HL-60 and MX2 cells were grown to 70% confluency and then exposed to either epirubicin (0–0.5 µM) or doxorubicin (0–0.5 µM) in the absence or presence of 20 µM genipin. Amount of cell death was determined by PI assay. Results were expressed as a percent of the control. A) ♦ epirubicin only, ▪ epirubicin + genipin. 1-way ANOVA with a post-hoc Tukey's test, n = 5, * p<0.05. * corresponds to statistical significance when epirubicin and genipin treated cells were compared to control. # corresponds to statistical significance when epirubicin-treated cells were compared to control cells. B) ♦ doxorubicin only, ▪ doxorubicin + genipin. 1-way ANOVA with a post-hoc Tukey's test, n = 5, p<0.05. * corresponds to statistical significance when epirubicin and genipin treated cells were compared to control cells.

ROS levels were assessed in intact HL-60 and MX2 cells using DCFH-DA. A) ROS levels in cells exposed to solely menadione (0–50 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 5, *p<0.05. B) ROS levels in cells exposed to 0 or 20 µM genipin and menadione (0–50 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 5, * p<0.05, **p<0.01.

A) The viability of HL-60 (filled diamond) and MX2 (filled square) following exposure to menadione. Cells were exposed for 24 h to different concentrations of menadione (0–100 µmol/L) followed by the assessment of the number of live cells. Viability was expressed as a percent of the control value. Kruskall-Wallis with a post-hoc Mann-Whitney test, n = 3, p<0.05. * indicates statistical significance for HL-60 cells compared to control and # indicates statistical significance for MX2 compared to control. B) Immunoblot analysis of anti-oxidant defense enzymes and UCP2. Mitochondria from MX2 (M) and HL-60 (H) cells were isolated and analyzed for UCP2, MnSOD, and GPx4. Cytosol was used for G6PDH determinations. NADP-ICDH served as the loading control for the cytosol and mitochondrial fractions. Spleen lysate (100 µg) was used as a UCP2 loading control. The molecular weight of UCP2 was confirmed using molecular mass markers. C) In situ analysis of the specificity of genipin for UCP2. Following the determination of basal oxygen consumption rates, the impact of genipin on OCR in CHO-UCP2 (white bars) cells was assessed. Cells were exposed to (0–50 µM) genipin for 15 min and then OCR was tested. Data was expressed as a % of CHO-EV OCR. INSET: immunoblot analysis of UCP2 in CHO-UCP2 and CHO-EV cells. SDH served as the loading control. Kruskall-Wallis with a post-hoc Mann-Whitney test, n = 3, p<0.05. * indicates statistical significance when compared to the control. D) Assessment of genipin toxicity and the response of drug-sensitive HL-60 and drug-resistant MX2 cells to simultaneous treatment with menadione and genipin. Cells were exposed to genipin (0–500 µmol/L) in the presence or absence of 20 µM menadione for 24 h. Following the exposure, cell viability was determined. ♦ and ▪ represent exposure to either genipin or genipin + menadione. Viability was expressed as a percent of the control. Kruskall-Wallis with a post-hoc Mann-Whitney test, n = 3, p<0.05. * corresponds to statistical significance when cells exposed to both menadione and genipin were compared to control. # corresponds to statistical significance when genipin-exposed cells were compared to the control cells. E) Treatment with genipin and menadione increases MX2 cell death. MX2 cells were exposed to 20 µmol/L menadione and genipin (0–500 µmol/L) for 24 h. The amount of cell death was determined using the PI assay. 1-way ANOVA with a post-hoc Tukey's test, n = 5, p<0.05. * represents significance when compared to 0 µM.

A) Oxygen consumption measurements in cells exposed to 20 µM genipin 24 h exposure. Oxygen consumption measurements were performed following a 30 min incubation in reaction medium in the absence (white bars) or presence (grey bars) of oligomycin. 1-way ANOVA with a post-hoc Tukey's test, n = 6, **p<0.01. Treated MX2 or HL-60 cells were only compared with their corresponding control mean values. B) Oxygen consumption measurements in cells exposed to genipin (20 µM) and/or menadione (20 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 4, *p<0.05,

ROS levels were assessed in intact HL-60 and MX2 cells using DCFH-DA. A) ROS levels in HL-60 cells and MX2 cells exposed to doxorubicin (0–1 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 5, *p<0.05, **p<0.01. B) ROS levels in HL-60 cells and MX2 cells exposed to 20 µM genipin and doxorubicin (0–1 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 5, *p<0.05, **p<0.01.