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Additional file 4: Figure S4. Gating strategy for Cyto-ID-based cell sorting. Debris and aggregates were excluded from the sorting using a sequential gating strategy relying on FSC-A versus SSC-A followed by FSC-H versus FSC-W and SSC-H versus SSC-W. Dead cells were excluded by gating on Sytox Blue-negative cells. Cells were sorted into three subpopulations of approximately equal number based on their Cyto-ID fluorescence intensities, i.e., into populations with low, medium and high Cyto-ID fluorescence. The numbers within the plots indicate the percentages of the respective parent population

Additional file 6: Figure S6. Metabolic phenotype of Cyto-ID-sorted cells. Cells were flow-cytometrically sorted on the basis of their Cyto-ID fluorescence intensity into subpopulations with low, medium and high Cyto-ID fluorescence (AutLO, AutME and AutHI, respectively). Oxygen consumption (OCR) and extracellular acidification rates (ECAR) were measured at basal conditions and after sequential injection of oligomycin (ATP synthase inhibitor), 2,4-dinitrophenol (DNP; oxidative phosphorylation uncoupler) and antimycin A (cytochrome c reductase inhibitor). (A) Higher basal mitochondrial respiration and ATP production in AutHI compared with AutLO and AutME. OCR as a proxy for oxidative metabolism was measured with the Seahorse Cell Mito Stress Test. (B) Higher glycolytic activity in AutHI compared with AutLO and AutME. ECAR was measured as a proxy for glycolytic activity. OCR and ECAR curves are representative of three independent measurements; each curve consisted of six readings.

Additional file 1: Figure S1. Autophagy inhibition affects etoposide-induced cell cycle arrest. Cells were exposed to etoposide with or without 3-MA for 24 h (Jurkat) or 48 h (MOLM-13). Cell cycle phases were determined by flow-cytometric analysis of DRAQ5-stained cells. Means ± SEM of each three separate measurements are shown.

Additional file 5: Figure S5. Prerequisites for Cyto-ID-based cell sorting. (A) Toxicity of Cyto-ID. Cells were incubated with Cyto-ID at a dilution of 1:1000 for 4 h. Cell death was determined by flow-cytometric analysis of PI uptake. (B) Stability of Cyto-ID fluorescence. Cells were incubated with Cyto-ID at a dilution of 1:1000 at 4, 23 and 37 °C for the indicated times. Autophagy was determined by flow-cytometric analysis of Cyto-ID-stained cells. (C) Effect of sorting on Cyto-ID fluorescence. Autophagy of unsorted and Cyto-ID-sorted cells was determined by flow-cytometric analysis of Cyto-ID-stained cells approximately 1 h after sorting. Cyto-ID fluorescence intensities were normalized to the mean Cyto-ID fluorescence intensities of unsorted cells. (D) Relationship of cell size and Cyto-ID fluorescence intensity. Cell size is proportional to FSC, autophagy was determined by flow-cytometric analysis of Cyto-ID-stained cells. Dot plots are representative of three independent measurements. Cyto-ID fluorescence intensities and FSC values were normalized to the mean Cyto-ID fluorescence intensities and mean FSC values, respectively, of "low Cyto-ID" cells. Means ± SEM of each three or two (B, MOLM-13) separate measurements are shown.

Additional file 2: Figure S2. Chloroquine enhances etoposide-induced increase in Cyto-ID fluorescence intensity. After a 24-h treatment with etoposide, cells were exposed to 10 µM (Jurkat) or 25 µM (MOLM-13) chloroquine for the indicated times. Autophagy was determined by flow-cytometric analysis of Cyto-ID-stained cells. Cyto-ID fluorescence intensities were normalized to the mean Cyto-ID fluorescence intensities of untreated cells. Means ± SEM of each four separate measurements are shown (etoposide versus control: *P < 0.05; etoposide plus chloroquine versus etoposide without chloroquine: #P < 0.05).

Additional file 8: Figure S8. Full immunoblot images. Black boxes indicate the cropped portion of each immunoblot shown in the corresponding main figures.

Additional file 7: Figure S7. Autophagy in DRAQ5-sorted cells. Cells were flow-cytometrically sorted on the basis of their DRAQ5 fluorescence intensity into G1, S and G2/M phase cells. Autophagy was determined by flow-cytometric analysis of Cyto-ID-stained cells. Cyto-ID fluorescence intensities were normalized to the Cyto-ID fluorescence intensities of G1 phase cells. Means ± SEM of each three separate measurements are shown (*P < 0.05).

