50 mg/kg/d, IP). (B) Imatinib or chloroquine-affected autophagy in primary bone marrow cells isolated from the mice with BCR-ABL-driven leukemia. Leukemic mice were treated with either imatinib (100 mg/kg/d) or chloroquine (50 mg/kg/every other day) by gavage. There are 3 or 4 mice per treatment group and error bars depict means ± s.d.; * P < 0.05.
Figure 10. Detection of autophagy in mice. (A) PP242 or chloroquine-mediated autophagy in mouse primary peripheral blood cells. Mice were treated with PP242 (60 mg/kg/d, IP) or chloroquine (50 mg/kg/d, IP). (B) Imatinib or chloroquine-affected autophagy in primary bone marrow cells isolated from the mice with BCR-ABL-driven leukemia. Leukemic mice were treated with either imatinib (100 mg/kg/d) or chloroquine (50 mg/kg/every other day) by gavage. There are 3 or 4 mice per treatment group and error bars depict means ± s.d.; * P < 0.05.CC BY-NC · PubMed Central

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Metadata

Authors
Guo, S., Liang, Y., Murphy, S. F., Huang, A., Shen, H., Kelly, D. F., Sobrado, P., & Sheng, Z.
Venue
Autophagy
Publisher
Informa UK Limited
Published
2015-02-25
Volume
11
Issue
3
Pages
560-572
ISSN
  • 1554-8627
  • 1554-8635
DOI
10.1080/15548627.2015.1017181
PMC
PMC4502761
Cited by
148
References
5
Language
en

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