Extended Data Figure 7 |. LACTB-induced effects on mitochondrial function.
a, Measurements of ATP levels in MCF7-RAS cells upon LACTB induction. b, Measurements of ROS levels in MCF7-RAS cells upon LACTB induction. Numbers within the graphs represent percentages of gated cells. c, Measurements of mitochondrial membrane potential, through incorporation of the cyanine dye DiIC1(5), by flow cytometry in MCF7-RAS cells upon LACTB induction. Numbers within the graphs represent percentages of gated cells. d, Immunofluorescence analysis of control MCF7-RAS cells mixed with MCF7-RAS–Tet/ON-LACTB cells, where LACTB was induced by addition of DOX for 1 day. Cells were stained with a mitochondrial marker (green), a LACTB marker (red) and DAPI (blue). Mitochondrial signal per area in control cells (n = 16) and in LACTB-expressing cells (n = 17) was calculated using ImageJ software. NS, not significant (P > 0.05). Scale bar 30 μm. e, Western blot analysis of sub-fractionated control MCF7-RAS cells and MCF7-RAS–Tet/ON-LACTB-expressing cells with 24 h of DOX treatment. CYT, cytosolic fraction, MITO, mitochondrial fraction. Membranes were probed for proteins involved in mitochondrial fusion (OPA1, MFN1, MFN2), fission (FISI, DRP1), composition of respiratory chain (individual OXPHOS components) and control antibodies: LACTB (to show the proper induction and localization of LACTB), actin (cytosolic marker) and COX4 (mitochondrial marker). The membrane presented here was also used in Fig. 5a, where it was probed with a different set of antibodies. Therefore the signal for the control antibodies is shared between these two figures. f, Electron microscopy images of mitochondria in control MCF7-RAS cells or MCF7-RAS cells where LACTB was induced for 1 or 3 days. Arrows indicate mitochondria. Scale bars, 600 nm. Data are mean ± s.e.m. (a, d).