Additional file 3: Figure S3. Cell cycle phase and autophagy are tightly associated. Autophagy and cell cycle phases were determined by flow-cytometric analysis of Cyto-ID + DRAQ5 double-stained cells. (A) Cell populations were grouped into quintiles based on their Cyto-ID fluorescence intensities. (B) Cell cycle histograms of each quintile of cells. Dot plots and histograms are representative of three independent measurements.

Additional file 6: Figure S6. Accumulation of intracellular defects in WE-68 cells. WE-68 cells were treated with 45 nM AUY922 (B), 2 µM VE821 (C) and their combination (D). DMSO was used for control (A). Intracellular structures were analyzed by transmission electron microscopy (TEM) and are labeled in red: lysosome (lyso), mitochondria (mito), endoplasmic reticulum (ER), nucleus (nuc), autophagosomes (auto), β-glycagon granule (glycagon), lipid droplets, vesicles and lipid-filled vesicles. Red arrows indicate sites of cell rupture. TEM pictures show one representative experiment.

Additional file 4: Figure S4. Analysis of molecular alterations. (A) Otherwise isogenic p53 wild-type (wt) and p53 null (p53-/-) HCT116 cells were treated with 45 nM AUY922 ± 2 µM VE821 for 24 h. Analysis of indicated proteins was done by Western blot. ⍺-tubulin and vinculin were used to control protein loading. Immunoblots are representative for at least two independent experiments. (B) Schematic workflow of the BALB/c cell transformation assay (BALB-CTA). WE-68 cells were treated with 30 nM of AUY922, 1 µM of VE821, 7.5 µM of KU55933 and their combinations; A673 cells were treated with 15 nM of AUY922, 1 µM of VE821, 5 µM of KU55933 and their combinations. DMSO was used for control. (C-D) The mRNA expression of indicated gene was analyzed after 24 h by qPCR. All graphs show the mean ± SEM of three independent experiments (* p

Additional file 3: Figure S3: Analysis of cell cycle distribution. (A) WE-68 and (B) A673 cells were treated with 15–45 nM of AUY922, 2 µM of VE821, 5 µM of KU55933 and their combinations for 24 h. (C-E) WE-68 and A673 cells were treated with indicated concentrations of AUY922 (C), VE821 (D) or KU55933 (E) for 48 h. Cell cycle distributions were determined by flow-cytometric analysis of PI-stained ethanol-fixed cells. All graphs show the mean ± SEM of at least two independent experiments.

Additional file 7: Figure S7. Accumulation of intracellular defects in A673 cells. A673 cells were treated with 45 nM AUY922 (B), 2 µM VE821 (C) and their combination (D). DMSO was used for control (A). Intracellular structures were analyzed by transmission electron microscopy (TEM) and are labeled in red: lysosome (lyso), mitochondria (mito), endoplasmic reticulum (ER), nucleus (nuc), autophagosomes (auto), β-glycagon granule (glycagon), lipid droplets, vesicles and lipid-filled vesicles. A673 cells were treated with 0.4–5 µg/ml of tunicamycin for 24 h. (E) Analysis of indicated proteins was done by Western blot. α-tubulin was used to control protein loading. TEM pictures show one representative experiment; immunoblots are representative for at least two independent experiments.

Additional file 10: Figure S10. Ingenuity pathway analysis of the proteome data set. A673 cells were treated with 45 nM AUY922 ± 2 µM VE821 for 24 h, and a quantitative whole proteome analysis was done by mass spectrometry from three individual experiments. The proteome data set was further processed by ingenuity pathway analysis (IPA; cutoff: q

Additional file 5: Figure S5. Analysis of ER stress. WE-68 and A673 cells were treated with 15–45 nM of AUY922, 2 µM of VE821, 5 µM of KU55933 and their combinations for 24 h. (A) Intracellular reactive oxygen species (ROS) level were analyzed by flow cytometry using CM-H2DCFDA. Both graphs show the mean ± SEM of three independent experiments. (B) Quantification of LAMP1 analyzed by Western blot (see Fig. 4) is shown representing the mean ± SEM of two independent experiments. (C) Otherwise isogenic p53 wild-type (wt) and p53 null (p53-/-) HCT116 cells were treated with 45 nM AUY922 ± 2 µM VE821 for 24 h. Analysis of indicated proteins was done by Western blot. ⍺-tubulin and vinculin were used to control protein loading. Immunoblots are representative for at least two independent experiments.

Additional file 2: Figure S2: Analysis of cell proliferation. (A) WE-68 and (B) A673 cells were treated with 15–45 nM of AUY922, 5 µM of VE821 and their combinations for up to 72 h. Cell densities were analyzed and quantified using crystal violet staining. A-B show the mean ± SEM of three independent experiments (*p