Fig. 5

Effects of over-expression of yeast ATPase proton pump, PMA1, in MCF-7 breast cancer cells in vitro. A Immunoblotting of protein lysates from empty vector (MOCK-1) or PMA1 vector (C1 and C5) transfected MCF-7 cells for PMA1 and GAPDH (loading control). B Representative immunocytochemistry image of PMA1 expression (yellow), overlayed with DAPI nuclear stain (blue), in PMA1-C5 non-permeabilized MCF-7 cells. C Seahorse extracellular flux analysis of glucose-induced proton production rate in MCF-7 parental, MOCK-1, PMA1-C1, and PMA1-C5 MCF-7 cells (n = 9 biological replicates). D Seahorse extracellular flux analysis of basal oxygen consumption in the presence of glucose for MCF-7 parental, MOCK-1, PMA1-C1, and PMA1-C5 MCF-7 cells (n = 20 biological replicates). E Glucose concentration in tumor-conditioned media collected after culturing to confluence from MCF-7 parental, MOCK-1, PMA1-C1, and PMA1-C5 MCF-7 cells for 24 h, measured using hexokinase activity assay (n = 9 biological replicates). F Lactate concentration in tumor-conditioned media collected after culturing to confluence from MCF-7 parental, MOCK-1, PMA1-C1, and PMA1-C5 MCF-7 cells for 24 h, measured using fluorescent lactate assay (n = 9 biological replicates). G Gel escape assay to measure migration and invasion in MOCK-1, PMA1-C1, and PMA1-C5 MCF-7 cells. Cells are embedded in a Matrigel droplet without serum, and they are surrounded by media containing serum. Droplets are monitored over 1 week for cell invasion out of the droplet and the area measured is normalized to cellular proliferation rates (n = 4 biological replicates). H Circular wound healing assay of MOCK-1, PMA1-C1, and PMA1-C5 MCF-7 cells. A circular wound is created with a rubber stopper and then cells are monitored migrating into the area to close the wound. The area healed is quantified in % relative to starting area and normalized to the cellular proliferation rate (N = 4 biological replicates). Data are shown as mean ± SD. Statistical analysis using ordinary one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001