Fig. 6: Tamoxifen reduces mitochondrial ATP generation by inhibiting respiratory complex I.
a Supra-resolution AiryScan2 confocal imaging of fluorescent tamoxifen derivative (FLTX1, green) colocalizing with the mitochondrial marker TOM20 (red) in HEL cells. Nucleus was stained with DAPI (blue). ImageJ plugin Colocalization Finder was used to generate a scatter plot of two selected channel intensity and calculate overlap coefficient for selected channels. R > 0.8 indicates significant colocalization. The analysis has been independently repeated three times, obtaining similar results. b 4OH-TAM dose-dependently inhibits mitochondrial respiration in permeabilized JAK2V617F-mutant cells (n = 10 independent experiments). Average oxygen consumption rate (OCR) after treatment with plasma membrane permeabilizer, 4OH-TAM or vehicle, and 5 mM Pyruvate (Pyr), 2.5 mM malate and 1 mM adenosine diphosphate (ADP). c Dose-dependent inhibition of complex I NADH:O2 oxidoreduction rate by 4OH-TAM in absence or presence of antimycin (anti) to inhibit complex III (n = 4 independent experiments). d, e Dose-dependent accumulation of 4OH-TAM in the mitochondria of MPN cell lines 24 h after 1 mM (d) and 10 mM 4OH-TAM treatment (e). Note 4-fold higher mitochondrial 4OH-TAM concentration in sensitive (serum-deprived), compared with resistant (grown in competent medium) UKE-1 cells (n = 3 independent experiments). f, g Complex II activation with monomethyl succinate rescues JAK2V617F-mutant HEL (f) and SET2 (g) cells from 4OH-TAM-induced cell death (n = 3 independent experiments). h, i OCR in HEL cells treated with 4OH-TAM or vehicle alone, or in combination with MMS (n = 8 independent experiments). The rescue of cell viability by complex II activation is explained by compensatory increase of mitochondrial respiration and ATP synthesis. d-i Data are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, Two-way ANOVA and Dunnett’s test.
