a Chemical structure of TMQ0153. b Time- and c dose-dependent effect of TMQ0153 on K562 cell proliferation (left panel) and viability (right panel). d Inhibitory effect of increasing concentrations of TMQ0153 on the colony forming capacities of the indicated CML cell lines. Upper panel: pictures representative of three independent experiments. Lower panel: quantification of the colony numbers. a–d All data represent mean (±S.D.) of three independent experiments. e Acute toxicity assay on zebrafish embryos after 24 h of treatment with increasing concentrations of TMQ0153. Pictures are representative of 10 fishes used for each condition (left panel) and the corresponding quantification of viable embryos (right panel). Ethanol (EtOH, 70%) was used as a positive control for toxicity. f Cytotoxicity of TMQ0153 on human peripheral blood mononuclear cells (PBMCs) by Trypan blue assay after 24, 48, and 72 h of treatment (PBMC data represent the mean (±S.D.) of five independent experiments). Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated cells. Two-way ANOVA (Cell viability); post hoc: Sidak’s test. One-way ANOVA (Colony formation); post hoc: Sidak’s test. Two-way ANOVA (PBMC toxicity); post hoc: Dunnett’s test.

a–d K562 cells were treated with various concentrations of TMQ0153 in presence or absence of the pan caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (z-VAD; 50 μM). a After 8, 24, 48, and 72 h of treatment the type of cell death triggered by TMQ0153 was characterized by fluorescence microscopy after Hoechst/propidium iodide (PI) staining. Pictures representative of three independent experiments (top panel). Etoposide (Eto; 100 µM, 24 h) was used as a positive control for apoptosis induction. Scale bar: 25 µM. b Analysis of caspase and poly [ADP-ribose] polymerase (PARP)-1 cleavage by western blot after 24 h of treatment. c Effect of 24 h of treatment on Mcl-1 and Bcl-xL protein expression levels (top panels) and the corresponding densitometric analysis (middle and lower panels). b, c β–actin was used as loading control. d Quantification of caspase-3/7 activity levels after 24 h of treatment. Etoposide (Eto; 100 µM, 24 h) was used as a positive control for apoptosis induction. All pictures are representative of three independent experiments and graphs represent the mean (±S.D.) of three independent experiments. Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated cells. Two-way ANOVA (microscopy analysis); post hoc: Sidak’s test. One-way ANOVA (caspase-3/7 assay); post hoc: Tukey’s test. One-way ANOVA (western blot quantification); post hoc: Dunnett’s test.

a K562 cells were incubated in presence or absence of 60 µM necrostatin (Nec)-1 for 1 h before a treatment with the indicated concentrations of TMQ0153. Shikonin (SHK; 5 µM) was used as a positive control for necrosis induction. a Nuclear morphology analyses by fluorescence microscopy following Hoechst/ propidium iodide (PI) staining after 8 and 24 h of treatment. Pictures representative of three independent experiments (left panels) and the corresponding quantification (right panels). b Measurement of intracellular ATP levels at the indicated concentrations and time points. c Receptor-interacting protein kinase (RIP)1 protein level and PARP-1 cleavage (left panel) were determined by western blotting using C2-10 antibody and the corresponding densitometric analysis (right panel). Shikonin (SHK; 5 µM, 24 h) and necrostatin (Nec)−1 (60 µM, 1 h) were used as a positive control and inhibitor for RIP1. Etoposide (Eto; 100 µM, 24 h) and ethanol (EtOH; 10 %, 2 h) were used as positive controls for apoptotic and necrotic PARP-1 cleavage, respectively. d Cytosolic Ca2⁺ levels were measured using Fluo-3-AM after 24 h (left panel) and time-dependently measured Ca²⁺ levels at 30 μM (right panel). Thapsigargin (TSG; 300 nM, 24 h) was used as a positive control for intracellular Ca²⁺ accumulation. β–actin was used as loading control. All pictures are representative of three independent experiments and all graphs represent the mean (±S.D.) of three independent experiments. Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons. Two-way ANOVA (microscopy analysis, cell viability); post hoc: Sidak’s test. Two-way ANOVA (Cell Titer Glo assay); post hoc; Tukey’s test. One-way ANOVA (intracellular Ca²⁺ assay); post hoc; Dunnett’s test. One-way ANOVA (western blot quantification); post hoc; Dunnett’s test.

a Confocal UV microscopy analysis after staining with Cyto-ID (left panel). Representative images of FITC stained images in the 4 groups accompanied by the corresponding pseudocolor masks (red: FITC signal, green: cell area) used for calculation and the corresponding quantification of fluorescence intensity (right panel). Statistical results were compared by Kruskal–Wallis test followed by Conover post-test further adjusted by the Benjamini-Hochberg FDR method. 0 h vs. 4 h and vs 8 h (p < 0.0004 and p < 0.00006, respectively), 4 h vs.8 h (p < 0.002). b Western blot detection of LC3 and p62 protein levels (left panel) and the corresponding densitometric analysis (right panel). c Similar analysis in cells pretreated with 40 nM bafilomycin A1 (baf-A1) for 1 h (upper panel) and the corresponding densitometric analysis (lower panel). 10 µM PP242 for 4 h was used as a positive control for autophagy induction. d Transmission electron microscopy at ×12.000 and ×25.000 magnification: arrows indicate autophagolysosomes. Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated cells unless otherwise specified. One-way ANOVA (western blot quantification); post hoc; Tukey’s test. Two-way ANOVA (mito stress test); post hoc; Sidak’s test.

