Fig. 3.

Experimental proof of concept. a pHi measurements of naïve and acid-adapted (AA) MCF7 breast cancer cells, under different oxygen availability, and following inhibition of MCT1/2. For pHi measurments at least 30 cells were analyzed. b Efficiency of knockdown of GAPDH and GPI mRNA and protein following transfection of MCF7 cells with the respective siRNAs (72 h). PCR was done in three separate biological replicate with three repeats. c Proliferation assays of cells (Methods) transfected with siRNA targeting GAPDH and GPI, across four conditions: normoxia, hypoxia, each at physiological pHe (7.4), or acidic pHe (6.7). Drop in proliferation following inhibition of MCT1/2 is shown as connected lines. Lowest values are obtained when pHi is low (yellow grids). The amplified effect of gene inhibition is seen relative to control (color vs. black). Viability assays is done in three replicates and three reads for each time point. d Viability assays (Methods) demonstrate that when pHi is sufficiently low the predicted strategy is particularly effective against AA cancer cells. The inhibition of GAPDH and GPI achieve efficient killing of cells at low pHi (yellow grids). Note the large slopes obtained for AA cells. The strategy is selective (Supplementary Figure 14), however, fails when sufficiently low pHi is unattainable, as in the case of triple negative breast cancer cells (Supplementary Figure 15). Bars depict the error of the mean over replicas. The experiments were repeated three times with three replicas for each condition