Fig. 4: Copper depletion disrupts mitochondrial respiration in SOX2/OCT4+ metastatic cells.

a Impact of TM (0.5 µM) on oxygen consumption rate of sorted GFP+ and GFP− ML1 cells as measured by Seahorse assay (n = 8/group). Quantification of basal respiration (b) and ATP-linked respiration (c) in GFP+ vs. GFP- ML1 cells with and without TM (0.5 µM). Significance was calculated using one-way ANOVA with Tukey post-test for comparing multiple groups. Center lines of box plots denote median values, top whiskers denote maxima and bottom whiskers minima. d Ratio of mitochondrial to glycolytic ATP production rate in GFP+ vs. GFP− ML1 cells, measured by Seahorse ATP production rate assay (n = 8/group). Significance was calculated using one-way ANOVA with Tukey post-test for comparing multiple groups. Representative data of two independent experiments are depicted. e Representative IF microscopy images from control and TM-treated (0.7 mg/day) primary LM2 tumors in the mammary gland (n = 5/group). Scale bar, 20 µM. f Flow cytometry analysis showing the percentage of GFP+ cells in primary tumors in the LM2 model (n = 12/group) of total mCherry+ tumor cells. Analysis was performed by unpaired two-sided t-test. p-value = 0.0111. g Disseminated tumor cells in early lungs of control and TM (0.7 mg/day) treated LM2 cohorts. Analysis was performed by unpaired two-sided t-test. p-value = 0.0150. Representative data of two independent experiments are depicted. Results are expressed as mean ± SD. Results are expressed as mean ± SD. **p < 0.01, ****p < 0.0001.