a Oxygen consumption rate (OCR) was measured by Seahorse XFp analyzer. b Caspase 8 analysis by western blot (upper panel) and the corresponding densitometric analysis (lower panel). Etoposide (Eto; 100 µM, 24 h) was used as a positive control for apoptotic caspase cleavage. c Caspase 8 analysis by western blot (upper panel) in the presence or absence of autophagy inhibitors: 40 nM baf-A1, 10 mM, 3-methyladenine (3-MA) and 75 μM chloroquine (CQ) and the corresponding densitometric analysis (lower panel). In western blot analyses, β-actin was used as a loading control. d Quantification of caspases-3/7 (left graph) and -9 activity (right graph) levels at 30 µM of TMQ0153 treatment. Etoposide (Eto; 100 µM, 24 h) was used as a positive control for apoptosis induction. All pictures are representative of three independent experiments and data represent mean (±S.D.) of three independent experiments. Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated cells unless otherwise specified. One-way ANOVA (Caspase-3/7 and -9 assay); post hoc; Tukey’s test. Two-way ANOVA (mito stress test); post hoc; Sidak’s test. One-way ANOVA (western blot quantification); post hoc; Dunnett’s test.

a Effect of autophagy inhibitors (40 nM baf-A1, 10 mM, 3-methyladenine (3-MA) and 75 μM chloroquine (CQ)) on death of K562 cells treated with 30 µM of TMQ0153 assessed by nuclear morphology analysis after 8 h of treatment. b-e K562 cells were transfected with specific small interfering (si)RNAs against beclin 1 [(SiBCN-1), 5 nM SiBCN-1_1 and 10 nM SiBCN-1_2] for 24 h. b Upper panel: effect of siRNA on beclin 1 protein expression level. After quantification of the bands, beclin 1 levels were normalized to β-actin. Lower panel: transfected cells were treated with 30 μM of TMQ0153 and a nuclear morphology analysis was carried out after 8 h of treatment. c-e Effect of siRNA on TMQ0153-induced autophagy quantified by flow cytometry after Cyto-ID staining (c); on caspase-3/7 activity (d), and PARP-1 cleavage using C2-10 antibody (e). Etoposide (Eto; 100 µM, 24 h) and EtOH (10 %, 2 h) were used as positive controls for apoptotic and necrotic PARP-1 cleavage (upper panel) and the corresponding densitometric analysis of necrotic cleavage (lower panel) is visualized. In western blot analyses, β-actin was used as a loading control. All pictures are representative of three independent experiments and data represent mean (±S.D.) of three independent experiments Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons. Two-way ANOVA (nuclear morphology analysis); post hoc; Sidak’s test. One-way ANOVA (Cyto-ID assay); post hoc; Dunnett’s test. One-way ANOVA (caspase 3/7 assay); post hoc; Tukey’s test. One-way ANOVA (western blot quantification); post hoc; Dunnett’s test.

a Cells were treated with TMQ0153 for 8 h and mitochondrial morphology was assessed by TEM at 12.000x and 25.000x magnification. Single arrows and double arrows indicate respectively dilated and giant mitochondria, asterisks indicate autophagophores. b Mitochondrial membrane potential (MMP) analysis in cells treated with increasing concentrations of TMQ0153 for 24 h. The fraction of low MMP presenting cells is depicted. c Cells were pre-incubated for 1 h in presence or absence of 50 mM N-acetyl-L-cysteine (NAC) followed by a treatment with the indicated concentrations of TMQ0153. After 24 h of treatment, MMP (upper panel) cell viability (bottom panel) were assessed by trypan blue assay and flow cytometry, respectively. All pictures are representative of three independent experiments and data represent the mean (±S.D.) of three independent experiments. Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated cells unless otherwise specified. One-way ANOVA (mitochondrial membrane); post hoc; Dunnett’s test. One-way ANOVA (cell viability); post hoc; Tukey’s test.

a Cells were pre-incubated for 1 h in presence or absence of 50 mM NAC or 60 µM necrostatin (Nec)−1. After 4 and 8 h of treatment with TMQ0153 at 20 and 30 µM, reactive oxygen species (ROS) levels were measured by flow cytometry following dichlorofluorescein diacetate (H₂DCFDA) staining. H₂O₂ was used as a positive control for ROS induction. b Quantification of total GSH levels (left panel) and GSH (glutathione)/glutathione disulfide (GSSG) ratio (right panel). 50 µM Buthionine sulfoximine (BSO) was used as a positive control for the inhibition of GSH synthesis. c Cells were stained with Hoechst and Lysotracker Red and analyzed by fluorescence microscopy. Lysotracker Red fluorescence intensity was quantified using Image J 1.8.0 software (upper panel). Lysotracker Red intensity was quantified by FACS (bottom panel). Chloroquine (CQ; 75 μM, 4 h), PP242 (PP, 10 μM, 4 h) and baf-A1 (40 nM, 4 h) were used as a positive and negative controls for autophagy inhibition and induction, respectively. All pictures are representative of three independent experiments and data represent the mean (±S.D.) of three independent experiments. Statistical significance was assessed as *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated cells unless otherwise specified. One-way ANOVA (LMP); post hoc; Dunnett’s test. One-way ANOVA (ROS, GSH assay); post hoc; Tukey’s test.